Patent Application: US-200913382686-A

Abstract:
the present invention relates to use of two micrornas in detection of lung cancer prognosis and in medicine preparation . particularly , the invention relates to a composition comprising two small rna molecules microrna - 150 and microrna - 886 - 3p , a device comprising the composition used in detection of lung cancer prognosis and in preparation of medicaments for inhibiting mammal and human lung cancer metastasis . specifically , the expression levels of microrna - 150 and microrna - 886 - 3p can be used as the prognostic criteria of lung cancer prognosis , wherein high expression level of the gene combination indicates favorable therapeutic effect . the invention also relates to a device detecting the expression levels of microrna - 150 and microrna - 886 - 3p in mammalian and human lung cancer and a method for detecting the expression levels of microrna - 150 and microrna - 886 - 3p in samples .

Description:
the present invention will be further described by use of the following examples , but not limited to these ones . in the following examples , unless specifically indicated , all the reagents used in the application are analytically pure and commercially available . unless otherwise indicated , rt - pcr , pcr and other operations as mentioned in the examples of the invention are performed in accordance with “ molecular cloning : a laboratory manual ( the 3rd edition )” ( j . sambrook and d . w . russell [ usa ], translated by peitang huang et al , science press , 2002 ) and the manufacturer &# 39 ; s instruction ; cell culture , cell passage , cell recovery and cryopreservation , cell transfection , immunofluorescence assay and other operations are carried out in accordance with “ culture of animal cells : a manual of basic technique ( the 4th edition )” ( r . ian freshney [ uk ], translated by jingbo zhang et al , science press , 2000 ) and the manufacturer &# 39 ; s instruction . the method for detecting gene expression status of microrna - 150 and microrna - 886 - 3p 1 . extraction of total rna of samples — extraction of total rna from tissues or cells with trizol method samples were collected from the cancer institute and hospital , chinese academy of medical sciences and peking union medical college . the inclusion criteria comprises : age ≦ 75 ; kps scores ≧ 80 ; the patients with small cell lung cancer in limited stage , receiving surgery ± chemotherapy ± radiotherapy ; sufficient formalin - paraffin - embedded tissues to obtain enough mirna ; complete written medical records and follow - up records . 42 cases of formalin - fixed and paraffin - embedded small cell lung cancer specimens between 2002 and 2005 which met the inclusion criteria were chosen for the microrna chip detection and screening of micrornas associated with prognosis . 40 cases of formalin - fixed and paraffin - embedded small cell lung cancer specimens between 2000 and 2001 were chosen for verification of the chip results . the characteristics of specific cases are shown in table 1 . the tissue sample with an area of about 1 cm 2 was broken in aluminum foil , then transferred to an eppendorf tube containing steel beads and ground with a grinding mill ( 30 l / s , 8 min ), note : this step should be operated as much as possible in a cryogenic liquid nitrogen environment , and grinding is not necessary for bacteria or cells ; ( 3 ) 1 ml of trizol was added into the eppendorf tube after the grinding step , and mixed by shaking ; ( 4 ) the mixed solution was transferred to a new eppendorf tube , and 200 μl of chloroform was added and mixed by shaking ; ( 5 ) the solution was centrifuged at 12000 rpm for 15 min at 4 ° c . ( 6 ) the obtained supernatant was transferred to a new eppendorf tube , and 500 μl of isopropanol was added , then gently mixed and placed for 15 min at room temperature ; ( 7 ) the solution was centrifuged at 12000 rpm for 15 min at 4 ° c . ( 8 ) the supernatant was removed , and then 1 ml of 75 % ethanol was added and mixed by shaking ; ( 9 ) the solution was centrifuged at 7500 rpm for 5 min at 4 ° q ( 10 ) the supernatant was removed , and the ethanol was totally evaporated in a laminar flow cabinet ; ( 11 ) 40 ˜ 60 μl of depc h 2 o was added , and the mixture was solubilized at 65 ° c . for 5 min ; ( 12 ) the obtained sample was frozen at − 20 ° c . for cryopreservation . ( 1 ) determination of total rna concentration by nanoprop ( loading 2 μl of total rna ), ( 2 ) determination of rna quality by means of 1 . 5 % formaldehyde denaturing agarose gel electrophoresis formaldehyde denaturing agarose gel : 0 . 45 g of agarose was added into 30 ml of 1 × tbe buffer , the mixture was heated to melt in a microwave oven and gently shaken to mix the agarose thoroughly ( no suspending granules can be visually observed ), 600 μl of formaldehyde was added when the mixture cooled to about 60 ° c ., mixed , and then poured into a special gel casting module for rna ( 7 . 5 × 5 . 5 cm ). after being placed for about 30 min at room temperature , the agarose gel would be ready to use . the quantity , degradation and size of the rnas extracted from the 40 specimens in the invention are shown in table 2 and fig4 . non denaturing agarose gel : 1 . 2 g of agarose was added into 80 ml of 1 × tbe buffer , the mixture was heated to melt in a microwave oven and gently shaken to mix the agarose thoroughly ( no suspending granules can be visually observed ), 2 μl of eb ( stock solution ) was added when the mixture cooled to about 60 ° c ., the solution was mixed , and then poured into a gel casting module ( 15 × 15 cm ). after being placed for about 30 min at room temperature , the agarose gel would be ready to use . the results of electrophoresis shows a good specificity in microrna realtime pcr reaction , as shown in fig1 . the expression values of micrornas were converted into codes , wherein they were divided into three equal parts according to the expression levels thereof . the first one third part was given a code “ 1 ” which corresponded to the low expression level among the total expressions , the second one third part was given a code “ 2 ” which corresponded to the median expression level , and the last one third was given a code “ 3 ” corresponding to the high expression level . then the code of each microrna was introduced into a univariate cox regression model to find the micrornas associated with prognosis . protective micrornas for prognosis were defined as those with hazard ratio for death & lt ; 1 . negative - associated micrornas for prognosis were defined as those with hazard ratio for death & gt ; 1 [ 18 ]. after the univariate cox proportional - hazards regression analysis was used to find micrornas , the expression values of each microrna were multiplied by the regression coefficients ( b value ) to form a linear combination used to be a prognosis risk score for each patient , wherein b value was given by the univariate cox regression analysis . the formula was given as follows : risk score = b1g1 + b2g2 + b3g3 + . . . + bngn ( b : regression coefficients , g : expression value of mirna , n : number of mirnas ). patients with higher risk score are expected to have poorer survival outcomes . then patients in different groups including training group and testing group were divided into high - risk and low - risk groups using the median microrna risk score as the cut - off point . the kaplan - meier method was used to estimate overall survival . differences in survival between the high - risk and the low - risk patients was analyzed . data were normalized using u6 rna as an internal standard . a univariate cox regression model was used to analyze the relationship between the abundance value of each microrna and the survival rate . a compound value was assigned to each patient according to a linear combination of the statistically significant signal value of the micrornas derived from the univariate cox regression analyses multiplied by the regression coefficients . the compound values were used to evaluate the prognosis of the patients . the patients were divided into two groups using the median microrna compound value as the cut - off point , and the low - risk group had a longer survival time compared to high - risk group ( p = 0 . 005 , see fig2 and fig3 ). as shown in fig2 , in the training group , the 3 - year and 5 - year survival of the high - risk group was 47 . 6 % and 28 . 6 % respectively , while the 3 - year and 5 - year survival of the low - risk group was 76 . 2 %. as shown in fig3 , in the testing group , the 3 - year and 5 - year survival of the high - risk group was 40 % and 33 . 6 % respectively , while the 3 - year and 5 - year survival of the low - risk group was 74 . 1 % and 68 . 8 %. study on the biological effect of microrna - 886 - 3p and microrna - 150 on inhibiting the invasion and adhesion of lung cancer cells human small cell lung cancer cell line nci - h446 was purchased from the cell center of basic medical sciences , chinese academy of medical sciences , and cultured in 1640 medium containing 10 % fetal bovine serum at 37 ° c . in a 5 % co 2 atmosphere . human non small cell lung cancer cell line nci - h1299 was kindly provided by professor weiguo zhu , department of biochemistry and molecular biology , peking university health science center , and cultured in 1640 medium containing 10 % fetal bovine serum at 37 ° c . in a 5 % co 2 atmosphere . a . preparation of mirna mother liquor : 250 μl 1 × universal buffer was added into 5 nmol mirna to obtain 20 μmol / l mirna mother liquor , b . the well grown cells were inoculated in a 6 - well plate ( without antibiotics ) on the day before transfection , and the transfection was carried out when the cell density reached about 70 %, c . preparation of the following complexes : solution a : the mirna at proper concentration was diluted into 250 μl of serum - free medium , and gently mixed . solution b : 6 μl of lipofectamine 2000 , which had been mixed thoroughly before use , was diluted in 250 μl of serum - free medium , mixed , and incubated for 5 minutes at room temperature , d . the dilution of liposome ( solution b ) was gently mixed with the dilution of mirna ( solution a ), and incubated for 20 minutes at room temperature , e . 500 μl of the mixed complexes was added into the 6 - well plate . 2 ml of serum - free medium was added and then gently mixed . the original medium was removed after 6 hours , and replaced with rpmi 1640 medium containing 10 % serum . a . extraction of total rna of samples - extraction of total rna from cells with trizol method ( i ) the well grown cells were employed . when the cell density reached 80 %- 90 %, the culture medium was poured out of the bottle , and the cells were washed twice with pbs ; ( ii ) 1 ml of trizol was added into the bottle , gently shaken , and placed on ice for 15 minutes ; ( iii ) the mixed solution was transferred to an eppendorf tube pretreated with depc , and 200 μl of chloroform was added and mixed by shaking ; ( iv ) the solution was centrifuged at 12000 rpm for 15 min at 4 ° c . ; ( v ) the obtained supernatant was transferred to a new eppendorf tube , 500 μl of isopropanol was added , gently mixed , then placed for 15 min at room temperature ; ( vi ) the solution was centrifuged at 12000 rpm for 15 min at 4 ° c . ; ( vii ) the supernatant was discarded , and then 1 ml of 75 % ethanol was added and mixed by shaking ; ( viii ) the solution was centrifuged at 7500 rpm for 5 min at 4 ° c . ( ix ) the supernatant was discarded , and ethanol was totally evaporated in a laminar flow cabinet ; ( x ) 40 ˜ 60 μl of depc h 2 o was added , and the pellets were solubilized at 65 ° c . for 5 min ; ( xi ) the obtained sample was frozen at − 20 ° c . for cryopreservation . ( i ) determination of total rna concentration by nanoprop ( loading 2 μl of total rna ), ( ii ) determination of rna quality by means of 1 . 5 % formaldehyde denaturing agarose gel electrophoresis formaldehyde denaturing agarose gel : 0 . 45 g agarose was added to 30 ml × tbe buffer , heated to melt in a microwave oven , gently shaken to thoroughly mix the agarose ( no suspended granules can be visually observed . ), then 600 ul formaldehyde was added when cooled to about 60 ° c . and the solution was mixed and then poured into a special gel casting module for rna ( 7 . 5 × 5 . 5 cm ). after being placed for about 30 min at room temperature , the agarose gel would be ready to use . ( ii ) program of reverse transcription was : 16 ° c . for 30 min , 37 ° c . for 30 min , 70 ° c . for 10 min , then kept at 4 ° c . ( iii ) detection of realtime pcr products by means of 1 . 5 % non - denaturing ( formaldehyde free ) agarose gel electrophoresis non - denaturing agarose gel : 1 . 2 g of agarose was added into 80 ml of 1 × tbe buffer , the mixture was heated to melt in a microwave oven and gently shaken to thoroughly mix the agarose ( no suspending granules can be visually observed ), 2 μl of eb ( stock solution ) was added when the mixture was cooled to about 60 ° c ., the solution was mixed , and then poured into a gel casting module ( 15 × 5 cm ). after being placed for about 30 min at room temperature , the agarose gel would be ready to use . the principle is based on the characteristics of motility and directivity of tumor invasion . after contacting with the surface of matrix , tumor cells can move in a certain direction through a series of mechanisms . a . for h446 and h1299 cells , matrigel was respectively diluted to 500 μg / ml and 1 mg / ml . 100 μl diluent was added into the upper chamber of the transwell insert of polycarbonate membrane ( with 8 μm pores ) and incubated for one hour at 37 ° c . in a 5 % co 2 incubator , and then the aqueous phase was aspirated , b . the well grown tumor cells were digested and re - suspended at a certain density after 48 hours post - transfection , c . 200 μl of cell suspension respectively containing 10 × 10 4 h446 cells or 5 × 10 4 h1299 cells was seeded in the upper chamber of each transwell insert , and 800 μl culture fluid containing 10 % serum was added into the bottom chamber , then cells were cultured for 12 hours at 37 ° c . in a 5 % co 2 incubator , d . the chamber was taken out and the upper layer of cells without migration were scrapped off , e . cells on the membrane were fixed with 70 % methanol for 15 min , f . cells were stained with 5 % crystal violet ( in methanol ) for 20 min , then washed with distill water , g . cells on the surface of bottom chamber were counted under a microscope , and statistically analyzed , and photographed at the same time . a . fibronectin was aspirated using precooling tips under aseptic operation , and diluted to 20 μg / ml , b . 50 μl diluted fibronectin was added into each well of a 96 - well plate , c . the 96 - well plate coated with fibronectin was dried in a sterile workbench , d . cells were digested , centrifuged and resuspended with culture fluid containing 10 % serum at 48 hours post - mirna transfection , e . 5 × 10 4 cells were seeded into each well of the 96 - well plate coated with fibronectin , and 5 parallel wells were set , f . after an incubation of 30 , 60 , 90 min or 30 , 60 min respectively , h446 and h1299 cells were washed with pbs to remove non - adherent cells , and the medium was discarded in completely adhesion groups after 3 hours . cells were fixed with 70 % methanol for 10 min and dried at room temperature , then stained with 0 . 1 % crystal violet for 20 min od 570 was determined after decolorization with 10 % sds , which represents the adherent cells at different time points . a completely adhesion group was set up in each experimental group , g . the adhesion rate was calculated with residual cells , and the cell adhesion rate =( od value of experimental group / od value of completely adhesion group )× 100 %. the experimental data were analyzed using spss10 . 0 software package ( spss , chicago , ill .) with two - sided student &# 39 ; s t - test , p & lt ; 0 . 05 as a significant difference . ( 1 ) transfection of lung cell lines with chemically synthetic mature mirnas to overexpress target mirna transfection was performed using lipofectamine 2000 . for a single transfection , mir - 886 - 3p , mir - 150 or mir - as - egfp at a final concentration of 50 nm was used to transiently transfect h446 and h1299 cells , while for a co - transfection mir - 886 - 3p and mir - 150 at a final concentration of 37 . 5 nm or mir - as - egfp at a final concentration of 75 nm were used to transfect h446 and h1299 cells with mir - as - egfp as a control . at 48 hours post - transfection , cells were collected . and the expression levels of mir - 886 - 3p and mir - 150 were determined by real - time pcr . in the single transfection in h446 cell line , the expression level of mir - 886 - 3p and mir - 150 was increased by 2 . 5 - fold and 2 . 9 - fold respectively as compared to the control . in the co - transfection in h446 cell line , the expression level of mir - 886 - 3p and mir - 150 was increased by 1820 . 6 - fold and 101 . 5 - fold respectively as compared to the control . in the single transfection in h1299 cell line , the expression level of mir - 886 - 3p and mir - 150 was increased by 235 . 4 - fold and 1723 . 3 - fold respectively as compared to the control . in the co - transfection in h1299 cell line , the expression level of mir - 886 - 3p and mir - 150 was increased by 2736 . 3 - fold and 2052 . 0 - fold respectively as compared to the control . the results showed that the expression levels of mir - 886 - 3p and mir - 150 in h446 and h1299 cell lines increased significantly after the transfection , indicating that the transfection procedure and system was suitable for the corresponding research of overexpression of mirna . ( 2 ) effect of high expression of mir - 886 - 3p and mir - 150 on in vitro invasion ability of cells in vitro invasion ability of tumor cells was studied using transwell invasion assays . h446 cells and h1299 cells were digested and resuspened with serum free rpmi 1640 at 24 hours post - transfection , and were respectively seeded at an amount of 1 × 10 5 cells and 5 × 10 4 cells in the upper chamber of a transwell insert , while 800 μl rpmi 1640 containing 10 % serum was added in the bottom chamber , then the cells were cultured for 12 hours at 7 to allow their entry into the lower layer of 8 m - pored polycarbonate membrane . following staining with 0 . 5 % crystal violet , cells stained purple were visible under a microscope ( fig5 a , fig6 a ), and cells on the lower surface of the polycarbonate membrane were counted . by calculation , the numbers of h446 cells transfected by mir - 886 - 3p , mir - 150 and mir - 886 - 3p / mir - 150 which had crossed the membrane were respectively 55 . 0 %± 8 . 5 %, 65 . 7 %± 8 . 5 %, 71 . 0 %± 8 . 5 % of that of the control . the numbers of h1299 cells transfected by mir - 886 - 3p , mir - 150 and mir - 886 - 3p / mir - 150 which had crossed the membrane were respectively 73 . 6 %± 3 . 5 %, 61 . 8 %± 11 . 1 %, 83 . 7 %± 8 . 3 % of that of the control . the results showed that the in vitro invasion ability of h446 and h1299 cells with high expression of mir - 886 - 3p , mir - 150 and mir - 886 - 3p / mir - 150 was obviously abated as compared to the control cells ( fig5 b , fig6 b ). and the difference was significant in the statistics analysis . ( 3 ) effect of high expression of mir - 886 - 3p and mir - 150 on extracellular matrix adhesion of h446 and h1299 cell lines cell adhesion ability plays an important role in the metastasis of tumor cells . at 48 hours post - transfection , 5 × 10 4 h446 and h1299 cells were seeded into a fibronectin ( 20 μg / ml ) extracellular matrix - coated 96 - well plate . the cells were washed at various time points , and then the residual cells were adherent cells . the cells were fixed with 70 % methanol for 10 min , stained with 0 . 1 % crystal violet for 20 min . od 570 value was determined after decolorization with 10 % sds . the adhesion rate was calculated , which reflected the adhesion ability to extracellular matrix . the results showed that the adhesion rate of h446 cells transfected by mir - nc , mir - 886 - 3p , mir - 150 and mir - 886 - 3p / mir - 150 respectively was 14 . 9 %± 0 . 9 %, 11 . 3 %± 0 . 3 %, 13 . 3 %± 0 . 1 %, 11 . 4 %± 0 . 3 % at 30 min ; respectively was 45 . 1 %± 1 . 9 %, 42 . 8 %± 2 . 2 %, 45 . 1 %± 1 . 8 %, 41 . 6 %± 1 . 1 % at 60 min ; respectively was 56 . 9 %± 1 . 0 %, 49 . 0 %± 2 . 0 %, 52 . 8 %± 0 . 5 %, 47 . 6 %± 0 . 5 % at 90 min ( fig7 a ). for h1299 cells transfected with mir - nc , mir - 886 - 3p , mir - 150 and mir - 886 - 3p / mir - 150 , the adhesion rate respectively was 37 . 6 %± 1 . 5 %, 34 . 7 %± 2 . 8 %, 24 . 6 %± 3 . 0 %, 23 . 4 %± 0 . 5 % at 30 min ; respectively were 47 . 1 %± 1 . 5 %, 40 . 0 %± 2 . 1 %, 36 . 6 %± 2 . 2 %, 29 . 9 %± 4 . 2 % at 90 min ( fig7 b ). as compared to the control cells , the adhesion rates of h446 and h1299 cells transfected with mir - 886 - 3p , mir - 150 and mir - 886 - 3p / mir - 150 were all reduced , and the difference was significant by a statistics analysis . the results showed that the extracellular matrix adhesion ability of h446 and h1299 cells with high expression of mir - 886 - 3p , mir - 150 and mir - 886 - 3p / mir - 150 was obviously diminished . fischer b , marinov m , arcaro a ( 2007 ). “ targeting receptor tyrosine kinase signalling in small cell lung cancer ( sclc ): what have we learned so far ?” cancer treatment reviews 33 : 391 - 406 . bonner j a , harari . p m , giralt j , et al ( 2006 ). “ radiotherapy plus cetuximab for squamous - cell carcinoma of the head and neck .” n engl j med 354 : 567 - 578 . xiao c , calado d p ., galler g , et al ( 2007 ). “ mir - 150 controls b cell differentiation by targeting the transcription factor c - myb .” cell 131 : 146 - 159 . sekine i , yamamoto n , kunitoh h , et al ( 2004 ). “ treatment of small cell lung cancer in the elderly based on a critical literature review of clinical trials .” cancer treatment reviews 30 : 359 - 368 . morita t , sugano h . 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