MRNA And Protein Expression Of Noxa In Lung Cancer

Objective To explore the correlation of noxa expression to tumorigenesis and development of lung cancer and regulation of p53 on noxa.Methods The quantitative fluorescence real-time PCR and Western Blot Analysis were used to detect the expression noxa mRNA and its protein in all 44 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, and used WB to detect p53 protein. Apoptosis of 20 NSCLC samples were detected by TUNEL technique.Results1. The quantitative fluorescence real-time PCR detect noxa mRNA Used ABI TaqMan Universal PCR amplification of pre-mixed reagents, specific primers and probe. The quantitative fluorescence real-time PCR reaction mixtures contained TaqMan Universal PCR Master Mix12.5μl,20×Assays-on-Demand^TM Gene ExpressionAssay Mix1.25ul(contained forward primer,reverse primer and TaqMan probe), cDNA template2.5 ul, nucleic acid-free water8.75 ul, the total reaction volume was 25μl. In addition to GAPDH amplification primers and probes, the other system components ibid. The RT-PCR was performed under the following conditions:50℃2 min; 95℃10 min; followed by 40 cycles of 95℃15 sec,60℃1 min.2. The quantitative fluorescence real-time PCR detect expression of noxa mRNA The relative rate of noxa mRNA in lung cancer tissue, paracancer lung tissue, farcancer lung tissue and non-cancer lung tissues were 0.52(0.31～0.80),1.49(0.51～2.56),1.00(0.52～1.54) by fluorescence quantitative RT-PCR. The data were evaluated by analysis of ranks test, there were significant difference among lung cancer tissue, paracancer lung tissue and farcancer lung tissue(χ²(H)=14.65, P< 0.05). Through pairwise comparisons, there were significant difference between lung cancer tissue and paracancer lung tissue,cancer tissue and farcancer lung tissue, but there were no any significant difference between paracancer lung tissue and farcancer lung tissue. 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(χ²(H)= 1.446, P>0.05; F=0.976, P> 0.05;F=3.082,P>0.05).3. Western Blot Analyzed NOXA protein In the 44 cases of primary lung cancer tissues, the medians of the relative rate of NOXA protein and GAPDH protein in lung cancer tissue, paracancer lung tissue and farcancer lung tissue were 0.77(0.40～1.59); 1.49 (0.79～2.69);1.40 (0.75～1.98) The data were evaluated by analysis of ranks test, here were significant difference among lung cancer tissue,paracancer lung tissue,farcancer lung tissue and benign lesions tissue(χ²(H)=23.211, P<0.05). Through pairwise comparisons, there were significant difference among lung cancer tissue and paracancer lung tissue,farcancer lung tissue,benign lesions tissue(F=9.064, P<0.05) but there were no any significant difference among paracancer lung tissue,farcancer lung tissue and benign lesions tissue. 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(χ²(H)=1.137, P>0.05;χ²(H)=0.000, P>0.05;χ²(H)=1.054, P>0.05).4. 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; also farcancer lung tissue and lung benign disease tissue. there was no any significant difference among paracancer lung tissue and farcancer lung tissue.5. The results of TUNEL 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(Z=-3.469, P=0.001).6 The relation of noxa and p53 noxa 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 noxa. Conclusions Our study showed that noxa mRNA, protein expression in lung cancer is reduced,it maybe did play a role in NSCLC initiation and progression of cancer, but there was not relationship between noxa mRNA and protein with different pathological types of NSCLC; NOXA and P53 of no correlation in NSCLC, suggesting that NOXA expression was not mediates by P53, which may be the result of high mutation. lung cancer tissue is increased in apoptosis, is not the result of NOXA expression, which may be caused by other mechanisms for DNA damage. In addition, This findings are inconsistent with in other tumor organization findings, explained that noxa in the tumor occurrence development, different organizations have different performance, and its occurrence in tumor development remains to be further research, whether as tumor diagnosis and treatment have yet to be a new target for further study.