MicroRNA let - 7c inhibits proliferation of hepatocellular carcinoma cells by targeting cyclin dependent kinase 6

Abstract

Objective To explore let-7c’s influence on the proliferation of human hepatocellular carcinoma cells and to clarify its target gene. Methods The let-7c expression in human hepatocellular carcinoma cells (MHCC-97L, HepG2, HCCLM3, SMMC-7721) and human hepatic cell L02 were detected by real-time quantitative polymerase chain reaction (Real-time PCR). MicroRNAs (miRNAs) were transfected into HCCLM3 cells via Lipofectamine™2000. The cells were divided into three groups: let-7c group transfected with let-7c, negative control group transfected with negative control miRNA, blank control group transfected with no miRNAs. The proliferation of cells was assessed via cell counting kit-8 (CCK-8), cell cycle of each group was detected by flow cytometry, Western blotting was used to evaluate the protein expression of cyclin dependent kinase 6 (CDK6) after transfection. The 3’untranslated region (3’-UTR) of CDK6 containing the putative binding sites of let-7c was inserted into a firefly luciferase reporter vector named pMIR-REPORT. MiRNAs, pMIR-REPORT and renilla luciferase control plasmid were transfected into 293T cells together, the Dual-Luciferase Reporter Assay System was used to assay the luciferase activity. Results The expression level of let-7c in MHCC-97L, HepG2, HCCLM3, SMMC-7721 were (3.22±0.08)×10-4, (2.34±0.11)×10-4, (1.85±0.03)×10-4 and (3.03±0.11)×10-4 respectively, lower than that in L02 [(4.37±0.09)×10-4,P 0.05). The percentage of G1 stage in let-7c group was (56.95±2.40)% at 72 h after transfection, higher than that of the other two groups (P 0.05). The protein level of CDK6 in let-7c group was lower than that of the other two groups at 48 h after transfection (P 0.05). The Dual-Luciferase Reporter Assay System revealed that let-7c could suppress the luciferase’s activity compared with negative control miRNA (P<0.05). Conclusion The expression level of let-7c is lower in the hepatocellular carcinoma cells compared with normal hepatic cell. let-7c can inhibit the proliferation of hepatocellular carcinoma cells and cause a delay in G1-S transition, CDK6 is its target gene. Key words: Carcinoma, hepatocellular; MicroRNAs; Cyclin-dependent kinase 6; Let-7c