The Role Of Hsa-miR-1246in EMT Of Gastric Adenocarcinoma

Background and ObjectiveIn clinical, gastric cancer (GC) is one of the most common malignant tumors, ranking the second of the causes of cancer-related death, and approximately95%of gastric cancer are gastric adenocarcinoma. Cancer metastasis is the major reason causing the death of gastric cancer patients. Epithelial to mesenchymal transition (EMT) is a biological process, in which malignant cells originated from epithelial cells acquire the migration and invasive abilities, functioning of great importance in promoting the tumor process. Enormous reports proved that miRNAs are involved in many pathophysiological processes, the function of miRNAs in the EMT process is important.Previous reports showed that serum miR-1246level is related with the TNM stage of esophageal squamous carcinoma (ESCC); six miRNAs (including miR-1246) expression level in serum can be used to predicting lymph node metastasis of cervical squamous cell carcinoma in early-stage. p53and its analog p63and p73target miR-1246to suppress DYRK1A (dual-specificity tyrosine-(Y)-phosphorylation regulated kinase1A), which in turn activates NFAT (nuclear factor of activated T cells), resulting in cell apoptosis and anti-cancer function. The relation between EGFL7and metastasis of gastric adenocarcinoma was discussed, in which the significant difference of hsa-miR-1246(hereafter termed miR-1246) expression level was found between different experiment groups. Therefore, we hypothesize that miR-1246might be involved in regulating the metastasis process in gastric cancer, which need to be described more. In our study, we use gastric adenocarcinoma cell line BGC823and paraffin-embedded sections of gastric carcinoma, pericarcinomatous tissue and benign lesions of stomach as study objects to analize the function of miR-1246in gastric adenocarcinoma.Methods(1)The relative expression level of miR-1246in well differentiated, moderately differentiated, poorly differentiated paraffin-embedded sections of gastric adenocarcinoma is analyzed by TaqMan real-time PCR. In each gastric adenocarcinoma group, corresponding pericarcinomatous paraffin-embedded sections are randomly chosen to analyze the relative expression level of miR-1246. Twelve gastric mucosa paraffin-embedded sections of benign lesions of stomach (gastritis or gastric ulcer) are chosen to analyze the relative expression level of miR-1246. Statistical analysis is applied to determine the relationship between miR-1246expression level and differentiation degree and clinical data, etc.(2) Immunohistochemistry and immunohistochemical score standard are used to analize the expression level of E-cadherin and vimentin in paraffin-embedded sections of gastric adenocarcinoma, which is further analized by studying its relationship with miR-1246expression level and corresponding clinical data.(3)Use lentiviral vector to deliver BGC823pre-hsa-miR-1246mimics and BGC823mimics negative control into BGC823respectively, and then two stable transfected cell lines are established by using puromycin. TaqMan real-time PCR is applied to quantitate the relative expression level of miR-1246in BGC823pre-hsa-miR-1246mimics, BGC823mimics NC and BGC823groups to make sure the transfection is successful. The three cell groups are harvested to detect the protein expression level of E-cadherin and vimentin by using Western blotting. Photographs are taken in the three cell groups to observe the morphological change.Results(1) The relative expression level of miR-1246increases successively in gastric adenocarcinoma group, pericarcinomatous tissue group and benign lesions of stomach group. While the difference between the gastric adenocarcinoma group and the benign lesions of stomach group is of statistical importance (P<0.05), the rest difference is of no statistical difference (P>0.05). The relative expression level of miR-1246in well-differentiated, moderately differentiated, poorly differentiated paraffin-embedded sections of gastric adenocarcinoma decreases successively, but the difference between any two of them is of no statistical importance (P>0.05). The relative expression level of miR-1246decreases as the TNM stage increases (P>0.05).(2) As the differenciation level of gastric adenocarcinoma increases, the protein expression level of E-cadherin increases, while that of vimentin decreases. However, the difference among each group is of no statistical significance (P>0.05). As the TNM stage increases, the protein expression level of E-cadherin decreases, while that of vimentin increases. The expression level of miR-1246in E-cadherin positive group is higher than that in E-cadherin negative group, the expression level of miR-1246in vimentin positive group is lower than that in vimentin negative group, both differences are of significant importance (P<0.05).(3) After BGC823cell line had be stably transfected with lentiviral vector pre-miR-1246mimics and lentiviral vector mimics negative control, stable cell lines BGC823miR-1246high expression and BGC823miR-1246negative control were established. MiR-1246expression level in BGC823miR-1246high expression cell line was found to be increased (P<0.05); E-cadherin expression level was relatively increased, while vimentin expression level was relatively decreased. The morphology of BGC823cell was changed between BGC823miR-1246negative control cell line (and BGC823cell line) and BGC823miR-1246high expression cell line, from a spindle shape to oval or circular shape. According to Chi-square test, the difference of spinder cell ratio between BGC823miR-1246negative control cell (and BGC823cell) and BGC823miR-1246high expression cell is of statistical significance (P<0.001).Conclusion(1) For the first time to prove the existence of abnormal expression of miR-1246in gastric adenocarcinoma.(2) The decreased expression of miR-1246may be function as a factor to promote the EMT process in gastric adenocarcinoma.