circ-PRKDC通过调控miR-505-3p影响肺癌细胞增殖、凋亡和放射敏感性机制研究

circ-PRKDC affects the proliferation,apoptosis and radiosensitivity of lung cancer cells by regulating miR-505-3p

目的探讨circ-PRKDC对肺癌细胞增殖、凋亡和放射敏感性的影响及分子机制。方法培养正常肺上皮细胞BEAS-2B和肺癌细胞系NCI-H1299、NCI-H2170、NCI-H1975。将NCI-H1299细胞分为si-NC、si-PRKDC、pcDNA-NC、pcDNA-PRKDC、miR-NC、miR-505-3p、anti-miR-NC、anti-miR-505-3p、si-PRKDC+anti-miR-NC、si-PRKDC+anti-miR-505-3p组。RT-qPCR检测circ-PRKDC和miR-505-3p的表达水平;蛋白质印迹法检测蛋白表达;MTT检测细胞增殖;流式细胞术检测细胞凋亡;平板克隆形成实验检测细胞放射敏感性;双荧光素酶报告实验检测circ-PRKDC和miR-505-3p的靶向关系。结果与BEAS-2B细胞相比,NCI-H1299、NCI-H2170、NCI-H1975细胞circ-PRKDC表达水平升高(3.65、3.10、2.67∶1.00,P<0.05),miR-505-3p表达水平降低(0.42、0.50、0.54∶1.02,P<0.05)。低表达circ-PRKDC后CyclinD1表达水平降低(0.42∶0.81,P<0.05),Cleaved-caspase-3和γ-H2AX表达水平升高[(0.71∶0.33,P<0.05)和(0.89∶0.465),P<0.05];细胞A值降低(0.413∶0.839,P<0.05);细胞凋亡率升高(20.35∶6.21,P<0.05);细胞存活分数降低(P<0.05);β-catenin表达水平降低(0.35∶0.73,P<0.05)。miR-505-3p高表达后CyclinD1表达水平降低(0.34∶0.83,P<0.05),Cleaved-caspase-3(0.65∶0.32,P<0.05)和γ-H2AX(0.96∶0.45,P<0.05)表达水平升高,细胞A值降低(0.386∶0.851,P<0.05),细胞凋亡率升高(16.38∶6.20,P<0.05),细胞存活分数降低(P<0.05)。与miR-NC比较,miR-505-3p组转染circ-PRKDC野生型报告质粒的细胞荧光素酶活性降低(0.44∶1.00,P<0.05)。下调miR-505-3p能逆转circ-PRKDC低表达对NCI-H1299细胞增殖、凋亡和放射敏感性以及β-catenin表达的影响。结论低表达circ-PRKDC可能通过上调miR-505-3p抑制肺癌细胞增殖,促进凋亡以及增强细胞的放射敏感性,且其可能与Wnt/β-catenin信号通路有关。

Objective To investigate the effect of circ-PRKDC on lung cancer cell proliferation,apoptosis and radiosensitivity and its molecular mechanism.Methods Normal lung epithelial cells BEAS-2B and lung cancer cell lines NCI-H1299,NCI-H2170,NCI-H1975 were cultured.NCI-H1299 cells were divided into the si-NC,si-PRKDC,pcDNA-NC,pcDNA-PRKDC,miR-NC,miR-505-3p,anti-miR-NC,anti-miR-505-3p,si-PRKDC+anti-miR-NC and si-PRKDC+anti-miR-505-3p groups.RT-qPCR was used to detect the expression levels of circ-PRKDC and miR-505-3p.Western blot was employed to measure the protein expression.MTT was used to detect cell proliferation.Flow cytometry was utilized to detect cell apoptosis.Plate clone formation assay was conducted to detect the cell radiosensitivity.Dual luciferase reporter assay was performed to analyze the targeting relationship between circ-PRKDC and miR-505-3p.Results Compared with normal lung epithelial cells BEAS-2B,the expression levels of circ-PRKDC in the lung cancer cell lines NCI-H1299,NCI-H2170 and NCI-H1975 were significantly up-regulated(3.65,3.10,2.67 vs.1.00,all P<0.05),whereas those of miR-505-3p were significantly down-regulated(0.42,0.50,0.54 vs.1.02,all P<0.05).After low expression of circ-PRKDC,the expression level of CyclinD1 was significantly down-regulated(0.42 vs.0.81,P<0.05),whereas those of Cleaved-caspase-3(0.71 vs.0.33,P<0.05)andγ-H2AX(0.89 vs.0.46,P<0.05)were significantly up-regulated,the cell A value was significantly decreased(0.413 vs.0.839,P<0.05),cell apoptosis rate was significantly increased(20.35 vs.6.21,P<0.05),cell survival fraction was significantly decreased(P<0.05),andβ-catenin expression was significantly down-regulated(0.35 vs.0.73,P<0.05).After high expression of miR-505-3p,the expression level of CyclinD1 was significantly down-regulated(0.34 vs.0.83,P<0.05),those of Cleaved-caspase-3(0.65 vs.0.32,P<0.05)andγ-H2AX(0.96 vs.0.45,P<0.05)were significantly up-regulated,the cell A value was significantly decreased(0.386 vs.0.851,P<0.05),the apoptosis rate was significantly increased(16.38 vs.6.20,P<0.05),and the cell survival fraction was significantly decreased(P<0.05).Compared with miR-NC,the luciferase activity of miR-505-3p group transfected with circ-PRKDC wild-type reporter plasmid was significantly decreased(0.44 vs.1.00,P<0.05).Down-regulation of miR-505-3p could reverse the effect of low expression of circ-PRKDC on the proliferation,apoptosis,radiosensitivity andβ-catenin expression of NCI-H1299 cells.Conclusion Low expression of circ-PRKDC may inhibit lung cancer cell proliferation,promote cell apoptosis and enhance cell radiosensitivity by up-regulating miR-505-3p,which is probably associated with the Wnt/β-catenin signaling pathway.