The Influence Of MiR-27b-5p On Human Hepatocellular Carcinoma Cells And Its Mechanism Analysis

Background&0bjective:Hepatocellular carcinoma(HCC) is one of the most common cancers in the world, which has a highly recurrence rate and very poor prognosis and remains one of the tumors most refractory to treat. The cause maybe related to the cancer stem cell which have a capable of self-renewal and multi-directional differentiation. mi RNAs are small(20-24 nucleotides) noncoding RNA molecules that act as negative regulators of gene expression at the post-transcriptional level and play an important role in tumorigenesis. It may be a potential therapeutic targets. In our previous study, we got the differential expression profiles of mi RNAs in liver cancer stem cells from cancer cells with mi RNAs microarray. In this study, we aimed to find out the mi RNAs associated with liver cancer stem cells, to study its effect on liver cancer cells and the mechanism of action, and to provide a new direction for the diagnosis and treatment of liver cancer.Methods: Part I: Reproducibility some liver cancer stem cell-specific micro RNAs in the different mi RNAs expression profiles by quantitative RT-PCR. 1 RT-PCR was applied to verify the mi RNAs chipresults in Huh7 cancer cells and cancer stem cells. 2 RT-PCR was applied to verify the mi RNAs chipresults in hepatocellular carcinoma and adjacent noncancerous liver tissue. Part II : The effect of mi R-27b-5p on the Huh7 cell and liver cancer stem cell biology behavior.1 Hepatoma cell Huh7 were transfected with lentiviruses vectors expressing mi R-27b-5p. 2 The functional effect of mi R-27b-5p on cancer cell survival, migration, invasion and the properties of stem cells were investigated by CCK-8 assay, colony formation assay, wound healing assay, tumorosphere formation assay and metrigel invasion assay.Part III: Predicted and validated the target genes of mi R-27b-5p. 1 The target genes of mi R-27b-5p were predicted by bioinformatics analysis. 2 The correlation between mi R-27b-5p and FOXO1 m RNA was detected by RT-PCR. 3 The correlation between mi R-27b-5p and FOXO1 protein was detected by Western blot. 4 Direct binding of mi R-27b-5p to the FOXO1 3’UTR was verified by Dual-luciferase reporter assay.Results: Part I 1 In our previous study, the result of mi RNA microarray showed the pattern of mi RNA expression in cancer stem cells was markedly different from Huh7 cancer cells. After normalization, the expression profiles of 35 mi RNAs were determined between cancer stem cells and Huh7 cancer cells, revealing 10 upregulations and 25 downregulations mi RNAs. mi R-27b-5p was overexpressed both in cancer stem cells and hepatocellular carcinoma tissues.Part II 1 We observed much expression of e GFP in 72 h after transfection both in mi R-27b-5p group and NC group, indicating successful transfection with lentivirus vectors. The RT-PCR results also showed that our aim gene mi R-27b-5p’s expression is significantly much higer than it in the NC and Blank group(P<0.01)which indicates expression of mi R-27b-5p in Huh7 cells are up-regulated successfully by lentivirus infection.2 CCK-8 assay showed that over-expression of mi R-27b-5p could improve the Huh7 cell’s proliferative capacity(P<0.05). Colony formation assay showed that over-expression of mi R-27b-5p can improve cloneforming ability of Huh7 cells(P < 0.01). Wound healing assay showed that over-expression of mi R-27b-5p can improve migration capacity of Huh7 cells in vitro(P<0.05). Metrigel invasion assay showed that over-expression of mi R-27b-5p can improve invasion ability of Huh7 cells in vitro(P<0.01). Tumorosphere formation assay showed that over-expression of mi R-27b-5p can improve self-renewal of cancer stem cells(P < 0.01). And RT-PCR suggested that over-expression of mi R-27b-5p can improve cancer stem cells biomarkers’ expression of Huh7 cells(P<0.01).Part III1 Prediction of mi R-27b-5p targets by several prediction softwares on line indicated that FOXO1 may be one of the potential downstream target molecule of mi R-27b-5p. FOXO1 has been reported that it was an important tumor suppressor gene in mammalian cells.2 RT-PCR assay showed that the FOXO1 m RNA expression levels between mi R-27b-5p group and NC group showed statistical significance(P<0.01). 3 Western blot results showed that the FOXO1 protein level of overexpression mi R-27b-5p group was much lower than the NC group. 4 Dual-luciferase reporter assay showed that the luciferase activity in FOXO1-WT group is much lower than the FOXO1-Mut group.Conclusion: 1 Over-expression of mi R-149-5p could significantly improved proliferation, invasion and metastasis of liver cancer cells in vitro. It also could improved self-renewal of cancer stem cells and improved cancer stem cells biomarkersp expression of Huh7 cells. 2 The prediction software indicted that FOXO1 might be a downstream target of mi R-27b-5p. 3 mi R-27b-5p could reduced FOXO1, mi R-27b-5p might affect biology function of Huh7 cells and cancer stem cells by targeting FOXO1.