Retracting and seeking movements during laparoscopic goal-oriented movements . Is the shortest path length optimal ? Aims Minimally invasive surgery -LRB- MIS -RRB- requires a high degree of eye -- hand coordination from the surgeon . To facilitate the learning process , objective assessment systems based on analysis of the instruments ' motion are being developed . To investigate the influence of performance on motion characteristics , we examined goal-oriented movements in a box trainer . In general , goal-oriented movements consist of a retracting and a seeking phase , and are , however , not performed via the shortest path length . Therefore , we hypothesized that the shortest path is not an optimal concept in MIS . Currently , there are various virtual-reality -LRB- VR -RRB- trainers available to train MIS skills -LSB- 1 , 2 -RSB- . These trainers have the advantage over box trainers of providing objective feedback about the performance of the user -LRB- performance parameters -RRB- , which motivates residents to learn -LSB- 3 -- 5 -RSB- . The most common parameter used to measure basic MIS skills objectively is task completion time . However , this parameter alone does not adequately evaluate the task performance -LSB- 6 -RSB- . Therefore , the time for completion is often used in conjunction with other parameters -LSB- 1 , 7 -- 12 -RSB- . One of the task-independent parameters that is used in each VR trainer is path length -- the length of the curve described by the tip of the instrument over time -LSB- 13 -RSB- . Intuitively , a longer path length indicates a less-efficient movement and is often interpreted as lack of experience . In MIS , goal-oriented movements -LRB- point-to-point movements -RRB- are very common -LRB- e.g. , during grasping , placing a clip on the vessel , or while using diathermy -RRB- . In general , a simple strategy to perform such a movement is to move along a straight line between an initial position and a specified target position -LSB- 14 , 15 -RSB- . However , it is hardly possible to make such a movement during MIS . Movements of MIS instruments are done through the incision point , which acts as a pivot point . Therefore , point-to-point aiming movements in MIS may simply result in a curved workspace path -LRB- Fig. 1 -RRB- . Both these ways of performing point-to-point movements are very efficient . In general , however , goal-oriented movements are not performed via the shortest path ; a pull-back movement is performed before the specified target position is approached -LSB- 16 , 17 -RSB- . Therefore , we hypothesized that the shortest path , as presently used to indicate more-efficient performance in MIS , is not a suitable reference . Fig. 1Three strategies of performing a goal-oriented movement in MIS . -LRB- a -RRB- a movement along the straight line between the initial and the end position ; -LRB- b -RRB- a movement along a curved path between the initial and the end position , which is the result of the movement of the MIS instrument through the incision point ; and -LRB- c -RRB- a realistic movement performed by the surgeon during the goal-oriented task . A -- the initial position ; B -- the end position ; P -- incision -LRB- pivoting -RRB- point The objective of this study was to examine goal-oriented movements during training of the basic MIS skills . The study consisted of two parts : first , we investigate whether goal-oriented movements can be split into two phases : a retracting phase and a seeking phase ; and , subsequently , we investigate whether movements of the MIS instrument in these two phases depend on the surgeon 's experience . The performance of the participants was analyzed and compared using normalized path lengths . Methods Participants Experienced gynaecologists -LRB- with the experience of more than 100 MIS procedures -RRB- , their residents -LRB- experience of 10 -- 100 MIS procedures -RRB- , and novices -LRB- medical students with no previous experience in MIS -RRB- were invited to participate in this study . Each participant , voluntarily enrolled in this study , was asked to complete a short questionnaire detailing demographic information and prior experience in laparoscopy . Task The hypothesis was tested using a simple one-hand positioning task , which requires touching the top of eight cylinders with the tip of the MIS instrument -LRB- Fig. 2 -RRB- . All cylinders were situated in a box trainer in various three-dimensional -LRB- 3D -RRB- positions . Every correctly touched cylinder resulted in the lighting of the lamp corresponding to this cylinder . The start/end point and the order of touching cylinders -LRB- indicated by the numbers located next to the cylinders -RRB- were the same for each participant . Fig. 2The positioning task . The task requires touching the top of the eight cylinders -LRB- of varying 3D position -RRB- with the tip of the MIS instrument . A correctly touched cylinder results in lighting up a lamp -LRB- above on the picture -RRB- corresponding to this cylinder . The start/end point and the order of touching the cylinders are indicated by the numbers located next to the cylinders To mimic the in vivo gynaecological MIS situation , all participants performed the task with their left hand , while the camera was held in the right hand . To provide the same conditions for each participant , the position of the task and the incision points for the camera and the MIS instrument were standardized in the box trainer . The image of a 0 ° laparoscope was presented on a monitor . Before the test , all participants were instructed how to perform the positioning task . Furthermore , they were allowed to make one trial before testing . Data analysis Movements of the MIS instrument were recorded with our newly developed TrEndo tracking system with a sample frequency of 100 Hz -LSB- 18 -RSB- . The data of the eight simple goal-oriented movements performed by each participant during the task were analyzed . The first goal-oriented movement was defined by the movement between the start point and first cylinder . Each successive goal-oriented movement was defined by the movement between two consecutive cylinders -LRB- Fig. 2 -RRB- . The retracting phase describes the pull-back movement of the MIS instrument in the direction of the pivoting point . Therefore , we analyzed the projected movements of the instrument in the ABP plane -LRB- Fig. 3 -RRB- , which passes through the begin point of the movement -LRB- point A -RRB- , the end point of the movement -LRB- point B -RRB- , and the pivoting point -LRB- point P -RRB- . To examine the deviation of the movement from the plane ABP , we also analyzed the movements projected in the plane AB , which is perpendicular to the plane ABP , and which passes through the points A and B -LRB- Fig. 3 -RRB- . Point M , defined as the point of the movement most distant from the line AB , was used to make a distinction between the retracting phase AM -LRB- from point A to point M -RRB- and the seeking phase MB -LRB- from point M to point B -RRB- . The analysis of each of the two phases was done using a normalized path length , which was derived as follows : where PLreal is the real path length -LRB- between A and M for retracting , and M and B for seeking phase -RRB- , and PL0 is the distance -LRB- length of the straight line -RRB- between A and M and M and B -LRB- for retracting , and seeking phases , respectively -RRB- . The normalized path lengths of all eight successive goal-oriented movements were averaged per participant and analyzed . Fig. 3The ABP -LRB- described by the points A , B , and P -RRB- and AB -LRB- that passes through points A and B , and is perpendicular to the plane ABP -RRB- planes presented in an orthographic projection : -LRB- a -RRB- the ABP and AB planes , and the goal-oriented movement presented in the three-dimensional space ; -LRB- b -RRB- front view : projection of the goal-oriented movement in the ABP plane ; -LRB- c -RRB- top view : projection of the goal-oriented movement in the AB plane ; and -LRB- d -RRB- side view : the ABP and AB planes , and the goal-oriented movement presented in a side view . A -- the initial position ; B -- the end position ; M -- a point , which is used to make a distinction between the retracting and the seeking phases ; P -- incision -LRB- pivoting -RRB- point Statistics Data were analyzed using the Statistics Toolbox of MATLAB 7 . Statistical analysis was performed using one-way analysis of variance -LRB- ANOVA -RRB- , and Wilcoxon tests . A probability p < 0.05 was considered to be statistically significant . Results Participants Thirty-eight participants completed the task and the questionnaire . The group of experts consisted of 9 experts -LRB- age 38 -- 59 years -RRB- . The group of residents consisted of 17 gynaecological residents -LRB- age 29 -- 41 years -RRB- . The group of novices consisted of 12 medical students -LRB- age 23 -- 28 years -RRB- . All participants were right-handed . Retracting and seeking phases Typical trajectories for an expert and a novice performing the goal-oriented movement are presented in Fig. 4 . The figure shows that the retracting and seeking phases can easily be distinguished from each other . No significant difference between the experts , residents , and novices was observed in the distance between point M and the line AB . Fig. 4The typical trajectories for an expert -LRB- left -RRB- and a novice -LRB- right -RRB- performing a goal-oriented movement . Top : projection of the goal-oriented movement in the plane ABP . Bottom : projection of the goal-oriented movement in the plane AB . A -- the initial position ; B -- the end position ; M -- a point , which is used to make a distinction between the retracting and the seeking phases Normalized path lengths of the two phases are presented in Fig. 5 . In 3D space , the experts ' normalized path length was shorter during the retracting phase -LSB- median -LRB- range -RRB- % : 152 -LRB- 129 -- 178 -RRB- -RSB- than during the seeking phase -LSB- 180 -LRB- 172 -- 247 -RRB- -RSB- . Similarly , the residents ' and novices ' path lengths were shorter during the retracting phase -LSB- 164 -LRB- 126 -- 250 -RRB- , and 168 -LRB- 136 -- 268 -RRB- -RSB- than during the seeking phase -LSB- 201 -LRB- 163 -- 287 -RRB- , and 290 -LRB- 244 -- 469 -RRB- , respectively -RSB- . Fig. 5Normalized path lengths of the retracting and the seeking phases for experts , residents , and novices . Upper : normalized path length in 3D space . Middle : normalized path length in the ABP plane . Lower : normalized path length in the AB plane . The results are presented as notched box-and-whisker plots , where every box has a line at every quartile , median , and upper quartile values . The whiskers are presented as lines that extend from each end of the box in order to show the extent of the rest of the data . The notches represent the 95 % confidence interval for the median . Boxes whose notches do not overlap are significantly different -LRB- p < 0.05 -RRB- . ** p < 0.01 ; *** p < 0.001 ; E -- experts ; R -- residents ; N -- novices ; 1 -- retracting phase ; 2 -- seeking phase In the ABP plane , the experts ' normalized path length in the retracting phase -LSB- 132 -LRB- 120 -- 194 -RRB- -RSB- was significantly shorter than in the seeking phase -LSB- 199 -LRB- 154 -- 234 -RRB- -RSB- . The normalized path lengths of residents and novices were also significantly shorter in the retracting phase -LSB- 133 -LRB- 108 -- 492 -RRB- , and 180 -LRB- 118 -- 287 -RRB- -RSB- than in the seeking phase -LSB- 208 -LRB- 162 -- 286 -RRB- , and 310 -LRB- 252 -- 469 -RRB- -RSB- . In the AB plane , the experts ' normalized path length was significantly shorter in the retracting phase -LSB- 122 -LRB- 100 -- 200 -RRB- -RSB- than in the seeking phase -LSB- 183 -LRB- 141 -- 209 -RRB- -RSB- . Normalized path lengths of residents and novices were also significantly shorter during the retracting phase -LSB- 147 -LRB- 107 -- 264 -RRB- , and 207 -LRB- 128 -- 271 -RRB- -RSB- than during the seeking phase -LSB- 199 -LRB- 162 -- 285 -RRB- , and 310 -LRB- 252 -- 469 -RRB- -RSB- . Influence of the experience The experts ' , residents ' , and novices ' normalized path lengths during the retracting and the seeking phases are presented in Fig. 6 . The data in Fig. 6 are the same as the data in Fig. 5 , but presented per phase for all groups . In 3D space , there was no significant difference between experts , residents , and novices for the retracting phase . The normalized path lengths of experts and residents were significantly shorter than the path length of the novices during the seeking phase . No significant difference between experts and residents was observed in that phase . Fig. 6The influence of the experience on the performance of the retracting and the seeking phases . The data presented in this figure are the same as the data in Fig. 7 , but presented per phase . * p < 0.05 ; *** p < 0.001 ; E -- experts ; R -- residents ; N -- novices ; 1 -- retracting phase ; 2 -- seeking phase In the ABP plane , we found no significant difference between experts , residents , and novices for the retracting phase . In the seeking phase , the normalized path lengths of experts and residents were significantly shorter than the path length of novices . No significant difference between experts and residents was observed in that phase . In the AB plane , a significant difference between experts , residents , and novices was found for both phases . In both the retracting and the seeking phases , the normalized path lengths of the experts and residents were significantly shorter than the path length of the novices . No significant difference between experts and residents was observed for both phases . Discussion Our findings indicate that goal-oriented movements are not performed via the shortest path . The movements clearly distinguish a retracting and a seeking phase . The results show that the normalized path length during the retracting phase is significantly shorter than during the seeking phase . Furthermore , experience in MIS does only influence the seeking phase . Experts ' and residents ' normalized path lengths were significantly shorter than the path lengths of the novices . The shorter path length in the seeking phase implies better performance ; therefore , the seeking phase is characteristic of the differences in performance . This finding confirms that the path length is an important measure in the assessment of basic MIS skills , in which the seeking phase is the only component that can distinguish between novice and expert . The retracting phase is essential in MIS , because it improves safety of the patient by avoiding unpredicted contact with the tissue . It seems that the best strategy to perform this phase safely is to pull back the instrument along its axis -LRB- in the direction of the pivoting point -RRB- and avoid any movements in the AB plane , which can cause unpredicted contacts with the tissue . The results of this study confirm this assumption ; experts ' and residents ' normalized path lengths are smaller in the AB plane than the path length of the novices . Movements in the AB plane can result in unintended tissue contact and be dangerous for the patient ; therefore , it would be beneficial to let novices learn how to perform a more-precise retracting movement . The experience in MIS does not influence the retracting phase in the ABP plane . Moreover , a longer retraction of the MIS instrument in the direction of the pivoting point does not denote worse or less-efficient performance . In contrast , it may be a sign of the safer -LRB- for the patient -RRB- use of the MIS instruments . This finding is important , because it shows that the shorter path length , as presently used to indicate more-efficient performance in MIS , is not a correct concept for analyzing optimal movements in laparoscopy . To investigate whether the retracting phase really represents safety , it is necessary to design a different study . For example , one possibility would be to include obstacles which , upon touching represent an error . With such a study design , it would be feasible to determine how much retraction is necessary and whether the experts have learnt the optimal retraction amount . The present study can not be used to decide on these aspects . The results of this study indicate that the comparison of the novices ' path length to the experts ' path length is an important and valid component of the overall criterion-based assessment of basic MIS skills . It is necessary to realize that this study was not designed to determine whether expert performance actually results in the optimal path length and , therefore , the results of this study can not be used to indicate that experts ' movements are optimal . In the literature , there is a tendency to assess MIS performance using very basic terms -LRB- e.g. , time , path length , penalty points -RRB- , which do not show `` the size and the nature of the gap between expert and novice performance '' -LSB- 13 -RSB- . The results of this study show that the analysis of the separate phases of the movement in various planes can give such details . For example , movements of the MIS instrument along its axis -LRB- in and out the trocar -RRB- during the seeking phase characterize the surgeon 's level of depth perception . Movements of the instrument in the AB plane may indicate eye -- hand coordination problems -LRB- e.g. , orientation -RRB- . Since every phase of the movement analyzed in different planes provides details about different limitations , it is crucial to analyze the movements in MIS separately for each phase and in both planes . Only then will the feedback about the performance and the nature of the limitations lead to goal-oriented training curricula . This study shows for the first time the importance of proper analysis of the instruments ' movements during training of MIS skills . The clinical impact of such extended analysis is that only in this way is it possible to implement a correct objective score that will measure and certify the competence of surgeons ' basic psychomotor MIS skills in addition to the existing criteria for the assessment of MIS performance . Moreover , this extended motion analysis can result in improvement of the training of basic MIS skills , since it will identify the differences between the experts ' and novices ' performance , and which areas require more training . Conclusions Movements during laparoscopic tasks are not performed via the shortest path . Therefore , the shortest path length , as presently used for the assessment of basic MIS skills , may be not a proper concept for analyzing optimal movements and therefore needs to be revised . Goal-oriented movements should be split into two phases : a retracting and a seeking phase . Novices are less efficient in the seeking phase . This finding confirms that path length is an important measure in the assessment of basic MIS skills , but that the seeking phase is the only component that distinguishes between novice and expert . Furthermore , the retracting phase is very important in MIS , since it improves safety by avoiding intermediate tissue contact . Analyzing motions in the separate phases provides greater insight into the nature of the gap between expert and novice performance . Motion analysis in MIS should be seen as an addition to the existing criteria in assessing performance .

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O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O

minimally invasive surgery
training
motion analysis
objective assessment
path length


MicroRNA -- implications for cancer MicroRNAs -LRB- miRNAs -RRB- are small RNA molecules that regulate gene expression post-transcriptionally . After the discovery of the first miRNA in the roundworm Caenorhabditis elegans , these short regulatory RNAs have been found to be an abundant class of RNAs in plants , animals , and DNA viruses . About 3 % of human genes encode for miRNAs , and up to 30 % of human protein coding genes may be regulated by miRNAs . MicroRNAs play a key role in diverse biological processes , including development , cell proliferation , differentiation , and apoptosis . Accordingly , altered miRNA expression is likely to contribute to human disease , including cancer . This review will summarize the emerging knowledge of the connections between human miRNA biology and different aspects of carcinogenesis . Various techniques available to investigate miRNAs will also be discussed . The `` small RNA revolution '' Small ribonucleic acid -LRB- RNA -RRB- can act as a specific regulator of gene expression . This discovery has been an exciting breakthrough in Biological Sciences of the past decade , culminating in last year 's Nobel Prize in Physiology or Medicine awarded to Andrew Fire and Craig Mello . Building on previous work mainly in plants -LSB- 50 -RSB- , Fire et al. -LSB- 23 -RSB- discovered that exogenous double-stranded RNA can be used to specifically interfere with gene function . This phenomenon was called RNA interference -LRB- RNAi -RRB- . They also speculated that organisms might use double-stranded RNA naturally as a way of silencing genes . It was then shown that RNA interference was mediated by 22 nucleotide single-stranded RNAs termed small interfering RNAs -LRB- siRNAs -RRB- derived from the longer double-stranded RNA precursors -LSB- 87 -RSB- . The small interfering RNAs were found to repress genes by eliminating the corresponding messenger RNA transcripts , and thus , preventing protein synthesis . Over the following years , many new small functional RNAs have been found . RNA is usually thought of as messenger RNA that serves as the template for translation of genes into proteins . In contrast , functional or non-coding RNA molecules are transcribed from a DNA sequence , but not translated into protein . The encoding DNA sequence is often referred to as an RNA gene . Functional RNA genes in the human genome include transfer RNA -LRB- tRNA -RRB- , ribosomal RNA -LRB- rRNA -RRB- , and various other small non-coding RNAs . Several hundred genes in our genome encode small functional RNA molecules collectively called microRNAs -LRB- miRNAs -RRB- . Precursors of these miRNA molecules form structures of double-stranded RNA that can activate the RNA interference machinery . MicroRNAs downregulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation . The first miRNA was discovered in 1993 by Victor Ambros and colleagues Rosalind Lee and Rhonda Feinbaum -LSB- 42 -RSB- . A genetic screen in the roundworm Caenorhabditis elegans , a millimeter-long animal used as a model organism in biological research , identified genes involved in developmental timing -LSB- 42 -RSB- . Surprisingly , one of the genes , termed lin-4 , did not encode a protein but instead a novel 22-nucleotide small RNA . Seven years later , Reinhart et al. -LSB- 70 -RSB- discovered a second 22-nucleotide small RNA of this type , let-7 , a gene also involved in C. elegans developmental timing . The lin-4 and let-7 small regulatory RNAs soon became very exciting for two reasons . Firstly , homologs of the let-7 gene were identified in other animals including humans -LSB- 65 -RSB- . The conservation of let-7 across species suggested an important and fundamental biological role for this small RNA . Secondly , the mechanism of RNA interference -LRB- RNAi -RRB- was discovered at that time , and it became clear that miRNA and RNAi pathways were intricately linked and shared common components . Within the following year , more than 100 additional small regulatory RNAs similar to lin-4 and let-7 were identified in worms , the fruit fly Drosophila , and in humans -LSB- 38 , 40 , 41 -RSB- . These small non-coding RNAs were named microRNAs -LRB- miRNAs -RRB- -LSB- 38 , 40 , 41 -RSB- . Subsequently , many more short regulatory RNAs were identified in almost all multicellular organisms , including flowering plants , worms , flies , fish , frogs , mammals -LSB- 38 , 40 , 41 , 48 , 71 -RSB- , and in single cellular algae and DNA viruses -LSB- 66 , 75 -RSB- . To date , more than 500 human miRNAs have been experimentally identified . Computational predictions of miRNA targets suggest that up to 30 % of human protein coding genes may be regulated by miRNAs -LSB- 46 , 68 -RSB- . This makes miRNAs one of the most abundant classes of regulatory genes in humans . MicroRNAs are now perceived as a key layer of post-transcriptional control within the networks of gene regulation . MicroRNAs are sequentially processed from longer precursor molecules that are encoded by the miRNA genes -LSB- 1 -RSB- -LRB- Fig. 1 -RRB- . MiRNA genes are referred to by the same name -LRB- termed mir -RRB- written in italics to distinguish them from the corresponding mature miRNA -LRB- termed miR -RRB- followed by a number , e.g. , mir-1 or miR-1 . The encoding DNA sequence is much longer than the mature miRNA . Two ribonuclease enzymes , Drosha and Dicer , subsequently process the primary transcripts -LRB- or pri-miRNA -RRB- to generate mature miRNAs . The primary transcripts contain one or more stem-loop structures of about 70 bases . Stem-loops are double-stranded RNA structures consisting of a nucleotide sequence that can fold back on itself to form a double helix with a region of imperfect base pairing that forms an open loop at the end -LRB- Fig. 1a -RRB- . The ribonuclease Drosha excises the stem-loop structure to form the precursor miRNA -LRB- or pre-miRNA -RRB- -LSB- 43 -RSB- . After export into the cytoplasm , the pre-miRNA is cleaved by the ribonuclease Dicer to generate a short RNA duplex -LSB- 6 , 28 -RSB- . After untwisting , one RNA strand becomes the mature single-stranded miRNA , while the complementary strand , termed miRNA * , is usually rapidly degraded -LRB- Fig. 1b -RRB- . Fig. 1The biogenesis and function of miRNAs . a Primary miRNAs -LRB- pri-miRNA -RRB- are transcribed from longer encoding DNA sequences -LRB- miRNA genes -RRB- . The pri-miRNA contains one or more stem-loop structures of about 70 bases . In the nucleus , the ribonuclease enzyme Drosha excises the stem-loop structure to form the precursor miRNA -LRB- pre-miRNA -RRB- . b After export into the cytoplasm , the pre-miRNA is cleaved by the ribonuclease Dicer to generate a short RNA duplex -LRB- miRNA : miRNA * -RRB- . c The mature single-stranded miRNA is incorporated into the RNA-induced silencing complex -LRB- RISC -RRB- , while the complementary strand -LRB- miRNA * -RRB- is usually rapidly degraded . The miRNA incorporated into the silencing complex can bind to the target messenger RNA by base pairing , causing inhibition of protein translation and/or degradation of the target messenger RNA MicroRNAs recognize their targets based on sequence complementarity -LSB- 10 -RSB- . The mature miRNA is partially complementary to one or more messenger RNAs . In humans , the complementary sites are usually within the 3 ′ - untranslated region of the target messenger RNA . To become effective , the mature miRNA forms a complex with proteins , termed the RNA-induced silencing complex . The miRNA incorporated into the silencing complex can bind to the target messenger RNA by base pairing . This base pairing subsequently causes inhibition of protein translation and/or degradation of the messenger RNA -LRB- Fig. 1c -RRB- . The potential mechanisms underlying this process were recently reviewed -LSB- 30 , 67 -RSB- . Protein levels of the target gene are consequently reduced , whereas messenger RNA levels may or may not be decreased . In humans , miRNAs mainly inhibit protein translation of their target genes and only infrequently cause degradation or cleavage of the messenger RNA -LSB- 1 -RSB- . The biological role and in vivo functions of most mammalian miRNAs are still poorly understood . In invertebrates , miRNAs regulate developmental timing -LRB- e.g. , lin-4 -RRB- , neuronal differentiation , cell proliferation , growth control , and programmed cell death -LSB- 9 , 33 , 42 -RSB- . In mammals , miRNAs have been found to play a role in embryogenesis and stem cell maintenance -LSB- 7 -RSB- , hematopoietic cell differentiation -LSB- 17 -RSB- , and brain development -LSB- 59 , 60 -RSB- . To date , knowledge of human miRNAs has been primarily descriptive . MicroRNA expression has been found to be deregulated in a wide range of human diseases including cancer . However , it remains uncertain whether altered miRNA expression is a cause or consequence of pathological processes . The underlying mechanisms of why and how miRNAs become deregulated are largely unknown . Although bioinformatics approaches can predict thousands of genes that are potentially targeted and regulated by miRNAs based on sequence complementarity , only very few miRNA target genes have been functionally validated . Our group is currently investigating the role of miRNAs in mammary gland development and breast cancer pathogenesis . A comparison of miRNA and gene expression identified miRNAs that classify molecular breast cancer subtypes -LSB- 8 -RSB- . As cancer is ultimately a consequence of disordered gene expression , miRNAs have been suggested to contribute to the development of cancer -LSB- 11 -RSB- . This review will focus on the connection between human miRNA biology and different aspects of carcinogenesis . Various techniques available to investigate miRNAs will also be discussed . MicroRNAs and cancer Three important observations early in the history of miRNAs suggested a potential role in human cancer . Firstly , the earliest miRNAs discovered in the roundworm C. elegans and the fruit fly Drosophila were shown to control cell proliferation and apoptosis -LSB- 9 , 42 -RSB- . Their deregulation may therefore contribute to proliferative diseases such as cancer . Secondly , when human miRNAs were discovered , it was noticed that many miRNA genes were located at fragile sites in the genome or regions that are commonly amplified or deleted in human cancer -LSB- 14 -RSB- . Thirdly , malignant tumors and tumor cell lines were found to have widespread deregulated miRNA expression compared to normal tissues -LSB- 12 , 24 , 52 -RSB- . The question remained whether the altered miRNA expression observed in cancer is a cause or consequence of malignant transformation . MicroRNAs as causal cancer genes at genomic breakpoints Five years ago , the first direct evidence for an involvement of miRNAs in cancer was reported -LSB- 13 -RSB- . Calin et al. studied a well-known deletion on chromosome 13 , which is the most frequent chromosomal abnormality in chronic lymphocytic leukemia -LRB- CLL -RRB- . This deletion had long been suspected to contribute to leukemogenesis . However , extensive studies had failed to identify a causal gene . Calin et al. -LSB- 13 -RSB- found that two miRNA genes , mir-15 and mir-16 , were located within this 30-kb deletion . They subsequently analyzed the expression of miR-15 and miR-16 in blood samples from patients with CLL . Both miRNAs were absent or downregulated in the majority -LRB- 68 % -RRB- of cases when compared to normal tissue or lymphocytes . This finding suggested that these two miRNAs were causally involved in the pathogenesis of chronic lymphocytic leukemia . In 2005 , three reports provided the first mechanistic insight into how miRNAs might contribute to carcinogenesis . Two independent studies described the relationship between a miRNA cluster , mir-17-92 , and the Myc oncogenic pathway -LSB- 27 , 63 -RSB- . A third report demonstrated an interaction between let-7 miRNA and the RAS proto-oncogene -LSB- 32 -RSB- . The mir-17-92 cluster -- small RNAs with oncogenic potential A cluster of six miRNAs , the mir-17-92 cluster , was found to be located within a region on chromosome 13 that is commonly amplified in human B-cell lymphomas -LSB- 64 -RSB- . He et al. -LSB- 27 -RSB- demonstrated that the miRNAs from the mir-17-92 cluster were overexpressed in lymphoma cell lines carrying this amplification , and expression levels correlated with gene copy number of the mir-17-92 locus -LSB- 27 -RSB- . Further , the miR-17-92 primary transcript was found to be overexpressed in tumor samples from lymphoma patients . To test their hypothesis that mir-17-92 actively contributes to lymphomagenesis , the authors took advantage of a mouse model of human B-cell lymphoma . These mice develop lymphomas due to an overexpression of the Myc oncogene . The Myc oncogene encodes the transcription factor c-Myc that regulates cell proliferation , growth , and apoptosis , and overexpression of c-Myc is common in cancer . He et al. -LSB- 27 -RSB- demonstrated that additional expression of the mir-17-92 cluster accelerated c-Myc-induced tumorigenesis in mice . The authors therefore suggested that mir-17-92 was the first potential non-coding oncogene , referred to as oncomir-1 . The cellular function of miR-17-92 was not identified in these experiments . Nevertheless , the pathology of the tumors indicated lower rates of apoptosis as compared to tumors with Myc overexpression alone . Three recent studies contributed towards our understanding of the oncogenic potential of miR-17-92 . Two reports demonstrated an anti-apoptotic effect of miR-17-92 through various pathways that promote cell proliferation and growth -LSB- 55 , 76 -RSB- . A third study identified mir-17-92 as a mediator of angiogenesis in tumors induced by the oncogene c-Myc -LSB- 19 -RSB- . O'Donnell et al. -LSB- 63 -RSB- independently identified the same cluster of miRNAs , mir-17-92 , to be regulated by the transcription factor c-Myc . The transcription factor Myc induces expression of E2F1 growth factor . The mir-17-92 cluster which is also induced by c-Myc does , in contrast , inhibit E2F1 expression . The authors therefore suggested a novel regulatory mechanism by which c-Myc fine-tunes gene expression by activating the transcription of target genes and by simultaneously inducing inhibitory miRNAs that reduce their translation . The example of the mir-17-92 cluster highlights that a distinction between oncogenic and tumor suppressor miRNAs is likely to be an oversimplification . The same miRNAs may have oncogenic or tumor suppressor activity depending on the context and the cell type they are expressed in . A single miRNA may regulate various unrelated target genes and thereby control opposing activities such as cellular proliferation and apoptosis . The ultimate function of a miRNA may depend on the tissue type they are expressed in and what target genes are present . MicroRNAs with tumor suppressor potential The let-7 family of miRNAs was the first group of miRNAs shown to regulate expression of a proto-oncogene , the RAS protein . RAS proteins are membrane-associated signaling proteins that regulate cell growth and differentiation . A miRNA that controls expression of these potentially oncogenic proteins would be predicted to possess tumor suppressor activity . Mutations in the RAS oncogene are present in approximately 15 -- 30 % of all human cancers , and overexpression of the RAS oncogene is common in lung cancer . Johnson et al. -LSB- 32 -RSB- showed that overexpression of RAS protein in lung cancer tissue correlated with reduced expression of let-7 miRNA . They experimentally confirmed that let-7 can inhibit RAS expression in human cancer cell lines . Loss or reduction of let-7 in lung cancer leads to RAS overexpression , thus , promoting cellular growth and contributing to tumorigenesis . The authors therefore suggested that let-7 acts as tumor suppressor -LSB- 32 -RSB- . Another group independently reported reduced expression of let-7 in lung cancers and found that this correlated with a poor prognosis -LSB- 77 -RSB- . Global loss of miRNA expression in cancer A global decrease in miRNA levels has been observed in human cancers , indicating that small RNAs may have an intrinsic function in tumor suppression . Lu et al. -LSB- 52 -RSB- were the first to show that the expression levels of many miRNAs were significantly reduced in cancers compared to the corresponding normal tissues . They analyzed a total of 217 human and mouse miRNAs across 334 human cancers , cancer cell lines , and normal tissues . Cancers had significantly reduced global miRNA expression . Poorly differentiated tumors had lower miRNA levels compared with more-differentiated tumors . The authors hypothesized that miRNAs can function to drive terminal differentiation and prevent cell division . Global changes in miRNA expression may reflect the degree of cell differentiation -LSB- 52 -RSB- . A recent study examined the expression of 241 human miRNAs in a comprehensive panel of human cancer cell lines , the NCI-60 panel , and in normal tissues -LSB- 24 -RSB- . The authors confirmed the finding that most miRNAs were expressed at lower levels in human tumor-derived cell lines compared with the corresponding normal tissue -LSB- 24 -RSB- . Until recently , considerable uncertainty remained as to whether the altered miRNA expression observed in cancer was a cause or consequence of malignant transformation . Earlier this year , a study by Kumar et al. -LSB- 37 -RSB- proved for the first time that widespread reduction in miRNA expression does , indeed , promote tumorigenesis . The authors globally reduced the production of mature miRNAs through a knockdown of the miRNA-processing enzymes Drosha and Dicer in cell lines . The mouse and human cancer cells consequently showed decreased steady-state miRNA levels . These cells with global miRNA loss showed enhanced cellular growth in vitro -LSB- 37 -RSB- . When injected into nude mice , these cells generated faster growing and more invasive tumors compared to controls . To assess the effect of global miRNA loss in vivo , the authors deleted the miRNA-processing enzyme Dicer in a mouse model of lung cancer . The Dicer mutant mice who had impaired miRNA processing developed an increased tumor burden , with an expansion in tumor number and tumor size , as well as tumors which were less well differentiated compared to controls -LSB- 37 -RSB- . Overall , these data clearly suggest that global miRNA loss enhances tumorigenesis . Kumar et al. demonstrated that loss of miRNAs leads to upregulation of proto-oncogenes such as RAS and c-Myc . However , it remains to be elucidated whether loss of all miRNAs is necessary or whether reduction of a subgroup of key tumor suppressor miRNAs , such as let-7 , is the event that promotes malignant transformation . MicroRNAs in the p53 tumor suppressor network Transcriptional networks are often deregulated in cancer cells and may lead to altered transcription of miRNA genes . Two recent studies identified a miRNA , miR-34 , to be regulated by the p53 transcription factor -LSB- 16 , 26 -RSB- . The p53 protein , also called `` the guardian of the genome '' , regulates the cellular response to stress and cancer-initiating events such as DNA damage . He et al. -LSB- 26 -RSB- found that a miRNA , miR-34 , is directly activated by the transcription factor p53 after DNA damage . Expression of miR-34 induces cell cycle arrest and thereby acts together with other effectors of the p53 tumor suppressor network to inhibit inappropriate cell proliferation . Another group independently demonstrated that miR-34 is upregulated by p53 upon DNA damage and promotes apoptosis -LSB- 16 -RSB- . Together , these data indicate that altered expression of miRNAs is not simply a secondary event that reflects the less differentiated state of cancer cells . In contrast , at least in some cases , miRNA expression is specifically driven by tumor suppressors and oncogenes . MicroRNAs with a role in tumor invasion and metastasis Transcriptional networks may drive miRNA expression in cancers . Recent work from Ma et al. -LSB- 54 -RSB- suggested a model by which a pleiotropic transcription factor , Twist , induces expression of a specific miRNA , which suppresses its direct target and in turn activates a pro-metastatic gene , leading to tumor cell invasion and metastasis . The expression of miR-10b induced by the transcription factor Twist promoted cell migration and invasion in mouse and human breast cancer cells . Furthermore , the expression level of miR-10b in primary human breast carcinomas correlated with clinical progression -LSB- 54 -RSB- . These findings , if confirmed , suggest that specific miRNAs may have a role beyond the tumor-initiating event and directly participate in tumor progression and metastasis . Regulation of miRNAs in cancer -- who regulates the regulators ? In few cases , the underlying cause of miRNA deregulation in cancer is clear . As discussed above , the overexpression of miR-17-92 correlates with amplification of its gene locus -LSB- 27 -RSB- . Similarly , decreased expression of miR-15 and miR-16 is associated with a corresponding chromosomal deletion -LSB- 13 -RSB- . Transcriptional or epigenetic regulation of miRNAs has been recently reported -LSB- 53 , 73 -RSB- . The transcription of a miRNA gene , mir-124a , was shown to be inactivated by hypermethylation of its promoter in various human tumors . This process of epigenetic silencing is a well-known mechanism to inactivate protein-coding genes in cancer cells and may similarly apply to miRNAs . The miRNA gene mir-127 is usually expressed in normal cells but not in cancer cells . Saito et al. -LSB- 73 -RSB- demonstrated that miR-127 was highly induced in cultured human cancer cells after treatment with demethylating drugs , suggesting that it is subject to epigenetic silencing through promoter hypermethylation . A novel mechanism of miRNA regulation was suggested by Mayr et al. -LSB- 56 -RSB- and Lee and Dutta -LSB- 44 -RSB- . They demonstrated that miRNA function could be regulated through loss of miRNA binding sites in the target gene . Both groups independently demonstrated that chromosomal translocations in a known oncogene , high mobility group A2 -LRB- Hmga2 -RRB- , led to loss of the let-7 miRNA binding sites in its messenger RNA . Disrupted repression of Hmga2 by let-7 promoted oncogenic transformation and growth in mammalian cells . These two studies provide the first evidence that disrupting the interaction of a single miRNA and its target can produce an abnormal phenotype in mammalian cells -LSB- 44 , 56 -RSB- . In addition , there is evidence that miRNAs are regulated indirectly through control of their processing enzymes . Thomson et al. -LSB- 81 -RSB- showed that a downregulation of miRNAs in human cancer was not associated with reduced levels of the primary miRNA transcripts . The authors therefore suggested regulation of miRNAs during subsequent processing steps , e.g. , through altered function of the enzyme Drosha -LSB- 81 -RSB- . MicroRNA profiling -- implications for cancer diagnosis Lu et al. -LSB- 52 -RSB- asked the question whether global miRNA expression profiles could classify human cancer . MicroRNA expression profiles clearly differentiated human cancers according to their developmental origin . Cancers of epithelial and hematopoietic origin had distinct miRNA profiles . A subgroup of gastrointestinal tumors , which arise from endoderm , was distinguished by miRNA expression patterns . Furthermore , tumors within a single cell lineage such as acute lymphoblastic leukemia were further differentiated according to their underlying genetic abnormality into BCR/ABL-positive tumors , T-cell tumors , and those with MLL gene rearrangement -LSB- 52 -RSB- . Finally , the authors applied the miRNA expression profiles they had established to an independent series of 17 poorly differentiated tumors of unknown origin . Based on the differential expression of 217 miRNAs , a correct diagnosis could be established in 12 out of 17 of the tumors . In contrast , gene expression profiling based on ∼ 16,000 messenger RNAs did not accurately classify the tumors -LSB- 52 -RSB- . This has potential important clinical implications . If miRNAs prove useful for clinical diagnosis , their key advantage might be their high stability . In contrast to most messenger RNAs , they are long-lived in vivo -LSB- 49 -RSB- and very stable in vitro -LSB- 78 -RSB- , which might allow analysis of paraffin-embedded samples for routine diagnostic applications . MicroRNAs -- novel therapeutic targets ? Regulatory RNAs may also have therapeutic applications by which disease-causing miRNAs could be antagonized or functional miRNAs restored . The most intuitive choice of molecules to correct altered miRNA -- messenger RNA interactions are RNA oligonucleotides . These oligonucleotides need to be chemically modified to allow for stability in serum and cellular uptake . Modified antisense oligonucleotides are already being developed to utilize the intrinsic RNAi pathway for delivery of gene therapy . If the delivery problem can be overcome , then miRNA therapies may also be possible . Two studies have successfully applied 2 ′ - O-Methyl-modified antisense RNAs to inhibit miRNA function in cultured cells -LSB- 29 , 57 -RSB- . Recent work by Krutzfeldt et al. -LSB- 36 -RSB- demonstrated that modified cholesterol-conjugated antisense RNAs designated `` antagomirs '' could effectively inhibit miRNA function in vivo in the adult mouse . The authors applied three daily intravenous injections of antagomirs and achieved effective inhibition of four miRNAs over a period of weeks in most tissues except brain -LSB- 36 -RSB- . A novel approach was recently reported by Ebert et al. -LSB- 21 -RSB- . They developed miRNA inhibitors that can be transiently expressed in cultured mammalian cells . These competitive inhibitors termed `` miRNA sponges '' derepressed miRNA targets at least as strongly as chemically modified antisense oligonucleotides -LSB- 21 -RSB- . A different approach was taken by Tsuda et al. -LSB- 83 -RSB- . The authors designed synthetic miRNAs to target overexpressed tumor proteins , such as HER-2 protein . A synthetic miRNA targeting HER-2 messenger RNA successfully inhibited HER-2 protein expression in ovarian cancer cells -LSB- 83 -RSB- . Together , these studies hold some promise of miRNAs as future therapeutic targets . One limitation of antisense RNA therapies is the restricted number of cells that can be targeted . Any approach to knock down a particular miRNA with antisense oligonucleotides will only result in partial knockdown . This may represent a limitation for cancer therapies . It remains to be seen whether indirectly mediated bystander effects on cancer cells that have not been directly targeted may partly overcome this limitation . In contrast , a partial effect on function may be of therapeutic value in neurodegenerative diseases , such as Parkinson 's or Alzheimer 's disease . A partial restoration of dopamine production by antisense therapy might result in a significant clinical improvement in Parkinson patients . Similarly , a partial reduction of the disease-causing proteins in Alzheimer 's disease may lead to a clinical improvement and might be achievable by RNA based or miRNA gene therapy . Techniques and approaches to study miRNAs All known miRNAs are registered in a public web-based registry , the `` miRBase '' database that provides up-to-date information on all published miRNAs -LSB- 25 -RSB- . Novel miRNA genes can be discovered by bioinformatics approaches searching for evolutionary conserved stem-loop structures in the genome -LRB- reviewed in -LSB- 3 , 5 -RSB- -RRB- . Experimentally , miRNAs are discovered by cloning all small RNAs from a certain tissue type or developmental stage and subsequent sequencing to identify the subgroup of small RNAs that fulfill the criteria for miRNAs -LSB- 38 , 51 -RSB- . Both computational and experimental approaches indicate that many more miRNAs are likely to be identified -LSB- 4 , 5 -RSB- , which is reflected by the rapidly increasing number of annotated miRNAs which increased from less than 300 to more than 4,000 over the past 4 years -LSB- 58 -RSB- . MicroRNA expression studies Northern blot analysis is a well-established technique for studying messenger RNA expression and was soon adapted to detect miRNAs in cells or tissues -LSB- 42 , 84 -RSB- . Subsequently , conventional DNA microarray technology was modified to form miRNA microarrays , allowing for the detection of multiple miRNAs simultaneously across various samples -LSB- 15 , 60 , 62 , 82 -RSB- . Lu et al. -LSB- 52 -RSB- developed a novel microarray strategy to improve probe specificity , which is critical due to the short nature of mature miRNAs . They performed hybridization in solution using polystyrene capture beads that are coupled to oligonucleotide probes complementary to the miRNAs of interest . The solution hybrids are then analyzed using a multicolor flow cytometer measuring bead color , denoting miRNA identity , and labeling intensity , denoting miRNA abundance -LSB- 52 -RSB- . In parallel to microarray platforms , commercial assays for quantitative reverse transcriptase polymerase chain reaction -LRB- RT-PCR -RRB- have become available . These allow for the analysis of miRNAs in small tissue samples or even single cells -LSB- 79 -RSB- , as well as validation of microarray data . In addition to mature miRNAs , these quantitative RT-PCR assays can be applied to analyze miRNA precursors and primary transcripts -LSB- 31 -RSB- . In situ hybridization for the detection of mature miRNAs has recently become possible by using special high-affinity locked nucleic acid -LRB- LNA -RRB- - modified DNA oligonucleotide probes and holds promise for the application on human formalin-fixed and paraffin embedded tissue -LSB- 35 , 61 -RSB- . Functional characterization of miRNAs Various strategies have been used to investigate the function of specific miRNAs . In worms and flies , loss-of-function mutants for specific miRNAs or miRNA families allow us to draw conclusions regarding possible physiological functions of miRNAs from the resulting abnormal phenotype -LSB- 34 , 39 , 86 -RSB- . The knockdown of miRNAs or pre-miRNAs using modified antisense oligonucleotides has proven particularly useful in cell lines -LSB- 29 , 45 , 57 -RSB- . LNA-modified antisense oligonucleotides have been successfully utilized to knock down specific miRNAs in cultured cells -LSB- 22 -RSB- . This approach allowed identification of a crucial role for a miRNA , miR-223 , in granulocytic differentiation -LSB- 22 -RSB- . In addition , the modified antisense RNAs -LRB- antagomirs -RRB- described by Krutzfeldt et al. -LSB- 36 -RSB- , which inhibit miRNA function in the adult mouse , may provide a potential research tool to study miRNA function in vivo . In mammals , induced defects in miRNA biogenesis are a useful tool for investigating the biological roles of miRNAs , as loss-of-function mutants are not available for most miRNA genes . Dicer knockout mouse models have revealed essential roles for miRNAs in murine organogenesis -LSB- 88 -RSB- . A recent study utilized a combined knockdown of the miRNA-processing enzymes Drosha , Dicer1 , and DGCR8 to study the consequences of a global decrease in mature miRNAs in cancer cell lines and in a mouse model for lung cancer -LSB- 37 -RSB- . Earlier this year , four independent groups have , for the first time , deleted genes for single miRNAs in mice -LSB- 72 , 80 , 85 , 88 -RSB- . Two of the groups deleted the same DNA sequence for mir-155 and described severe immune defects -LSB- 72 , 80 -RSB- . Mice lacking miR-155 showed impaired function of B and T lymphocytes and dendritic cells -LSB- 72 -RSB- . In particular , T helper cell differentiation and the germinal center reaction to produce a T-cell-dependent antibody response were defective -LSB- 80 -RSB- . Together , these two studies demonstrated a key role for miR-155 in normal immune function . The two other groups deleted different miRNAs , miR-1-2 and miR-208 , and reported cardiac defects . Mice lacking miR-208 showed inadequate cardiac growth in response to stress -LSB- 85 -RSB- , while mice lacking miR-1-2 had defects in cardiac morphogenesis and electrical conduction -LSB- 88 -RSB- . MicroRNA target sites A validated biochemical strategy for identifying miRNA targets would be highly desirable . Two groups have recently reported promising approaches to experimentally identify miRNA targets . Both approaches apply biochemical methods to purify the effector complexes of miRNAs associated with proteins and bound messenger RNA targets -LSB- 2 , 20 -RSB- . An increasing number of sophisticated bioinformatics approaches are being developed to predict putative miRNA target genes -LSB- 3 , 10 , 69 , 74 -RSB- . This is based on the fact that miRNA target recognition is at least partly based on simple sequence complementarity . Interestingly , exact base pairing between miRNAs and their targets commonly appears to be required only in the first six to eight bases from the 5 ′ end of the miRNA . The short nature of this designated `` seed region '' allows a single miRNA to act on up to a hundred different target sites , and all human miRNAs together may regulate up to one third of protein coding genes -LSB- 10 , 47 -RSB- . A different approach to discovering miRNA target genes is to knock out or overexpress a particular miRNA and use conventional microarrays to identify genes that show changes in expression . This approach is based on the observation that some miRNAs can also downregulate messenger RNA levels in addition to downregulating protein levels of their target genes -LSB- 49 -RSB- . Experimental validation of miRNA target sites has been limited to date . A common approach has been to express a miRNA in vivo while simultaneously expressing and monitoring the target messenger RNA linked to a reporter gene , i.e. , Luciferase -LSB- 10 , 18 , 55 , 56 , 76 -RSB- . The fact that a single miRNA can regulate multiple targets and a particular target may be regulated by various miRNAs suggests a highly complex network of miRNA-target interactions , which is only beginning to be unraveled . Conclusions Over recent years , miRNAs have emerged as major players in the complex networks of gene regulation and have been implicated in various aspects of human disease . Only 5 years after the first study reported a direct involvement of miRNAs in cancer , these small RNAs have already significantly improved our understanding of carcinogenesis . In addition to protein-coding oncogenes and tumor suppressor genes , we will have to take into account miRNAs and their regulatory networks if we aim to understand the complex processes underlying malignant transformation .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O B B O O O O B O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O B O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

microrna mirna cancer pathogenesis development