[Molecular mechanisms of TPT1-AS1 in regulating epithelial ovarian cancer cell invasion, migration, and angiogenesis by targeting the miR-324/TWIST1 axis]

Abstract

Objective To explore the mechanism of TPT1-AS1 targeting miR-324/TWIST1 axis to regulate the proliferation, invasion, migration and angiogenesis of epithelial ovarian cancer (EOC) cells, thereby affecting ovarian cancer (OC) progression. Methods RT-qPCR was used to detect the expression of TPT1-AS1 and miR-324 in 29 OC lesions and adjacent tissue samples. The two OC cell models of TPT1-AS1 overexpression and miRNA324 knockdown were constructed, and the cell proliferation, invasion and migration abilities were detected by CCK-8, Transwell^TM and scratch test. Western blot analysis was used to detect the protein expression levels of TWIST1, epithelial cadherin (E-cadherin), Vimentin, and vascular endothelial growth factor A (VEGF-A) in OC cells. Fluorescence in situ hybridization (FISH) and RNA pull-down experiments were used to verify the interaction between TPT1-AS1 and miR-324. Immunohistochemistry and Targetscan bioinformatics analysis were used to verify the negative regulatory role of miR-324 in the epithelial-mesenchymal transition (EMT) process. Results The TPT1-AS1 expression was significantly higher in OC tissues than that in para-cancerous tissues, while the miR-324 expression was significantly lower. In SKOV3 cells with TPT1-AS1 overexpression, the miR-324 expression decreased significantly, and TPT1-AS1 was negatively correlated with miR-324. It was also found that TPT1-AS1 and miR-324 were co-expressed in OC cells, and there was a direct binding relationship between them. Down-regulation of miR-324 significantly promoted the proliferation, invasion and migration of SKOV3 cells. Further studies revealed that miR-324 had a binding site at the 3'-UTR end of the TWIST1, a key transcription factor for EMT. Inhibiting miR-324 expression increased the transcription level of TWIST1, leading to a decrease in E-cadherin protein expression and an increase in Vimentin protein expression. Additionally, the downregulation of miR-324 resulted in an increased expression level of VEGF-A protein, which in turn enhanced angiogenesis of OC. Conclusion TPT1-AS1 promotes EOC cell proliferation, invasion, migration and angiogenesis by negatively regulating the miR-324/TWIST1 axis, thus promoting the development of OC. These findings provide new potential targets for the diagnosis and treatment of OC.