| BackgroundGastric Cancer (GC) is one of the most common malignant tumors in China, with the highest incidence in gastrointestinal cancer and the second mortality rate among malignancies. Many factors may affect the development of GC. However, the exact pathogenesis remains to be elucidated. Therefore, clarifying the molecular mechanisms of GC development as well as exploring novel early diagnosis markers and potential therapeutic targets has very important significance for the treatment of GC.microRNAs are a class of non-coding RNA that has regulatory functions. They have been reported to involve in various signaling pathways and their target genes are related to the regulation of tumor differentiation, proliferation, apoptosis, invasion, migration, angiogenesis, and so on. Angiogenesis is an essential part of tumor development. A large number of studies have shown that miRNAs also involved in the tumor angiogenesis and played an important role in this process.miR-125a-5p is one of the mature forms of miR-125a. MiR-125a must be processed into miR-125a-5p and miR-125a-3p to exert its biological function. It has already been demonstrated that miR-125a-5p involved in the regulation of HER-2 signaling pathway in GC. Our study was designed to further investigate the function and possible mechanisms of miR-125a-5p in gastric cancer.Subject:Our aim was to investigate the expression level of miRNA-125a-5p in human gastric cancer tissues and cells, analyze its association with clinicopathological parameters, and clarify the influence and mechanism of miR-125a-5p-modified AGS cells on HUVECs proliferation, migration capabilities and tumor growth in gastric cancer model of nude mice.Methods:1. miR-125a DNA copy number was analyzed by bioinformatics analysis from more than 40 kinds of gastric cancer cell lines; RT-PCR was used to verify the miR-125a-5p expression in gastric cancer cell lines SGC-7901, AGS and BGC-823; Moreover, RT-PCR, Western blot and ELISA were used to detect VEGF-A mRNA and protein levels in miR-25a-5p mimics or inhibitors transfected AGS cells; Luciferase reporter analysis was used to verify whether VEGF-A gene was a target gene of miR-125a-5p.2. HUVECs were co-cultured with miR-125a-5p mimics or inhibitors modified AGS cells. Then, CCK-8 proliferation assay and colony formation assay, transwell migration assay as well as tube formation assay were performed to detect the influence of miR-125a-5p on HUVECs proliferation, migration and tube formation capabilities; MK-2206 2HCI (Akt activity inhibitor) was used to detect the influence of miR-125a-5p on VEGF-A downstream signaling pathways.3. Gastric cancer model was constructed in nude mice. RT-PCR was used to detect the mRNA expression levels of miR-125a-5p and VEGF-A in tumor tissues; Western blot and IHC were conducted to exam VEGF-A protein levels in tumor tissues; IHC was used to detect the expression of CD31 in tumors.4. RT-PCR was performed to detect the miR-125a-5p expression levels in 73 cases of gastric cancer specimens. Then determinate the expression of VEGF-A, HER-2, Ki-67, E-cadherin and CD31 in tissue samples by IHC and analyze their correlation with clinicopathological parameters. Retrospective analysis was also conducted.5. Bioinformatics analysis was used to investigate the related miRNAs with VEGF-A expression in gastric cancer.Results:1. Bioinformatics data showed quite different DNA copy number of miR-125a in each cell line. RT-PCR showed that the expression of miR-125a-5p in SGC-7901, AGS and BGC-823 were lower than normal gastric epithelial cells GES-1(p<0.05). In miR-125a-5p mimics modified AGS, VEGF-A mRNA and protein expression were reduced relative to the control group and had statistical difference (p<0.05); while in miR-125a-5p inhibitors modified AGS, VEGF-A mRNA and protein expression were increased compare to the control group (p<0.05). Predictive analysis suggested that VEGF-A maybe a target gene of miR-125a-5p, and luciferase reporter analysis verified this hypothesis.2. HUVECs were co-cultured with miR-125a-5p mimics or inhibitors modified AGS cells. Functional analysis showed that miR-125a-5p mimics can reduce the secretion of VEGF-A in AGS cells and inhibit HUVECs proliferation, migration and tube formation ability; while miR-125a-5p inhibitors can increase the VEGF-A secretion and enhance HUVECs proliferation, migration and tube formation ability, and Akt inhibitors can reverse this effect (p<0.05).3. We successfully built gastric cancer model in nude mouse using AGS cells. Control agomir and miR-125a-5p agomir were injected into the tumor and surrounding areas for 4 weeks when the tumors grew up to 50 mm3. The results showed that the average tumor weight and size in control group were significantly larger than that in experimental groups and had a significant difference (P< 0.05). RT-PCR revealed that the expression of miR-125a-5p in tumor tissues of control group was significantly lower than that in experimental groups (p< 0.05). Meanwhile, western blot showed a higher expression of VEGF-A in tumor tissue of control group than that in experimental groups (p< 0.05). Microvascular density analysis displayed that MVD was positively correlated with the expression of VEGF-A (r= 0.5495), and negatively related to the expression of miR-125a-5p (r=-0.7419).4.73 cases of gastric adenocarcinoma tissue samples analysis showed that the expression of miR-125a-5p in tumor tissues was reduced compared with normal tissue adjacent to the tumor, and the expression was correlated with lymph node metastasis (P=0.019), clinical stage (P=0.001), MVD (P= 0.002), expression of HER-2 (P= 0.007) and expression of VEGF-A (P= 0.001). A quantitative analysis of VEGF-A expression indicated that expression of VEGF-A was negatively correlated with miR-125a-5p expression (r=-0.5382) and positively correlated with MVD (r= 0.7226); meanwhile, the expression of miR-125a-5p was negatively correlated with MVD (r=-0.4554). Finally, survival analysis suggested that low expression of miR-125a-5p, high expression of VEGF-A and high MVD were related with bad prognosis. Moreover, meta-analysis demonstrated that miR-125-5p was an independent prognostic factor.5. Bioinformatics analysis revealed that let7c, miR-100 and miR-143 were negatively correlated with VEGF-A expression (r<0.4, p<0.05); while let-7g, miR-15b, miR-16-1, miR-30b, miR-126, miR-191, miR-200c, miR-455 and miR-500a were positively correlated with VEGF-A expression (r> 0.4, p<0.05).Conclusion:1. miR-125a-5p can influence tumor angiogenesis by regulating the secretion of VEGF-A in gastric cancer cells.2. miR-125a-5p can inhibit tumor growth by targeting VEGF-A and may be a potential therapeutic targets of gastric cancer.3. miR-125a-5p expression was decreased in human gastric cancer and was negatively correlated with VEGF-A expression. It may influence angiogenesis and was not conducive to the prognosis of patients.4. We conducted a preliminary screening on VEGF-A-related miRNAs in gastric cancer to lay the foundation for the later study. |
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