The Construction and Functional Analysis of the Lentivirus Mediated Low Expression RNF20 Hepatocellular Carcinoma Cell Lines
CUI Yani1,2, WANG Yanfeng2, SUN Junning2, DUAN Jingjing2, REN Laifeng2, SUWen2*
The purpose of this research was to investigate the effects of RNF20 (ring finger protein 20) deficiency on both of proliferation and migration in hepatocellular carcinoma cells, and to explore its mechanism. Three sets of short hairpin RNA targeting RNF20 gene were designed which were RNF20-shRNA1, RNF20-shRNA2 and RNF20-shRNA3. The lentiviral vector pLent-U6-GFP-Puro-shRNF20 was constructed. The RNF20 knockdown SMMC-7721 and Huh7 cell lines were established by packaging lentivirus and infecting hepatocellular carcinoma cells. The infected cells were divided into two groups. Group 1: the control group was infected with control lentivirus pLV-shCtrl-EGFP. Group 2: the defective group was infected with virus pLV-shRNF20-EGFP. The RNF20 mRNA was detected by qPCR. The expression of GFP (green fluorescence protein) was observed under fluorescence microscope after the screen by puromycin. The level of RNF20, T-Akt and p-Akt protein was detected by immunofluorescent staining and Western blot. The proliferation of hepatocellular carcinoma cells was detected by BrdU incorporation assay and cell counting kit-8 assay. The migration of hepatocellular carcinoma cells was detected by scratch test. The gene transcription levels were detected by RNA-seq. The infecting efficiency of the two groups was more than 85%. The expressions of RNF20, Wee1, p27, p53 gene and RNF20 protein of defective groups were significantly lower than that in the control groups. The expression of p-Akt protein and the ability of both proliferation and migration of the RNF20 defective group were higher than those in control group. The ability of both proliferation and migration of the RNF20 defective group which treated by Perifosine was higher than those in control group. The deficiency of RNF20 may enhance the proliferation and migration of hepatocellular carcinoma cells by regulating Akt signaling pathways in vitro.
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