Effects Of Downregulated EEF1A1 On Apoptosis,Proliferation And Migration Of Hepatocarcinoma Cells And Interaction Between EEF1A1/A2 And ANXA7

Background:Hca-F and Hca-P cell lines are highly homologous cell lines with different lymph node metastasis ability established by our labrotary.They were gained by the foot pads of mice inoculated with mouse hepatocarcinoma ascites cells,and repeatedly screened through lymphatic system.Hca-F cell lines perform high metastatic potential,while Hca-P cell lines perform low metastatic potential.Previous study shows that the expression of ANXA7 at both gene and protein levels in Hca-F cells were significantly higher than that in Hca-P cells.Furthermore,we found that expression levels of ANXA7 are positively correlated with cell proliferation and metastasis,implicating that ANXA7 is possibly an critical gene affecting lymphatic metastasis ability of hepatocarcinoma cells.e EF1A1/A2(Eukaryotic translation elongation factor 1A1/A2)are important molecules involved in the process of protein translation.Recently,it has been realized that they are highly expressed in various malignant tumors,closely associated with the proliferation,invasion and metastasis of tumor cells.Protein is the executor of cell function,which regulates a variety of biological processes by the way of protein-protein interaction.Therefore,investigating the correlation of ANXA7 and e EF1A1/A2 is of great significance for further explore the mechanism of proliferation and metastasis of hepatocarcinoma cells.Objective: To investigate the effect of downregulating e EF1A1 gene on the apoptosis,proliferation and migration of hepatocarcinoma cells,and to discuss the association with their expression as well as to analyze the possible interactions between e EF1A1/A2 and ANXA7 in hepatocarcinoma cells by respectively downregulating the expression of e EF1A1 and ANXA7.Method:The p GPU6/GFP/Neo-e EF1A1-sh RNA and p GPU6/GFP/Neo-NC-sh RNA were established and transiently transfected into Hca-P cells,and transfection efficiency was determined after 48 h.Hca-P cells were divided into three groups,namely that blank control group(CON),e EF1A1-sh RNA group(e EF1A1-sh RNA)transfected with p GPU/GFP/Neo-e EF1A1-sh RNA and negative control group(NC)transfected with p GPU/GFP/Neo-NC.Cell proliferation,apoptosis and migration were detected by CCK-8 method,flow cytometry and Transwell test respectively.The expression of e EF1A2 and ANXA7 at m RNA and protein levels were detected by q RT-PCR and Western blot.Then we revived the Hca-P cells with stable low expression of ANXA7 preserved by our group.Similarly,the expression of e EF1A1,e EF1A2 at m RNA and protein levels of three groups were measured by q RT-PCR and Western blot.Results:The sh RNA targeting e EF1A1 gene successfully knocked down its expression at both m RNA and protein levels.As the q RT-PCR and Western blot results showed,compared with the CON group and NC group,the expression levels of e EF1A2 and ANXA7 genes of e EF1A1-sh RNA group were decreased(P<0.05).It was also found that the ratio e EF1A1 to e EF1A2 in Hca-P cells rise following downregulation of e EF1A1.While the expression levels of e EF1A1/A2 of ANXA7-sh RNA group reduced at both m RNA and protein levels,compared with CON group and NC group(P<0.05),but there was no apparent difference in their degree of decline.Flow cytometry assay suggested that the apoptotic rates of the three groups were 2.85±0.37%(CON),4.71±0.21%(e EF1A1-sh RNA)and 3.01±0.33%(NC)(P<0.05).We found that cell proliferation obviously declined after e EF1A1-sh RNA transfection by CCK-8 method.It also indicated that cell migration in e EF1A1-sh RNA group was lower than that of the untreated group and the negative control group through Transwell test,as the number of migrating cells was 123.75±9.26,66.84±17.47,115.31±12.59(P<0.05).All the results illustrated that with the inhibition of the expression of e EF1A1 by sh RNA,cell proliferation and migration declined(P<0.05),and the number of apoptotic cells increased(P<0.05).No significant difference was found between the blank control group and the negative control group(P>0.05).Conclusion: Knocking down the expression of eEF1A1 gene by shRNA targeting it can induce apoptosis,inhibit proliferation and migration on Hca-P cells.It is suggested that the expression of e EF1A1,e EF1A2 and ANXA7 are closely related,and some interaction exists between e EF1A1 and ANXA7,which may jointly involved in the regulation of tumor occurrence and development.