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lncRNA NEAT1 regulates the proliferation, apoptosis and invasion of ovarian cancer SKOV3 cells through the miR-1287-5p/DDIT4 axis
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Abstract:
[Abstract] Objective: To explore the effects and mechanisms by which lncRNA nuclear enriched transcript 1 (NEAT1) regulating the miR-1287-5p/DNA damage inducible transcript 4 (DDIT4) axis on the proliferation, apoptosis and invasion of ovarian cancer SKOV3 cells. Methods: Tissue samples of cancer and adjacent tissues from 27 patients with ovarian cancer who underwent surgery at Suizhou Hospital Affiliated to Hubei University of Medicine between June 2023 and June 2024 were collected. Normal human ovarian epithelial cells IOSE80 and ovarian cancer cells SKOV3, CAOV3 and A2780 were also collected. RT-qPCR was used to detect the expressions of lncRNA NEAT1, miR-1287-5p and DDIT4 mRNA in ovarian cancer tissues and cells. SKOV3 cells were divided into the Ctrl group, the si-NC group, the si-NEAT1 group, the si-NEAT1 + anti-miR-NC group, the si-NEAT1 + anti-miR-1287-5p group, the si-NEAT1 + vector group, and the si-NEAT1 + OE-DDIT4 group. The CCK-8 assay, colony formation assay, flow cytometry, and Transwell assay were used to detect the proliferation, apoptosis, and invasion abilities of cells in each group. The WB method was used to detect the protein levels of DDIT4, cyclin D1, p53, and migration-invasion enhancer 1 (MIEN1) in the cells. The dual-luciferase reporter gene was used to verify the targeted binding relationship between lncRNA NEAT1 and miR-1287-5p/DDIT4. The RNA pull-down experiment and RNA immunoprecipitation experiment were used respectively to verify the target binding relationships between lncRNA NEAT1 and miR-1287-5p, and between miR-1287-5p and DDIT4. Additionally, the pcDNA group (transfected with empty vector pcDNA3.1) and the pc-NEAT1 group (transfected with pcDNA-NEAT1) were established to validate the overexpression effects of NEAT1. Results: The expression levels of lncRNA NEAT1 and DDIT4 mRNA in ovarian cancer tissues were significantly higher than those in adjacent tissues (P < 0.05), while the expression level of miR-1287-5p was significantly lower than that in adjacent tissues (P < 0.05). After knockdown of lncRNA NEAT1, compared with those in the Ctrl group and the si-NC group, the expressions of lncRNA NEAT1 and DDIT4 mRNA, the number of colony formation, the cell proliferation activity, the number of cell invasion, and the expressions of DDIT4, cyclin D1, and MIEN1 proteins in the si-NEAT1 group all decreased significantly (all P < 0.05), while the expression of miR-1287-5p, the apoptosis rate, and the expression of p53 protein increased significantly (all P < 0.05). Conversely, overexpression of lncRNA NEAT1 decreased miR-1287-5p expression and increased DDIT4 expression, showing opposite regulatory effects to the knockdown experiment. Further experiments proved that anti-miR-1287-5p or OE-DDIT4 might weaken the inhibitory effect of si-NEAT1 on the proliferation and invasion of SKOV3 cells, as well as its promoting effect on cell apoptosis. lncRNA NEAT1 targetedly regulates miR-1287-5p/DDIT4. Conclusion: lncRNA NEAT1 enhances SKOV3 cell proliferation and invasion, and inhibits cell apoptosis by targeting regulating the miR-1287-5p/DDIT4 axis.
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