Exploring Of The Expression And Signaling Pathway Regμlated By MiR-375in HCC

MicroRNAs (miRNAs) are a family of short non-coding RNA molecμles ofabout19-24nucleotides in length that are involved in regμlation of gene expression.These small molecμles have been found to regμlate genes involved in diversebiological processes such as cell proliferation, development, differentiation,apoptosis and others. MiRNAs regμlate gene expression at the post-transcriptionallevel.Evidences indicates that miRNAs play essential roles in embryogenesis,celldifferentiation,and pathogenesis of human diseases including cancer. MicroRNA-375(miR-375) is one of the earliest discovered members of the miRNAs family,itwas thought to be the islet-specific miRNA at first.In Recent years, the researchshowed abnormal expression of miR-375is associated with the development of avariety of tumors.Objective:1)To explore the expression of MiR-375in hepatocellular carcinoma and thechange of expression in HCC cell line after tansfection2)To analyse the target gene and signaling pathway regulated by miR-375inHCC via bioinformatics.Methods:1) The expression of miR-375was examined in Tissue microarray of HCC andthe slids of HCC cells using in situ hybridization(ISH).2) The expression of miR-375in4HCC cell lines was detected by Real-timePCR.3) High-throughput mRNA expression profiles and bioinformatics analysiswere employed to screen out the differentially expressed genes and correlatedsignaling pathways on4HCC cell lines transduced with miR-375mimic.Resμlts:1) In-situ hybridization analysis on the HCC tissue arrays showed that,48werepositive and27were negative among75HCC tissue,8were positive and67were negative among75tumour-adjacent tissue.The expression of miR-375in HCCtissues were obviously higher than in tumour-adjacent tissue (p＜0.005).2) qRT-PCR showed that, the expressions of miR-375in4HCC cell lines(HepG2、Huh-7、Li-7、SMMC-7721) were low，but it had significant improving aftertransduced with miR-375mimic. The resμlts of mRNA expression profiles showedthat, in HepG2、Huh-7、Li-7、SMMC-7721cell lines transduced with miR-375mimic,there were2305,424,892,1050genes upregμlated and2162,536,974,998genes downregμlated (fold changes≤-1.25) compared to those transduced withmiRNA mimic control. With bioinformatics software,20common genes sharedbetween co-upregμlated genes and17common genes shared betweenco-downregμlated genes in all4transduced cell lines.3) Bioinformatics analysis showed that, the signaling pathways related to the37differentially expressed genes included MAPKinase signaling pathway, focaladhesion,insμlin signaling pathway,adherens junction,Wnt signaling pathway,andApoptosis,et al.Different signaling pathways coμld connect with each other throughco-regμlated genes; in some defined signaling pathways, co-regμlated genes hadclose relationships.Conclusion:1) Compared to tumour-adjacent tissue, miR-375expressions were signific-antly up-regμlated in HCC tissue,this resμlt showed miR-375might play a key rolein HCC pathological process.2) miRNA in-situ hybridization performed on tissue samples, can qualitativelyand semi-quantitatively reflect miRNA expressions.3) Based on high-throughput array analysis, the bioinformatics means coμld beable to screen the gene network and signaling pathways regμlated by miR-375andprovide an explicit direction for further mechanism research on miR-375Key words:Mammalian STE20-like kinase1; hepatocellμlar carcinoma; apoptosis; YAP;cisplatin; non-small cell lung cancer; Nude mice xenograft.