👤 Arash Javeri

METTL3, an m6A methyltransferase, is integral to the regulation of messenger RNA (mRNA) biogenesis, degradation, and translation through the N6-methyladenosine (m6A) modification. Alterations in m6A homeostasis have been implicated in the development, progression, invasion, and metastasis of certain cancers. The present research aims to examine the consequences of METTL3 knockdown using short hairpin RNA (shRNA) on the proliferation and invasive capabilities of human colorectal and melanoma cancer cell lines. A specific shRNA against METTL3 mRNA was designed and inserted into an expression vector. Highly invasive colorectal cancer cell line SW480 and melanoma cell line A375 were cultured and transfected by METTL3-shRNA and scramble-control vectors and kept under culture condition for 2 weeks. The cells were harvested for analysis of gene expression by quantitative polymerase chain reaction (qPCR), invasion assay using three-dimensional (3D) spheroid assay and cell cycle and apoptosis analyses. In the METTL3-shRNA transfected cells, the expression of METTL3, VIM, SNAI1, SNAI2, ZEB1, CDH1, and TGFB1 genes were downregulated significantly compared with the scramble-control transfected cells. Expression of b-catenin, N-cadherin, vimentin, ZEB1, pro- and active MMP2, OCT4A, SOX2, and MYC proteins were also downregulated following METTL3 knockdown. Transfection by METTL3-shRNA reduced proliferation rate of the cells and increased the apoptotic rate significantly. Both migration and invasion rate of the cancer cells transfected with METTL3-shRNA were significantly decreased. These findings highlight the pro-oncogenic function of METTL3 in colorectal and melanoma cancer cells, indicating that inhibiting METTL3 could be a promising approach for tumor suppression across multiple cancer types; nonetheless, further investigation is essential to confirm these observations. Show less

CXCR4 plays an important role in colorectal cancer (CRC) development and metastasis. Some previous studies have indicated CXCR4 as a therapeutic target in cancer. CXCR4 is known as a direct target of miR-146a. The present study aimed to investigate how exogenous induction of miR-146a affects CXCR4 gene and protein expression and also proliferation, apoptosis and migration of CRC cells. Transfection of Caco-2 and SW480 cells by a synthetic miR-146a mimic led to downregulation of CXCR4 expression at both gene and protein levels. It also downregulated expression of several miR-146a targets, including GSK3B, IRAK1, TRAF6, AKT2, SMAD4, EGFR and NFKB1, mostly in SW480 cells. Overexpression of miR-146a resulted in a partial cell cycle arrest in the both cell lines, while the apoptotic rate was also decreased. In regards to epithelial-mesenchymal transition factors, VIM was downregulated in the both cell lines, but SNAI1 was upregulated in Caco-2 cells. The wound closure assay showed a reduction in cell migration in SW480 cells, but an opposite effect was detected in Caco-2 cells following transfection with miR-146a mimic. Therefore, our results are indicating that overexpression of miR-146a, despite downregulation of oncogenic CXCR4, may not lead to a universal tumor suppressive effect in all CRC cells, and this is possibly due to differences in miR-146a effects on signaling pathways in each cell type. Selection of miR-146a for tumor suppression requires enough details regarding the signaling profile of cancer cells otherwise it may produce unexpected outcome. Show less