摘要
目的 观察肺腺癌细胞lncRNA MANCR表达情况,探讨lncRNA MANCR对肺腺癌细胞增殖、侵袭、迁移及凋亡的影响.方法 对数生长期A549细胞分为sh-MANCR组(转染shRNA-MANCR慢病毒)、sh-NC组(转染shRNA-NC慢病毒)、对照组(不转染慢病毒).转染48 h后,采用实时荧光定量PCR法检测细胞lncRNA MANCR相对表达量,培养24、48、72、96 h时采用CCK-8法检测3组细胞增殖吸光度(OD)值.转染48 h后,采用克隆形成实验检测细胞克隆形成率,采用流式细胞术检测细胞周期及细胞凋亡率,采用细胞划痕实验检测细胞迁移距离、划痕愈合率,采用Transwell小室实验检测侵袭细胞数;采用Western blot法检测细胞周期和增殖相关蛋白[周期蛋白D1(Cyclin D1)、周期蛋白依赖性激酶(CDK)2、CDK4、增殖细胞核抗原(PCNA)]、凋亡相关蛋白[B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、caspase-3、caspase-9]及迁移和侵袭相关蛋白[环氧化酶-2(Cox-2)、基质金属蛋白酶(MMP)-2、MMP-9]相对表达量.结果 转染48 h后,sh-MANCR组细胞lncRNA MANCR相对表达量(0.31±0.01)低于对照组(1.00±0.03)、sh-NC组(0.98±0.02)(P＜0.05).培养 48、72、96 h 时 sh-MANCR 组(0.29±0.03、0.38±0.01、0.51±0.03)细胞增殖 OD 值低于对照组(0.40±0.02、0.65±0.02、0.91±0.04)、sh-NC 组(0.37±0.03、0.58±0.02、0.79±0.08)(P＜0.05).转染48 h 后,sh-MANCR 组细胞克隆形成率[(2 483.33±225.46)％]、S 期比率[(17.11±1.74)％]、G2/M 期比率[(10.54±0.95)％]均低于对照组[(4 600.00±132.29)％、(28.21±2.26)％、(17.53±1.09)％]、sh-NC 组[(4 650.00±150.00)％、(31.16±2.01)％、(17.50±1.87)％](P＜0.05),G0/G1 期比率[(63.54±2.16)％]、细胞凋亡率[(18.24±0.49)％]均高于对照组[(44.86±1.65)％、(8.43±0.45)％]、sh-NC 组[(44.71±2.26)％、(8.03±0.25)％](P＜0.05),细胞迁移距离[(108.85±8.04)μm]短于对照组[(240.10±7.17)μm]、sh-NC 组[(224.60±9.83)μm](P＜0.05),划痕愈合率[(12.05±1.00)％]低于对照组[(24.16±0.84)％]、sh-NC 组[(23.70±1.51)％](P＜0.05),侵袭细胞数[(45.33±1.53)个]少于对照组[(138.33±2.52)个]、sh-NC 组[(135.33±1.53)个](P＜0.05),以上指标sh-NC 组与对照组比较差异均无统计学意义(P＞0.05)o sh-MANCR 组 Cyclin D1、CDK2、CDK4、PCNA、Bcl-2、Cox-2、MMP-2、MMP-9蛋白相对表达量均低于对照组、sh-NC组(P＜0.05),Bax、caspase-3、caspase-9蛋白相对表达量均高于对照组、sh-NC组(P＜0.05);sh-NC组Cyclin D1、MMP-2蛋白相对表达量均高于对照组(P＜0.05),CDK2、PCNA、MMP-9蛋白相对表达量均低于对照组(P＜0.05),CDK4、Bcl-2、Bax、caspase-3、caspase-9、Cox-2蛋白相对表达量与对照组比较差异均无统计学意义(P＞0.05).结论 下调lncRNA MANCR表达可抑制肺腺癌细胞增殖、侵袭及迁移,促进细胞凋亡.
Abstract
Objective To observe the expression of lncRNA MANCR in lung adenocarcinoma cells,and to explore the influence of lncRNA MANCR on the proliferation,invasion,migration and apoptosis of lung adenocarcinoma cells.Methods A549 cells in logarithmic growth phase were divided into sh-MANCR group(transfected with MANCR-shRNA),sh-NC group(transfected with shRNA-NC lentivirus),and control group(transfected with no virus).After transfection for 48 h,the relative expression of lncRNA MANCR was detected by real-time fluorescence quantitative PCR.After culture for 48,72 and 96 h,the optical density(OD)values were detected by CCK-8 assay.After 48-h transfection,the cell clone formation rate was detected by clone formation assay,the cell cycle and apoptosis rate were detected by flow cytometry assay,the cell migration distance and wound healing rate were detected by wound healing assay,the number of invading cells was detected by Transwell assay,and the relative expressions of cell cycle-and proliferation-related proteins[Cyclin D1,cyclin-dependent kinase(CDK)2,CDK4,and proliferating cell nuclear antigen(PCNA)],apoptosis-related proteins[B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),caspase-3,and caspase-9],and migration/invasion-related proteins[cyclooxygenase-2(Cox-2),matrix matelloproteinase(MMP)-2,and MMP-9]were detected by Western blot.Results After 48-h transfection,the relative expression of lncRNA MANCR was lower in sh-MANCR group(0.31±0.01)than that in control group(1.00±0.03)and sh-NC group(0.98±0.02)(P＜0.05).The OD values of cell proliferation were lower in sh-MANCR group(0.29±0.03,0.38±0.03,0.51±0.03)than those in control group(0.40±0.02,0.65±0.02,0.91±0.04)and sh-NC group(0.37±0.03,0.58±0.02,0.79±0.08)after culture for 48,72 and 96 h(P＜0.05).After 48-h transfection,the clone formation rate,S phase ratio and G2/M phase ratio were lower in sh-MANCR group[(2 483.33±225.46)％,(17.11±1.74)％,(10.54±0.95)％]than those in control group[(4 600.00±132.29)％,(28.21±2.26)％,(17.53±1.09)％]and sh-NC group[(4 650.00±150.00)％,(31.16±2.01)％,(17.50±1.87)％](P＜0.05),the G0/G1 phase ratio and cell apoptosis rate were higher in sh-MANCR group[(63.54±2.16)％,(18.24±0.49)％]than those in control group[(44.86±1.65)％,(8.43±0.45)％]and sh-NC group[(44.71±2.26)％,(8.03±0.25)％](P＜0.05),the migration distance was shorter in sh-MANCR group[(108.85±8.04)μm]than that in control group[(240.10±7.17)μm]and sh-NC group[(224.60±9.83)μm](P＜0.05),the wound healing rate was lower in sh-MANCR group[(12.05±1.00)％]than that in control group[(24.16±0.84)％]and sh-NC group[(23.70±1.51)％](P＜0.05),and the number of invading cells was lower in sh-MANCR group[(45.33±1.53)％]than that in control group[(138.33±2.52)％]and sh-NC group[(135.33±1.53)％](P＜0.05).All the above indexes showed no significant differences between sh-NC group and control group(P＞0.05).The relative expressions of Cyclin D1,CDK2,CDK4,PCNA,Bcl-2,Cox-2,MMP-2 and MMP-9 were lower in sh-MANCR group than those in control group and sh-NC group(P＜0.05).The relative expressions of Bax,caspase-3 and caspase-9 proteins were higher in sh-MANCR group than those in control group and sh-NC group(P＜0.05).The relative expressions of Cyclin D1 and MMP-2 proteins were higher in sh-NC group than those in control group(P＜0.05).The relative expressions of CDK2,PCNA and MMP-9 proteins were lower in sh-NC group than those in control group(P＜0.05).There were no significant differences in the relative expressions of CDK4,Bcl-2,Bax,caspase-3 and caspase-9 proteins between sh-NC group and control group(P＞0.05).Conclusion The proliferation,invasion and migration can be inhibited,and the cell apoptosis of lung adenocarcinoma cells can be promoted via down-regulating lncRNA MANCR expression.
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