Suppression Of Cell Proliferation And Migration By MiRNA-494in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of cancer deaths in the world. More than half of those people are in China. The prognosis of patients with advanced HCC is poor and most of them die within months after diagnosis. The overall5-year survival rate for HCC patients is less than5%. Treatment strategy is tailored on the basis of the extent of tumor burden, liver function, physical status and potential treatment efficacy. In very early and early stages, HCC may be treated with curative intent in selected cases. However, only about30%of cirrhotic patients enrolled in surveillance programs are eligible for these types of intervention and, even after a curative treatment, the recurrence rate approaches70%at5years. In advanced HCC, treatment options are even more limited:curative treatments are not available and traditional chemotherapy proved to be only arginally effective or even toxic. However, the molecular pathogenesis, including alterations of oncogenes and tumor suppressor genes during this transformation is still unclear. Thus, the identification of new possible targets for the development of non conventional treatments is needed and will necessarily take advantage of progresses in the comprehension of the molecular pathogenesis of HCC.MicroRNAs (miRNAs) are a diverse class of highly conserved small RNA molecules that function as critical regulators of gene expression in multicellular eukaryotes and some unicellular eukaryotes. miRNAs are initially transcribed as long primary transcripts (pri-miRNAs) that undergo sequential processing by the RNase III endonucleases Drosha and Dicer to yield the mature20-23nucleotide species. Mature miRNAs associate with the RNA-induced silencing complex (RISC) and interact with sites of imperfect complementarity in3’untranslated regions (UTRs) of target mRNAs. Targeted transcripts subsequently undergo accelerated turnover and translational repression. Importantly, the ability of individual miRNAs to regulate hundreds of transcripts allows these RNAs to coordinate complex programs of gene expression and thereby induce global changes in cellular physiology. Indeed, a growing body of evidence has documented that miRNAs provide functions essential for normal development and cellular homeostasis and, accordingly, dysfunction of these molecules has been linked to several human diseases.Recently, a particularly important role for miRNAs in cancer pathogenesis has emerged. Virtually all examined tumor types are characterized by globally abnormal miRNA expression patterns. Profiles of miRNA expression are highly informative for tumor classification, prognosis, and response to therapy. Moreover, recent results have documented a functional contribution of specific miRNAs to cellular transformation and tumorigenesis. For example, miRNAs are known targets of genomic lesions that frequently activate oncogenes and inactivate tumor suppressors in cancer cells, such as amplification, deletion, and epigenetic silencing. Additionally, miRNAs provide critical functions downstream of classic oncogenic and tumor suppressor signaling pathways such as those controlled by Myc and p53. Finally, functional studies have directly documented the potent pro-and antitumorigenic activity of specific miRNAs both in vitro and in vivo.It has also been reported that the global miRNA expression profiles of HCC are substantially different from those of non-tumorous liver tissues. However, studies on the deregulation of miRNAs in the early stages or subsequent steps of hepatocarcinogenesis are inadequate. Whether miRNAs play roles in the various steps of hepatocarcinogenesis is unclear and the clinical and diagnostic values of miRNAs in human HCC remain elusive.The down regulation of MicroRNA-494(miR-494) occurred at the early phase of liver regeneration, and there are some reports that miR-494can suppress some cancer cells proliferation. These suggested that miR-494may be involved in HCC pathogenesis.In this present study, we examined the expression level of miR-494in HCC cell lines, and34pairs of HCC and adjacent non-tumorous tissue(NT)by real-time RT-PCR. The biological functions and targets of miR-494have been detected by a serial of analysis. This information may help clarify the molecular mechanisms involved in the progression of HCC. This potential will need to be addressed in a prospective study. First, to explore the relationship between miR-494and HCC, real-time RT-PCR analysis was performed to detect the expression level of miR-494in HCC cells and HCC samples. A significant reduction in the expression of miR-494was observed in both all HCC cell lines examined. And down-regulation of miR-494expression was highly significant in24of34(70%) human hepatocellular carcinoma (HCC) tissues compared with adjacent non-tumorous liver tissues. The distribution pattern of miR-494was further analyzed by dividing the expression levels of miR-494according to the clinicopathological parameters. The downexpression levels of miR-494cases were found to be significantly associated with tumor size. The miR-494expression was lower in tumors more than5cm than less than5cm in diameter(P<0.05). However, there was no significant difference between miR-494expression and other clinicopathological parameters in HCC.Second, to clarify the biologic function of miR-494in HCC, its mimics was transfected into Huh7cells. Subsequently, HCC cell proliferation and migration were examined by MTT assay, colony formation assay, nude mouse xenografts and wound healing assay, respectively. Apoptosis assay and cell cycle analysis were performed48h post-transfection using a flow cytometer. These data show that enhanced miR-494expression in cultured Huh7cells by transfection with miR-494mimics decreased tumor cell proliferation, migration, and the results of flow cytometry suggest that miR-494mimics induce the G0/G1phase cell cycle arrest and have no effect on cell apoptosis in Huh7cells. The proportion of G0/G1phase cells treated with miR-494mimics increased. At the same time, the fraction of cells in S phase decreased accordingly. Together, these results suggest that miR-494may be a tumor suppressor in HCC.Third, to identify the target genes and explore the molecular mechanisms of miR-494, a search was conducted to locate putative targets of miR-494through silico analysis. We looked for the target genes of miR-494with the application of bioinformatics tools. Potential target genes expression were assessed by western blot for proteins, real-time PCR for miRNAs. Luciferase repot gene assays were carried out using target genes3’UTR reporter constructs or its miR-494binding site mutant construct co-transfected with miR-494mimics.Bioinformatics analysis deduced CDK6and ROCKl, as the targets of miR-494. Real-time PCR and western blot results showed that Huh7cells transfected with miR-494mimics have reduced expression of target genes CDK6and ROCK1. Further investigation revealed that miR-494significantly repressed the activity of luciferase carrying the3’ UTR of CDK6and ROCK1.In summary, these findings suggest the involvement of miR-494in the growth and migration of HCC partly by down-regulating CDK6and ROCK1expression. Thus, it may be useful as a novel therapeutic target in HCC.