Influence of lncRNA MANCR expression on biological behaviors of lung adenocarcinoma cells
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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