长链非编码RNA NR2F1-AS1靶向微小RNA-493-5p表达调控卵巢癌细胞增殖及凋亡的机制研究

Mechanism of long non-coding RNA NR2F1-AS1 regulates proliferation and apoptosis of ovarian cancer cells by targeting microRNA-493-5p expression

  • 摘要:
    目的 探讨长链非编码RNA NR2F1-AS1(LncRNA NR2F1-AS1)靶向微小RNA-493-5p(miR-493-5p)的表达调控卵巢癌细胞增殖及凋亡的机制。
    方法 采用实时荧光定量聚合酶链反应(qRT-PCR)检测NR2F1-AS1与miR-493-5p在不同卵巢癌细胞株中的表达水平; 采用双荧光素酶报告基因检测NR2F1-AS1与miR-493-5p的相互作用; 采用MTT增殖实验检测干扰NR2F1-AS1表达或miR-493-5p过表达后卵巢癌细胞增殖能力的变化; 采用流式细胞术检测细胞凋亡率; 采用Western blot检测CyclinD1、p21、p27、Bcl-2、Bax、cleaved-caspase 3蛋白表达。
    结果 与正常卵巢上皮细胞比较, NR2F1-AS1在卵巢癌细胞中的表达水平升高,而miR-493-5p的表达水平则降低,差异有统计学意义(P < 0.05)。双荧光素酶实验证实NR2F1-AS1能与miR-493-5p特异性结合,并可负性调控miR-493-5p的表达及活性。干扰NR2F1-AS1表达或miR-493-5p过表达后可抑制卵巢癌细胞增殖能力,促进细胞凋亡,并可显著上调p21、Bax、p27、cleaved-caspase 3蛋白的表达,下调CyclinD1、Bcl-2蛋白的表达。抑制miR-493-5p表达可逆转干扰NR2F1-AS1表达对卵巢癌细胞增殖及凋亡的作用。
    结论 干扰NR2F1-AS1表达可通过上调miR-493-5p的表达抑制卵巢癌细胞增殖及诱导细胞凋亡。

     

    Abstract:
    Objective To explore the mechanism of long non-coding RNA NR2F1-AS1 (LncRNA NR2F1-AS1) in regulating proliferation and apoptosis of ovarian cancer cells by targeting microRNA-493-5p (miR-493-5p) expression.
    Methods The quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of NR2F1-AS1 and miR-493-5p in different lines of ovarian cancer cells; the dual luciferase reporter gene was used to detect the interaction between NR2F1-AS1 and miR-493-5p; the MTT proliferation assay was used to determine the change in the proliferation ability of ovarian cancer cells after interference with NR2F1-AS1 expression or miR-493-5p overexpression; the apoptosis rate of ovarian cancer cells was detected by flow cytometry; the Western blot was used to detect the protein expression levels of CyclinD1, p21, p27, Bcl-2, Bax and cleaved-caspase 3.
    Results Compared with normal ovarian epithelial cells, the expression level of NR2F1-AS1 in ovarian cancer cells increased significantly, while the expression level of miR-493-5p decreased significantly (P < 0.05). The dual luciferase assay confirmed that NR2F1-AS1 could specifically bind to miR-493-5p, and the expression and activity of miR-493-5p were negatively regulated by NR2F1-AS1. Interference with NR2F1-AS1 expression or overexpression of miR-493-5p could inhibit the proliferation of ovarian cancer cells, promote apoptosis, significantly up-regulate the expression of p21, Bax, p27 and cleaved-caspase 3, and down-regulate protein expression levels of CyclinD1 and Bcl-2. Inhibiting the expression of miR-493-5p was able to reverse the effect of interference with the expression of NR2F1-AS1 on the proliferation and apoptosis of ovarian cancer cells.
    Conclusion Interference with the expression of NR2F1-AS1 can inhibit the proliferation of ovarian cancer cells and induce apoptosis by up-regulating the expression of miR-493-5p.

     

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