微小 RNA-301b-3p靶向第 10号染色体缺失的磷酸酶及张力蛋白同源物对骨肉瘤细胞增殖、凋亡的影响

目的 探讨微小RNA-301b-3p(miR-301b-3p)对骨肉瘤细胞增殖、凋亡的影响及其作用机制.方法 本研究起止时间为2019年3—9月.人成骨细胞(hFOB)和骨肉瘤细胞U2OS、MG63购自美国菌种保藏中心.U2OS细胞分为miRNA抑制物阴性对照(anti-miR-NC)组、miR-301b-3p抑制物(anti-miR-301b-3p)组、anti-miR-301b-3p+小干扰RNA阴性对照(si-NC)组、anti-miR-301b-3p+第10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)小干扰RNA(si-PTEN)组;实时荧光定量PCR(RT-qP-CR)检测miR-301b-3p表达水平;四甲基偶氮唑盐比色法(MTT)检测细胞活性;流式细胞术检测细胞凋亡;双荧光素酶报告实验检测miR-301b-3p和PTEN的靶向关系.结果 与hFOB细胞相比,U2OS、MG63中miR-301b-3p表达水平(0.89±0.09),(0.70±0.07)比(0.20±0.02)]显著升高.与anti-miR-NC组比较,anti-miR-301b-3p组U2OS细胞活性(0.59±0.06)比(1.33±0.13)]显著降低,而凋亡率(22.06±2.31)％比(7.48±0.78)％]、PTEN蛋白表达(0.69±0.07)比(0.35±0.03)]显著升高.PTEN是miR-301b-3p的直接靶基因.与anti-miR-301b-3p+si-NC组比较,anti-miR-301b-3p+si-PTEN组U2OS细胞活性(1.16±0.12)比(0.56±0.06)]显著升高,而凋亡率(10.28±1.16)比(22.12±2.34)]显著降低.结论 抑制miR-301b-3p表达可通过调控PTEN抑制骨肉瘤细胞增殖,促进细胞凋亡.

Effect of miR-301b-3p targeting PTEN on proliferation and apoptosis of osteosarcoma cells

Objective To explore the effect of microRNA-301b-3p (miR-301b-3p) on proliferation and apoptosis of osteosarcoma cells and its mechanism.Methods The start and end time of this research was from March to September 2019. Human osteoblasts(hFOB) and osteosarcoma cells U2OS and MG63 were purchased from the American Tissue Culture Collection. U2OS cells were assigned into miRNA inhibitor negative control (anti-miR-NC) group, miR-301b-3p inhibitor (anti-miR-301b-3p) group, anti-miR-301b3p+small interfering RNA negative control (si-NC) group, anti-miR-301b-3p+phosphatase and tensin homolog deleted on chromosome 10 (PTEN) small interfering RNA (si-PTEN) group. Real-time quantitative PCR (RT-qPCR) was used to detect the expression of miR301b-3p, methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability, flow cytometry was used to detect apoptosis, and dual luciferase reporter assay was used to detect the targeting relationship between miR-301b-3p and PTEN.Results Compared with hFOB cells, the expressions of miR-301b-3p (0.89±0.09),(0.70±0.07) vs (0.20±0.02)] in U2OS and MG63 cells were significantly increased. Compared with the anti-miR-NC group, the cell viability (0.59±0.06) vs (1.33±0.13)] of U2OS cells in the anti-miR-301b-3p group was significantly reduced, while the apoptosis rate (22.06±2.31)% vs(7.48±0.78)%] and PTEN protein expression (0.69±0.07) vs (0.35±0.03)] were significantly increased. PTEN is the direct target gene of miR-301b-3p. Compared with the anti-miR-301b-3p+si-NC group, the cell viability (1.16±0.12)vs (0.56±0.06)] of U2OS cells in the anti-miR-301b-3p+si-PTEN group was significantly increased, while the apoptosis rate (10.28±1.16)% vs (22.12±2.34)%] was significantly reduced.Conclusion Inhibition of miR-301b-3p expression can inhibit osteosarcoma cell proliferation and promote cell apoptosis by regulating PTEN.