Metformin Affects Colorectal Cancer Cell Migration by Regulating TMEM16
HAN Yu, LI Siyuan, TAN Yao, DING Kaizhi, ZHANGSUN Dongting, LUO Sulan*, ZHU Xiaopeng*
The purpose of this study was to investigate the molecular mechanism through which metfor min inhibits TGF-β1 (transforming growth factor-β1)-induced migration of colorectal cancer cells, focusing on its regulation of TMEM16A (transmembrane protein 16A). HCT116 cell proliferation was assessed using the CCK-8 assay. Changes in ROS (reactive oxygen species) levels were evaluated using DCFH2-DA fluorescent probes. Cell migration and invasion capabilities were determined by scratch wound healing assays and Transwell migration as says, respectively. Plate colony formation assays were employed to assess cell proliferation potential. RT-qPCR (real-time quantitative reverse transcription PCR) was used to detect the expression of EMT (epithelial-mesenchymal transition)-related genes, including CDH1 (cadherin 1), CDH2 (cadherin 2), SNAIL (snail family transcriptional re pressor 1), SLUG (snail family transcriptional repressor 2), and VIM (Vimentin). Immunofluorescence staining visu alized the expression and distribution of E-cadherin and Vimentin proteins. Western blot analysis measured the ex pression of E-cadherin, Vimentin, and Slug proteins. Bioinformatics analysis utilizing TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) databases screened for potential metformin targets. The patch-clamp technique recorded the effect of metformin on TMEM16A whole-cell chloride currents. TGF-β1 treatment signifi cantly promoted HCT116 cell migration and invasion, enhanced colony formation capacity, and increased ROS release. These effects were accompanied by hallmark EMT marker changes: decreased CDH1 gene and E-cadherin protein expression, alongside significant upregulation of CDH2, SNAIL, SLUG, VIM genes and Vimentin, Slug pro tein expression (P<0.05). Metformin intervention effectively reversed the TGF-β1-induced EMT progression and its pro-migratory effects. Bioinformatics analysis revealed high TMEM16A expression in colorectal cancer. Elec trophysiological recordings demonstrated that metformin potently inhibited the TGF-β1-induced enhancement of TMEM16A channel currents (P<0.05). Metformin likely inhibits TGF-β1-induced EMT and migration in colorectal cancer cells by downregulating TMEM16A expression and suppressing its mediated chloride channel activity. This study provides novel insight into the molecular mechanism underlying metformin’s anti-tumor effects.
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