Effect And Mechanism Of Let-7c On Proliferation Of Hepatocellular Carcinoma Cells

Background and ObjectiveMicroRNAs are a class of small single-stranded RNAs about 22nt. The expressions of miRNAs in tumors are different from those in surrounding normal tissues. These miRNAs play an important role in the proliferation, differentiation, apoptosis and invasion of tumors. The let-7 family was first found in the lung tumor with low expressions. They can inhibit the proliferation of lung cancer cells by regulating the expression of oncogene RAS. But effect of let-7 on liver cancer cells is not clear, and new target genes should be discovered. This study tries to explore the effect of hsa-let-7c on the proliferation of hepatoma cells and discover the new target genes.MethodsThe lipofectamine 2000 was used to transfect miRNAs into the HCCLM3 cells. Cells were divided into three groups:let-7c group where let-7c was transfected, negative control group where negative control miRNA was transfected, blank control group where nothing was transfected. The proliferation of HCCLM3 cells was evaluated using Cell Counting Kit-8(CCK-8). The differences of cell cycle in each group were assayed by flow cytometry. Cyclin D1, cyclin D2 and CDK6 were predicted as target genes of let-7c at bioinformatics sites. Western blot and real time PCR were used to analyze the protein and mRNA expressions of each target genes in different groups. The pMIR-REPORT luciferase vectors which contain the let-7c binding sequence at the 3’-UTR of cyclin D1 and CDK6 were used to verify the combination of let-7c and the 3’-UTRs.Results1. CCK-8 results showed that there were no differences in the absorbances of each group at 24h after transfection (P>0.05), but the absorbances of let-7c group were all lower than the other two groups at 48h、72h、96h respectively (P<0.05). There were no differences in absorbances between negative control group and blank control group at these three time monitoring points (P>0.05).2. The flow cytometry at 72h after transfection revealed that the percent of cells in G1 phase of let-7c group was 54.52%±0.13%, blank control group was 43.53%± 0.86%, negative control group was 44.82%±0.77%. Obviously, the ratio of let-7c group was high than other two groups (P<0.01), and there were no difference between negative control group and blank control group (P>0.05).3. Cyclin D1, cyclin D2 and CDK6 were predicted as target genes of let-7c by querying in the bioinformatics sites.4. The western blot results showed that the expressions of cyclin D1, cyclin D2, and CDK6 at protein levels of let-7c group at 48h post-transfection were all lower than negative control group and blank control group.5. The real time PCR results showed that the mRNA level of cyclin D1 in let-7c group decreased at 24h post-transfection (P<0.05), the mRNA level of CDK6 in let-7c group decreased at 48h (P<0.05). There were no differences in the expressions of the two mRNA between negative control group and blank control group respectively (P>0.05).6. The luciferase reporter assay results showed let-7c inhibited the luciferase activity when the reporter contained the 3’UTR of cyclin D1 or CDK6 compared with the negative control miRNA (P<0.05).Conclusion1. let-7c can inhibit proliferation of the hepatoma cell HCCLM3 and increase the proportion of cells in G1 phase.2. let-7c can repress expressions of cyclin D1, cyclin D2 and CDK6 at protein and mRNA levels. This may be the potential mechanisms for the first conclusion.3. The luciferase reporter assay confirms let-7c can combine with 3’UTRs of cyclin D1 and CDK6. This demonstrates cyclin D1 and CDK6 are target genes of let-7c.