摘要
目的研究长链非编码RNA DNA损伤诱导的非编码RNA(NORAD)在肺癌中的表达及其对肺癌细胞增殖的影响。方法RNA-seq分析肺癌组织中LncRNA及miRNA的表达改变,根据表达量确定候选LncRNA;q RT-PCR检测分析肺癌组织及肺癌细胞中候选NORAD的表达;免疫荧光检测分析肺癌组织中细胞增殖核抗原Ki-67的表达;通过线性回归分析肺癌组织中NORAD与Ki-67的表达相关性。生物在线软件联合miRNA测序结果,并通过荧光素酶检测分析NORAD与miR-26b-5p、miR-654-5p结合情况。将A549细胞分成空白对照组(Control)、si-NORAD组、miR-26b-5p mimics、miR-654-5p mimics、si-NORAD与miR-26b-5p mimics联合组、si-NORAD与miR-654-5p mimics联合组,利用EdU检测细胞增殖能力、Annexin V-FITC/PI双染检测细胞凋亡、蛋白印迹(Western blot)检测细胞凋亡相关蛋白(Bad、Bcl-2、Cleaved Caspase-3)的表达。结果NORAD在肺癌组织及肺癌细胞中的表达均显著升高(均P<0.01),其表达量与Ki-67的表达呈正相关(r=0.646,P<0.01)。miR-26b-5p、miR-654-5p在肺癌组织及细胞中的表达均显著降低(均P<0.01),生物在线软件及荧光素酶检测验证,NORAD与miR-26b-5p、miR-654-5p存在结合位点;在肺癌组织中,miR-26b-5p(r=-0.403,P=0.000)、miR-654-5p(r=-0.423,P=0.000)的表达与Ki-67的表达均呈负相关;miR-26b-5p(r=-0.435,P<0.05)与miR-654-5p(r=-0.395,P<0.05)的表达与NORAD表达均呈负相关。与Control组比较,si-NORAD可显著抑制A549细胞的增殖(P<0.01)、促进其凋亡(P<0.01),Bad及Cleaved caspase-3的表达均显著升高(均P<0.01),Bcl-2的表达显著降低(P<0.05);与si-NORAD组比较,si-NORAD与miR-26b-5p mimics联合组、si-NORAD与miR-654-5p mimics联合组可进一步抑制细胞增殖(P<0.01),促进细胞凋亡(P<0.01)。结论肺癌中NORAD的表达显著上调,可能通过抑制mi R-26b-5p、mi R-654-5p的表达而促进肺癌细胞增殖,NORAD可能作为肺癌的诊断标志物。
Objective To investigate the expression of long non-coding RNA DNA damage induced non-coding RNA(NORAD)in lung cancer and its effect on the proliferation of lung cancer cells.Methods The expression changes of LncRNA and miRNA in lung cancer tissues were analyzed by RNA-seq,and candidate LncRNA were identified according to the expression levels.qRT-PCR was used to detect the expression of candidate NORAD in lung cancer tissues and lung cancer cells.Immunofluorescence assay was used to detect the expression of Ki-67 in lung cancer tissues.The correlation between NORAD and Ki-67 expression in lung cancer tissues was analyzed by linear regression.The results of miRNA sequencing were combined with biological online software,and the possibility of NORAD binding to miR-26b-5p and miR-654-5p was analyzed by lucifase detection.A549 cells were divided into blank control group(Control),si-NORAD group,miR-26b-5p mimics,miR-654-5p mimics,si-NORAD and miR-26b-5p mimics combined group,si-NORAD and miR-654-5p mimics combined group.Cell proliferation was detected by EdU,apoptosis was detected by Annexin V-FITC/PI double staining,and expression of apoptosis-related proteins(Bad,Bcl-2,Cleaved caspase-3)was detected by Western blot.Results The expression of NORAD in lung cancer tissues and lung cancer cells was significantly increased(all P<0.01),and the expression level of NORAD was positively correlated with the expression of Ki-67(r=0.646,P<0.01).The expressions of miR-26b-5p and miR-654-5p in lung cancer tissues and cells were significantly decreased(all P<0.01).Biological online software and luciferase detection verified that NORAD had binding sites with miR-26b-5p and miR-654-5p.In lung cancer tissues,the expression of miR-26b-5p(r=-0.403,P<0.01)and miR-654-5p(r=-0.423,P<0.01)was negatively correlated with the expression of Ki-67.The expression levels of miR-26b-5p(r=-0.435,P<0.05)and miR-654-5p(r=-0.395,P<0.05)were negatively correlated with NORAD.Compared with the Control group,si-NORAD significantly inhibited the proliferat
作者
王靖
杨利杰
宋政
王瑞宇
张钰璐
李俊
WANG Jing;YANG Li-jie;SONG Zheng;WANG Rui-yu;ZHANG Yu-lu;LI Jun(College of Clinical Medicine,Dali University,Dali 671000,Yunnan,CHINA;Cardio-Thoracic Surgery,the First Affiliated Hospital of Dali University,Dali 671000,Yunnan,CHINA;College of Public Health,Dali University,Dali 671000,Yunnan,CHINA)
出处
《海南医学》
CAS
2023年第9期1217-1224,共8页
Hainan Medical Journal
基金
大理大学博士科研启动基金项目(编号:KYBS2018018)。
