Cell biologic changes in thd cells which PTEN gene activated by double-stranded RAN / 中华胸心血管外科杂志

ABSTRACT

ObjectiveTo evaluate thd cell biologic changes in thd non-small-cell lung cancer(NSCLC) which PTEN gene were activated by double-stranded RNA(dsRNA).MethodsSpecific dsRNA was designed.First,the promoter region of PTEN gene was determined by Promoter 2.0 program,then the CpG island in the promoter was found by CpGisland searcher software and the possible target non-CpG sequence that dsRNA might activate were defined by SiRNA Target Finder software.dsRNA were synthesized at Genechem Company( Shanghai,China).Then the specific dsRNA was transfected into A549 and H292 cells which were stored in our laboratory using Lipofectamine 2000 ( Invitrogen,USA) according to manufacture's instruction.Total celluar RNA was isolated.The expression of PTEN mRNA in transfected,control and mock group were determined by real-time quantitative polymerase chain reaction.Cell profiferation was investigated on days 1 to 5 by using Cell Counting Kit-8 according to the manufature's technical manual.Cell invasion ability was assessed by Transwell method that transmembrane cells were counted,and cell bycle distribution were studied by flow cytometer(FCM) using CycleTESTTM PLUS DNA Reagent Kit.ResultsAfter the introduction of dsRNA into the A549 cells,the PTEN mRNA expressin was upregulated to (4.35 ±0.42) folds compared with the mock and control cells.And in H292 cells,the mRNA expression of PTEN was upregulated to (3.92 ± 0.20) folds.It confirmed the RNA activation phenomenon in the PTEN gene in NSCLC cells.Compared with the control group,the number of alive transfected cells did not decreased in the cell proliferation assay.In the cell invasion test we found that the transmembrane A549 cells were 122.4 ±11.2 vs.150.7 ±13.1 in transfected group and control group respectively.In the cell cycle distribution we found dsRNA in duced part ofthe transfected cells arrested in G1 phase and a corresponding decrease in S-phase population was observed,though this change was not statistically significant.Conclusion The expression of PTEN mRNA could by enhanced by inducing the specific dsRNA into the A549 and H292 cells,though no evidence was found that after the activation of silenced PTEN,the cell proliferation and invasion ability were significantly changed.