LncRNA-ZFAS1 contributes to colon cancer progression through the miR-150-5p/VEGFA axis
Authors: X. Chen, S. Wang
Journal: Annals of Oncology (2018)

Introduction: Increasing long noncoding RNAs (lncRNAs) have been reported to play key role in the development and progression in many types of malignancies. ZNFX1 antisense RNA1(ZFAS1) has been reported to be aberrant expression and suggested as a tumor suppressor or oncogene in many cancers. However, the biological role and underlying molecular mechanism of ZFAS1, especially the miRNA sponge role of which in colon cancer remain largely unknown. Methods: Quantitative PCR was employed to evaluate the level of ZFAS1, miR-150-5p and VEGF-A mRNA expression in colon cancer tissues and cell lines. CCK-8, colony formation, wound-healing, transwell chamber and tube formation assays were used to detect the effects of ZFAS1 on colon cancer cell proliferation, migration, invasion and angiogenesis, respectively. Luciferase reporter assays were applied to confirm the miR-150-5p target genes, and RIP assay was used to determine whether ZFAS1 binds to miR-150-5p in an AGO2-dependent manner. In vivo tumor growth and metastasis were conducted in nude mice, and in vivo tumor angiogenesis was conducted using chicken chorioallantoic membrane model. The markers of metastasis and angiogenesis- VEGFA, CD31, MMP-9, E-cadherin, N-cadherin and Vimentin, VEGFR2, Akt, mTOR and phosphorylation of VEGFR2(p-VEGFR2), phosphorylation of Akt (p-Akt), phosphorylation of mTOR(p-mTOR) were detected by using immunohistochemistry and western blot analysis. Results: In our study, by using TCGA database, we identified a group of differentially expressed lncRNAs in human colon cancer, including ZFAS1, which mainly located in the cytoplasm with RNA-FISH. We found that ZFAS1 expression was higher in colon cancer tissues, where it was associated with TNM stage, pTstage, distant metastasis and poor overall survival. Moreover, ZFAS1 and VEGF-A are target genes of miR-150-5p, while ZFAS1 binds to miR-150-5p in an AGO2-dependent manner. Additionally, ZFAS1 knockdown significantly suppressed colon cancer cell proliferation, migration, invasion, EMT and angiogenesis in vitro, and also inhibited tumor growth, metastasis and angiogenesis in vivo. While antagomiR-150-5p or VEGF-A could reverse ZFAS1 knockdown- induced suppressing of proliferation, metastasis and angiogenesis both in vitro and in vivo. Lastly, ZFAS1 knockdown could markedly reduce the expression of VEGF-A, p-VEGFR2, p-Akt and p-mTOR, VEGFR2 inhibitor Ki8751 could obviously decrease the expression of p-VEGFR2, p-Akt and p-mTOR and inhibited colon cancer cell proliferation, metastasis and angiogenesis. Based on above data, we could conclude that ZFAS1 promoted colon cancer growth, metastasis and angiogenesis via activating VEGFA/VEGFR2 and downstream Akt/mTOR signaling pathway. Conclusion: ZFAS1 promoted tumor progression by upregulating VEGF-A via competitively binding to miR-150-5p, which acts as a tumor suppressor by targeting VEGF-A in colon cancer. Furthermore, we verified that ZFAS1 promoted colon cancer cell proliferation, metastasis, angiogenesis and EMT process via activated VEGFA/VEGFR2/Akt/mTOR signaling pathway in colon cancer.