| Gastric cancer (GC) is one of the most common malignancies and the second leading cause of incidence following lung cancer, moreover GC is the second leading cause of cancer-related death worldwide. There is very high incidence of GC in China, and WHO reported that the42%of new incidence occured in China every year. It is difficult to diagnose GC early owing to lack of effective diagnosis marker, and most of patients showed the advanced GC at first diagnosis thus lost the chance of operation; In addition, GC is not sufficiently sensitive to present radiotherapy and chemotherapy resulting in poor prognosis; Previous studies showed that the5-year survival rate of GC patients was only24%. So, it is essential to explore the mechanism of gastric carcinogenesis to seek for effective diagnosis marker and to improve therapeutic effect.The hallmark of malignant tumor is the unlimited cell proliferation. There are two factors affecting cell proliferation, one is positive factor promoting cell proliferation and the other is negative factor inhibiting cell proliferation. They synchronously afftect the genes associated with cell cycle, cell proliferation, cell differentiation, cell apoptosis and so on. Usually, the cell growth is under control because of the balance between positive and negative factors; once the balance is lost then the cell growth is out of control to resulting in carcinogenesis. Ornithine decarboxylase (ODC) is the first key enzyme to catalyze the biosynthesis of polyamines, which can promote cell proliferation and is closely associated with tumorigenesis. RUNX3is an important transcription factor regulating the growth of gastric epithelial cells. It can suppress cell proliferation by inhibting the progress of cell cycle and promoting cell apoptosis. In this research, we explored the dysregulation mechanism of ODC and RUNX3in gastric carcinogenesis to improve the prevention and cure. It is well known that Helicobacter pylori (H. pylori) infection increased the risk of GC. And the infection could induce chronic gastric inflammation that stimulated excessive cell proliferation resulting in gastric carcinogenesis. H. pylori has many virulent factors, and cytotoxin-associated gene A (cagA) is the factor most closely associated with gastric carcinogenesis. Previous studies reported that ODC enzyme activity was increased in the patient with chronic gastritis and gastic cancer, and ODC enzyme activity in H.pylori-positive patient with chronic gastritis was much higher than that in negative control, but the molecular mechanism was not clear; c-Myc could activate ODC gene transcription in the fibroblasts, but the relationship between them in gastric epithelial cells was not clear. So, we deeply explored that whether ODC could be regulated by H. pylori and its virulent factor CagA, whether c-Myc functions in the regulation in gastric epithelial cells and the influence of ODC on GC cell proliferation.Objective:To explore the mechanism of ODC expression upregulation induced by H. pylori infection and the role of ODC in gastric carcinogenesis.Methods:(1) Immunohistochemistry (IHC) was used to detect ODC and c-Myc protein expression in clinical specimens including chronic superficial gastritis, chronic atrophic gastritis and primary GC. The IHC results of c-Myc and ODC were quantified and assayed for their correlation by SPSS software.(2) Different strains of H. pylori (including wild-type and mutant cagA strains) were used to infect GC cell AGS and immortalized gastric epithelial cell GES-1at a bacteria-to-cell ratio of100:1. Western blot was used to detect ODC protein expression. Transfection of CagA eukaryotic expression vector (pcDNA3.1-WT-cagA) into the AGS, GES-1and GC cell BGC-823,24h later the cells were harvested and assayed for ODC mRNA and protein expression, c-myc mRNA and protein expression by QRT-PCR and Western blot. (3) The luciferase reporter vector containing wild-type ODC gene promoter (including two c-Myc binding elements) was constructed. After identification by dual RE digestion and DNA sequencing, it was used as the template for constructing the luciferase reporter vector containing mutant ODC gene promoter (c-Myc binding elements were deleted), which was identified by dual RE digestion and DNA sequencing. Wild-type or mutant ODC gene promoter-luciferase reporter and pcDNA3.1-WT-cagA were cotransfected into the BGC-823cells and the lucifease activity was detected.(4) The c-myc siRNA expression vector (pSUPER-c-myc) was constructed, confirmed by DNA sequencing, and transfected into the BGC-823cells to inhibit cellular c-Myc protein production. After transfection of pcDNA3.1-WT-cagA, western blot was used to detect ODC protein expression.(5) After treatment with four specific signal protein inhibitors (PP1to block Src family kinase activity, PD98059to block MEK1kinase activity, LY294002to block PI3K kinase activity and BAY11-7082to inhibit NF-κB activity), pcDNA3.1-WT-cagA was transfected into the cells. In addition, the mutant CagA expression vector (pcDNA3.1-PR-cagA) was transfected into the cells.24h later the cells were harvested and assayed for ODC protein expression by Western blot.(6) After infecting with a green fluorescent protein (GFP)-expressing adenovirus vector harboring ODC antisense RNA, the BGC-823cells were co-cultured with H. pylori for2h. After digestion by trypsin and counting, the cells were assayed for proliferation by colony foci formation.Results:(1) The IHC results showed that the immunostaining intensity and positive rate of ODC protein in gastric epithelial cells increased from chronic superficial inflammation, chronic atrophic inflammation to primary GC. Moreover, ODC protein was expressed in all patients with chronic gastritis accompanying with intestinal metaplasia (IM).(2) The western blot assay showed that ODC protein level was upregulated apparently in H.pylor-infected GES-1at1,2,4,8h after infection, and the effect was time-dependent. GES-1cells incubated with H. pylori NCTC11637for4h showed similar ODC protein elevation. Additionally, similar results were also obtained in AGS cells incubated with H. pylori11637and26695. But, the cagA mutant H. pylori26695infection could not induce ODC protein upregulation in AGS and GES-1cells.(3) Transfection of pcDNA3.1-WT-cagA into AGS, BGC-823and GES-1could induce ODC mRNA and protein expression upregulation. Dual luciferase reporter analysis showed that CagA could enhance the luciferase activity mediated by wild-type ODC gene promoter.(4) c-myc mRNA and protein level were also upregulated in the pcDNA3.1-WT-cagA transfected cells. The IHC results of c-Myc and ODC were quantified and analyzed by Spearman's correlation, which showed that the expression of both proteins was positively correlated in clinical gastric specimens (p=0.635,P<0.001). Dual luciferase reporter assay showed that CagA could not enhance the luciferase activity mediated by mutant ODC gene promoter. After inhibition of c-Myc expression by c-myc siRNA in BGC-823, CagA-mediated ODC protein expression upregulation was attenuated.(5) Western blot analysis showed that PP1, PD98059, LY294002and BAY11-7082could inhibit CagA-induced ODC protein level upregulation. Transfection of pcDNA3.1-PR-cagA (expressed CagA protein can not be phosphorylated by cellular kinase) could not induce ODC protein level upregulation.(6) Colony foci number was diminished after ODC inhibition by ODC antisense RNA both before and after H. pylori infection.Conclusions:(1) ODC protein participated in gastric carcinogenesis and IM was closely correlated with ODC protein expression.(2) The luciferase reporter vectors containing wild-type or mutant ODC gene promoter and c-myc siRNA expression vector were constructed successfully.(3) H. pylori infection induced ODC protein level upregulation dependent on CagA, and CagA could induce ODC gene expression by enhancing ODC gene promoter activity.(4) The c-Myc binding elements were necessary for ODC gene promoter enhancement induced by CagA, and cellular c-Myc protein was necessary to CagA-induced ODC protein level upregulation.(5) Src, MEK, PI3K and NF-κB pathway participated in CagA-mediated ODC protein level upregulation, and CagA required phosphorylation to exert its function.(6) Inhibition of ODC protein expression decreased the proliferation potential of GC cell before and after H. pylori infection. As a transcription factor, RUNX3regulates multiple gene expression to inhibit cell proliferation, promote cell apoptosis and inhibit cancer cell migration, which functions as a tumor suppressor. Previous studies showed that RUNX3was inactivated in human primary GC due to hypermethylation in RUNX3gene promoter, loss of allele heterozygosity and mislocaliztion of RUNX3in cytoplasm. However, some patients showed downregulated RUNX3, but lacked upper inactivation mechanisms. This implies that other factor could regulate RUNX3expression. Recently, discovery of miRNA and rise of its research open an new era for study on RUNX3downregulation. Based on the post-transcriptional regulation by miRNA, we explored the mechanism of RUNX3regulation by miR-532-5p and the influence of miR-532-5p on the biological functions of gastric epithelial cells.Objective:To explore the regulatory mechanism of RUNX3by miR-532-5p in GC and the influence of miR-532-5p on the biological functions of GC cells.Methods:(1) The expression of miR-532-5p, RUNX3mRNA and protein level in seven GC cell and immortalized gastric epithelial cell GES-1were detected by QRT-PCR and Western blot. The results of protein expression were quantified and SPSS software was used to analyze the correlation between miR-532-5p and RUNX3mRNA or protein.(2) The miR-532-5p eukaryotic expression vector (pSilencerTM4.1-CMV-532) was constructed, identified by dual RE digestion and DNA sequencing, and transfected into the gastric epithelial cells. Meanwhile, the specific miR-532-5p mimics and inhibitor were transfected into the cells individually. QRT-PCR and Western blot were used to detect the expression of miR-532-5p, RUNX3mRNA and protein, and the RUNX3targets p21and Bim protein.(3) The wild-type RUNX33'UTR-luciferase reporter vector containing miR-532-5p binding sequence (pMIR-REPORTTM-RUNX3WT UTR) was constructed. After identification by dual RE digestion and DNA sequencing, it was used as the template for constructing the mutant RUNX33'UTR-luciferase reporter vector containing mutant sequence for miR-532-5p binding (pMIR-REPORTTM-RUNX3Mut UTR). Dual RE digestion and DNA sequencing confirmed the inserting sequence in the luciferase reporter vector.(4) Then the specific miR-532-5p mimics and pMIR-REPORTTM-RUNX3WT UTR or pMIR-REPORTTM-RUNX3Mut UTR were cotransfected into the GES-1, and at the same time the pMIR-REPORTTM-(3-gal Control was cotransfected to rectify the differences caused by transfection efficiency.48h later, luciferase activity and β-galactosidase activity were detected.(5) The BGC-823cells were transfected by the specific miR-532-5p mimics and control, collected48h later and assayed for cell cycle and apoptosis by flowcytometry.Results:(1) The expression of miR-532-5p, RUNX3mRNA and protein in GC cells and immortalized cells were detected by QRT-PCR and Western blot. The results showed that, there were much higher miR-532-5p level in AGS, KATO-III and NUGC-3cells than other cells, but there were absense of RUNX3expression in these cells. There were much higher RUNX3expression in BGC-823, GES-1, SGC-7901and MKN-45cells, but there were lower miR-532-5p expression. The results of protein expression were quantified and analyzed for correlation between miR-532-5p and RUNX3by SPSS software. The results showed that miR-532-5p expression was negatively correlated with RUNX3mRNA and protein expression, and the correlation coefficient was-0.738and-0.776respectively which was significant (p<0.05).(2) Dual RE digestion and DNA sequencing confirmed the correctness of pSilencer4.1-CMV-532. The result of QRT-PCR showed that miR-532-5p expression was increased apparently in the transfected cells, confirming the successful construction of pSilencerTM4.1-CMV-532.(3) Transfection of the specific mimics into BGC-823, GES-1and SGC-7901could increase miR-532-5p expression resulting in decreased RUNX3protein expression. And transfection of pSilencerTM4.1-CMV-532could inhibit RUNX3protein expression, but the inhibition effect was less apparent than that caused by mimics. In contrast, transfection of miR-532-5p inhibitor decreased the level of miR-532-5p resulting in increased RUNX3protein expression.(4) The analysis of luciferase reporter activity showed that miR-532-5p mimics could decrease the luciferase activity mediated by WT RUNX33'UTR, but had no effect on the luciferase activity mediated by mutant RUNX33'UTR.(5) The Western blot assay showed that the cell cycle associated p21and apoptosis associated Bim protein were decreased after transfection of miR-532-5p mimics, whereas p21and Bim protein expression were increased after transfection of miR-532-5p inhibitor. The flowcytometry analysis showed that miR-532-5p mimics could decrease the percentage of cell apoptosis, increase the ratio of G2-M phase cells and decrease the ratio of G1-phase cells resulting in increased GC cell survival and accelerated cell cycle progression.Conclusions:(1) miR-532-5p could negatively regulate RUNX3protein expression in GC cells and gastric immortalized cells.(2) The plasmids pSilencerTM4.1-CMV-532, pMIR-REPORTTM-RUNX3WT UTR and pMIR-REPORTTM-RUNX3Mut UTR were constructed successfully.(3) miR-532-5p negatively regulated RUNX3protein expression depending on the interaction of its seed sequence and RUNX33'UTR. RUNX3was the direct target of miR-532-5p.(4) miR-532-5p decreased apoptosis, and accelerated cell cylce progression of GC cells by decreasing Bim and p21protein expression. Thus, miR-532-5p could increase the survival and promote the proliferation of GC cells.
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