Patent Application: US-201013639664-A

Abstract:
key genes , micrornas , other non - coding rnas and a combination thereof for identifying and regulating pluripotency of cells are provided . the key genes , micrornas , other non - coding rnas and a combination thereof are highly expressed in full pluripotent stem cells but are significantly suppressed or silenced in partially pluripotent stem cells . the genes , micrornas and other non - coding rnas are those of a chromosome imprinted dlk1 - dio3 region located on the long arm of mouse chromosome 12 , and are homologous genes , micrornas and other non - coding rnas having 70 - 100 % homology with them in genomic syntenic regions of other mammals . also provided are uses of the genes , micrornas , other non - coding rnas and combination thereof in identifying the pluripotent status of stem cells and regulating pluripotency of cells ; in typing stem cells ; regulating pluripotency of cells , pluripotent states and levels of cells ; treating diseases ; and developing drug targets for tumor treatment and antitumor drugs .

Description:
the research of the inventors is based on rigorous animal experiments , identifies and classifies es cells and pluripotent stem cells with different developmental potentialities , and traces the difference between these pluripotent stem cells through massive solexa sequencing and gene chip data , and thus discovers that genes , micrornas and other non - coding rnas located in the dlk1 - dio3 region are highly expressed in fully pluripotent cells with highest developmental potentiality but are significantly suppressed or silenced in partially pluripotent stem cells with lower developmental potentiality ; this conclusion totally coincides with the result , which is obtained by first detecting the cluster of genes and non - coding micrornas and then validating pluripotency . therefore , in mice , the expression of the cluster of genes is an important marker for the pluripotent status of stem cells . microrna ( mirna ) is a short - chain rna has a length of an average of 22 nucleotides , which has a hairpin - shaped secondary structured precursor , can be complementary to the sequence of a corresponding target messenger rna ( mrna ) and regulate gene silencing at translational level post - transcription . at present , the standards for judging the developmental potentiality of pluripotent stem cells of mice include : 1 . expression of pluripotency markers , for example , pou5f1 , nanog , rex1 , etc . ; 2 . formation of embryoid body ( eb ) and teratoma ; proving that it can differentiate into three germ layers ; 3 . inducing to differentiate into a plurality of cell types in vitro , for example , nerve cells , cardiac muscle cells , islet cells , etc . ; 4 . obtaining chimeric animals ( 2n ); 5 . capable of obtaining germ - line chimeric animals , that is , it can develop into a germ - line and into a sperm or egg ( glt ); 6 . healthy birth of tetraploid complementation animals ( 4n ); the latter is the strictest standards ( gold standards ); however , due to ethical problems , only the former three standards are applicable to the judgement of human pluripotent stem cells . the inventors selected the fourth , fifth and sixth levels of stem cells of mice to conduct experiments . this experiment first analysed the expression of pluripotent genes and cell karyotype according to a conventional method ; then performed in vitro and in vivo examination experiments , including the formation of eb and teratoma , the formation of chimera and the tetraploid embryo complementation assays . the inventors injected the pluripotent stem cells into a cd - 1 diploid mouse embryo to obtain chimeric mice ; when these mice grew up , they were mated with cd - 1 mice to produce infant mice ; the production of germ - line chimeric mice was determined by their fur colors . the tetraploid embryo complementation is the strictest standard for detecting the pluripotent status of cells and the inventors obtained mice totally from the es cell or ips cell source by injecting pluripotent stem cells into tetraploid embryos . the following cell lines were selected to extract total rna and perform sequencing : 1 . two es cell lines and six ips cell lines ( 4n ) with the ability for germ - line chimerism and tetraploid complementation ; 2 . one ips cell line ( glt ) only with the germ - line chimeric ability ; 3 . one ips cell line ( 2n ) without germ - line chimeric ability but can produce chimera animals . the total rna of cells were extracted using trizol ( invitrogen ); the extracted total rna was detected for completeness through agilent 2100 , wherein the qualified rna should satisfy rin value being greater than or equal to 8 . 0 , and the amount of 28s : 18s being greater than or equal to 1 . 10 ug of qualified total rna was taken and isolated on denatured page gel of 15 % polyacrylamide ( 7m carbamide ); small rnas with lengths between 18 - 30 nt were recovered by gel extraction . the recovered non - coding small rnas were connected to the 5 ′ end joint ; the connection product was isolated on the denatured page gel of 15 % polyacrylamide ( 7m carbamide ); 40 - 60 nt of connection product was recovered by gel extraction . the recovered connection product was connected to the 3 ′ end joint gain ; the connection product was isolated on the denatured page gel of 15 % polyacrylamide ( 7m carbamide ); 70 - 90 nt of connection product was recovered by gel extraction . the recovered connection product was subjected to inverse transcription through specific primers to obtain cdna , wherein the cdna was subjected to pcr amplification for 15 amplification cycles and the product obtained by amplification was isolated on non - denatured page gel of 10 % polyacrylamide ; 90 bp of specific band was recovered by gel extraction . sequencing of the recovered pcr product was performed through a high - throughput sequencing instrument of illumina company . original data returned from the illumina company was obtained ; the data were primarily processed using biological information methods ; reads with sequencing quality not meeting standards were removed to obtain high - quality non - redundant sequences and expression values thereof . the sequences were located to the genome through sequence alignment ; between pluripotent stem cells with different pluripotent status ( based on whether a tetraploid complementation animal can be obtained ), specific non - coding microrna clusters were compared and screened by segments according to the location on the genome , to obtain features capable of indicating whether es cells or ips cells meet the standards of highest tetraploid - complementation pluripotency . the expression profiling microarray hybridization results of all cell lines was processed using the bio - chip data and genome data analysis software package bioconductor in r language environment . the original hybridization signal value of the chip was normalized by the rma method and then converted into base - 2 logarithmic values . in conjunction with fdr calibration , genes having expression differences between two cell lines were screened using student &# 39 ; s t - test ( p & lt ; 0 . 05 ). a thermograph of expressions of studied genes and non - coding micrornas in the imprinted region was drawn using the software package “ heatmap . 2 package ” in r . this research finds that the cluster of genes , micrornas and other non - coding rnas specifically expressed in mammals have significant effects on the pluripotency of mouse es cells . all genes , micrornas and other non - coding rnas in the imprinted dlk1 - dio3 region of mouse chromosome 12 ( containing dlk1 and dio3 genes and intergenic regions from this region to the upstream gene begain and to the downstream gene ppp2r5c ) are highly expressed in fully pluripotent stem cells with the ability to produce animals through tetraploid complementation , but are suppressed or silenced in partially pluripotent stem cells without the ability to produce animals through tetraploid complementation . the genes , micrornas and other non - coding rnas are highly conserved in mammalian genomes . the ips cells and es cells used in the experiments were examined for the expression of pluripotent genes , karyotype analysis , eb and teratoma formation , diploid chimeric formation ability and tetraploid embryo complementation ability and the detection results are shown in table 1 . in order to find a key indicator for judging whether the pluripotent stem cells have the tetraploid complementation ability at molecular level , 18 nt to 30 nt of non - coding small rnas were collected from two es cell lines , six 4n - ips cell lines and two 2n - ips cell lines and sequenced in the invention . the cluster analysis method proves that the pluripotent stem cells with the tetraploid complementation ability have great difference from the 2n - ips cells , for both the es cells and the ips cells ; however , the 4n - es cells and the 4n - ips cells have no obvious difference ( see fig1 ). a further research found that 75 micrornas had more than two - time expression changes between 2n and 4n cells , wherein 62 micrornas were generally lowly expressed or not expressed in the 2n - ips cell line , but were highly expressed in 4n - es / 4n - ips cell lines generally . high activity of the imprinted region dlk1 - dlo3 is an important characterization of pluripotency fig2 shows all genes , micrornas and other non - coding rnas in the imprinted region dlk1 - dio3 of mouse chromosome 12 ( containing dlk1 and dio3 genes and the intergenic regions from this region to the upstream gene begain and to the downstream gene ppp2r5c ). the expression of genes of this region is different in 2n - ips cells and 4n - es / ips cells . green rectangles are used to mark the genes and other non - coding rnas with up - regulated expression in 4n - es / ips cells ; genes with no expression differences are marked by grey rectangles outside the imprinted region ; blank squares are used to mark undetected genes . hairpin - shaped structure represents large amount of micrornas generated by this region ; the deeper the color is , the higher the expression level of the microrna is in 4n - es / ips cells . pentagons indicate other non - coding small rnas , the shade of color and the change tendency of expression level thereof are consistent with the above micrornas . all genes , micrornas and other non - coding rnas of the imprinted dlk1 - dio3 region are in an activation state in 4n - es / ips cells . it should be noted that most of the detected differentially expressed mirnas are the lowly expressed micrornas in 2n - ips cells are located in the imprinted dlk1 - dio3 region of mouse chromosome 12 . this region is of about 1380kb , including five genes of dlk1 , rtl1 , 1110006e14rik , b830012l14rik and dio3 , three non - coding genes of meg3 , rian and mirg , a gene cluster of c / d box snornas , 72 micrornas and a plurality of other non - coding small rnas ( see fig2 ). a previous research shows that the expression of the genes and micrornas of this region is only in the mouse embryo and the adult - mouse brain , but their specific functions are not clear . the foregoing five genes and three non - coding genes are as shown in table 2 . the micrornas of the dlk1 - dio3 region mentioned above generally are lowly expressed in the 2n - ips cell lines ; compared to 2n - ips cells , the micrornas are generally highly expressed in the 4n - es / ips cell lines . another two micrornas mir - 342 and mir - 345 of adjacent regions also have similar tendency ( see fig3 a ). more notably , massively endogenous sirnas of this region also have the same expression trend ( see fig3 b ). in addition , using expression profiling microarray to analyze transcription abundance , it is discovered that all genes of this region except for b830012l14rik are highly expressed in the 4n - es / ips cell lines but are suppressed in the 2n - ips cells ( see fig3 c ). therefore , the inventors conclude that high activity of the imprinted dlk1 - dio3 region is an important characterization of true pluripotency . small expression differences of partially reprogrammed cell lines with or without germ - line chimeric ability in this research , the pluripotency of two 4n - ips cell lines has difference too ; ip20d - 3 has a germ - line chimeric ability , while ip36d - 3 does not have this ability . as deduced by the inventors , although the encoded micrornas of the dlk1 - dio3 region are lowly or not expressed in the two 2n - ips cell lines , the expression level in the ip20d - 3 generally is higher than that in the ip36d - 3 among these limited expressions ( see fig4 ). corresponding to the germ - line chimeric ability of the ip20d - 3 , it is very likely that the higher expression of micrornas from this imprinted region endowed this cell line with a higher developmental potentiality . activated microrna clusters positive - feedback regulate the prc2 complex and caused demethylation of the dlk1 - dio3 region the expression profiling microarray of 2n - ips cells and es cells and 4n - ips cell lines shows that 1638 genes have expression up - regulated in es and 4n ips cell lines relative to 2n - ips cell lines and 3467 genes have expression down - regulated . since the microrna generally has a negative regulation on the target gene thereof , the genes with expression down - regulated in es and 4n - ips cells should include the target genes of the micrornas cluster identified and obtained by the inventors . the inventors analyzed the 3 ′ utr regions of mrnas with at least two loci which can be in complement combination with the 2 - 8 nucleic acid seed regions of microrna 5 ′ end , and found that 28 micrornas , which are moderately or highly expressed , have 717 potential target genes with down - regulated expression in the es and 4n - ips cell lines . gene ontology ( go ) analysis shows that the target genes deducted from the micrornas with high abundance are related to growth , differentiation and metabolism , and development process regulation and other phenotypes , and that the microrna cluster highly expressed in fully pluripotent cells may function by suppressing development related genes . signalling pathway analysis shows that three elements ( hdac2 , rbap48 and eed ) forming the polycomb repressive complex 2 ( prc2 ) are predicted microrna target genes . the prc2 can trigger trimethylation of lysine at position 27 of histone h3 , thereby causing gene silencing . before the prc2 triggers methylation , it is needed to activate histone deacetylase 2 ( hdac2 ) to remove the acetylation modification of histone h3 . our research result indicates that , in es and 4n ips cells , the expression of encoded micrornas of the dlk1 - dio3 region suppresses the expression of hdac2 , rbap48 and eed , thereby resulting in the reduction of formation of prc2 and demethylation of genome . in es and ips cells , the reduction of formation of prc2 would cause demethylation of the dlk1 - dio3 region , which coincides with our research result that the expression of genes and micrornas is increased . ( see fig5 ) the expression of micrornas further suppresses the formation of prc2 by targeting the transcription of hdac2 , rbap48 and eed , and finally a positive feedback regulation signalling pathway is formed . fig5 shows a mechanism sketch model of micrornas of the dlk1 - dio3 region regulating a prc2 complex . a . the prc2 complex suppresses the expression of genes and micrornas through tri - methylation of histone h3k27 locus . b . in partially pluripotent stem cells , the expression of micrornas of the dlk1 - dio3 region is suppressed by the methylation of histone h3k27 mediated by the prc2 complex . in fully pluripotent stem cells , micrornas of the dlk1 - dio3 region are in an activation state . therefore , the mrna used for guiding the synthesis of the three proteins of hdac2 , rbap48 and eed can not function normally ; cells can not form the prc2 complex , thereby causing the demethylation of the dlk1 - dio3 region and further facilitating the expression of micrornas of the dlk1 - dio3 region to form a positive feedback regulation pathway . the expression of genes and micrornas of the dlk1 - dio3 region is detected in six 4n - es cell lines , seven 4n - ips cell lines , four glt - ips cell lines , one 2n - ips cell and one 2n - es cell by methods of real - time qpcr and northern blot hybridization . the result shows that the genes and micrornas of the dlk1 - dio3 region are highly expressed in the six 4n - es cell lines and seven 4n - ips cell lines ; are moderately expressed in the four glt - ips cell lines , without a high expression level ; and are not or lowly expressed in the 2n - ips cells and 2n - es cells . it is proved that the genes and micrornas of the dlk1 - dio3 region regulate the pluripotency of cells . if the genes and micrornas of this region are highly expressed in a cell , it is indicated that this cell has a better developmental potentiality . hu , b . y ., weick , j . p ., yu , j ., ma , l . x ., zhang , x . q ., thomson , j . a ., and zhang , s . c . neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency . proc natl acad sci u s a 107 , 4335 - 4340 . judson , r . l ., babiarz , j . e ., venere , m ., and blelloch , r . 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