Effect Of Exogenous P51A Gene On The Growth And Chemosensitivity Of Human Lung Adenocarcinoma Cell Lines

Objective:p53 is one of the most popular genes which has been studied extensively in the field of tumor biology,and its loss of function will always relate to tumorigenesis.Current study has revealed that p53 mutation exists in over 50%of all human tumors,including 50%of non-small cell lung cancer and 70%of small cell lung cancer.The dysfunction of p53-mediated apoptosis is the key to tumorigenesis,thus most gene therapy programs concentrate on improving the expressing level of wild-type p53 in tumor cells.However the p53 gene therapy has mostly succeeded in tumor cells with p53dysfunction,and has not yielded satisfactory results in normal ones.Therefore many research programs have been carried out to make up for the limitation of p53 gene therapy.The new target gene should be able to induce cell apoptosis in spite of p53 status.p63,a new member of p53 gene family,has provided new hopes.Human p63 is located in 3q27-3q29 area of chromosome 3,including two independent promoters and 15 exons.Due to the changes of promoter and the variability of splicing,p63 gene can encode at least eight kinds of protein forms.The upper promoter of intron 1 can lead to four kinds of isoforms-TAp63α（p51B,KET）,TAp63β,TAp63γ（p51A）and TAp63δ,referred to as TAp63.TAp63 has three similar domains as p53:N-transcriptional activation domains,DNA-binding domain and C-oligomerization domain.The lower promoter of intron 3 triggers the synthesis of mRNA without the N-transcriptional activation domains,referred to asΔNp63,includingΔNp63α（CUSP,p73L）,ΔNp63β,ΔNp63γ（p40）andΔNp63δ.Due to the importance of p53 in tumor research,p63 gene has become hot spots in the field of cell apoptosis and tumor from the very beginning.The current study has showed that,p63 mutation rarely occurs in tumors,while expression abnormity can always be found.Therefore p63 may not be the classic tumor suppressor gene as p53.But p63 has many isoforms:ΔNp63 may not be able to activate the p53 target genes,due to its lack of N-transcriptional activation domains,while TAp63,especially p51A（TAp63γ）resembles p53 the most,thus it might mimic the tumor suppression function of p53,induce cell cycle arrest and apoptosis,so as to become a new promising therapeutic gene of tumors.Methods:1.Construction of pcDNA3.1/myc-His-p51A:We designed the primer pairs of p51A（synthesized by Invitrogen）,and amplified the p51A cDNA sequence from human skeletal muscle poly A+RNA by RT-PCR.After identification by BglⅠenzyme digestion and sequencing,we constructed the recombinant plasmid through BamHⅠ+XhoⅠdouble enzyme digestion,purification and T₄DNA ligase reaction.The recombinant plasmid was then transformed into DH5αand screened for positive clones.Plasmid was extracted from positive clones and underwent identification by double enzyme digestion and sequencing.2.Cell culture:Human adenocarcinoma cell lines A549 and NCI-H1299 were grown in RPMI-1640 medium with 10%fetal bovine serum at 37℃,5%CO₂ and saturated humidity.1.5g/L sodium bicarbonate and 10mM HEPES were added into both medium.And 4.5g/L glucose and 1.0mM sodium pyruvate were added for NCI-H1299.0.25%trypsin was used for cell passage.3.Gene transfection and clone screening:Recombinant plasmid and empty plasmid were transfected into A549 and NCI-H1299 respectively by TransLipid reagent,followed by G418 screening and culture of positive clones.4.RT-PCR analysis:Total RNA were extracted from every cell group by Trizol,RT-PCR and agarose gel electrophoresis were used for analysis.5.Western blot analysis:Anti-myc antibody was used to detect whether the recombinant plasmid was transfected into cells successfully or not.Anti-p51A antibody and anti-p21 antibody were used to detect P51A and P21 protein level.ECL chemiluminescence imaging and image scan analysis were performed.6.Cell proliferation capacity was detected by MTT assay and cell growth curves were made.7.Cytotoxicity of drug:Different concentration of cisplatin or adriamycin were added into every cell group and the IC50 were detected by MTT assay.8.Colony formation assay:The survival fraction of each cell line was calculated9.Cell apoptosis was analyzed by flow cytometry（AnnexinⅤ-FITC and PI）.10.Statistical analysis:The data were analyzed by SPSS 15.0 software.The results were expressed as mean±SD.Absorbance was examined by variance analysis.Statistical significance was determined by t test.A value of P<0.05 was considered significant.A value of P<0.01 was considered distinctly significant.Results:1.pcDNA3.1/myc-His-p51A constructed successfullyWe amplified a single 1371bp band from human skeletal muscle poly A+RNA with self-designed primer pairs by RT-PCR,which accorded with the theoretical expected length of the p51A cDNA fragment.BglⅠenzyme digestion and sequencing confirmed the RT-PCR product,and the recombinant plasmid was also confirmed by BamHⅠ+XhoⅠdouble enzyme digestion and sequencing.2.p51A transfection and stable expressionA549/pcDNA3.1 and NCI-H1299/pcDNA3.1 could survive G418 screening,and western blot analysis showed that myc-tag was expressed both in A549/p51A and NCI-H1299/p51A,implicating successful transfection of plasmid.p51A mRNA,P51A protein and P21 protein level arised significantly after p51A transfection.3.Cell proliferation capacityMTT assay showed that the proliferation capacity of A549/p51A and NCI-H1299/p51A decreased significantly.4.Cytotoxicity of drugThe sensitivity of both cell lines to chemotherapy was increased significantly after p51A transfection.A549 IC50 of cisplatin and adriamycin decreased by 91.10%and71.43%,respectively;NCI-H1299 IC50 of cisplatin and adriamycin decreased by 52.27%and 64.29%,respectively.5.Colony formation assayThe survival fraction of A549/p51A and NCI-H1299/p51A decreased significantly,and even more when combined with chemotherapy.6.Cell apoptosis analysisFlow cytometry analysis revealed that the apoptosis index of A549 and NCI-H1299increased significantly after p51A transfection with or without chemotherapy.Conclusion:We amplified p51A gene sequence from human skeletal muscle poly A+RNA with self-designed primer pairs by RT-PCR,and constructed pcDNA3.1/myc-His-p51A successfully for continued research.Exogenous p51A gene can increase its expression in human lung adenocarcinoma cell lines A549 and NCI-H1299,suppress cell growth and proliferation,reduce colony formation rate and induce cell apoptosis.More over,it can also cooperate with chemotherapy,increase the sensitivity of both cell lines to cisplatin and adriamycin,and thus reduce the dose and the side-effect of chemotherapy.p51A gene can suppress tumors in spite of p53 status.And p21 gene might be involved.p51A might become a new promising therapeutic gene of tumors,which will make up for the limitation of p53 gene therapy.