Effect Of ARID4B Silencing On Proliferation And Apoptosis Of Human Hepatoma HepG2 Cell Line

Hepatocellular carcinoma(HCC)is one of the most common and aggressive malignancies worldwide.Due to the low rate of early diagnosis of HCC and the loss of surgical opportunity at late stage,the five-year survival rate of HCC has been greatly improved by the combined treatment of multiple disciplines focusing on surgery,but the high incidence and mortality of HCC are still a difficult problem to be solved urgently.The most important reason for the poor prognosis of HCC patients is that the specific molecular mechanism of the etiology,occurrence and development of HCC is still poorly understood.Therefore,it is of great significance to explore the molecular biological mechanisms closely related to the development and progression of HCC to improve the level of diagnosis and treatment and improve the prognosis.The AT-rich interaction domain 4B(ARID4B)gene is a member of the ARID(AT-rich interaction domain)family.In recent years,more and more studies have shown that ARID4B is highly expressed in lung,breast,colon,ovarian and other tissues,which may play the role of oncogene.It has been found that the high expression of ARID4B in HCC tissues is closely related to the number of tumors,vascular infiltration,Edmondson-Steiner grading and lymp H node metastasis stage,but the role of ARID4B in the occurrence and development of HCC remains unclear.In this study,we used RNA interference technique to silence the expression of ARID4B gene in hepatocellular carcinoma cells,to investigate its effect on proliferation and apoptosis of hepatocellular carcinoma cells and its possible molecular mechanism,so as to provide a new potential molecular target for the early diagnosis and treatment of hepatocellular carcinoma.Part ? Expression of ARID4B in human hepatocellular carcinoma cellsObjective:To detect the expression of ARID4B in different hepatocellular carcinoma cell lines at the cell level.Methods:The mRNA and protein expression levels of ARID4B gene in human normal liver cell line LO2,human hepatocellular carcinoma cell lines HCCLM3,Hep3 B,HepG2 and Huh-7 were detected by RT-qPCR and Western blot.Results:The results showed that the mRNA and protein expression levels of ARID4B gene in human hepatocellular carcinoma cells Hep3 B and huh-7 were relatively low compared with normal human hepatocytes LO2(P<0.01,P<0.001).The relative expression level of human hepatocellular carcinoma cells HepG2 was higher(P<0.001,P<0.01),while the relative expression level of mRNA and protein in human hepatocellular carcinoma cells HCCLM3 showed no significant difference(P>0.05).Conclusion:Compared with human normal liver cells LO2,ARID4B gene was expressed at different levels in different human hepatocellular carcinoma cell lines,and with higher expression in HepG2.Part ? The identification of interference effect of specific interference sequence siRNA on ARID4BObjective:To identify the interference effect of siRNA and screen the optimal interference sequence,action time and concentration.Methods:Two siRNA sequences targeting ARID4B gene siRNA-1,siRNA-2 and siRNA-1+2 which in equal proportion with siRNA-1 and siRNA-2 were designed and transfected into HepG2 cells by the liposome LipofectamineTM 2000.Interference efficiency was detected by RT-qPCR and Western blot,and the siRNA with the optimal interference sequence and the optimal action time and concentration was selected for the next part experiment.Results:1.siRNA optimal interference sequenceCompared with the blank control group,there was no significant difference in the mRNA and protein expression of ARID4B after the negative control sequence transfection into HepG2(P>0.05);after transfection with interfering sequences siRNA-1,siRNA-2 and siRNA-1+2,the expression of ARID4B mRNA and protein in HepG2 was inhibited to varying degrees(P<0.001),and the interference effect of sequence siRNA-1+2 was the best.2.Optimal time of siRNA actionAfter transfection with specific interference sequence siRNA-1+2 into HepG2,RT-q RCR and Western blot were used to detect the interference effect 24,48 and 72 hours later,respectively.Compared with the negative control group,after transfection 48 hours,the relative mRNA and protein expression levels of ARID4B were the lowest(P<0.001,P<0.01).3.Optimal siRNA concentrationSiRNA-1+2 at different concentrations(0,15 nmol/L,30 nmol/L,60 nmol/L)were transfected into HepG2,and the interference effects of siRNA-1+2 at different concentrations were detected by the above methods 48 hours later.The results showed that siRNA-1+2(60 nmol/L)could significantly down-regulate the mRNA and protein expression of ARID4B in HepG2,compared with the negative control group(P<0.001).The inhibition rate of ARID4B mRNA was(70.54±0.53)% and the protein inhibition rate was(60.00±9.17)%.Conclusion:RNA interference technology was applied to transfection hepatocellular carcinoma cells HepG2 with specific targeted siRNA sequence of ARID4B,which could successfully mediate ARID4B gene silencing and achieve targeted down-regulation of ARID4B gene expression.Part ? The effect of ARID4B gene silencing on the proliferation and apoptosis of HepG2Objective:To investigate the effect of ARID4B gene on proliferation and apoptosis of human hepatocellular carcinoma cell line and its possible molecular mechanism.Methods:1.After transfection with siRNA and targeting silencing ARID4B gene,the cell growth was detected by CCK-8 method;2.The effect of ARID4B silencing on cell cycle and apoptosis of hepatocellular carcinoma cells were detected by flow cytometry.3.Western blot was used to detect the changes in expression levels of proliferation-related proteins Cyclin D1,c-Myc,ERK1/2,p-ERK1/2,and apoptosis-related proteins Cleaved Caspase-3,Bax,and Bcl-2 after silencing ARID4B.Results:1.Cell proliferation was detected by CCK-8 methodCompared with the negative control group,there was no significant difference in the number of living cells in siRNA-1+2 group at 24 h after transfection(P>0.05),while the number of living cells in siRNA-1+2 group decreased significantly at 48 h and 72 h after transfection(P<0.01,P<0.05).2.Cell cycle distribution and apoptosis were detected by flow cytometry after transfectionHuman hepatocellular carcinoma cells HepG2 of was detected 48 hours after transfection with the interfering sequence siRNA-1+2.Compared with negative control group,there was no significant difference in the distribution of cells in G0/G1 phase,S phase and G2/M phase(P>0.05).Compared with the negative control group(5.42±0.408)%),the transfected siRNA-1+2 group(6.71±0.575)%)had a relatively high rate of early apoptosis(P<0.05).3.Western blot was used to detect the expression of proteins related to cell proliferation and apoptosisCompared with the negative control group,the expression levels of cell proliferation-related proteins Cyclin D1,c-Myc,ERK1/2 and p-ERK1/2 had no significant change in siRNA-1+2 cells(P>0.05).Compared with the negative control group,the expression levels of apoptosis-related protein Cleaved Caspase-3 and Bax in siRNA-1+2 group were significantly increased(P<0.01,P<0.05),and the expression levels of Bcl-2 were significantly decreased(P<0.01).Conclusion:1.The silencing of ARID4B gene had no significant effect on the proliferation of human hepatocellular carcinoma cells HepG2.2.Silencing ARID4B gene can lead to an increase in the apoptosis rate in human hepatocellular carcinoma cells HepG2,which may be related to the up-regulation expression of Cleaved caspase-3 and Bax protein and the down-regulation expression of Bcl-2 protein,suggesting that ARID4B may become a new target for the treatment of HCC.