| Objectives：Studies have shown that miR-148a was proved to be silenced while DNAmethyltransferase1(DNMT1) was over expressed in gastric cancer. But themechanism of aberrant expression of miR-148a and DNMT1, and their relationshipsin gastric cancer are still unknown. The aim of this study was to investigate theexpression profile of miR-148a and DNMT1, and reveal whether they have anyrelationships.Methods：We used reverse transcriptase quantitative real-time PCR, methylation-specific PCRand Western blot to measure the level of miR-148a expression, DNA methylationlevel and DNMT1expression, respectively. Gastric cancer cells were transfected withplasmid or siRNA or treated with5-aza-2’-deoxycytidine. Cell proliferation andapoptosis was detected by cell counting and flow cytometric analysis. Results：1.Among38cases of gastric cancer patients,32(84%) cases of gastric cancer patientsshowed down regulation of miR-148a in cancer tissues compared with their normalmucosae, the expression level of miR-148a in tumor tissues was significantly lowerthan that in normal mucosal tissues (p<0.01,Δ CT of miR-148a were4.27±1.67and2.97±1.79).2. MiR-148a was significantly down regulated in gastric cancer cell lines comparedwith GES-1(MGC-8030.19±0.03-fold, p<0.01; SGC-79010.29±0.03-fold, p<0.01and BGC-8230.37±0.03, p<0.01).3. Among38cases of gastric cancer patients,26/38(68.4%) cases of gastric cancertissues showed visible methylation strips, while only7/38(18.4%) cases of normalmucosal tissues showed the strips (p<0.05), in26cases of methylation group,miR-148a expression level was significant higher than that in12cases ofunmethylation group(p<0.01).4. MiR-148a was significantly up-regulated when the5-aza-dC concentrations were5μ/L(7.6±1.1-fold, p<0.01) and10μ/L (10.4±1.50-fold, p<0.01).22/38(57.9%) caseshad a higher expression of DNMT1when compared with their normal mucosae,while in22cases of DNMT1over-expression group, miR-148a expression level wassignificantly lower than that of non-high expression group(p<0.05). In SGC-7901,MGC-803and BGC-823cell lines, DNMT1protein was also higher than that ofGES-1.5. Down-regulation of DNMT1could decrease hypermethylation of miR-148a geneand increase its expression in BGC-823and SGC-7901cells lines.6. The over expression of miR-148a caused reduction of DNMT1protein expressionin gastric cancer cells.7. Cell count of experimental groups of cell lines was significant lower than that ofcontrol groups48h after transfected with miR-148a overexpression plasmid. ButFCAS showed no obvious change in apoptosis either in BGC-823(p=0.14, t-test) or inSGC-7901(p=0.12) Conclusion：We described here the identification of a tumor suppressor gene, miR-148a, whichwas frequently silenced by DNA hypermethylation through DNMT1over-expressionin gastric cancer. Moreover, we identified that DNMT1was a potential target ofmiR-148a, the silence of miR-148a seemed to repress its suppression of DNMT1,thus causing the higher level of DNMT1expression and further silence of miR-148a.Our data suggests that there may have a circulating regulation between the silence ofmiR-148a and the up-regulation of DNMT1in gastric cancer. This work will deepenour knowledge of how miRNA-mediated mechanisms contribute to the pathogenesisof gastric cancer and help us to identify new therapeutic approaches for the patients. |
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