Dentification And Validation Function Of MiR-139-5p Target Genes In Mammary Epithelial Cell MCF-10A

Searching for the target genes of miRNAs is still an important direction for clinical and scientific research beacause each miRNA has many target genes.The mammary gland tissue of in few animals were periodicly varied with the corresponding of age ?oestrous cycle and breeding conditions.Therefore,to identify and validate miR-139-5p target genes were critical for human mammary gland.Mi R-139 and its target genes could affect a number of biological functions,such as protein localization?cell cycle regulation and inhibition of tumor invasion et al.It is well known that the orderly development of cell cycle was the basis of growth,development and reproduction of organisms,but accelerating or retardanting cell cycle may affected cell proliferation and migration ability,and then leadrd to tumors?cancer?and genetic mutations et al.In order to fully study the role and function of miR-139-5p,the identification and validation of target genes have become an urgent problem to be solved.In this study,MCF-10 A cell lines were used to identify and validate the target genes of miR-139-5p by RNA binding protein immunoprecipitation(RIP)and combining with a new generation of sequencing technology.At first,the corresponding RNA-protein complex was precipitated by using the antibody against the target protein Ago2.Then,the RNA was bound to the compound by sequencing analysis to find out the difference peak in the pair of samples,and confirming the target genes of miR-139-5p in the MCF-10 A cells and analysising the target genes by KEGG and GO.Finally,the target genes of miR-139-5p function were verified by q PCR.These results will provide a new reference for the study of miR-139-5p in human mammary gland.The results are as follows:(1)The data processing and bioinformatics were analysised by RIP-seq:We comparing the 608 peaks(Negative Control group)and 1000 peaks(miR-139-5p mimic)from RIP-seq sequencing,then obtained the 484 different peaks.These different peaks were different target genes of miR-139-5p in MCF-10 A cells,which may be important target genes in mammary gland development or mammary gland evolution;Then,using KEGG to analyze the target genes,we obtained 204 pathways of which 21 were significant pathways(p <0.05)with involving 120 target genes;In the GO analysis,it was found that 448 significant GO terms which were involved protein localization?regulation of cell cycle?organelle part?RNA binding and nucleic acid binding;(2)Validation of miR-139-5p target genes functionThe 18 selected target genes were significantly down-regulat by miR-139-5p at mRNA level,and indicating that their expression were regulated by miR-139-5p.Mi R-139-5p gene functional validation showed that miR-139-5p significantly inhibited G1/S cell cycle and then inhibiting cell proliferation;Thus outcomes were interacted from many target genes.