The Molecular Mechanism Of RUNX3 Inhibiting The Proliferation And Migration Of Gastric Cancer Cells Via Upregulating MiR-29b

Backgrounds:Gastric cancer(GC) is one of the most common malignancy worldwide, especially in China. The incidence of GC is high. The patients suffer from poor prognosis and short survival time. So, it is important to explore the molecular mechanism of GC and look for effective diagnostic marker and therapeutic targets for the early recognition and intervention of GC.RUNX3 belongs to the Runt-related transcription factor family and contains highly conserved Runt-domain that mediates the interaction between the RUNX3 protein and specific DNA binding element to regulate the transcription of target genes. Present study suggests that RUNX3 is a tumor suppressor and could cooperate with Smad3/4 in the downstream of the TGF-β signal. RUNX3 cooperates with Smad3/4 to induce the expression of cell cycle inhibitor p21 and apoptotic factor Bim, resulting in the growth inhibition and apoptosis, respectively. RUNX3 could also directly activate the expression of Claudin-1, inhibit the expression of Aktl and vascular endothelial growth factor VEGF to inhibit tumourigenesis, metastasis and angiogenesis of gastric cancer. But it is still unknown whether RUNX3 as a transcription factor could regulate noncoding microRNAs expression to exert its biological function.In this report, we firstly used miRNA microarray to identify the miRNAs that are regulated by RUNX3 and found that miR-29b was the most up-regulated miRNA after RUNX3 overexpression. We found there are putative RUNX3 binding sites in the promoter of miR-29b using the bioinformatics analysis. Therefore, we focused our attention on miR-29b. We used qRT-PCR to verify the microarray results and investigated the regulatory mechanism of RUNX3 on miR-29b expression and further explored the biological function of the miR-29b in gastric cancer.Objective:We explore the molecular mechanisms of RUNX3 regulating miR-29b expression and analyse the biological function and the downstream target genes of miR-29b in GC cell lines. And we also clarify whether RUNX3 could inhibit the proliferation and migration of gastric cancer cells by regulating miR-29b.Methods:1. We transfected RUNX3 overexpression vector or siRNA and the corresponding control vector in GC cell lines to detect the expression of miR-29b using qRT-PCR.2. Chromatin immunoprecipitation (ChIP) assay was used to to detect the interaction between RUNX3 protein and the corresponding binding element in the promoter region of miR-29b in GC cell lines.3. We constructed miR-29b promoter luciferase reporter vector and RUNX3 binding site mutation vector, using Luciferase assay to detect the effect of RUNX3 on the promoter activity of miR-29b.4. We transfected Smad3 overexpression vector and the corresponding control vector into GC cells, using qRT-PCR and Luciferase assay to detect the effect on the expression and the promoter activity of miR-29b. We co-transfected RUNX3 and Smad3 into GC cells, using Luciferase assay to detect whether RUNX3 could cooperate with Smad3 to increase the promoter activity of miR-29b.5. We used qRT-PCR to detect the expression level of RUNX3 and miR-29b in gastric cancer and the corresponding non-cancerous tissues and analyzed the correlation between RUNX3 and miR-29b expression.6. We transfected miR-29b mimics or inhibitor in GC cell lines, using Transwell, EdU to detect the effect on the cell proliferation, invasion and metastasis of miR-29b.7. We transfected miR-29b mimics or inhibitor in GC cell lines, using qRT-PCR, western blot to detect the regulation of miR-29b on the target gene DNMT3A, DNMT3B, KDM2A. We constructed 3’-UTR luciferase reporter vector and mutation vector, using Luciferase assay to detect whether miR-29b could directly target the 3’UTR of targets.Results:1. The level of miR-29b expression was significantly increased in gastric cancer cells transfected with RUNX3 over-expression plasmid; whereas the level of miR-29b expression was significantly decreased in GC cells transfected with RUNX3 siRNA to knock down the expression of RUNX3. However, the Runt domain mutant vector of RUNX3 transfected into GC cells, miR-29b expression level has no significant change.2. ChIP assay result showed that RUNX3 could directly bind to the promoter region of miR-29b; Luciferase assay result showed that RUNX3 could significantly increase the promoter activity of miR-29b.3. Smad3 overexpression significantly increased the expression and the promoter activity of miR-29b in GC cell lines; the co-transfection of RUNX3 and Smad3 could coordinate to increase the promoter activity of miR-29b.4. The clinical GC specimens and surrounding non-cancerous tissues assay showed that 76% of the clinical GC specimens showed significantly low expression of miR-29b; Statistical analysis showed that the average expression level of miR-29b in tumor tissues was significantly lower than that in surrounding non-cancerous tissue, and the expression level was correlated with tumor size, local lymph node metastasis, etc. Futher statistical analysis showed that RUNX3 expression was positively associated with miR-29b expression in human gastric cancer samples.5. miR-29b mimics significantly decreased the proliferation and metastasis of the GC cell lines. In contrast, miR-29b inhibitor could promote the proliferation and metastasis of the GC cell lines.6. We found that KDM2A was a novel direct target of miR-29b in GC cell lines. miR-29b could significantly decrease the mRNA and protein level of KDM2A and could inhibit the expression via directly binding to the 3’-UTR of KDM2A.7. EdU and Transwell assay were used to detect the proliferation and migration of the GC cell lines transfected with KDM2A siRNA. The results showed that KDM2A siRNA could significantly decrease the proliferation and migration of the GC cell lines. We knocked down KDM2A in the cells transfected with miR-29b inhibitor and detected cell proliferation and migration and found that the increase of cell proliferation and migration mediated by miR-29b inhibitor was partially abrogated by the KDM2A siRNA. These results demonstrated that miR-29b inhibit cell proliferation and migration by targeting KDM2A partially.8. We futher detected whether miR-29b is involed in RUNX3 mediated biological functions, the results showed that the cell proliferation and migration were significantly decreased after transfection with RUNX3 overexpression vector. miR-29b inhibitor partially abrogated RUNX3-mediated inhibition of cell proliferation and migration in gastric cancer cells.Conclusion:RUNX3 could cooperate with Smad3 to induce the expression of miR-29b in gastric cancer. RUNX3-mediated up-regualtion of miR-29b inhibited the proliferation and migration of GC cells by targeting KDM2A, representing a novel molecular mechanism of RUNX3 regulating cell biological functions.