Expression And Role Of Golgi Integral Membrane Protein 4(GOLIM4) In Human Head And Neck Cancer

Objective To investigate the expression of GOLIM4(Golgi integral membrane protein 4)in human head and neck cancer cells and its effects on proliferation,apoptosis and cell cycle of human head and neck cancer cells.Methods 1.Cultured hypopharyngeal carcinoma FaDu cells and tongue squamous cell carcinoma Tca-8113 cells,which were subjected to lentivirus infection according to the purpose of the experiment.The cell group infected with candidate gene-shRNA lentivirus was the experimental group,and the cell group infrcted with sh-Ctrl negative lentivirus was control group.2.The high-content screening assay was used to select the positive gene,and the effect of candidate genes(including GOLIM4)after knockdown on the growth of FaDu cells,and the effect of GOLIM4 after knockdown on the growth of Tca-8113 cells were tested by Celigo test.3.Real time PCR and Western blot were used to detect lentiviral infection efficiency at mRNA and protein levels.4.FACS analysis after PI staining was used to detect changes in cell cycle of FaDu cells and Tca-8113 cells after GOLIM4 knockdown.5.The BrdU assay was further applied to test the effect of GOLIM4 after knockdown on the number of cells in S phase.6.Finally,the effect of GOLIM4 after knockdown expression on apoptosis was detected by FACS analysis after staining with Annexin V.7.Statistical analysis was performed using SPSS 16.0 software.Results 1.GOLIM4 is a selected proliferation-related positive gene.According to the analysis of Celigo experimental data,the active cells of the shGOLIM4 group were lower than those of the shCtrl group in the two cells from the fourth day(P < 0.001).GOLIM4 could maintain cell proliferation activity,and low expression of GOLIM4 could inhibit the growth of human head and neck cancer cells.2.After the cells were infected with lentivirus,real-time qPCR showed that the expression level of shCtrl group was 1.000±0.038 in FaDu cells,and the expression level of shGOLIM4 group was 0.180±0.000.The GOLIM4 in the FaDu cells of the experimental group was significantly inhibited at the mRNA level(p<0.01),and the knockdown efficiency reached 82.0%.In Tca-8113 cells,the expression level of shCtrl groupwas 1.010±0.179,and the expression level of shGOLIM4 group was 0.431±0.051.The GOLIM4 in the Tca-8113 cells of the experimental group was significantly inhibited at the mRNA level(p<0.01),and the knockdown efficiency reached 56.9%;Western blot analysis showed that the expression of GOLIM4 at protein level in shGOLIM4 group of FaDu cells and Tca-8113 cells were significantly inhibited(p<0.001).3.FACS analysis using PI staining showed that,after GOLIM4 knockout,the percentage of cells in the G2/M phase of shCtrl group and shGOLIM4 group in FaDu cells was 15.45±0.382 and 10.51±0.1665,respectively,and in the Tca-8113 cells were 19.88±0.3214 and 10.77±0.4325,respectively,the number of G2 phase cells in both experimental groups were reduced(P<0.01).4.BrdU incorporation assay showed that,in FaDu cells,the proliferation multiple of shCtrl group and shGOLIM4 group in day4 were 1.412±0.007 and 1.076±0.059,respectively,and 2.805±0.044 and1.707±0.027 in Tca-8113 cells,respectively.That is,after GOLIM4 was knocked down,it can decrease markedly the cells in S-phase(P <0.01).5.FACS analysis by Annexin V staining showed that the apoptosis rates of shCtrl group and shGOLIM4 group in FaDu cells were4.45±0.1684 and 17.97±0.4258,respectively.The apoptosis rate of shCtrl and experimental group shGOLIM4 in Tca-8113 cells were 4.23±.0.275 and 10.43±0.4965,the apoptosis rate of the two experimental group significantly increased(P <0.01).Conclusion Low expression of GOLIM4 can inhibit the growth of human head and neck cancer cells,suggesting that GOLIM4 may have a tumor-promoting effect and provide a new potential target for the clinical treatment of human head and neck cancer.