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OBJECTIVE To investigate the effect of eukaryotic translation elongation factor 1A1(eEF1A1)on the replication of vesicular stomatitis virus(VSV)and Herpes simplex virus 1(HSV-1)to identify a potential target for broad-spectrum regulation of viruses.METHODS Small interfering RNA(si-eEF1A)was transfected into human skin fibroblasts(BJ-5ta)to inhibit the expression of eEF1A1,and the negative control group was set up.The transfection efficiency was detected by real-time fluo-rescence quantitative PCR(RT-qPCR)and Western blotting,the cell model of eEF1A1 gene silencing was constructed.The cell model was infected with VSV,the gene copy number and protein expression of VSV in the cells were detected.The cell model was infected with HSV-1,the mRNA and protein expres-sion of HSV-1 in the cells were detected.The cell models were transfected with polyinosinic acid[Poly(I:C)]or sodium deoxyribonucleic acid(HT-DNA),respectively.The mRNA expression of interferon-β(IFN-β),C-X-C Motif Chemokine 10(CXCL10)and interferon-stimulated gene 56(ISG56)were detected by RT-qPCR.The phosphorylation expression of interferon regulatory factor 3(IRF3)and TANK binding kinase 1(TBK1)were detected by Western blotting.RESULT Compared with the negative control group,the mRNA and protein expression of eEF1A1 in the eEF1A1 gene silencing group were signifi-cantly decreased(P<0.01),the cell model of eEF1A1 gene silencing was successfully constructed.Compared with the negative control group,the VSV gene copy number of the eEF1A1 gene silencing group decreased by 70%-80%.The VSV protein expression decreased significantly(P<0.01).The mRNA expression of HSV-1 was decreased by 50%-60%,and the protein expression of HSV-1 was significantly decreased(P<0.01).After stimulation with Poly(I:C)or HT-DNA,compared with the negative control group.there was no significant difference in the mRNA expressions of IFN-β,ISG56 and CXCL10 and the protein phosphorylation expression of IRF3 and TBK1 in the eEF1A1 gene silencing group.CONCLUSION eEF1A1 silencing can inhibit VSV and HSV-1 virus replication,suggesting that eEF1A1 has a potential broad-spectrum regulatory effect on RNA viruses and DNA viruses,and may not recog-nize activated immune pathways through intracellular nucleic acid recognition.
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<p><b>OBJECTIVE</b>To explore the role of eukaryotic translation elongation factor 1A1 (eEF1A1) in regulating the invasion and metastasis of hepatocellular carcinoma (HCC) cells and the possible mechanism.</p><p><b>METHODS</b>qRT-PCR and Western blotting were used to detect the mRNA and protein expression of eEF1A1 and NOB1 in different HCC cell lines and normal liver cells. The invasion and migration abilities of HCC cells with eEF1A1 knockdown or overexpression were examined using Transwell chamber assay and RTCA assay, and the changes in NOB1 mRNA and protein expressions in the cells were detected. The effects of increasing NOB1 expression in HCCLM3-sheEF1A1 cells and decreasing NOB1 expression in eEF1A1-overexpressing MHCC97h cells on eEF1A1 expression and cell invasion and migration abilities were analyzed using Western blotting, Transwell chamber assay and RTCA assay.</p><p><b>RESULTS</b>The expressions of eEF1A1 and NOB1 were significantly increased in positive correlation in HCC cells as compared with normal hepatocytes. Knockdown of eEF1A1 significantly decreased the invasion and migration of HCC cells and reduced the mRNA and protein expression of NOB1 ( < 0.01). Overexpression of eEF1A1 significantly enhanced invasion and migration of HCC cells and increased NOB1 mRNA and protein expressions ( < 0.01). Increasing NOB1 expression in HCCLM3-sheEF1A1 cells led to the restoration of NOB1 expression and cell invasion and migration abilities ( < 0.01), whereas decreasing NOB1 in MHCC97h-eEF1A1 cells resulted in inhibition of NOB1 expression and cell invasion and migration ( < 0.01).</p><p><b>CONCLUSIONS</b>eEF1A1 positively regulates the expression of NOB1 to promote the invasion and migration of HCC cells .</p>
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Objective: To study the effects of eukaryotic translation elongation factor 1A1 (eEF1A1) on the cell cycle and proliferation of hepatocellular carcinoma (HCC) cells, and to explore the molecular mechanism. Methods: The expression of eEF1A1 protein in 101 HCC tissues and their adjacent tissues were detected by immunohistochemistry, and the correlations between the expression of eEF1A1 protein and the clinicopathologic parameters of HCC patients was further analyzed. The expressions of eEF1A1 mRNA and protein in HCC cells (including Huh7, SMMC7721, MHCC97H and Hep3B) and the normal liver cells HL-7702 were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The specific shRNA-1/2 targeting eEF1A1 gene (eEF1A1-shRNA-1/2) and the overexpression plasmid pcDNA3.1- eEF1A1 were respectively transfected into Huh7 and SMMC7721 cells, and the alteration of eEF1A1 expression was verified by real-time fluorescent quantitative PCR and Western blotting, respectively. Then the proliferation and cell cycle of HCC cells were detected by EdU and FCM assays, respectively. In addition, the expressions of cyclin D2 and the signal transducer and activator of transcription 1 (STAT1)-related pathway proteins in Huh7 cells transfected with eEF1A1-shRNA-1/2 and in SMMC7721 cells transfected with pcDNA3.1- eEF1A1 were detected by Western blotting. Results: The expression level of eEF1A1 protein in HCC tissues was markedly higher than that in the adjacent tissues (P 0.05) in SMMC7721 cells after eEF1A1 overexpression and treatment with STAT1 inhibitor fludarabine. Conclusion: eEF1A1 regulates the expression of cyclin D2 through STAT1 pathway, thereby promoting the cell cycle progression and proliferation of primary HCC cells.
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Objective: To investigate the effects of RNA interference targeting eukaryotic translation elongation factor 1A1(eEF1A1) expression on the proliferation of hepatocellular carcinoma Hep3B cells, and to explore its possible mechanism. Methods: A recombinant vector pGPU6/GFP/Neo-eEF1A1-shRNA containing short hairpin RNA (shRNA) targeting eEF1A1 was constructed, and then it was transfected into the Hep3B cells. After tansfection with pGPU6/GFP/Neo-eEF1A1-shRNA, the expressions of eEF1A1mRNA and protein were examined by real time fluorescence quantitative PCR and Western blotting, respectively, the cellular growth ability was examined by cell counting kit 8 (CCK-8) assay, the colony formation ability was detected by colony formation assay, and the cell cycle distribution of Hep3B cells was detected by flow cytometry (FCM). The expression levels of cyclin D1 and cyclin-dependent kinase 4 (CDK4) proteins in Hep3B cells were examined by Western blotting. Results: The recombinant vector pGPU6/GFP/Neo-eEF1A1-shRNA was successfully constructed, and the Hep3B cells with stable expession of eEF1A1 were established. The expression levels of eEF1A1mRNA and protein in Hep3B cells after transfection with pGPU6/GFP/NeoeEF1A1-shRNA were lower than those in the negative control cells (Hep3B cells transfected with a negative control vector pGPU6/GFP/Neo-NC) and the blank control cells (Hep3B cells without any transfection) (P < 0.01, P < 0.05). As compared with the negative control, the cellular growth ability and the colony formation ability of Hep3B cells after tranfection with pGPU6/GFP/Neo-eEF1A1-shRNA were obviously decreased (P < 0.05, P < 0.01); the percentage of the cells in G1 phase was increased (P < 0.01), and which in S-phase was decreased (P < 0.05); the expression levels of cyclin D1and CDK4 proteins were down-regulated (both P < 0.05). Conclusion: Down-regulation of eEF1A1 expression can inhibit the proliferation of Hep3B cells.