MRNA And Protein Expression Of Puma In Lung Cancer

Objective to explore the correlation of puma expression to tumorigenesis and development of lung cancer.Methods By adjusted 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 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 exploring the conditions of fluorescence quantitative RT-PCR,we showed that the optimized fluorescencequantitative RT-PCR reaction mixtures contained the forward and reverse primers at 500nmol/L each;800μmol/L each of dNTP;1.5mmol/L MgCl₂;1×Enhancer solution 5.0μl;10×PCR buffer 5.0μl;0.5×SYB GREENâ… 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.2.The means of the relative rate of puma mRNA and GAPDH mRNA in lung cancer tissue,paracancer lung tissue,farcancer lung tissue and non-cancer lung 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 were 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).3.In the 40 cases of primary lung cancer tissues,the medians of the relative rate of PUMA protein andβ-actin 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 the relative rate of PUMA protein andβ-actin protein in non-cancer 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).4.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).Conclusions Our study showed that puma expression maybe did not play a role in NSCLC initiation and progression of cancer.There was no evident relationship of puma mRNA and protein among different pathological types of lung cancer.There was not relationship between puma mRNA and protein with different pathological types of NSCLC. 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.