| Objective:To investigate expression and function of miRNA-146a in gastric cancer tussue samples and cell lines. The biological behaviors and mechanism of miR-146a in gastric cancer was also studied.Methods:Quantitative real-time polymerase chain reaction was used to detect the levels of miR-146a expression in gastric cancer tissue samples and cell lines. The relationships between miR-146a expression patterns in human gastric cancer tissue samples with clinicopathological features and overall survival was also assessed. The growth of MKN-45 cells transfected with miR-146a mimics was examined by MTT assay.The effects of miR-146a on cell cell cycle and apoptosis were assessed by FACS analyses in MKN-45 cells. The proportion of apoptotic cells induced by chemotherapeutic agents oxaliplatin and docetaxcel in MKN-45 cells transfected with miR-146a mimics or control mimics were also assessed by FACS analyses."Scratch wound healing"and Transwell with or without matrigel was used to investigated the the impact of miR-146a on migratory and invasive abilities of MKN-45 cells. Using Label-free quantitative shotgun proteomics approach to investigate the direct target and undirected target of miR-146a. Microrna.org predictions were used to predict potential targets of miR-146a and the prediction results were compared with our quantitative proteomic approach results. Western blot was used to confirm the proteins expression patterns identified by proteomic approach. Results:1. MiR-146a expression was significantly lower in gastric cancer samples than in non-tumor samples. The average Delta Ct (ΔCt) value of miR-146a in gastric cancer samples was 5.18 and that in non-tumor samples was 3.51 (P=0.001). Thirty six of 43 gastric cancer samples (84%) showed decreased expression of miR-146a with a median fold change at 0.34.2. We found low expression of miR-146a was correlated with increased tumor size (p=0.006) and poor cell differentiation (p=0.010) in gastric cancer.3. The potential correlation between expression of miR-146a and gastric cancer prognosis was addressed in the present study. Follow-up was available in 30 patients in the present study. Patients were classified into 2 groups according to the median miR-146a expression in gastric cancer. Kaplan–Meier curves suggested that overall survival time of patients with high miR-146a expression was significantly longer than that of patients with low expression of miR-146a (P = 0.011).4. We also compared the expression of miR-146a among several gastric cancer cell lines and one normal gastric epithelial cell line,GES-1. We recorded a significantly lower expression of miR-146a in gastric cancer cell lines than GES-1(p=0.000). Compared to GES-1 cells,the expression of miR-146a in highly differentiated MKN-28 cells (Media fold,0.742) was higher than moderately differentiated SGC-7901 cells (Media fold,0.013),poorer differentiated MKN-45 cells (Media fold,0.004) and BGC-823 cells (Media fold,0.005). The expression of miR-146a was lowest in MKN-45 cells,and this cell line was selected for further experiment.5. In order to investigate the role of miR-146a in gastric cancer carcinogenesis,we tested the effect of miR-146a overexpression on the proliferation of MKN-45 cells derived from gastric cancer. The MTT assay showed that introduction of miR-146a caused a remarkable inhibition of cell proliferation in MKN-45 cells (P<0.05).6. To understand whether the reduced cell proliferation was due to cell cycle arrest or apoptosis,we performed FACS analyses. The proportion of apoptotic cells induced by transfection of miR-146a mimics was greater than that induced by transfection of the negative control mimics (11.9% vs 5.9%). Cells over-expressing miR-146a did not show significant changes in the cell cycle distribution.7. Transwell with or without matrigel results showed that overexpression of miR-146a significantly inhibited both migratory and invasive numbers of MKN-45 cells.We assayed the polarized migration of cells using"scratch wound healing"assay.Similar to Transwell without matrigel analysis,MKN-45 cells transfected with miR-146a mimics closed the scratch wound more slowly than cells transfected with negative controls.8. The proportion of apoptotic cells induced by chemotherapeutic agents oxaliplatin and docetaxcel in MKN-45 cells transfected of miR-146a mimics was greater than that induced by transfection of the negative control mimics,which increased 13.1% and 18.6% individually. Overexpression of miR-146a sensitized MKN-45 cells to chemotherapeutic agents oxaliplatin and docetaxcel.9. We identifed 116 candidate targets of miR-146a using a Label free shotgun quantitative proteomic approach. Relative changes in the levels of identified proteins were determined by the ratio of intensity from miR-146a transfected samples to negative control mimics samples. Forty-nine proteins was downregulated in MKN-45 cells transfection with miR-146a mimics,including 8 predictive targets. Sixty-six proteins was upregulated in MKN-45 cells transfection of miR-146a mimics,including 15 predictive target. We obtained differential expression patterns for 5 proteins (Upregulation of PDCD6,PDCD4,DDX3 and downregulation of EGFR,VDAC1) related to cell proliferation and apoptosis and 6 protein (Upregulation of Dsc2,SELENBP1, SNX1 and downregulation of L1CAM,ROCK2,RBBP7) related to cell migratory and invasive abilities. In additional,there are 5 proteins and (Upregulation of PDCD4,SELENBP1 and downregulation of IMPDH2,RPN2,PRKDC) may involved in modulating cell sensitivity to oxaliplatin and docetaxcel. Quantitative proteomic approach also identifed downregulation of GART,which is required for de novo purine biosynthesis. Western blot confirmed the downregulation of EGFR,VDAC1 in MKN-45 cells transfected with miR-146a mimics identified by proteomic approach.Conclusion:In the present study, we showed that the expression of miR-146a was down-regulated in gastric cancer tissue samples and gastric cancer cell lines. Moreover, the expression of miR-146a was associated with tumor size and cell differentiation in gastric cancer. Down-regulation of miR-146a is further suggested to correlate with shorter survival of gastric cancer patients. Next investigations revealed that overexpression of miR-146a substantially inhibited proliferation, induced cell apoptosis and inhibited cell migration and invision in gastric cancer MKN-45 cells. Overexpression of miR-146a also increased the proportion of apoptotic cells induced by chemotherapeutic agents oxaliplatin and docetaxcel in MKN-45 cells. Label free shotgun quantitative proteomic approach obtained differential expression patterns for several proteins involved in cell proliferation and apoptosis, cell migratory and invasive abilities, and modulating chemotherapeutic drug sensitivity. The direct and indirect targets identified by quantitative proteomic approach shed light on the potentially pathways and molecular mechanisms of miR-146a in gastric cancer. |
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