miR-20b acts as potential oncogenic miRNA in gastric cancer derived cell lines by targeting phosphatase and tensin homolog (PTEN) and thioredoxin-interacting protein (TXNIP) genes

Abstract

Recently discovered microRNAs (miRNAs) post-transcriptionally regulate gene expression and play important role in a variety of processes. The aim of a study was to examine hsa-miR-20b-5p (miR-20b) function in gastric carcinogenesis by experimentally determining miRNA target-genes and impact to physiological cell processes in vitro and in vivo. After bioinformatics analysis five potential target genes were selected for miR-20b (EREG, FAT4, IRF1, TXNIP, PTEN) and analyzed for expression changes in mRNA and protein levels after transfection with miR-20b inhibitor. Cell viability was assessed using a MTT assay, colony formation and proliferation was evaluated using clonogenic assay, and cell migration was determined using wound healing assay. INS-GAS mice model was used to evaluate the miR-20b alterations in vivo following H.pylori infection with follow up to 50 weeks. Inhibition of miR-20b expression significantly increased IRF1, PTEN, TXNIP genes expression level in AGS (p=0.008, p=0.021, p=0.046, respectively) and MKN28 cell line (p=0.003, p=0.005, p=0.0004, respectively) 24 h after transfection. IRF1, TXNIP genes expression remained increased 48 h post-transfection in AGS cell line (p=0.001, p=0.006, respectively), while only TXNIP - in MKN28 cell line (p=0.001). Secondly, miRNA target-genes were analyzed for protein expression changes. Analysis revealed that PTEN protein expression statistically significantly increased in AGS cell culture (p= 0.020) and TXNIP protein expression increased in MKN28 72 h after transfection (p=0.036). Finally, miR-20b inhibition reduced cell viability in AGS cell line (p= 0.029). Furthermore, number of colonies reduced dramatically in both cell lines (p=0.0002, p= 0.021; AGS and MKN28 respectively) compared to cells transfected with control miRNA. INS-GAS mice showed gender specific miR-20b expression pattern following H.pylori infection. Only male mice showed significantly higher miR-20b expression for all time points (p=0.028