| Background: Gastric cancer is the second leading cause of cancer-related deathworldwide, and the five-year survival rate was unsatisfactory after surgery inadvanced gastric cancer. Risk factors of gastric cancer mainly included H. pyloriinfection, Nitrite and nitrate diet, smoking and alcohol habits. Even though geneticaberrant plays critical roles, it’s not enough to explain the mechanism of gastriccarcinogenesis. Epigenetics is becoming one of the important field of cancer research.Increasing evidence indicates that abnormal epigenetic change may involved ingastric carcinogenesis. Homobox containing genes A11(HOXA11) are evolutionarilyconserved genes encoding transcription factors that regulate embryonic development,cell differentiation and proliferation.Reduced expression was found frequently inhuman tumors, including ovarian cancer, cervical cancer and endometrial carcinoma.However epigenetic changes and the effect of HOXA11on human gastric cancerremain unclear.Objective: To explore the possibility of HOXA11methylation serving as a gastriccancer detection marker, epigenetic changes were detected in gastric cancer cells andprimary cancer samples, and the association of HOXA11methylation with clinicfactors was analyzed. To see if HOXA11may serve as a therapeutic target in gastriccancer, the regulation of HOXA11expression and the function of HOXA11in humangastric cancer was investigated.Method: The expression of HOXA11was examined by RT-PCR in six gastric cancercell lines (AGS, NCI-N87, SGC7901, MGC803, BGC823, HGC27and MKN45)before and after5-aza-2’-deoxycytidine treatment. The methylation status of sixgastric cancer cell lines, five cases of normal gastric samples and112cases of primary gastric cancer tissues were detected by methylation specific PCR (MSP). BisulfiteSequencing was employed to validate the MSP results and further evaluate themethylation density. The association of HOXA11methylation and clinical pathologyfactors in gastric cancer patients was evaluated by Chi-Square test, and P<0.05wasconsidered as significanctly difference. The expression of HOXA11andphosphorylation-beta-catenin were analyzed by immunohistochemistry (IHC) in45paires available matched gastric cancer and adjacent tissue samples. MTT and colonyformation assay were employed to examine the role of HOXA11in gastric cancer cellproliferation. Flow cytometry assay was used to detect cell cycle status and apoptosis.Microaray assay was applied to screen the expression changes of HOXA11downstream genes before and after reexpression of HOXA11. Luciferase reporterassay and western blot were used to analyze the potential function of HOXA11inWNT signaling pathway and the effect on downstream protein expression.Results: HOXA11expression was found in MGC803, SGC7901, MKN45, BGC823and HGC27cells. Loss of HOXA11expression was found in AGS, and reducedexpression was found in MGC803. Complete methylation of HOXA11was found inAGS cells and partial methylation was found in MGC803and SGC7901cells.Unmethylation was found in MKN45, BGC823and HGC27cells. Loss of HOXA11expression is correlated to promoter region completely methylation. Restoration ofHOXA11expression was induced by5-AZA treatment. Above results suggest thatHOXA11expression was regulated by promoter region methylation. Sodium bisulfitesequencing conformed MSP results in AGS, SGC7901and BGC823cells.81.25%(91/112) of primary gastric cancer was methylated and no methylation was found in5cases of normal gastric mucosa. Methylation of HOXA11is related to male gender(p<0.05), tumor size (p<0.05) and positive lymph node metastasis (p<0.05). Lost orreduced HOXA11expression was found in cancer significantly by comparing theexpression of HOXA11in45cases of available matched gastric cancer with adjacenttissue with IHC (P<0.001). Cell proliferation, colony formation, cell migration andinvasion were inhibited, apoptosis and G2/M arrest was induced after re-expression in AGS cell. Dual-luciferase assay microarray Analysis combined and western Blotdemonstrated that Wnt signaling was inhibited by HOXA11up-regulting NKD1geneexpression.Conclusion: HOXA11is frequently methylated in human gastric cancer andHOXA11expression was silenced by promoter region hypermethylation. Wntsignaling was inhibited by HOXA11up-regulating NKD1gene expression in gastriccancer. |
PDF Full Text Request