Increasing the Treatment Efficacy of CDK Inhibitor-Resistant Genes in Breast Cancer by miRNA Silencing
Project no. 30785 · Status: Kapanmış (Yayın bekleniyor) · 2023
Summary
<p>Objective: This study aims to investigate the potential of miR-640 to downregulate genes associated with CDK4/6 inhibitor resistance; and to investigate miR-640's apoptotic, anti-metastatic, anti-proliferative effects and synergistic effects with abemaciclib through functional analyses to examine miR-640's effects on resistance to the CDK4/6 inhibitor abemaciclib.</p><p>Method: miRNA-640 was identified by analyzing the data set with accession number GSE126125 in the GEO database using the GEO2R tool. Subsequently, a list of potential target genes for miR-640 was generated from databases such as miDB, TargetScan, and TargetMiner. Genes associated with CDK inhibitor resistance were filtered from this list. The IC50 values of abemasib and the percentage of viability in combination with miR-640 were calculated using the MTT method and dose-response curve analysis. The effects of miR-640 were analyzed in MCF7 and MDA-MB-231 cell lines using Annexin V (apoptosis), cell cycle, and wound healing (cell migration) functional assays. Subsequently, the expression levels of potential target genes of miR-640 associated with CDK4/6 inhibitor resistance were analyzed using the RT-qPCR method. Experiments were performed in triplicate. Gene expression analyses were performed using the 2-ΔΔCT method. One-way ANOVA was used to compare samples treated with abemaciclib, miRNA, and the combination of abemaciclib+miRNA with untreated samples. All analyses and graphs were performed using GraphPad Prism software.</p><p>Result: Successful transfection was achieved in both cell lines, and a significant increase in miR-640 expression was observed (~50-fold in MCF7, ~42-fold in MDA-MB-231). Following transfection, miR-640 and its combination with abemaciclib were observed to inhibit cell migration and induce apoptosis, but did not arrest the cell cycle at any specific phase. Additionally, gene expression analyses showed that in the MCF7 cell line, CDK4, CDK6, CDKN2B, CDK9, E2F2, SMAD2, WEE1, WNT7b, ZFP91, PDK1, MAP3K1, and AURKA; In the MDA-MB-231 cell line, strong downregulation was observed in the PDK1 gene, along with downregulation in the CDK6, CDKN2B, SMAD2, ZFP91, MAP3K1, and AURKA genes.</p><p>Conclusion: This study is the first to demonstrate that miR-640 plays a tumor-suppressive role in breast cancer. The findings indicate that miR-640 suppresses cell proliferation and migration by downregulating genes associated with resistance to CDK4/6 inhibitors, while also inducing apoptosis. Based on these results, it is suggested that miR-640 could be used as a potential therapeutic agent in breast cancer treatment, either alone or in combination with abemaciclib.</p>