The Mechanisms Of MiR-129on The Growth Of Gastric Cancer Cells

ObjectiveGastric cancer is a malignant tumor. Recent research showed that there is a close relationshipbetween the occurrence, development of gastric cancer and microRNA. MicroRNA throughvarious pathways to affect the development of gastric cancer. Its mechanism is complex. MiR-129,which has three mature products, has important biological function. Reduced expression ofmiR-129has been reported in multiple tumor cell lines and in primary tumors. However, theexpression of miR-129-1-3p, miR-129-2-3p and miR-129-5p in gastric carcinoma and their effecton the proliferation of gastric cancer cells is not clear. We study the function of three matureproducts of miR-129and their effects on the growth of gastric cancer cell line. The study will haveimportant significance for improve the level of gastric cancer treatment.Methods1. To identify the miR-129-1-3p, miR-129-2-3p and miR-129-5p expression level in normalgastric epithelial GES-1cells and two human gastric cancer cell lines (MGC-803, SGC-7901), weused real-time RT-PCR.2. We transfected five concentrations of miR-129-5p mimic into MGC-803cells byLipofectamine2000reagent. By means of real-time cell analyzer (RTCA) to screen the optimaltransfection concentration3. We transfected miR-129-1-3p, miR-129-2-3p and miR-129-5p mimics into normal gastricepithelial GES-1cells and human gastric cancer cell line MGC-803. Real-time PCR wasperformed to detect the corresponding miRNA levels in cells.4. Used RTCA to monitor miR-129-1-3p, miR-129-2-3p and miR-129-5p mimics effect onthe proliferation of normal gastric epithelial cells and human gastric cancer cell lines5. We transfected miR-129-1-3p, miR-129-2-3p and miR-129-5p mimics into two kinds ofgastric cancer cells which had different differentiation degree. Then cell cycle was analyzed on aFlow Cytometer. 6. Bioinformatics analysis was used to predict the target genes of miR-129-1-3p,miR-129-2-3p and miR-129-5p. Then we used Western blot to analysis the expression changes oftumor associated targets, CDK6and SOX4.Results1. RT-PCR results showed that the level of miR-129-1-3p, miR-129-2-3p and miR-129-5p inall gastric cancer cells detected were significantly lower than those in normal gastric epithelialGES-1cells. Besides, the miR-129-1-3p, miR-129-2-3p and miR-129-5p levels in MGC-803(thelow differential gastric cancer cell lines) were lower than those in SGC-7901(the middledifferential gastric cancer cell lines). Further analysis found that miR-129-5p levels were higherthan miR-129-1-3p and miR-129-2-3p levels in gastric cancer cells.2. RTCA showed that the concentration of150nM could significantly suppress the growth ofhuman gastric carcinoma cell line MGC-803.3. Real time RT-PCR results showed that150nM concentration of miR-129-1-3p,miR-129-2-3p and miR-129-5p mimics could successfully transfected into cells.4. RTCA results showed that the proliferation of gastric cancer cells were significantlyinhibited by miR-129-1-3p, miR-129-2-3p and miR-129-5p mimics, especially by miR-129-2-3pand miR-129-5p, while the growth of normal gastric epithelial cells GES-1had no significantchange.5. Flow Cytometer analyzed showed that miR-129-1-3p, miR-129-2-3p and miR-129-5pmimics arrested gastric cancer cells at the G0/G1phase.6. Bioinformatics analysis showed that miR-129three mature producs had putativecomplementary binding sites in3’UTR of CDK6mRNA and SOX4mRNA. Western blot resultshowed that miR-129-1-3p, miR-129-2-3p and miR-129-5p mimics suppressed the expression ofCDK6in gastric cancer cells, miR-129-2-3p and miR-129-5p mimics suppressed the expression ofSOX4in gastric cancer cells.ConclusionsFirst of all, RT-PCR technology found that reduced expression of miR-129-1-3p,miR-129-2-3p and miR-129-5p in gastric cancer cell lines. Secondly, three miR-129matureproducts could significantly inhibit the growth of gastric carcinoma cells, resulted in significantG0/G1phase arrest. Finally, Western blot results elucidate the tumor suppressor mechanism ofmiR-129three mature products in gastric carcinoma may be through inhibiting the expression ofCDK6cause cell cycle arrest in G0/G1phase. miR-129-2inhibit the proliferation of gastric cancercells more obviously may be attributed to influence both the expression CDK6and SOX4.