| Background and Objective:As known, mutation, phenotypic drift, epithelial-mesenchymal transition (EMT), collective migration, resistance to apoptosis and anoikis, vascularization, and lymphangiogenesis are the probable mechanisms of metastasis. Evolving evidence suggests that microRNAs (miRNAs) play an important role in carcinogenesis by affecting the expression of genes that regulate cancer progression, such as inhibited cancer cell proliferation and migration, blocked the cell cycle, and activated apoptosis. When specific miRNAs involved in the process of metastasis are identified, therapeutic strategies can be developed to silence oncogenes or up-regulate tumor suppressor genes. MicroRNA-206 (miR-206), as one members of MyomiRs family, is frequently down-regulated in many human malignancies, including CRC, has an association with a more malignant phenotype and plays important roles in tumorigenesis and tumor progression of various human malignancies. Colorectal cancer (CRC) is the most common cancer diagnosed worldwide, and the development of metastases is the major cause of death. Therefore, it is of great significance to identify the regulation mechanism of miR-206 in colorectal cancer. Recently, several reports showed HeLa and C2C12 cells had validated the inhibitory mechanism of miR-206 via NOTCH3 targeting. Whether or not the interplay between miR-206 and NOTCH3 also occurs in CRC is unknown. Therefore, we sought to investigate the tumor suppressive and metastasis effects of miR-206 and its target, NOTCH3, in CRC.Materials and Methods:49 sequential patients who underwent surgery for the first manifestation of CRC were included. According to the occurrence of metastasis, patients were enrolled in the following categories:stage I and II with non-metastatic disease (n=28); and stage III and IV with metastatic disease (n=21). The tissue sections of CRC patients, both tumors and normal tissues, were examined tissue distributions for miR-206 by in situ hybridization and NOTCH3 by immunohistochemistry. The levels of miR-206 were quantified by RT-qPCR and normalized to RNU6-1, the relative levels of NOTCH3 were determined by western blotting normalized by ACTB.Colon cancer cell line (SW480) and a metastatic strain (SW620) were cultured for subsequent experiments. After transfecting cells with miR-206 mimic, N0TCH3 mRNA and protein levels were detected in SW480 and SW620 cells. Following miR-206 mimic transfection, the cell viability, apoptotic cells and caspase-3 expression were measured by MTT, flow cytometry and western blotting, respectively. The cell cycle distribution was detected by flow cytometry and the proportions of cells within GO/Gl, S, G2/M phases were determined. To identify whether or not miR-206 affects migration in CRC, two methods (wound healing and Boyden chamber assays) were applied to perform cell motility measurements. Furthmore, the mRNA levels of two NOTCH transcriptional targets (JAG1 and HEY1) and two metastasis-related genes (CDH2 and MMP9) were evaluated by RT-qPCR after post-transfection with miR-206 mimic in SW480 and SW620 cells.Results:The miR-206 expression of patients was distinct in stage I-II or stage III-IV disease between tumors and normal tissues of CRC. MiR-206 expression of patients was significantly decreased in tumors, While NOTCH3 wasincreased in tumors and minimally detected in normal tissues. The tissue distribution of miR-206 was weak-to-moderate in normal mucosa by ISH,, while absent in tumor tissues. Tissue distribution of NOTCH3 was predominantly localized in cytoplasm and nucleus of tumor cells with strong staining by ICH, while rarely visible in normal mucosa.After transfecting SW480 and SW620 cells with miR-206 mimic, NOTCH3 mRNA and protein levels were significantly reduced compared to the medium group. Based on the MTT assay, cell viability was significantly reduced after transfecting with a miR-206 mimic when compared to the medium group. The cell cycle showed that the percentage of transfected cells in the G0/G1 phase was significantly increased and accompanied a reduction in the S and G2/M phases in both cell lines. In addition, miR-206 overexpression increased the apoptotic cell number and increased caspase-3 content in both cell lines. The wound closure and cell migration were redused significantly in miR-206 overexpressed cell lines though the Boyden chamber assay.The mRNA levels of two NOTCH transcriptional targets (JAG1 and HEY1) and two metastasis-related genes (CDH2 and MMP9) were all significantly down-regulated after post-transfection with miR-206 mimic in SW480 and SW620 cells.Conclusions:Overexpressed miR-206 could inhibit CRC cancer cell proliferation, migration, blocked the cell cycle, and activated apoptosis. The tumor suppressive capacity of miR-206 had a similar effect on CRC cells with a different metastatic potential, and may be explained by its direct NOTCH3 signaling inhibition and indirect cross-talk with other signaling pathways involving CDH2 and MMP9. These implied that miR-206 was a tumor suppressor in CRC and a potential therapeutic target for clinical intervention. |
PDF Full Text Request