Abstract 2443: miR-146a methylation regulates proliferation and metastasis by targeted activation of VASN/TGF-beta signaling pathway in colorectal cancer

Tumor specific alterations in miRNA provide ideal biomarkers for early diagnosis of colorectal cancer(CRC). Methylation changes play an important role in the miRNA regulation mechanism involved in tumorigenesis. Therefore, it is important to find miRNA methylation markers related to tumor proliferation and metastasis, and to explore its key regulatory mechanisms. Previous integrated bioinformatics analysis suggested that reduced expression and hypermethation of miR-146a have detected in majority of CRC. However, its biological behavior in CRC remains unclear. This article aims to explore its epigenetic regulation and mechanism in the development of CRC. We collected peripheral blood and tissues from 60 patients with advanced primary CRC, analyzed their clinicopathological characteristics. The mRNA and methylation difference was detected by qPCR and MSP respectively, and the significance in the diagnosis of CRC was analyzed. The research was further confirmed in SW480, SW620, HCT116 and HT29 CRC cell lines, as well as CRC animal models treated with demethylated drugs–5-aza-miR2-oxyeytidineine(5-AZA) and various gene transfection, in order to explore the key downstream regulatory network of miR-146a methylation changes and the effect of methylation inhibitors in the development of CRC. We found that the percentage of down-regulated and methylation of miR-146a in both tissues and plasmas of CRC was significantly higher than that of normal samples, and the expression of miR-146a could be recovered after demethylation treatment in cancer, which was associated with clinical stage. miR-146a methylation was significantly correlated with grading, but not gender, tumor location, histological type or TNM, it may be used as a predictor of poor prognosis. Diagnostic evaluation of miR-146a methylation in tissue and plasma was evaluated using ROC curve, area under regional curve, which indicated that miR-146a methylation could be used as a biomarker for the diagnosis of CRC. Fluorescein report system showed that miR-146a targeted VASN protein, and while miR-146a was overexpressed after transfection into CRC cells, the expression levels of VASN, TGF-β, VEGF-C, MMP9, and Vimentin were significantly reduced, while the expression of TIMP1 and Ecadherin were up-regulated. Additionally, the miR-146a mRNA in CRC tissues was also negatively correlated with the expression of VASN. VASN Silencing down-regulated TGF-β expression, accompanied by a decrease of MMP9. At the same time, after 5-aza demethylation or siVASN, proliferation activity of CRC cells decreased, accompanied by varying degrees of decline in membrane penetrating and migration ability, suggesting that miR-146a may regulate the proliferation and metastasis by regulating VASN/TGF-β relatived pathway. In vivo animal models, it was further confirmed that overexpressed miR-146a was consistent with the effect of 5-aza intervention or siVASN, which could significantly inhibit the occurrence of liver metastasis of CRC. Demethylated drugs treatment or VASN knockdown also significantly inhibited tumor growth in nude mice with CRC. miR-146a was a potential epigenetic silencing tumor suppressor in CRC, and the methylation of miR-146a may play an important role in the diagnosis and prediction of tumor progression in CRC as a new tumor marker. Citation Format: Weiwei Tang, Dan Zhou, Hanxiang An, Jiapeng Kang, Mingquan Cai, Ru Zeng, Jing Song, Bin Hu, Jiabian Lian, Qin Lin, Lilin Chen, Feng Ye. miR-146a methylation regulates proliferation and metastasis by targeted activation of VASN/TGF-beta signaling pathway in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2443.