MiR-543 Promotes The Development Of Gastric Cancer By Targeting SIRT1

Background:Gastric cancer is one of the most common malignant tumor in the global scope, it is the third most common cause of cancer death. Although gastric cancer incidence and mortality has been reduced year by year, but it is still an important public health problem in the worldwide.Our previous study also found that SIRT1 in gastric cancer is low expressionby inhibiting cell cycle D1 protein and induced the stagnation of G1 phase, thus inhibiting the occurrence of gastric cancer development.However, the regulatory mechanism of SIRT1 low expression is still unknown.Objective:We explore the role of the miR-543 for SIRT1 in the development of gastric cancer and its molecular regulation mechanism, provide new targets for the treatment of gastric cancer.Results:1. Using three different types of bioinformatics software, such as Pictar、miRanda and TargetScan, we selected 8miRNAs may be targeted for the regulation of SIRT1, such as miR-92a-3p、miR-128、miR-132-3p、miR-135、miR-138、miR-181、 miR-199a-5p and miR-543. We found that miR-543 can combine with SIRT1 mRNA 3’-UTR region by two different binding sites. Then, we transfected mimics into BGC-823 respectively,found that miR-543reduced significantly protein levels of SIRT1, and the expression of other miRNAs had no significant effect on SIRT1.2.At the molecular level, after transfecting miR-543 mimics/inhibitor into human gastric mucosa epithelial cell line GES-1 and the gastric cancer cell line AGS、 BGC-823,we confirmed the regulation function of miRNA-543targeting for SIRT1 through qRT-PCR、 Western blotting and dual luciferase reporter assays. We found that miR-543 reduced the mRNA and protein expression level of SIRT1 in BGC-823 and GES-1.Although the mRNA was not significantly change, but the protein expression level of SIRT1 was lowered in AGS.Then, we transfected miR-543 mimics/inhibitor and SIRT1 mRNA 3’-UTR wild type vector WT、SIRT1 mRNA 3’-UTR mutation vectorMut-1 (mutation of the first site)、SIRT1 mRNA 3’-UTR mutation vector Mut-2(mutation of the second site)、SIRT1 mRNA 3’-UTR mutation vector Mut-bo(mutation of the both sites) respectively into BGC-823 cells, we found that after the transfection of miR-543 mimics in BGC-823, WT reduced the luciferase activity of BGC-823 cell obviously; Mut-1、Mut-2 had a different degree recovery of the luciferase activity in BGC-823 cells;Mut-bo had a full recoveryof the luciferase activity in BGC-823 cells. On the contrary,after the transfection of miR-543 inhibitor, WT increased the luciferase activity of cells obviously; Mut-1、Mut-2 had a different degree recovery of the luciferase activity in cells;Mut-bo had a full recoveryof the luciferase activity in cells.3.At the cellular level, we detected effects of candidate miRNAs on cell proliferation through the MTS experimental method.MTS assay show that after transfected miR-543 mimics in human gastric mucosa epithelial cell line GES-1 and the gastric cancer cell line AGS、BGC-823cells,the ability of cell proliferation was increased obviously. On the contrary, after transfected miR-543 inhibitor, the ability of cell proliferation was significantly suppressed.Then, wetransfected miR-543 mimics and SIRT1 vector in BGC-823 cells and found that SIRT1 can reverse the ability that the miR-543 promote cell proliferation.4.At the cellular level, wedetected effects of candidate miRNAs on cell clone formation ability through the clone formation assay. Clone formation assay show thatafter the transfection of miR-543 mimicsinto human gastric mucosa epithelial cell line GES-1 and the gastric cancer cell line AGS、BGC-823, the clone formation ability was increased obviously. On the contrary, after the transfection ofmiR-543inhibitor, the clone formation ability was significantly suppressed. Then, we transfected miR-543 mimics and SIRT1 vector in BGC-823 cells and found that SIRT1 can reverse the ability that the miR-543 promote cell the clone formation ability.5.At the cellular level,wedetected effects of candidate miRNAs on the cell cycle progression through through the flow cytometry. The flow cytometry assay show thatafter the transfection of miR-543 mimicsinto the gastric cancer cell line AGS、 BGC-823, the cell cycle progression was promoted obviously.On the contrary, after the transfection ofmiR-543 inhibitor, the cell cycle progression was significantly suppressed. Then, we transfected miR-543 mimics and SIRT1 vector in BGC-823 cells and found that SIRT1 can reverse the ability that the miR-543 promote the cell cycle progression ability.6.At the tissueslevel, we used qRT-PCR method to detect the expression level of miR-543 and SIRT1 in gastric cancer tissues and the corresponding non-cancerous normal mucosa tissues.Compared with the matched non-cancerous normal tissues, the expression of miR-543 is markedly increased in GC tissues, the expression of SIRT1 is reduced, and the expression of SIRT1 was negatively associated with the expression of miR-543 in patient tissues. The expression of miR-543is correlation with tumor size, clinical stage, TNM stage and lymphatic metastasis, and it has nothing to do with sex, age and tumor infiltration.Conclusion:1. In gastric cancer cells, miR-543 can combine with two different positions of SIRT1 mRNA 3’-UTR region so as to regulate targeted for the expression of SIRT1.2. The miR-543 can significantly promote the cell proliferation ability, cell clone formation ability and promote cell cycle progression in gastric cancer cells by targetting for SIRT1.3. In gastric cancer, the expression of miR-543 increased significantly and the expression of SIRT1 reduced significantly compared with normal gastric mucosa tissues, miR-543 and the SIRT1 expression was significantly negative correlation. The expression of miR-543 is correlation with tumor size, clinical stage, TNM stage and lymphatic metastasis.