Effects Of Long Non-coding RNA PVT1 On The Proliferation,migration Of Lung Adenocarcinoma A549 Cells

ObjectiveThe objective of this study is to investigate the effects of Long non-coding RNA Plasmacytoma variant translocation gene 1 on the proliferation,migration and other biological behaviors of lung adenocarcinoma A549 cells,which provides the experimental basis for exploring the role of LncRNA PVT1 in the development of lung adenocarcinoma.Methods1.Construction of PVT1 low expression cell line:The small interfering RNA(PVT1siRNA)was transfected into A549 cells using Lipofectamine 2000 to knock down the expression of LncRNA PVT1.The expression level of LncRNA PVT1 was measured by real-time PCR.2.Construction of PVT1 overexpression cell line:Human LncRNA PVT1 overexpression plasmid was transfected into A549 cells by Lipofectamine2000.Stable cell lines with high expression of PVT1 were selected by G418,and PVT1 expression was verified by real-time PCR.3.Experimental groups:① Untreated normal A549 cells(blank control);②A549 cells transfected with PVT1-siRNA(PVT1 low expression group);③ A549 cells transfected with NC-siRNA(negative control of PVT1 low expression group)④A549 cells transfected with overexpression plasmid of PVT1(PVT1 overexpression group);⑤ A549 cells transfected with empty vector(negative control of PVT 1 overexpression group).4.Cell viability was detected by CCK-8 assay:cells were seeded in 96-well plates,and the absorbance value of each group were detected at 12h,24h,48h and 72h,respectively.5.Colony formation assay was used to observe the colony formation ability:cells were seeded in 6-well plates,and visible clones were fixed,stained and counted.6.Transwell assay was used to detect capability of cell migration:Cells were seeded in the upper chambers of the Transwell chambers.After 24 hours’ culture,cells penetrating the membrane of the transwell compartment were fixed,stained,and then counted.7.Xenograft experiments:The A549 cells with or without PVT 1 overexpression were subcutaneously injected into the left and right side of six BALB/c-nu male nude mice aged 4-5 weeks old.The length and diameter of the nodules were measured every three days.Results1.CCK-8 results:Compared with untreated normal A549 cells and NC-siRNA group A549 cells,the proliferation activity of the A549 cells with down-regulated LncRNA PVT1 expression level was decreased.The decreased proliferation was more significant with longer culturing time(P<0.01).On the other hand,compared with the control group,the proliferation of A549 cells with PVT1 overexpression was significantly increased(P<0.01).2.The results of colony formation experiment:the colony formation of A549 cells transfected with PVT1-siRNA was significantly lower than that of untreated normal A549 cells,and the colony formation ability was weakened(P<0.01).A549 cells with PVT1 overexpression formed the most colonies among the experimental groups,which was significantly higher than that in the empty vector group and the blank control group,therefore the colony forming ability was enhanced(P<0.01)in cells with PVT1 overexpression.3.Results of Transwell experiment:Compared with untreated A549 cells,the number of transmembrane penetrating cells in the PVT1 knockdown group decreased significantly,and the migration ability decreased(P<0.01).Compared with untreated A549 cells,the number of transmembrane penetrating cells in the PVT1 overexpression group increased significantly,and the migration ability was enhanced(P<0.01).4.Results of subcutaneous transplantation in nude mice:the subcutaneous tumors in PVT1 overexpression group grew more rapidly,and the exfoliated tumor tissues were larger and heavier.Conclusion:1.The expression of LncRNA PVT1 is an important factor to maintain the active proliferation of A549 cells,knockdown LncRNA PVT 1 decrease the viability of A549 cells,while overexpression of PVT 1 promotes proliferation.2.The expression of LncRNA PVT1 is an important factor to maintain the migration ability of A549 cells,knockdown LncRNA PVT1 attenuates the migration ability of A549 cells,while overexpression of PVT1 enhances its migration ability.3.The expression of LncRNA PVT1 accelerates the growth of non-small cell lung cancer in vivo.