The Mechanism Study Of SAMHD1 Inhibits The Proliferation Of HCC Cells By Regulating P27 Expression

Objective:Hepatocellular carcinoma(HCC),as an aggressive malignant tumor with rapid clinical progress,and hepatocellular carcinoma cells are easily disseminated and metastasize.However,the current understanding of the molecular mechanism of HCC is still not clear.Chemotherapy resistance of liver cancer is also facing severe challenges,and many other factors have limited the improvement of treatment effects.Therefore,it is of great significance to investigate the mechanism of liver cancer development and find new biomarkers and therapeutic targetsMethods:Part ?:The effect of SAMHD1 regulateing the biological function of hepatocellular carcinoma cells1.Firstly,we compared the relateive expression of SAMHD1 protein in normal immortalized liver cell line(L02)and four liver cancer cell lines(PLC/PRF/5,Hep3B,Huh7,HepG2)employing Western blotting(WB).2 We constructed a stable cell line that knocking out SAMHD1 on hepatocellular carcinoma cells Huh7 and HepG2 using CRISPR/Cas9 technology,and constructed stable cell lines that overexpressing SAMHD1 wide type and mutants(SAMHD1-D207N and SAMHD1-T592E)on Huh7 cells.3.The effects of SAMHD1 on the proliferation of hepatocellular carcinoma cells were detected by MTT assay and clone formation experiments on the constructed stable cell lines;the effects of SAMHD1 on the cycle distribution and apoptosis of hepatocellular carcinoma cells were detected by flow cytometryPart ?:The molecular mechanism of SAMHD1 inhibits cell proliferation of hepatocellular carcinoma cells1:RT-qPCR and Western blot were employed for detection of cyclin(cyclinA,cyclinB,cyclinD and cyclinE),cyclin-dependent kinases(CDK1,CDK2,CDK4 and CDK6),cyclin inhibitor/kinase inhibitors(p21 and p27),Rb,and phosphorylated CDK2 and Rb.2.Analysis of the correlation between p27 expression and SAMHD1 in 373 HCC tissue samples using Pearson correlation in TCGA database.3.Separation of cytoplasm and nuclear proteins in HepG2-KO SAMHD1 cells.Western blot was used to detect the changes in the distribution of p27 between in the nucleus and the cytoplasm.4.Western blot analysis of protein expression levels of Akt and activated p-Akt in HepG2-KO SAMHD1 cells.5.The HepG2-KO SAMHD1 cells were treated with Akt inhibitor LY294002,then the MTT assay was used to detect cell proliferation,and Western blot was used to detect p-Akt protein expression levelsResults:Part ?:The effect of SAMHD1 regulateing the biological function of hepatocellular carcinoma cells1.The protein expression levels of SAMHD1 in four liver cancer cell lines(PLC/PRF/5,Hep3B,Huh7,HepG2)were up-regulated by 3.7,2.3,1.7 and 4.3 times compared with normal L02 cell lines,respectively.2.Knockout of SAMHD1 can promote the proliferation and colony forming ability of hepatocellular carcinoma cell lines HepG2 and Huh7.The proportion of G2/M in the cell cycle increases,and the proportion of G1/G0 phase decreases There was no significant change in early and late apoptosis;Conversely,overexpression of wild-type SAMHD1,SAMHD1 dNTPase mutant(SAMHD1-D207N)and SAMHD1T592 phosphorylation site mutant(SAMHD1-T592E)can inhibit the proliferation of hepatocellular carcinoma cell Huh7,and the proportion of the cell cycle G1/G0 phase increased,the proportion of G2/M decreased,and there was no change in apoptosisPart ?:The molecular mechanism of SAMHD1 inhibits cell proliferation of hepatocellular carcinoma cells1.Western blot results showed that the expression of p27 mRNA and protein levels were reduced,and the expression levels of CDK1,cyclinD,p-CDK2 and p-Rb were increased in Huh7-SAMHD1 KO cells that the SAMHD1 was knocked out.The conversed effect was observed in overexpression SAMHD1 Huh7 cells.2.Pearson correlation analysis in the 377 clinical HCC cases showed that the expression levels of p27 was positively correlated with SAMHD1(r=0.2812,P<0.0001).3.Western blot results showed that Akt protein expression was unchanged,and the level of phosphorylated p-Akt protein in its active state increased in Huh7-KO SAMHD1 cells.4.After the cytoplasmic nucleus protein was isolated,Western blot results showed that part of p27 was transferred from the nucleus to the cytoplasm in HepG2-KOSAMHD1 cells.5.The HepG2-KO SAMHD1 cells were treated with PI3K inhibitor LY294002,and the results of MTT experiments showed that LY294002 can reverse the effect of SAMHD1 on liver cancer cell proliferation.In addition,the p-Akt protein level decreased with the increasing concentration of LY294002Conclusion:SAMHD1 protein is highly expressed in hepatocellular carcinoma cell lines;SAMHD1 can inhibit the proliferation of hepatoma cells(Huh7 and HepG2),this inhibitory effect does not depend on the form of its mutants(SAMHD1-D207N and T592E),and SAMHD1 does not affect cell apoptosis;The molecular mechanism of SAMHD1 reguating the cell proliferation is:SAMHD1 can act on the PI3K/Akt-p27 signaling pathway,inhibit Akt activity,reduce the expression of p-Akt,and increasing the expression level of p27,thereby increasing the proportion of cells in G1/G0 phase,the cell cycle arrest at G1/G0,and finally inhibit the proliferation of hepatocellular carcinoma cell.