POLE2 Gene Silencing Inhibits The Proliferation Of Lung Adenocarcinoma Cells By Regulating PLK1 Expression

Objective:Through screening the downstream key genes of POLE2(DNA polymerase ? B subunit),further studying the proliferation and apoptosis function of PLK1 Gene,and verifing the functional correlation between PLK1 gene and POLE2 gene,which colud lay a foundation for clarify the molecular mechanism of the antitumor effect of POLE2 gene.Method:(1)We designed and synthesized the oligonucleotide single chain for the target sequence of POLE2 siRNA,finally infected the packed virus particles with lung adenocarcinoma A549 cells.(2)After 72 hours,the cells of control group(Group shCtrl)and experimental group(Group shPOLE2)were observed under inverted fluorescence microscope.The results showed that the transfection efficiency(TE)was over 80%,and the next experiment could be carried out when the cells were in normal condition.(3)The total RNA of shCtrl and shPOLE2 group was respectively extracted.Real time quantitative PCR detection system(qPCR)was used to detect the silencing efficiency of POLE2 gene at mRNA level.At the same time,Western blot was used to detect the interference target.(4)Trizol method was used to extract the total RNA of 8 samples.After the samples passed the quality control,they were put into the chip experiment.The whole human genome expression chip scanner 3000 was used to scan the original data of the chip.(1)Screening criteria of differential gene: Fold Change(FC)is more than 2,and P value is less than 0.05.(2)The volcano map,scatter map and cluster map were drawn for the selected genes.(5)Using the technology of bioinformatics to carry out IPA analysis(Ingenuity Pathway Analysis)of differential genes,30 downstream key genes that may be regulated by POLE2 gene were screened comprehensively.(6)30downstream key genes were detected by qPCR,and the PLK1 gene with the most obvious down-regulation of mRNA expression was screened out after the POLE2 gene was silenced.(7)We designed and synthesized the oligonucleotide single chain for the target sequence of PLK1 siRNA,annealed and digested it to produce GV248-shPLK1 lentivirus vector,and co infected 293 T cells with GV248-shPLK1,help-erl.0 and helper2.0 plasmids,and packed them into pseudovirus particles and infected A549 cells and NCI-H1299 cells of non-small cell lung cancer.(8)After 72 hours of shCtrl group and the shPLK1 group cell culturing,We further compared the fluorescent field and dark field pictures of the two cells,and carried out the next experiment after the transfection efficiency reaches 80%.(9)Western blot was used to detect changes of PLK1 gene expression in foreign protein level.(10)The effect of PLK1 gene silencing on the proliferation of lung adenocarcinoma A549 and NCI-H1299 cells was verified by CCK-8assay.(11)Flow cytometry was used to verify the effect of PLK1 gene silencing on the cell cycle of lung adenocarcinoma A549 and H1299.(12)Flow cytometry was used to verify the effect of PLK1 gene silencing on apoptosis of lung adenocarcinoma A549 and H1299 cells.Result:(1)The expression vector of POLE2 siRNA lentivirus was successfully constructed and infected with A549 cells.The preliminary determination of infection efficiency under fluorescence microscope reached 80%.(2)qPCR and Western blot further separately confirmed that silencing efficiency of POLE2 gene at mRNA and protein levels.(3)All eight samples were qualified in quality control.935 differentially expressed genes caused by POLE2 gene change were screened by human whole gene expression profile chip technology,which includes 403 up-regulated genes and 532 down-regulated genes.30down-regulated key genes may be regulated by POLE2 gene were screened out from the down stream genes by using the IPA(Ingenuity Pathway Analysis)technology of bioinformatics analysis.(4)qPCR further confirmed the expression of the above 30 genes at mRNA level,and the results showed that PLK1 gene expression was significantlydown regulated.(5)Secondly,The expression vector of PLK1 siRNA lentivirus was successfully constructed and infected with adenocarcinoma of lung A549 and H1299 cells.The preliminary determination of infection efficiency under fluorescence microscope over 80%.(6)Western blot was used to detect PLK1 gene protein level expression.(7)Flow cytometry detected the apoptosis of lung adenocarcinoma A549 and NCI-H1299 cells,and the results showed that the apoptosis rate of silencing PLK1 gene was increased;(8)Determination of cell viability in 96 hours after virus transfection by CCK-8 assayand and drawing cell proliferation function curve,the results showed that PLK1 gene silencing significantly inhibited the proliferation of A549 and NCI-H1299cells;(9)Flow cytometry was used to detect the effect of PLK1 gene on cell cycle of lung adenocarcinoma A549 and NCI-H1299 cells,and the results showed that the number of S-phase cells was significantly reduced and G2 / M-phase cells were significantly increased comparring with the control group.(10)Flow cytometry was detected the effect of PLK1 gene on apoptosis of lung adenocarcinoma A549 and NCI-H1299 cells,and the results showed that the apoptosis rate was significantly increased comparring with the control group.Conclusion:1.Through the screening of the downstream key genes of POLE2 gene,the downstream key gene PLK1 was determined,which lays the foundation for the downstream molecular mechanism research of POLE2 gene.2.PLK1 gene silencing can induce apoptosis and anti proliferation of human lung adenocarcinoma cell lines A549 and NCI-H1299,and cause G2 / M phase arrest of lung cancer cells,which is consistent with the function of POLE2 gene,revealing that POLE2 gene and PLK1 Gene have functional correlation,which provides basis for further functional recovery experiment.