ECRG4Gene Expression In Gastric Cancer Tissues And The Intervention Study Of Gastric Cancer Cell Lines

Objective:To study the gene ECRG4in gastric adenocarcinoma and its expression differences with the patient of the clinical and pathological features of relevance.Clear gastric cancer cells and normal gastric epithelium cells, ECRG4mRNA proteins quantitative difference, and with different concentrations of demethylation drug5-aza-2’-deoxycytidine (5-Aza-CdR) intervention gastric cancer cells, in different time points, from cell growth status, the cell vitality, the cell cycle, proliferation index to judge demethylation drugs to gastric cancer the influence of the cell lines.Finally identificate the expression of ECRG4mRNA and protein after intervention.Methods:We collected from year2009to2011preserved stomach surgery pathological paraffin specimens,gastric cancer58cases as gastric cancer group, collect tissues adjacent to cancer15cases as adjacent normal gastric cancer tissues, the benign disease that in same period for the gastric tissues15cases as normal group.We use immunohistochemical method to detect ECRG4gene expression and analyze the clinical parameters of the relationship. With different concentrations (0μM,5μM,10μM)5-Aza-CdR intervention,gastric cancer cells SGC7901, in different time points (24h,48h,72h) to observe cell growth situation with microscope, the determination of the cell vitality with MTT, to analyze the cell cycle changes with PI flow cytometry.With RT-PCR and Western blot methods to quantitative detect ECRG4mRNA its encoded protein expression of gastric cancer cells SGC7901and normal gastric epithelium cells GES-1.Again with different concentration (0μM,5μM,10μM)5-Aza-CdR intervention, after48hours to detect SGC7901ECRG4mRNA and coding protein expression recovery in RT-PCR and Western blot methods.Results:1.ECRG4protein expression in the tissue of gastric cancer obviously down-regulated or missing, the cancer adjacent tissues cut or missing partly, and normal tissues common expression in the stomach.The three comparing statistical significance (F=42.819, P<0.001). Two comparison between two groups, have normal group and gastric cancer group, the adjacent normal gastric cancer group and gastric cancer group are different, the difference was statistically significant (P<0.001). But the normal group and the adjacent normal gastric cancer group compared, P>0.05, protein expression is no statistical difference. Using optical microscope to observe,we can see visible light yellow to tan positively for color expression, the main role in the cytoplasm and cell membrane. There is a relationship between ECRG4expression and gastric cancer patients about the differentiation degree (χ2=8.357, P<0.05). There is no relationship between ECRG4expression and patient age, sex, tumor location and whether lymph node metastasis (χ2=0.284～2.636, P>0.05).2.5-Aza-CdR intervention in48h,72h, inverted microscope observation found SGC7901cells are arranged close degree is abate, intercellular broadens, and the growth of stack scattered trend, and10μM drug treatment group than in the5μM drug treatment group effect more apparent.3. MTT test showed that cells SGC7901in5-Aza-CdR intervention after24hours,10μM drug concentration compared with a control group, OD570value reduced a statistically significant (P<0.05);48,72hours after the intervention, drug concentration5μM,10μM compared with the control group, were significantly reduce OD570value, a statistically significant difference (P <0.01); And48hours from the start,compared to5μM drug concentration group,the10μM group OD570evidently decreased value, a statistically significant difference (P<0.01).4.5-Aza-CdR treatment gastric cancer cell lines SGC7901, along with the increase of concentration and processing time, the cell cycle changes, the present G1phase block, cell proliferation index also gradually decrease. Among them and control group and5μM5-Aza-CdR intervention group compared to10μ M5-Aza-CdR intervention48hours after the cell proliferation index reduce are statistically significant (P<0.05).5.With5-Aza-CdR intervention SGC7901cells48hours, RT-PCR shows0μM group,5μM group and GES-1group ECRG4mRNA relative express should have statistical difference (P<0.05),10μM group and GES-1group will not have a statistics difference (P>0.05);0μM group and5μM group did not have statistical differences (P>0.05);0μM group,5μM group and10μM group has statistics difference (P<0.05). Western blot display,5-Aza-CdR intervention48hours, with the increase of drug concentration, SGC7901cells ECRG4protein expression gradually rise, than5μM group,the10μM group more obvious, the difference was statistically significant (P<0.01).Conclusion:1. ECRG4protein downregulated in gastric cancer tissues, normal tissues, gastric cancer tissues and adjacent normal gastric cancer tissues,ECRG4protein expression are differences; There is a relationship between the expression of the ECRG4and gastric cancer patients about the differentiation degree.2.5-Aza-CdR inhibit SGC7901cell growth and produce the G1phase block, with the drug concentration and the processing time increases,it shows more obvious inhibition. After48hours the inhibition get the strongest.3. The stomach cancer cells SGC7901ECRG4mRNA and protein expression quantity was significantly lower than the normal gastric epithelium cells GES-1. Processing after48h with5-Aza-CdR,SGC7901cells ECRG4mRNA and protein expression are raised, and10μM drug treatment group than in the5μM drug treatment group restore their expression effect more apparent.