The Effect Of TRIM67 On Proliferation And Apoptosis Of Gastric Cancer Cell

Objective: Tripartite motif containing 67(TRIM67)is a member of the TRIM protein family,the expression of this gene is silenced or down-regulated in the gastric cancer(GC),but the mechanism of TRIM67 is unknown in GC.A study was undertaken to examine the relationship between TRIM67 and cell growth,apoptosis in gastric cancer cell by real-time PCR,western blot analysis and flow cytometry.We mainly clarified its biological function as a tumour suppressor in gastric cancer.Design:1 In the study,21 paired biopsy specimens of gastric tumour and adjacent non-tumour sites from patients with gastric cancer were obtained during endoscopy.Real-time PCR was used to detect the expression level of TRIM67 mRNA in 21 paired sample,and we analysed the difference between the expression of TRIM67 mRNA in gastric tumour and adjacent non-tumour.2 Reverse transcription PCR was used to detect the expression of TRIM67 mRNA in gastric cancer cell lines(AGS,BGC823,MGC803,MKN1,MKN45,MKN74,SNU1,SNU638).Methylation status of the ZNF545 promoter confirmed by bisulfite genomic sequencing(BGS).After cells were treated with 2 mM of the DNA demethylating agent 5-Aza for 96 h,we examined the expression of TRIM67 compare with control group which is without 5-Aza treatment.3 Recombinant plasmid of pcDNA3.1-Flag-TRIM67 was constructed and transfected into AGS and MKN1 GC cell lines.The stable transfected cells was screened by G418.Effect of ectopic TRIM67 expression on tumour growth.Ectopic expression of TRIM67 mRNA and protein in AGS and MKN1 cell lines was evidenced by RT-PCR and western blot analysis,respectively.The MTT assay was preformed to determine effect of TRIM67 relative to cell viability in gastric cancer cell lines.The effect of TRIM67 on cancer cell growth was further confirmed by colony formation assay.We also used MKN74 GC cell line which is higher expression of TRIM67 to be stably transfected with shRNA in order to knocked down TRIM67.MTT assay and colony formation assay was preformed to determine effect of TRIM67 relative to cell viability and cell growth in MKN74 GC cell line.4 We based on the stable cell lines transfected pcDNA3.1-Flag-TRIM67 compared with empty vector.After staining with PI,the effect of TRIM67 on cell cycle distribution was determined by flow cytometry and analysed by Flowjo software.The expression of CyclinD1,CDK4,P21 and P27 were detected by western blot analysis to identify the relatioship between TRIM67 and these proteins.5 Apoptosis of stable cell lines transfected pcDNA3.1-Flag-TRIM67 was assessed by flow cytometry after staining with Annexin V(FITC-conjugated)and 7-amino-actinomycin(7-AAD).The expression of apoptosis-related proteins(Caspase-3,Caspase-7,Caspase-8,Caspase-9,poly ADP-ribose polymerase)were detected by western blot analysis in order to demonstrate the effect of TRIM67 on apoptosis of Gastric Cancer cell.Results:1 The expression level of TRIM67 in primary gastric cancers compared with adjacent normal tissuesWe detected the mRNA expression of TRIM67 in primary gastric cancers and adjacent normal tissues.The expression of TRIM67 were downregulated in primary gastric tumours compared with adjacent normal tissues of 21 gastric cancers(P < 0.001,n=21).2 The expression level of TRIM67 gene and methylation levels of its promoter in gastric cancer cell linesWe first determined the expression level of TRIM67 in gastric cancer cell lines,TRIM67 was silenced or downregulated in 7(AGS,BGC823,MGC803,MKN45,MKN1,SNU1,SNU638)of the 8 gastric cancer cell lines but was readily expressed in normal human gastric tissue.and the normal human gastric epithelial cell line(GES1).The promoter methylation status of TRIM67 was further evaluated by BGS,CpG sites of TRIM67 promoter region were densely methylated in the 7 silenced cell lines,but only sparsely methylated in MKN74 and GES1 cells and no methylation was found in normal human gastric tissue.GC cell lines(AGS,BGC823,MKN45,SNU1,SNU638)were treated with the DNA methyltransferase inhibitor 5-Aza.Re-expression of TRIM67 was observed in all five cell lines examined.3 The effects of TRIM67 on GC cell proliferation and cell growthThe frequent silencing of TRIM67 in GC cell lines and tissues suggests that TRIM67 is probably a tumour suppressor.TRIM67 expression vector was stably transfected into the TRIM67-silenced AGS and MKN1 cells.Re-expression of TRIM67 in the stably transfected AGS and MKN1 cells was evidenced by RT-PCR and western blot analysis.Ectopic expression of TRIM67 caused a significant decrease in cell viability in AGS(P < 0.0001)and MKN1(P < 0.0001)and significantly inhibited cell growth,as evidenced by colony formation assays in AGS(P < 0.01)and MKN1(P < 0.01).To further confirm the tumour suppressive role of TRIM67 in gastric cancer,TRIM67 was knocked down by shRNA in MKN74 cells which showed endogenous TRIM67 expression.Knockdown of TRIM67 markedly enhanced cell viability(P < 0.01)and clonogenicity(P < 0.01).4 The impacts of TRIM67 on cell cycle in GC cell linesTo determine the mechanism by which TRIM67 inhibited cell growth,we analysed the effect of TRIM67 on cell cycle distribution and apoptosis by flow cytometry.The ectopic expression of TRIM67 led to a significant increase in cells of the G1 phase population,and a corresponding reduction in the S phase population.G1 cell cycle arrest by TRIM67 was evidenced by reduced protein expression of key G1 cell cycle regulators(cyclin D1 and CDK4)and elevated G1 cell cycle inhibitors(p21 and p27)by western blot analysis.5 The effects of TRIM67 on GC cell apoptosisWe therefore examined the contribution of apoptosis to the observed growth inhibition of TRIM67-transfected cells using flow cytometry with Annexin V and 7-AAD double staining.Our results showed an increase in the numbers of total apoptotic cells(P < 0.05)in TRIM67-transfected AGS cells compared with AGS cells transfected with empty vector.This effect was also observed in TRIM67-transfected MKN1 cells,the proportions of both early apoptotic cells(P < 0.001)and late apoptotic cells(P < 0.05)being significantly increased compared with the control vector-transfected MKN1 cells.We examined the key cell apoptosis regulators and found that TRIM67 significantly enhanced the protein levels of the active forms of caspase-3,caspase-7,caspase-8,caspase-9 and poly ADP-ribose polymerase(PARP)by western blot analysis.Conclusions:1 The expression mRNA of TRIM67 in adjacent normal tissues is higher than the expression in primary gastric tumours.2 As an important factor,Promoter hypermethylation of TRIM67 can silence an down-regulate the expression of TRIM67 in gastric cancer cell lines.3 TRIM67 maybe as a novel tumour suppressor gene,which can inhibited cell viability and colony forming activity in GC cell lines.4 TRIM67 inhibits G1/S cell cycle transition by up-regulating p21,p27 and down-regulating cyclin-D1,CDK4.5 TRIM67 enhances the protein levels of the active forms of caspase-3,caspase-7,caspase-8,caspase-9 and PARP to induces GC cell apoptosis.