长链非编码RNA细胞周期依赖性激酶抑制基因2B-AS1与特发性肺纤维化合并肺癌的关系探讨  

To investigate of relationship between long non-coding RNA CDKN2B-AS1 and idiopathic pulmonary fibrosis coexisting with lung cancer

作　　者：罗潇[1] 刘学军[1] 杜毓锋[1] Luo Xiao;Liu Xuejun;Du Yufeng(Department of Geriatrics,the First Hospital of Shanxi Medical University iTaiyuan 030001,China)

机构地区：[1]山西医科大学第一医院老年病科,太原030001

出　　处：《中华老年医学杂志》2020年第8期905-909,共5页Chinese Journal of Geriatrics

摘　　要：目的探讨长链非编码RNA细胞周期依赖性激酶抑制基因(CDKN)2B-AS1及其邻近基因CDKN2A在特发性肺纤维化(IPF)合并肺癌中的作用及机制。方法收集8例肺腺癌患者手术中切除的少许癌组织标本及癌旁的正常肺组织标本,实时定量反转录聚合酶链反应(RT-qPCR)检测CDKN2B-AS1及其邻近基因CDKN2A的含量。选择人肺成纤维细胞系MRC-5为研究对象,将细胞分为正常组、干预组[转化生长因子β1(TGF-β1)干预]、阴性siRNA干预组(TGF-β1干预,转染无义序列小干扰RNA)和阳性siRNA干预组(TGF-β1干预,转染CDKN2B-AS1的小干扰RNA)。干预24 h后倒置显微镜下观察细胞形态变化,RT-qPCR方法检测CDKN2B-AS1及CDKN2A的表达,蛋白质印迹法检测CDKN2A和P53蛋白的含量。结果与对照组21.90±19.83、19.83±7.67比较,肺癌组织中CDKN2B-AS1及CDKN2A均低表达,分别为2.60±1.33和0.34±0.10(t=2.747、7.187,P=0.016、0.000),与在IPF中的结果一致。在细胞试验中,观察到TGF-β1的干预可促使MRC-5肺成纤维细胞从多突的纺锤形或星形结构,逐渐转化为扁平状纤维结构的肌成纤维细胞,并出现不同程度的分化;与正常组56.12±2.46、64.54±3.89比较,TGF-β1干预后CDKN2B-AS1及CDKN2A mRNA的表达均明显减少,分别为6.80±0.30和9.39±0.37(t=47.746、33.797,均P<0.001);转染CDKN2B-AS1的siRNA后,与干预组6.80±0.30、9.39±0.37比较,CDKN2B-AS1及CDKN2A的表达进一步下调,分别为2.38±0.29、2.81±0.36(t=4.279、4.032,P=0.003、0.004),且两者表达量呈正相关(r=0.988,P=0.000);此外,蛋白水平干预组CDKN2A及P53的表达3.12±0.06、1.12±0.07,较正常组4.12±0.59、2.11±0.06均减少,差异有统计学意义(t=2.921、19.599,P=0.043、0.000),且两者表达量呈正相关(r=0.772,P=0.000)。结论长链非编码RNA CDKN2B-AS1在IPF和肺癌中均低表达,通过调节其邻近基因CDKN2A的表达,参与了P53通路,可能是IPF患者肺癌高发的原因之一。Objective To investigate the role and its mechanism of long non-coding RNA cell cyclin-dependent kinase inhibitor gene CDKN2B-AS1 and its adjacent gene CDKN2A in idiopathic pulmonary fibrosis coexisting with lung cancer.Methods The cancerous lung tissue specimens and adjacent normal lung tissue specimens were collected from 8 patients with lung adenocarcinoma.The expression level of CDKN2B-AS1 and CDKN2A mRNA were determined by reverse transcription quantitative real-time PCR(RT-qPCR)assays.Pulmonary fibrosis cell model was established by treating human fetal lung fibroblast MRC-5 cell line that was selected as the study object.And the cells were randomly divided into 4 groups:the normal group,the intervention group[induced by transforming growth factou-β(TGF-β1)],the negative siRNA intervention groupCinduced by TGF-β1 and transfected by nonsense sequence siRNA)and the positive siRNA intervention group TGF-β1 and transfected by siRNA of CDKN2B-AS1).The morphological changes of each group were observed by using the inverted phase contrast microscope after 24 hours intervention.The expression levels of CDKN2B-AS1 and CDKN2A mRNA were determined by RT-qPCR,and the protein amounts of CDKN2A and P53 were measured by Western blotting.Results The expression levels of CDKN2B-ASl and CDKN2A mRNA were lower in lung cancer tissue than in adjacent normal lung tissues(2.60±1.3321.90±19.83,0.34±0.1019.83±7.67,t=2.747 and 7.187,P<0.05),which were consistent with the results in IPF.In cell experiments>we observed that TGF-β1 intervention gradually transformed MRC-5 cells from multi-spindled or stellate structures to flattened fibroblasts with varying degrees of differentiation.Compared with the normal group,TGF-β1 intervention group showed that the expression levels of CDKN2B-AS1 and CDKN2A mRNA were significantly decreased(6.80±0.30m.56.12±2.46,9.39±0.37,t=47.54±3.89,z=47.746 and 33.797,both P<0.001).Compared with the intervention group,the expression levels of CDKN2B-AS1 and CDKN2A mRNA were further decreased in

关 键 词：肺纤维化 肺肿瘤 细胞周期蛋白质依赖激酶类 

分 类 号：R734.2[医药卫生—肿瘤] R563.9[医药卫生—临床医学]