Microvesicle-delivery MiR-29a/c Inhibits Angiogenesis By Targeting VEGF In Gastric Cancer

Background:Gastric cancer is one of the most commom malignant tumors in the world. Same as the majority of malignant tumors, gastric cancer cells need continuous supply of oxygen and nutrients during their growth process. So the cancer tissues need to form new blood vessels constantly to meet the need of their growth. If we can block the process of angiogenesis, the growth process and the metastasis of tumors can be suppressed at a certain level. The development of anti-angiogenesis drugs are based on the theory above. But the benefit of the nowadays angiogenesis therapy is limited. Vascular endothelial growth factor(VEGF) and its receptor VEGFR(Vascular endothelial growth factor receptro) play a key role in angiogenesis of tumors among the current identified factors related to the formation of vessels. Though anti-angiogenesis drug--beacizumab, targeted at VEGF, brings no survival benefit in patients with gastric cancer, it could prolong progress free survival. And the subgroup analysis showed the non-Asian people could benefit from the target therapy. Therefore, it needs further exploration of the anti-angiogenesis therapy in gastric cancer and there is also clinical value for the study of VEGF.Micro RNAs(miRNAs) are endogenous, noncoding, single-stranded RNAs with around 22 nucleotides. It regulates gene expression by repressing translation through its ability to bind with the complementary sequences in 3’-untranslated regions(3’-UTR) of target m RNAs. Mi RNAs possess various normal biological function and process of carcinogenesis and progression including angiogenesis.Microvesicles(MVs) are membranous structure secreted by kinds of cells actively. It can carry a variety of bioactive substances such as protein, m RNA and miRNAs and serves as a new way for information transmission and signal conduction between cells. Objective:This study will explore the miRNA- VEGF vascular regulating pathway and identify its role on angiogenesis in gastric cancer. And then explore the miRNA-carrier role of microvesicles on blocking the angiogenesis though both in vitro and in vivo study, which will provide new mentality in anti-angiogenesis therapy for gastric cancer.Methods: 1. The expression of VEGF in gastric cancer tissues and the paired non-cancerous tissues was detected by Western blot. The prediction and analysis of the potential upstream miRNA of VEGF were performed with the algorithms from Target Scan(http://www.targetscan.org/) Pic Tar(http://pictar.mdc-berlin.de/) and miRanda(http://www.microrna.org/). The luciferase reporter assay confirmed the direct binding of potential miRNA and VEGF. Then the regulatory effect of the potential upstream miRNA on the expression and the release of VEGF were detected by Western blot and ELISA assay.2. The microenvironment model gastric cancer was conducted through cells co-cultured technology to detect the interaction between gastric cancer cells and vascular endothelial cells. The CCK-8 assay and in–vitro matrigel tube formation assay were performed to detect the proliferation and the cyclization ability of the vascular endothelial cells co-cultured with gastric cancer cells treated with miR-29a/c mimics or inhibitors.3. Microvesicles were separated from cultured HEK293 T cell line and the phenotypic characteristics were confirmed through the detection of the specific membrane marker by Western blot. And the artificial modification of MVs was performed to upregulated the carried miR29a/c in MVs. Then the in-vivo and in-vitro experiments were performed to identify if the microvesicles can be used as a transporter which can carry miR-29a/c to the target cells to adjust the expression of VEGF thus inhibiting of angiogenesis, and finally inhibit the growth of tumor.Results: 1. Compared to the paired non-cancerous tissues, the expression level of VEGF was upregulated in gastric cancer tissues. The result of the prediction of the potential upstream miRNA showed that miR-29a/c can bind with the 3 ’- UTR region of VEGF; Luciferase reporter gene confirmed the direct recognition and regulation of miR-29a/c with VEGF; The result of cell transfection showed that, the expression and release of VEGF of gastric cancer cells were significantly downregulated when gastric cancer cells were transfected with miR-29a/c mimics. However, the expression and release of VEGF of gastric cancer cells were significantly upregulated when gastric cancer cells were transfected with miR-29a/c inhibitors.2. The results of co-cultured assay showed that the proliferation and the ability of cyclization of vascular endothelial cell co-cultured with gastric cancer cells treated with miR-29a/c mimics were significantly suppressed. However, the proliferation and the ability of cyclization of vascular endothelial cell co-cultured with gastric cancer cells treated with miR-29a/c inhibitors were significantly accelerated.3. Microvesicles derived from HEK293 T cells can be isolated through low temperature ultracentrifugation. Quantitative PCR confirmed that the carried miR-29a/c level of MVs can be changed through cell transfection. The expression and the release level of VEGF of gastric cancer cells treated with MVs derived from HEK293 T cells with high level of miR-29a/c was significantly downregulated. Then the treated gastric cancer cells were co-cultured with vascular endothelial cells. The results showed that the ability of proliferation, migration and cyclization of vascular endothelial cells co-cultured gastric cancer cells treated with MVs derived from HEK293 T cells with high level of miR-29a/c with was obviously restrained. MVs derived from HEK293 T cells with high level of miR-29a/c were injected into the gastric cancer tumor-burdened mice through tail veins. The results showed miR-29a/c delivered by MVs can significantly inhibit the expression of VEGF and the growth of tumor.Conclusion: 1. Mi R-29a/c can inhibit the tumor growth by downregulating the expression and the release of VEGF;2. Overexpression of miR-29a/c can significantly inhibit the proliferation and the ability of cyclization of the co-cultured vascular endothelial cells.3. The MVs can act as transporters of miRNAs. And the MV-delivery miR-29a/c can inhibit the proliferation and the ability of cyclization of vascular endothelial cells by directly inhibiting the expression and the release of VEGF of gastric cancer cells, thus inhibiting tumor growth of gastric cancer.