Expressional And Mutational Analysis Of Pro-apoptotic PUMA In Non-small-cell Lung Cancer

Objective to explore the correlation of the mutation and expression of puma gene to tumorigenesis and development of lung cancer and the relation between PUMA and p53.Methods Explored the condition of PCR and optimized amplification system and reaction conditions of three high GC content Exon of puma,it make the exon 2 that GC content 82%was amplificationed succeefully.Explored the reaction conditions of fluorescence quantitative reverse transcript-polymerase chain reaction,the FQ- RT-PCR assay was established to examine the puma mRNA.The quantitative fluorogenic real-time PCR and Western Blot Analysis were used to detect the mutation of puma,the expression puma mRNA and its protein in all 40 cases of primary lung cancer tissues,para-cancer lung tissues and far-cancer lung tissues,10 control cases of non-cancer lung tissues were also detected. Apoptosis of 20 NSCLC samples were detected by TUNEL technique.Results1.By explored the experiment conditions of PCR,the ultimate amplification system and react contions of three exon of puma were eatablished.(1) The optimized reaction system of exon2 were:dNTP 200μmol/L,enhancer system MgSO4 3.0mmol/L,each primers 500nmol/L,Taq DNA polymerase 0.1U/μl,template more than 4000ng,10×buffer 5μl,ehancer solution 10μl,the total volume was 50μl.The amplificat condition was:After 5 min at 95℃,reactions were cycled through 45s denaturation at 95℃,45s annealing at 64℃,and 45s extension at 72℃for 33 cycles;followed for 5 min.(2) Exon3 and exon 4 conditions were:MgSO4 2.0mmol/L and 1.0mmol/L,other components were same as exon2.2.By using the optimized amplification systems and reaction conditions,the exon 2, exon 3 and exon 4 of puma gene in all 40 cases of lung cancer tissues,paracancer lung tissues and farcancer lung tissues,10 cases of lung benign disease were also amplificationed.The amplification productions were depurated and sequenced both mutation and small deleted fragments.The sequencing results compare to GeneBank sequence by Blast 2,non of mutation and small fragments were found,and BH3 domain is normaol.3.By exploring the conditions of fluorescence quantitative RT-PCR,we showed that the optimized fluorescence quantitative RT-PCR reaction mixtures contained the forward and reverse primers at 500nmol/L each;dNTP 800μmol/L;1.5mmol/L MgCl₂;1×Enhancer solution 5.0μl;10×PCR buffer 5.0μl;0.5×SYB GREEN I 1.0μl;Taq DNA polymerase 0.1U/μl; template 1.0μl;the total reaction volume was 50μl.The RT-PCR was performed under the following conditions:94℃for 5 minutes;followed by 40 cycles of 94℃for 45 seconds,56℃for 45 seconds and 72℃for 45 seconds;and 72℃for 5 minutes.4.The means of the relative rate of puma mRNA and GAPDH mRNA in lung cancer tissue,paracancer lung tissue,farcancer lung tissue and lung benign disease tissues were 1.17±0.08,1.16±0.07,1.18±0.07 and 1.17±0.11 which detected by fluorescence quantitative RT-PCR.The data were evaluated by analysis of variance.There was no any significant difference among lung cancer tissue,paracancer lung tissue and farcancer lung tissue (F=0.416,P＞0.05).Also,there was no significant difference among squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma in the group of lung cancer tissue,paracancer lung tissue and farcancer lung tissue(F=0.311,P＞0.05;F=0.338,P＞0.05;F=0.329,P＞0.05).5.In the 40 cases of primary lung cancer tissues,the medians of PUMA protein in lung cancer tissue,paracancer lung tissue and farcancer lung tissue were 1.30(0.98～1.74), 1.11(0.72～1.69) and 1.01(0.71～1.49) which detected by Western Blot Analysis.The mean of PUMA protein in benigh disease lung tissues was 11.17±0.11 The data were evaluated by analysis of Krusdal-Wallis H test.There were no any significant difference among lung cancer tissue,paracancer lung tissue and farcancer lung tissue(x²(H) =4.918,P＞0.05).Also,there was no significant difference among squamous cell carcinoma,adenocarcinoma and adenosquamous carcinoma in the group of lung cancer tissue,paracancer lung tissue and farcancer lung tissue(F=0.101,P＞0.05;F=0.599,P＞0.05;F=1.054,P＞0.05).6.Western Blot Analyzed p53 protein:the p53 protein in cancer tissue is significant difference in paracancer lung tissue,farcancer lung tissue and lung benign disease tissue; there was no any significant difference among paracancer lung tissue and farcancer lung tissue.7.The results of TUNEL were:The apoptosic index(AI) in lung cancer tissue,paracancer lung tissue and farcancer lung tissue were 4.96%±0.1.63%,1.45%±0.46%and 1.26%±0.42%. AI in lung cancer was significant difference between lung cancer tissue with paracancer lung tissue and farcancer lung tissue(Z=-3.920,P=0.000),AI of paracancer lung tissue was higher than farcancer lung tissue,and has significant difference of them(Z=-3.469,P=0.001).8 The relationship of PUMA and p53:PUMA protein expression was not correlate with p53 protein expression.The cause of it may be that the p53 has high mutation in lung cancer and the mutated p53 can not imediate expression of PUMA.Conclusions Mutational analysis revealed that there was no PUMA BH3 domain and exon mutation in the 40 NSCLC,suggesting that PUMA BH3 domain mutation and exon mutation are not a direct target of inactivation in NSCLC development.PUMA expression may be not play a role in NSCLC initiation and progression of cancer.There was not relationship between puma mRNA and protein with different pathological types of NSCLC. PUMA expression was not mediates by p53,and also induced from various apoptotic stimuli. In the course of the occurrence and development of lung cancer,abnormal apoptosis,caused by excessive cell proliferation and to reduce the sensitivity of radiotherapy and chemotherapy, leading to lung cancer has been the global morbidity and mortality are highly malignant.This findings are inconsistent with in vitro cell line findings,but is consistent with other tumor organization findings,explained that PUMA in the tumor occurrence development,different organizations have different performance,and its occurrence in lung cancer development remains to be further research,whether as a lung cancer diagnosis and treatment have yet to be a new target for further study.