SMAD基因家族PCR引物设计及其在非小细胞肺癌中的表达  

Design of PCR primers for SMAD gene family and its expression in non-small-cell lung cancer

作　　者：林高阳 高大登 袁方 刘振波 LIN Gaoyang;GAO Dadeng;YUAN Fang;LIU Zhenbo(Qingdao Hiser Medical Group Affiliated to Qingdao University,Qingdao 266033,China)

机构地区：[1]青岛大学附属青岛市海慈医疗集团,山东青岛266033

出　　处：《山东医药》2018年第24期26-30,共5页Shandong Medical Journal

摘　　要：目的自行设计并验证SMAD基因家族PCR引物,探讨应用上述引物进行RT-PCR检测SMAD基因在非小细胞肺癌中表达的可行性。方法自主设计并验证SMAD基因家族SMAD1~SMAD8特异引物,确定SMAD1-1、SMAD2-1、SMAD3-1、SMAD4-2、SMAD5-1、SMAD6-1、SMAD7-1、SMAD8-2引物的扩增效率更高、特异性更强。采用RT-PCR技术检测SMAD1~SMAD8在人正常支气管上皮细胞系BEAS-2B,肺腺癌细胞系A549、H1299,肺鳞癌细胞系SK-MES-1中的表达变化,引物为SMAD1-1、SMAD2-1、SMAD3-1、SMAD4-2、SMAD5-1、SMAD6-1、SMAD7-1、SMAD8-2。结果以正常支气管上皮BEAS-2B细胞中SMAD1表达量为基准(校正RQ约为1),SMAD2相对表达量为6.590±0.459、SMAD3为10.977±0.312、SMAD4为4.651±0.115、SMAD5为16.762±0.389、SMAD6为3.248±0.108、SMAD7为2.007±0.012、SMAD8为1.144±0.103,均较SMAD1表达升高,其中SMAD5表达最高,SMAD3次之。SMAD2、SMAD4、SMAD5、SMAD6、SMAD7、SMAD8在肺腺癌A549、H1299细胞及肺鳞癌SK-MES-1细胞中的表达均低于人正常支气管上皮BEAS-2B细胞;在H1299、SK-MES-1细胞中SMAD1表达高于BEAS-2B和A549细胞,且在A549细胞中SMAD1表达低于BEAS-2B细胞;在SK-MES-1细胞中SMAD3表达升高,且在H1299、A549细胞中SMAD3表达较BEAS-2B细胞下降;在H1299细胞中SMAD2、SMAD6、SMAD7表较其他三种细胞均下降;各细胞间比较P<0.05或<0.01。结论成功自主设计SMAD基因家族特异引物,并用于检测非小细胞肺癌中SMAD基因表达;SMAD5可能参与了肺癌的发生、发展,SMAD3可能与上皮相关性疾病相关。Objective To design and verify the PCR primers for SMAD gene family independently,and to explore the feasibility of using these primers for detection of SMAD gene expression in non-small-cell lung cancer(NSCLC)by RT-PCR.Methods We independently designed and verified SMAD1-8 specific primers for the SMAD gene family,and identified that the amplification efficiency of SMAD1-1,SMAD2-1,SMAD3-1,SMAD4,SMAD5-1,SMAD6-1,SMAD7-1,and SMAD8-2 primers was higher and their specificity was stronger.The expression of SMAD1-8 in the normal bronchial epithelial cell line BEAS-2B,lung adenocarcinoma A549,H1299 cells,and lung squamous-cell carcinoma SK-MES-1 cells was detected by RT-PCR.The primers were SMAD1-1,SMAD2-1,SMAD3-1,SMAD4-2,SMAD5-1,SMAD6-1,SMAD7-1,and SMAD8-2,respectively.Results The expression of SMAD1 in the normal bronchial epithelial BEAS-2B cells was taken as the baseline[the revised relative expression(RQ)is 1],the relative expression of SMAD2 was 6.590±0.459,SMAD3 was 10.977±0.312,SMAD4 was 4.651±0.115,SMAD5 was 16.762±0.389,SMAD6 was 3.248±0.108,SMAD7 was 2.007±0.012,and SMAD8 was 1.144±0.103,all were higher than SMAD1 expression,and SMAD5 expression was the highest,followed by SMAD3 expression.The expression of SMAD2,SMAD4,SMAD5,SMAD6,SMAD7 and SMAD8 in the lung adenocarcinoma A549,H1299 cells and squamous-cell lung carcinoma SK-MES-1 cells was lower than that of the normal bronchial epithelial cell line BEAS-2B.The expression of SMAD1 in the H1299 and SK-MES-1 cells was higher than that in the BEAS-2B and A549 cells,and the expression of SMAD1 in the A549 cell was lower than that in the BEAS-2B cells.SMAD3 expression increased in the SK-MES-1 cells,and decreased in the H1299 and A549 cells as compared with that in the BEAS-2B cells.In H1299 cells,the expression of SMAD2,SMAD6 and SMAD7 were lower than that in the other three kinds of cells;significant difference was found between every two kinds of cells(P<0.05 or P<0.01).Conclusions The specific primers for SMAD gene family are successfully designed and

关 键 词：非小细胞肺癌 聚合酶链式反应 果蝇母源抗生物皮肤生长因子基因 

分 类 号：R734.2[医药卫生—肿瘤]