ABSTRACT
Objective To investigate the influence of GTPBP4 gene expression down-regulation in hepatocellular carcinoma (HCC) cell line SMMC-7721 on the intracellular and extracellular biological behaviors and its action mechanism.Methods The differences of GTPBP4 expression level in HCC tissues and paracancerous tissues were compared by using the immunohistochemical method.The expression of GTPBP4 mRNA in 4 kinds of HCC cell line(SMMC-7721,HEPG2,HUH-7,HEP3B) was detected by using the real-time fluorescent quantitative PCR(RT-PCR).The expression of GTPBP4 in HCC cell line SMMC-7721 was downregulated by using the RNA interference technique and the cellular biological behavior change was observed.Results In HCC histological chip,the expression level of GTPBP4 protein in HCC tissue was significantly higher than that in para-cancerous tissue.GT-PBP4 was also significantly up-regulated in 4 kinds of HCC cell line.After the expression of GTPBP4 was down-regulated,the proliferation ability of HCC cells was weakened and apoptosis was increased.The cells in S phase and G1.phase had no significant changes,but which in G2 phase were increased,the ability of in vitro clone formation was weakened,the nude mouse in vivo tumor formation capacity was weakened.The expressional profiles microarray results showed that 333 genes were changed in SMMC-7721 cells after GTPBP4 knockdown,in which the up-regulated genes were 134,the down-regulated genes were 199.The channel enrichment analysis found 10 signal transduction pathways of the enriched differential genes.Western blot showed that the expression levels of CCND1,CCND2,CDK6 and MDM2 were changed significantly after GTPBP4 expression down-regulation.Conclusion GT-PBP4 as a promoting HCC gene may influence HCC biological behavior possibly by regulating the expression of key gene in cell cycle.
ABSTRACT
Objective:To investigate the protein expression of GTPBP4 in human hepatocelluar carcinoma (HCC) tissues and the influ-ence of GTPBP4 silencing by siRNA on the proliferation and cell cycle of HCC cell line Hep G2. Methods:Western blot analysis was per-formed to observe the protein expression of GTPBP4 in 24 cases of HCC tissues compared with their adjacent noncancerous liver tis-sues. Lentivirus-mediated RNA interference (RNAi) was used to silence GTPBP4 expression in Hep G2, and the infection efficiency was observed under a fluorescence microscope. The silencing effect was tested by Western blot and real-time PCR. After silencing the GT-PBP4 gene, cell proliferation was detected using CCK-8 assay, and the cell cycle was observed using flow cytometry. Results:(1) GT-PBP4 was overexpressed in 21 cases (87.5%) of HCC tissues (P<0.000 1). (2) After the lentivirus with GFP reporter infected the Hep G2 cells, 90%of the cells showed green fluorescence. LV-GTPBP4-RNAi effectively inhibited the expression of GTPBP4 at both mRNA (70%) and protein (67%) levels. (3) The proliferation ability of the LV-GTPBP4-RNAi group significantly decreased after 96 h (inhibition rate:54.51%). Flow cytometry showed that the LV-GTPBP4-RNAi group significantly increased at the G0/G1 phase, whereas the S phase de-creased and arrested at the G0/G1 phase. Conclusion:GTPBP4 overexpression in HCC tissues was associated with hepatocarcinogenesis and promoted tumor cell proliferation, but the specific molecular mechanisms remain to be investigated.