微RNA-132-3p靶向第10号染色体缺失的磷酸酶和张力蛋白同源物调控葡萄膜黑色素瘤细胞增殖与凋亡

MicroRNA-132-3p targets phosphatase and tensin homolog to regulate proliferation and apoptosis of uveal melanoma cells

目的 研究微RNA(miR)-132-3p在葡萄膜黑色素瘤细胞增殖、凋亡中的作用及其作用机制。方法 该研究起止时间为2018年2月至2019年7月。定量聚合酶链反应(qPCR)检测正常葡萄膜上皮细胞ARPE-19和葡萄膜黑色素瘤细胞SP6.5、M23中miR-132-3p表达。SP6.5细胞中转染miR-132-3p干扰质粒(anti-miR-132-3p)、第10号染色体上缺失的磷酸酶和张力蛋白同源物(PTEN)过表达质粒(pcDNA3.1-PTEN)或共转染anti-miR-132-3p和PTEN干扰质粒(si-PTEN),MTT法和流式细胞术分别检测细胞增殖与凋亡,蛋白质印迹法检测PTEN、细胞周期蛋白D1(cyclin D1)、周期素依赖激酶抑制剂p21(P21)、B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达,生物信息学预测结合双萤光素酶报告实验分析miR-132-3p与PTEN的靶向关系。结果 与ARPE-19细胞相比,SP6.5、M23细胞中miR-132-3p表达量(0.26±0.02比0.94±0.09、0.81±0.08)明显升高(P<0.05)。与anti-miR-132-3p阴性对照(anti-miR-NC)组相比,anti-miR-132-3p组24 h、48 h、72 h的细胞活性(0.51±0.05比0.30±0.03、0.97±0.09比0.45±0.05、1.40±0.14比0.76±0.07)、cyclin D1、Bcl-2蛋白表达量显著降低(P<0.05),细胞凋亡率[(8.03±0.68)%比(21.51±2.06)%]、P21、Bax水平明显提高(P<0.05),与过表达PTEN相同。miR-132-3p与PTEN之间有靶向调控关系。抑制PTEN能逆转抑制miR-132-3p对SP6.5细胞增殖的抑制作用及对细胞凋亡的促进作用。结论 miR-132-3p通过直接靶向PTEN调控葡萄膜黑色素瘤细胞增殖与凋亡。

Objective To investigate the role of microRNA-132-3p(miR-132-3p) in the proliferation and apoptosis of uveal melanoma cells and its mechanism.Methods The study started and ended from February 2018 to July 2019. Real-time quantitative polymerase chain reaction(qPCR) was used to detect the expression of miR-132-3p in normal uveal epithelial cells ARPE-19 and uveal melanoma cells SP6.5 and M23. SP6.5 cells were transfected with miR-132-3p pnterfering plasmid(anti-miR-132-3p), phosphatase and tensin homolog deleted on chromosome ten(PTEN) overexpression plasmid(pcDNA3.1-PTEN) or co-transfected with anti-miR-132-3p and PTEN interfering plasmid(si-PTEN), and cell proliferation and apoptosis were determined by methyl thiazolyl tetrazolium(MTT) assay and flow cytometry, respectively. Western blot was applied to detect the expression of PTEN, cyclin D1, P21, B cell lymphoma/lewkmia-2(Bcl-2) and Bcl-2 associated X protein(Bax) protein. The targeting relationship between miR-132-3p and PTEN was detected by bioinformatics prediction and dual luciferase reporter.Results Compared with ARPE-19 cells, the expression of miR-132-3p(0.26 ± 0.02 vs. 0.94 ± 0.09, 0.81 ± 0.08) in SP6.5 and M23 cells were significantly increased(P<0.05). Compared with the anti-miR-132-3p negative control(anti-miR-NC) group, the cell viability(0.51 ± 0.05 vs. 0.30 ± 0.03, 0.97 ± 0.09 vs. 0.45 ± 0.05, 1.40 ± 0.14 vs.0.76 ± 0.07), cyclin D1 and Bcl-2 protein expressions of the anti-miR-132-3p group at 24 h, 48 h and 72 h significantly were significantly decreased(P<0.05), while the apoptosis rate [(8.03 ± 0.68)% vs.(21.51 ± 2.06)%], P21 and Bax levels were evidently increased(P<0.05), which was the same as overexpression of PTEN. There was a targeted regulatory relationship between mir-132-3p and PTEN.Inhibition of PTEN reversed the inhibitory effect of miR-132-3p on the proliferation of SP6.5 cells and the promotion effect of cell apoptosis.Conclusion miR-132-3p regulates proliferation and apoptosis of uveal melanoma cells by directly targeting PTEN.