| Introduction14-3-3σwhich is one of 14-3-3 proteins series and has direct relation to human tumor always expresses abnormally in human carcinomas in respects with gene and protein. Its expression in different lung cancer is special,such as its expression in squamous cell caicinoma is higher than that in adenocarcinoma,but little or even no in lung neuroendocrine tumors. hASHl gene which is in short of human achaete-scute homologue 1 is one of bHLH gene series which play important roles in the development of embryo and tumors. In recent it has been identified that the expression of hASH1 is very high in lung neuroendocrine tumors but extremely low in lung noneuroendocrine tumors.14-3-3a gene in breast cancer, ovarian cancer, endometrial cancer, prostate cancer, skin cancer and liver cancer is expressed lowly and even silenced which is induced by the methylation of promoter.The lack of 14-3-3σprotein which has relation to G2 checkpoint in cell cycle will induce malignance by promoting mitosis. The hypermethylation of promoter of 14-3-3a gene in lung neuroendocrine tumors is sspecial. hASH1 is a methyltransferase which can keep genes silenced by transporting methyl to the site of 4,9,20,27 and 36 of lysine of histone 3. Our investigation puts emphasis on the correlation between 14-3-3aand hASH1 in lung cancer cell lines and the relation of 14-3-3σwith clinical pathologic parameters of lung adenocarcinoma. Materials and methods一,SamplesCell lines:BE-1(gaint cell carcinoma cell), NCI-H460 (large cell undifferentiated carcinoma cell), LTE and A549(adenocarcinoma cell),NCI-H446(small cell lung cancer cell)47 fresh lung adenocarcinoma tissue samples and healthy tissue (obtained from patients who had surgery in the first Affiliated Hospital Of China Medcial University)二,ReagentThe anti-human 14-3-3σgoat polyclonal antibody (SC-7683,Santa Cruz); The anti-human hASH1 rabbit polyclonal antibody (ab-38557,abCam); Easy RT-PCR Kit(AE401, TRANS); TRIzol Reagent (15595-026, invitrogen); hAshl primer; 14-3-3σprimer, p-actin primer(JINSITE BIOTECHNOLOGY)三,Methods(一)RT-PCRTotal RNA was isolated from cells in logarithmic growth phase and fresh lung adencarcinoma tissue samples using TRIZOL.The PCR primers were as follows:14-3-3σforw 5'-TGCGAAGAGCGAAACCTGC-3'(19bp), 14-3-3σrev 5'-CTGTTGGCGATCTCGTAGTGG-3'(21bp) (the length of product is 446bp).hASHl forw 5'-TCCCCCAACTACTCCAACGAC-3'(21bp), hASH1 rev 5'-CCCTCCCAACGCCACTG-3'(17bp) (the length of product is 233bp).β-actin forw 5'-AAATCGTGCGTGACATTAA-3'(19bp),β-actin rev 5'-CTCGTCATACTCCTGCTTG-3'(19bp) (the length of product is 513bp).The temperature for RT:30℃10min,42℃40min,99℃5min,5℃5min。 The temperature for PCR(14-3-3aand hASH1):94℃5min,94℃30sec,55℃30sec, 72℃40sec,72℃10min。(二)Western BlotLung cancer cells were extracted with lysis buffer(150mMNacl,1%NP40, 0.1%SDS,2ug/ml aprotinin,1M PMSF)for 30 min at 4℃.The supernatants were centrifuged at 15000xg for 15 min at 4℃.Aliquots containing 100ug of protein were separated on 15% SDS-PAGE and transferred to PVDF membranes at 40v for 90 min at low temperature.The membranes were blocked with 5% skimmed milk.Then goat anti-human 14-3-3a polyclonal antibody(1:500 dilution), rabbit anti-human hASH1 polyclonal antibody(1:500 dilution) and anti-β-actin antibody(1:400) immunodetection was performed.The ECL Imaging System was used to catch up the bands.四,Statistical analysisALL the data are analyzed with SPSS for Windows 13.0 software. We use x2 test to analyze the correlation between clinic pathologic parameters of lung cancer and expression of 14-3-3a and hASH1,We use the permutation test for the Spearman correlation coefficient to analyze the correlation between expression of 14-3-3σand hASH1. The statistical significance is defined as P＜0.05.Result一,The expression of 14-3-3σand hASHl in five lung cancer cells(一)RT-PCRthe expression of 14-3-3σmRNA in five lung cancer cells is different and the highest is in 446 followed by LTE and A549(no expression in healthy lung tissue). The expression of hASHl mRNA is higher in 446 and 460 which are two kinds of lung neuroendocrine tumors.But perhaps there is no correlation between 14-3-3σand hASH 1 judging from their mRNA expression. (二)Western Blotthe expression of 14-3-3σprotein in five lung cancer cells is different and the highest in 446 followed by LTE and A549 (no expression with healthy lung tissue). The expression of hASH1 protein is higher in 446 and 460 which are two kinds of lung neuroendocrine tumors.But perhaps there is no relation between 14-3-3aand hASH1 judging from their protein expression.二,14-3-3σmRNA expression in 47 lung adenocarcinoma correlate with tumors differentiation,clinical stages and lymph node metastasisThe expression of 14-3-3amRNA is obviously higher in 47 lung adenocarcinoma than that in healthy lung tissue by RT-PCR detect. Furthermore, the expression of 14-3-3σmRNA in undifferentiated tumors is higher than that in differentiated tumors. As to say the relation between the expression of 14-3-3σmRNA and degree of differentiation of lung adenocarcinoma is negative.Also patients with high 14-3-3σmRNA expression were more likely than low expression patients have advanced disease (p=0.001) and lymph node metastasis(p=0.004). But there is no correlation between 14-3-3omRNA expression and patients age (p=0.496) and patients gender(p=0.765).Finally these results probably means 14-3-3σplays an important role in the development, differentiation and lymph node metastasis of lung adenocarcinoma.Conclusions14-3-3σprotein probably plays an important role in the development, differentiation and lymph node metastasis of lung adenocarcinoma.But the relation of higher hASH1 with lower 14-3-3σexisting in other lung neuroendocrine tumor cell lines does not exist in 446 cell line.
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