LncRNA PVT1调控miR-1207-5p/FMNL2对HL-60细胞凋亡及化疗敏感性的影响

Effect of LncRNA PVT1 on Apoptosis and Chemosensitivity of HL-60 Cells by Regulating miR-1207-5p/FMNL2

目的探讨长链非编码RNA浆细胞瘤转化迁移基因1(LncRNA PVT1)对HL-60细胞生物学行为及柔红霉素敏感性的影响。方法将HL-60细胞分为对照组、miR-NC组、PVT1-siRNA组、miR-1207-5p inhibitor组和miR-1207-5p mimic组。除对照组细胞不处理外,其余各组细胞分别将miR-NC,PVT1-siRNA,miR-1207-5p inhibitor,miR-1207-5p mimic转染到HL-60细胞中。采用萤光素酶检测试剂分析LncRNA PVT1对微小RNA-1207-5p(miR-1207-5p)和同源形成素样蛋白2(FMNL2)的调控作用,CCK-8法检测细胞存活率,改良Matrigel Boyden室测定法测定细胞侵袭性,划痕实验检测细胞迁移力,流式细胞术检测细胞凋亡率,MTT法检测细胞对柔红霉素敏感性,实时逆转录定量聚合酶链式反应(RT-qPCR)法检测miR-1207-5p和FMNL2 mRNA表达水平,免疫印迹(Western blot)法检测FMNL2蛋白表达水平。结果萤光素酶分析结果显示,LncRNA PVT1与miR-1207-5p、miR-1207-5p与FMNL2均存在靶向调节作用;与对照组比较,PVT1-siRNA组LncRNA PVT1表达水平显著降低(P<0.05),miR-1207-5p表达水平显著升高(P<0.05)。与对照组比较,miR-1207-5p inhibitor组细胞存活率、迁移率、侵袭数均显著降低,细胞凋亡率、FMNL2 mRNA和蛋白表达水平均显著升高(P<0.05);miR-1207-5p mimic组细胞存活率、迁移率、侵袭数均显著升高,细胞凋亡率、FMNL2 mRNA和蛋白表达水平均显著降低(P<0.05)。与对照组比较,相同质量浓度(0,0.5,1,2,4,8μg/mL)柔红霉素作用下PVT1-siRNA组HL-60细胞活力均显著升高(P<0.05)。结论沉默LncRNA PVT1可使miR-1207-5p表达增加,从而抑制FMNL2表达,导致HL-60细胞恶性生物学行为概率升高,同时降低细胞对柔红霉素的敏感性。

Objective To investigate the effect of the long non-coding RNA plasmacytoma variant translocation 1(LncRNA PVT1)on the biological behavior and the sensitivity to daunorubicin of HL-60 cells.Methods HL-60 cells were divided into the control group,miR-NC group,PVT1-siRNA group,miR-1207-5p inhibitor group and miR-1207-5p mimic group.Except for the control group,the other groups were transfected with miR-NC,PVT1-siRNA,miR-1207-5p inhibitor and miR-1207-5p mimic,respectively,and the control group was given no treatment.The regulatory effects of LncRNA PVT1 on the microRNA-1207-5p(miR-1207-5p)and formin-like 2(FMNL2)were analyzed by the luciferase reporter gene assay kit,the survival rate of cells was detected by the CCK-8 method,the invasiveness of cells was detected by the modified Matrigel Boyden chamber assay,the migration of cells was detected by the scratch test,the apoptosis rate of cells was detected by the flow cytometry,the sensitivity of cells to daunorubicin was detected by the MTT method,the expression levels of miR-1207-5p and FMNL2 mRNA were detected by the real-time reverse transcription-quantitative polymerase chain reaction(RT-qPCR),and the expression level of FMNL2 protein was detected by the Western blot.Results The results of luciferase analysis showed that LncRNA PVT1 had targeted regulatory effects on miR-1207-5p,and miR-1207-5p had targeted regulatory effects on FMNL2.Compared with those in the control group,the expression level of LncRNA PVT1 significantly decreased(P<0.05)and the expression level of miR-1207-5p significantly increased(P<0.05)in the PVT1-siRNA group.Compared with those in the control group,the survival rate,migration rate and invasion number in the miR-1207-5p inhibitor group significantly decreased,and the apoptosis rate,FMNL2 mRNA and protein expression levels in the miR-1207-5p inhibitor group significantly increased(P<0.05).Compared with those in the control group,the survival rate,migration rate and invasion number in the miR-1207-5p mimic group significantly increased,and the apoptosis rate,FMNL2 mRNA and protein expression levels in the miR-1207-5p mimic group significantly decreased(P<0.05).Compared with that in the control group,the viability of HL-60 cells in the PVT1-siRNA group increased significantly with the same mass concentration(0,0.5,1,2,4,8μg/mL)of daunorubicin(P<0.05).Conclusion The silence of LncRNA PVT1 can increase the expression of miR-1207-5p,thereby inhibiting the expression of FMNL2,which leads to an increase in the probability of malignant biological behavior of HL-60 cells,and a decrease in the sensitivity to daunorubicin.