Effect Of Helicobacter Pylori On Expression Of Pro-Apoptosis Gene PUMA

Helicobacter pylori(H.pylori,Hp),a spiral,gram-negative and microaerophilic bacterium,is one of the major factors inducing pathological changes in human gastric mucosa,and about half of the people in the world are infected with it.The World Health Organization (WHO) and International Agency for Research on Cancer consensus group(IARC) classified this bacterium as definite biological carcinogen. However,its role in generation of gastric cancer with H.pylori remains not well understood.PUMA(p53 up-regulated modulator of apoptosis) is BH3-only member of the Bcl-2 family which has owned powerful effect to promote apoptosis,which played a significant role in the priming of apoptosis and development of tumor by p53-dependent and p53-independent pathway. It was located on chromosome 19q.The full cDNA was 1.9Kb in size and encoded a 193-amino acid protein.The gene contained four exons(1a or 1b,2,3,4) and three introns,which transcripted four isoforms(PUMA-α,-β,-γand-δ).The distribution of expression and functions of four PUMA isoforms in tumor cells are still misunderstood now.The subject was designed to study the expression of four isoforms of PUMA gene in gastric cancer tissues and cells and the effect of two isolated H.pylori causing ulceration and gastric cancer,respectively,on the changes of expression of four isoforms of PUMA in human gastric cancer BGC-823 cells or human gastric epithelial GES-1 cells.Besides,it also aimed at exploring the mechanism underlying the effect of H.pylori on PUMA and the relationship between H.pylori and gastric cancer.Methods:1.The expression of four isoforms of PUMA gene in gastric cancer tissues,normal tissues and two gastric cancer cell lines were detected by semi-quantitative RT-PCR.The PCR products were then cloned and sequenced.2.Different H.Pylori strains were isolated from human gastric mucosa and added to gastric cancer cell line BGC-823 cells or gastric epithelial cell line GES-1 cells respectively.Total RNA was extracted using Trizol regent.We observed the changes of mRNA expression levels of PUMA-α,-β,-γ,-δat different time points.3.MTT assay was used to detect the proliferation of BGC-823 cells or GES-1 cells after H.pylori stimulating for 3 h,6 h and 9 h.4.The two H.pylori strains stimulated BGC-823 cells or GES-1 cells for 3,6,9 hours,then the change of mitochondrial membrane potential was detected by JC-1.5.Meanwhile,SYBR GreenⅠreal-time PCR was applied to assess the mRNA expression levels of p53 after H.Pylori co-cultured with BGC-823 cells or GES-1 cells to study the correlation with four PUMA isoforms. Results:1.PUMA-αand -βwere positively expressed in normal tissues,but hardly detected in cancer tissues(P＜0.05).PUMA-γand -δwere expressed both in cancer tissues and normal tissues and the expression in normal tissues was significantly higher in cancer tissues(P＜0.05).Expression of the four PUMA isoforms was detected in the two gastric cancer cell lines.Expression of the PUMA-βin low differentiated BGC-823 cells significantly was higher than in moderately differentiated SGC-7901 cells(P＜0.05).The sequencing results showed that they were PUMA-α,-β,-γand -δ,when compared with the sequences in GenBank. they displayed 100%,100%,99%,100%separately in homology.2. Compared with un-stimulant cell,the four isoforms of PUMA mRNA were all down-regulated in BGC-823 cells which were treated with two H.pylori strains respectively at three time points(P＜0.05).What is more, the expression of PUMA isoforms had their own characteristics:PUMA-βwas difficult to be detected.In the group of BGC-823 cells being treated with ulcerated H.pylori strain,PUMA-αmRNA was the most significant down-regulation at 6 hours.There were no remarkable differences of the decrease among the expression rates of -γat the three time points.-δmRNA decreased more notably at nine hours.But in the tumorous H.pylori group,we could not detect the expression of -αat 3 hours,-γat 3, 6 hours and -δat 9 hours.3.The results by two H.pylori strains which stimulated GES-1 cells showed that PUMA isoforms mRNA were up-regulated at three and six hours after being treated with ulcerated H.pylori strains.However,they were down-regulated at nine hours. Compared with control group,the expression levels of PUMA isoforms were down-regulated at 3 hours in GES-1 cells which stimulated with tumorous H.pylori and then had the recovery trend at 6 and 9 hours.4. The MTT assay showed that the two H.pylori strains could stimulate the proliferation of BGC-823 cells.However,to GES-1 cells,the ulcerated H.pylori strain induced apoptosis firstly and then enhanced proliferation. Tumorous H.pylori promoted proliferation of GES-1 cells.5.It was found in BGC-823 or GES-1 by JC-1 fluorescent staining that the two H.pylori strains all promoted proliferation.6.The results of SYBR GreenⅠreal-time PCR indicated that p53 mRNA significance up-regulated(P＜0.01) in BGC-823 cells which were treated with the two H.pylori strains at nine hours in comparison with control group.But there were no changes at three and six hours.Compared with control group,the expression of p53 mRNA was increased at three time points in GES-1 cells with the two H.Pylori strains and there were time-effect relationship(r_{ulcerated H.pylori}=-0.978,r_{tumorous H.pylori}=0.943,p＜0.01).Conclusion:1.The expression of PUMA isoforms was down-regulated in gastric cancer tissues,which was negative correlation to cancer,and the expression of PUMA-βwas also involved in cell differentiation.2. H.pylori could cause proliferation of BGC-823 cells and,downregulate the expression of PUMA isoforms.But this down-regulation was independent with p53.The expression of PUMA was negative correlation to proliferation.3.The ulcerated H.pylori strain induced apoptosis at first and then promoted proliferation in GES-1 cells.The alteration of PUMA was coincidental with the proliferation variation of cells.The expression of p53 was rapidly up-regulated at the prophase of infection,and then recovered gradually.There was similar tendency between p53 and PUMA.It indicated that the expression of PUMA might be dependent with p53.4.Tumorous H.pylori was able to promote CES-1 growth, inhibit the expression of PUMA and then recover,and increase the p53 mRNA.The effect of PUMA by tumorous H.pylori might be dependent with p53 in GES-1 cells.In a word,the results,stimulating cells by different H.pylori strains,were disparate,and tumorous H.pylori had a more remarkable effects on inducing cell proliferation.