The Preliminary Study On MiR-187-3p In Promoting The Formation Of Cancer Stem Cell Stemness Of HCC Cell Line Huh7

Objective:In our previous study, we have successfully enriched and expanded hepatoma stem-like cells from Huh7 cell line by using non-adherent culture method, and have screened the different expression profiles of miRNAs between hepatoma stem-like cells and hepatoma cells by using Exiqon micro RNAs chip hybridization technology. We performed this study to screen and validate these miRNAs, which promoted the formation of cancer stem cell stemness of HCC cell line Huh7, and to make a further study on the influence of this miRNA on the CSCs characteristics of hepatoma cells, and to provide research foundation for the molecular target therapy for HCC.Methods:Ⅰ. Screening and validation of miRNAs, which promoted the formation of cancer stem cell stemness of hepatocellular carcinoma cell line Huh71.Non-adherent culture method was used to enrich and expand hepatoma stem-like cells from Huh7 human hepatocellular carcinoma cells; Quantitative real time polymerase chain reaction(q PCR) was performed to validate the different expression profiles of miRNAs between hepatoma stem-like cells and hepatoma cells.2.QPCR was performed to validate the different expression of miRNAs in hepatocellular carcinoma tissues and para-carcinoma tissues.Ⅱ. The influence of miR-187-3p on the CSCs characteristics of Huh7 cells and its potential molecular mechanisms1.miR-187-3p was cloned into lentivirus vectors and were infected into Huh7 human hepatocellular carcinoma cells to get stably expressed cell line. Inverted fluorescence microscope was used to observe the expressions of e GFP in miR-187-3p group, NC group and Blank group, which were infected with lentivirus for 72 h, and then q PCR was performed to detect the expression of miR-187-3p in these three groups.2.Tumorsphere assay and q PCR technology were used to detect the influence of miR-187-3p on cancer stem cell characteristics of human hepatocellular carcinoma cells.3.CCK-8 assay, plate clone assay, transwell assay, wound-healing assay in vitro and tumor xenograft model in vivo assay were separately performed to investigate the influence of miR-187-3p on proliferation, clone formation, invasion and migration of HCC cells.4.Bioinformatic software was performed to predicte target genes of miR-187-3p.Result:Ⅰ. Screening and validation of miRNAs, which promoted the formation of cancer stem cell stemness of hepatocellular carcinoma cell line Huh71. The hepatoma stem-like cells were enriched from Huh7 human hepatocellular carcinoma cells by using non-adherent culture method in vitro. The q PCR result showed that miR-187-3p was overexpressed in hepatoma stem-like cells and hepatocellular carcinoma tissues, which was consistent with our previous miRNA microarray result.Ⅱ.The influence of miR-187-3p on the CSCs characteristics of Huh7 cells and its potential molecular mechanisms1. We observed much enhanced Green Fluorescent Protein(e GFP) expression in miR-187-3p group and NC group infected by lentivirus after 72 h, indicating that we have successfully infected the lentivirus into cells. The q PCR results showed that the expression of our aim gene miR-187-3p was significantly higher than that in NC group and Blank group(P<0.01), indicating that the expression of miR-187-3p was successfully up-regulated in Huh7 cells by lentivirus infection.2. Tumorosphere formation assay showed that the sphere formation efficiency of miR-187-3p group was significantly higher than that of control groups(P<0.01). The q PCR result also showed the expression of marker genes of CSCs such as BMI1,CTNNB1,KLF4 and PROM1, was obviously higher in miR-187-3p group than that in NC group and Blank group(P<0.05).3.CCK-8 assay and colony formation assay showed that overexpression of miR-187-3p could promote the proliferation of Huh7 cells in vitro(P<0.01). Transwell assay showed that overexpression of miR-187-3p could promote the invasion of Huh7 cells in vitro(P<0.01). Transwell assay and Wound healing assay showed that overexpression of miR-187-3p could promote the migration of Huh7 cells(P<0.01). Nude mice experiment showed that tumor volume of the nude mouse in miR-187-3p group was significantly larger than that in NC group and Blank group(P<0.01); Weight of tumor in miR-187-3p group was significantly heavier than that in NC group and Blank group(P<0.01).4. Target genes of miR-187-3p were predicted by several bioinformatic softwares on line, which showed that UCHL1 might be one of the potential downstream targets of miR-187-3p.Conclusion:1. Overexpression of miR-187-3p could promote self-renewal of hepatoma stem-like cells and the expression of the marker genes of CSCs on Huh7 cells.2. Overexpression of miR-187-3p could significantly promote proliferation, invasion and migration of HCC cells in vitro and vivo, and miR-187-3p may function as an oncogene by targeting UCHL1 in HCC.