Cadherin 1 Expression And Aberrant Methylation In Lung Cancer.

Objective This study was to explore the correlation of E-cadherin 1(CDH1)expression and aberrant methylation to tumorigenesis and development of lung cancer.Methods The semiquantitative real-time MSP assay.was developed to detect CDH1 aberrant methylation.The nested RT-PCR assay was established was to examine the CDH1 mRNA.We investigated lung cancer tissues,para-cancer lung tissues,far-cancer lung tissues and 5 control cases of non-cancer lung tissues for aberrant methylation,mRNA and protein expression of E-cadherin 1 from 50 patients with primary lung tumors by quantitative fluorogenic real-time PCR,nested RT-PCR and Western Blot Analysis.The immunohistochemical method was used to confirm some sample with negative results of Western Blot Analysis.Lung tissue from 5 noncancer patients was as a control.Results1.By developing the conditions of semi-quantitative real-time MSP,we showed that the optimized reaction mixtures contained the forward and reverse primers at 600 nmol/L each;200μmol/L each of dATP,dCTP and dGTP;400μmol/L dUTP;3.0mmol/L MgCl₂; 2.5%DMSO;1×SYBGREENI;1×PCR buffer;hot start Taq DNA polymerase 0.04U/μl; templates 3μl;he total reaction volume was 25μl.The semi-quantitative real-time MSP was performed under the following conditions:95℃for 12 minutes,followed by 2 cycles of 95℃for 15 seconds,62℃for 1 minute and 69℃for 15 seconds;3 cycles of 95℃for 15 seconds,60℃for 1 minute and 69℃for 15 seconds;and 45 cycles of 95℃for 15 seconds, 58℃for 1 minute and 69℃for 15 seconds.2.The medians of the relative rate of CDH1 aberrant methylation lung cancer tissue, paracancer lung tissue,farcancer lung tissue and non-cancer lung tissues were 0.2723(0.0013～4.5067),0.2268(0.0000～0.7545),0.1191(0.0000～1.4516)and 0.0000 (0.0000～0.0007)which detected by semi-quantitative real-time MSP.The data were evaluated by rank sum test.There were significant different among lung cancer tissue, paracancer lung tissue,farcancer lung tissue and non-cancer lung tissues(P＜0.01).Also,there was no significant different among squmaous cell carcinoma,adenocarcinoma and adenosquamous carcinoma in the group of lung cancer tissue,paracancer lung tissue and farcancer lung tissue.3.By exploring the conditions of nested RT-PCR,we found that the optimized nested RT-PCR reaction mixtures contained the forward and reverse primers at 600 nmol/L each; 200μmol/L each of dNTP;1.5mmol/L MgCl₂;1×PCR buffer;Taq DNA polymerase 0.1U/μl; templates 50ng;the total reaction volume was 25μl.The first round PCR of RT-PCR was performed under the following conditions:95℃for 12 minutes,followed by 25 cycles of 95℃for 1 minute,66℃for 1 minute and 72℃for 1 minute.But it was 25 cycles for the second round PCR of RT-PCR.4.The means of the relative rate of E-cadherin mRNA in lung cancer tissue,paracancer lung tissue,farcancer lung tissue and non-cancer lung tissues were 1.33±0.53,1.65±0.60, 2.01±0.46 and 2.09±0.09 which detected by nested RT-PCR.The data were evaluated by analysis of variance.There were significant different among lung cancer tissue,paracancer lung tissue and farcancer lung tissue(F=18.810,P＜0.01),but there was no significant difference between farcancer tissues and non-cancer lung tissues(P＞0.05).Also,there was no significant different among squmaous cell carcinoma,adenocarcinoma and adenosquamous carcinoma in the group of lung cancer tissue,paracancer lung tissue and farcancer lung tissue.5.The positive expression rate of E-cadherin mRNA in lung cancer tissue,paracancer lung tissue,farcancer lung tissue and non-cancer lung tissues were 38.0%(19/50), 70.0%(35/50)and 96.0%(48/50)which detected by Western Blot Analysis.The data were evaluated by chi-square test.There were significant different among lung cancer tissue, paracancer lung tissue and farcancer lung tissue(x²=38.79,P=0.000,＜0.05).Also,there was no significant different among squmaous cell carcinoma,adenocarcinoma and adenosquamous carcinoma in the group of lung cancer tissue,paracancer lung tissue and farcancer lung tissue.6.Using immunohistochemistry staining to confirm the results of 5 samples with loss of E-cadherin in lung cancer tissue in Western Blot Analysis.The results were as same as those in Western Blot Analysis. Conclusions Our study showed that E-cadherin 1 aberrant methylation and protein reduced expression would be an important role in tumorigenesis and development of lung cancer,and no evident relationship of E-cad mRNA among different pathological types of lung cancer.Our study also suggested that E-cadherin 1 aberrant methylation would restrain the transcription of CDH1 mRNA.