The Role Of NFAT2 And Its Mechanism In Hepatocellular Carcinoma Cells

Objectives: To study the role of three isoforms B,D,E of NFAT2 in hepatocellular carcinoma(HCC);and to study the mechanism of pro-apoptotic effects of NFAT2 isoformB in hepatocellular carcinoma cells.Methods:(1)Western blotting was used to detect NFAT2 protein in hepatocellular carcinoma cell lines(PLC,HepG2,Huh7,Hep3B)and normal liver cell lines L02(2)The target gene was obtained by extracting RNA from liver cancer cells,reverse transcription and PCR.The target gene was ligated with vector DNA and plasmid DNA was extracted from DH5? to construct different pcDNA3.1-NFAT2 isoformB,isoformD,isoformE eukaryotic vector plasmid?(3)The constructed pcDNA3.1-NFAT2 isoformB,isoformD,isoformE vector plasmids were transfected into Huh7 and PLC hepatoma cell lines respectively and the experimental group and the control group were established.The effect of overexpression of NFAT2 was detected by flow cytometry.(4)To further explore the mechanism by which NFAT2 exerts its tumor suppressor,we transfected the NFAT2 isoformB plasmid and detected mRNA changes in potential downstream target genes by qRT-PCR.(5)The different protein including FasL,Caspase-8,Caspase-3 and Caspase-9 was detected by overexpression of NFAT2 isoformB to understand the effect of NFAT2 on hepatocellular carcinoma.(6)Through previous studies,it was found that NFAT2 can reduce the proliferation and increase apoptosis of hepatocellular carcinoma,and NFAT2 can upregulate FasL expression.To further clarify the role of FasL in NFAT2 gene,we constructed a siRNA targeting FasL gene and used Huh7 and PLC cell lines to cotransfect NFAT2 overexpression plasmid and FasL siRNA respectively.Cell proliferation was measuredby CCK-8 assay and apoptosis was detected by flow cytometry.Results:(1)Western Blot showed that NFAT2 protein was decreased in the four hepatocellular carcinoma lines PLC,HepG2,Huh7 and Hep3 B compared with the normal liver cell line L02,and the relative expression of protein was lower than that of L02(P < 0.05).(2)The results of cell flow experiments showed that the apoptosis rates of all isoforms were increased when Huh7 and PLC cell lines were transfected with NFAT2 isoformB,isoformD and isoformE respectively(P<0.05).(3)Overexpression of NFAT2 isoformB in hepatocellular carcinoma cell line Huh7,we found that FasL gene mRNA increased among several candidate downstream target genes by qRT-PCR(P <0.05).(4)Western Blot results showed that the expression of FasL,Caspase-8 and Caspase-3 apoptotic proteins increased after transfection of NFAT2 isoformB overexpression plasmid(P<0.05),but Caspase-9 did not change significantly(P>0.05).(5)After cotransfection of NFAT2 isoformB overexpression plasmid and FasL siRNA,the si-FasL+NFAT2 group had a higher proliferation rate and a lower apoptotic rate than the si-NC+NFAT2 group(P <0.05).Conclusions:(1)NFAT2 expression in hepatocellular carcinoma is lower than that in normal hepatocytes.(2)Various isoforms of NFAT2 have a consistent pro-apoptotic effect on hepatocellular carcinoma.(3)In hepatocellular carcinoma,NFAT2 induces apoptosis by activating the FasLmediated extrinsic signaling pathway.(4)In hepatocellular carcinoma,siRNA silencing FasL-mediated pathways can abolish antiproliferative and pro-apoptotic effects of NFAT2 in HCC cells.