The Study Of MiR-520d-5p Function In Gastric Cancer

ObjectiveTo investigate the effect of mi R-520d-5p with down-regulated expression on the proliferation, metastasis and invasion of human gastric cancer cells. MethodsTotal RNA can be extracted from cancer tissue and adjacent noncancerous tissue after surgical resection using Trizol reagent. The mi RNA expression profiles in 3 pairs of gastric cancer and adjacent nontumorous tissue were analyzed using a mammalian mi RNA microarray. Aberrant mi RNA were then verified by Real-time RT-PCR(Taqman) in 47 pairs of gastric cancer and adjacent nontumorous. The results presented over-expression of mi R-520d-5p in gastric cancer tissue. To investigate the role of aforementioned mi RNA in gastric cancer cells, mi R-520d-5p inhibitor were transfected into human gastric cancer cell lines AGS and HGC-27 by liposomes to reduce the mi R-520d-5p expression. The cells tranfected by mi RNA inhibitor negative control were cultured as a negative control group(NC group). After 48 h transfection, the expression level of mi R-520d-5p was examined by Real-time RT-PCR. Cell proliferation was verified by WST-1 assay and cell colony formation assay. Cell cycle was observed by flow cytometry. Cell migrating property was detected by transwell migration assay and wound healing assay. Cell invasion ability was evaluated by transwell invasion assay. Results1. Results of mi RNA array analysis showed that there were 62 mi RNAs dysregulated expression in gastric cancer tissues compared to their normal counterparts, including 42 over-expression and 20 low expression of mi RNAs. Real-time RT-PCR test showed that the expression level of mi R-520d-5p was increased in gastric cancer significantly(P = 0.032).2. Real-time RT-PCR test showed that the expression level of mi R-520d-5p increased significantly in gastric cancer(P = 0.032).3. mi R-520d-5p expression level after the transfection of mi R-520d-5p inhibitor in AGS and HGC-27 was detected by real-time RT-PCR. The results showed that the corresponding expression had the 62-fold(p<0.001)and 88-fold(p<0.001)decrease in AGS and HGC-27 respectively, compared with NC group. These results suggest that mi R-520d-5p inhibitor can effectively down-regulate the expression of mi R-520d-5p in gastric cancer cells.4. The result of WST-1 assay showed that the down-regulation of mi R-520d-5p in gastric cancer cells can inhibit the cell growth by 35.08%(in AGS) and 21.03%(in HGC-27) after 96 hours post-transfection of inhibitor(F =51.496, P =0.000; F =56.689, P =0.000). Cell colony formation assay showed that the colony number of in AGS/NC group was 137.7±13.1,while that of the AGS/mi R-520d-5p inhibitor group was 56.7±8.5. The difference was statistically significant(t=9.006, P=0.001). The colony number of in HGC-27/NC group was 111.7±10.0,whereas that of the HGC-27/mi R-520d-5p inhibitor group was 58.3±5.5. Apparent difference was also observed(t=8.081, P=0.001). These results suggest that the decrease of the mi R-520d-5p expression can inhibit the ability of colony formation in gastric cancer cells.5. The cell cycle distribution obtained by flow cytometry showed that the proportion of S phase of AGS and HGC-27 increased by 11.88%(T=-20.193,P<0.001)and 13.94%(T=-6.578,P=0.003) respectively, compared with the NC group. The differences were all statistically significant. These results suggest that the down-regulated expression of mi R-520d-5p can inhibit the ability of gastric cancer cell colony formation.6. The results of transwell invasion assay showed that the number of invaded cells of the AGS/ NC group was 353.3±22.0,while that of the AGS/mi R-520d-5p inhibitor group was 148.0±8.5. The difference was statistically significant(t=-15.051,p<0.001). The number of invaded cells of the HGC-27/NC group was 336.7±12.1,while that of the HGC-27/mi R-520d-5p inhibitor group was 224.3±17.5. The difference was statistically significant(t=-9.155,p=0.001). These results suggest that the down-regulated expression of mi R-520d-5p can inhibit the ability of invasion in gastric cancer cell.7. The results of transwell migration assay showed that the number of migrated cells of the AGS/NC group was 382.0±12.1, whilst that of the AGS/mi R-520d-5p inhibitor group was 189.3±19.6. The difference was statistically significant(t=-14.464,p<0.001). The number of migrated cells of the HGC-27/NC group was 400.0±19.1,whereas that of the HGC-27/mi R-520d-5p inhibitor group was 215.3 ± 16.6. The difference was statistically significant(t=-12.660,p=0.001). These results suggest that the down-regulated expression of mi R-520d-5p can inhibit the ability of the cell colony formation and the migration of gastric cancer cell.8.The result of wound-healing assay showed that In 24 h after scratch, the healing rate of the mi R-520d-5p inhibitor transfection group were 12.24%±1.6% and 9.74%±1.6%in AGS and HGC-27 respectly.The healing rate of the inhibitor NC transfection group were 19.77%±1.4% and 45.22%±4.0% in AGS and HGC-27 respectly.The difference were all statistically significantless( p<0.05).In 48 h after scratch, the healing rate of the mi R-520d-5p inhibitor transfection group was32.82%±1.2% and 40.46%±1.2%,in AGS and HGC-27 respectly.The healing rate of the inhibitor NC transfection group was 42.79%±3.3% and 67.58%±2.1% in AGS and HGC-27 respectly.The difference were all statistically significantless(p<0.05). Conclusion1. The expression level of mi R-520-d-5p in gastric cancer tissue was significantly higher than that of normal gastric tissue.2. mi R-520d-5p inhibitor has the ability to inhibit the proliferation, metastasis and invasion of gastric cancer cells. The cell cycle was also arrested in S phase.