标题：长链非编码RNA NORAD在肺癌中的表达及其对肺癌细胞增殖能力的影响

      作者：王靖 1，杨利杰 2，宋政 2，王瑞宇 3，张钰璐 3，李俊 1    王靖 1，杨利杰 2，宋政 2，王瑞宇 3，张钰璐 3，李俊 11.大理大学临床医学院，云南 大理 671000；2.大理大学第一附属医院心胸外科，云南 大理 671000；3.大理大学公共卫生学院，云南 大理 671000

      卷次： 2023年34卷9期

      【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；qRT-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miR-26b-5p、miR-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。
      【关键词】 肺癌；长链非编码RNA；DNA损伤诱导的非编码RNA；细胞增殖；微小RNA
      【中图分类号】 R734.2 【文献标识码】 A 【文章编号】 1003—6350（2023）09—1217—08

Expression of long non-coding RNA NORAD in lung cancer and its effect on lung cancer cell proliferation.
WANGJing 1, YANG Li-jie 2, SONG Zheng 2, WANG Rui-yu 3, ZHANG Yu-lu 3, LI Jun 1. 1. College of Clinical Medicine, DaliUniversity, Dali 671000, Yunnan, CHINA; 2. Cardio-Thoracic Surgery, the First Affiliated Hospital of Dali University, Dali671000, Yunnan, CHINA; 3. College of Public Health, Dali University, Dali 671000, Yunnan, CHINA
【Abstract】 Objective To investigate the expression of long non-coding RNA DNA damage induced non-cod-ing RNA (NORAD) in lung cancer and its effect on the proliferation of lung cancer cells. Methods The expressionchanges of LncRNA and miRNA in lung cancer tissues were analyzed by RNA-seq, and candidate LncRNA were identi-fied according to the expression levels. qRT-PCR was used to detect the expression of candidate NORAD in lung cancertissues and lung cancer cells. Immunofluorescence assay was used to detect the expression of Ki-67 in lung cancer tis-sues. 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 bind-ing to miR-26b-5p and miR-654-5p was analyzed by lucifase detection. A549 cells were divided into blank controlgroup (Control), si-NORAD group, miR-26b-5p mimics, miR-654-5p mimics, si-NORAD and miR-26b-5p mimicscombined group, si-NORAD and miR-654-5p mimics combined group. Cell proliferation was detected by EdU, apopto-sis 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 lungcancer cells was significantly increased (all P<0.01), and the expression level of NORAD was positively correlated withthe expression of Ki-67 (r=0.646, P<0.01). The expressions of miR-26b-5p and miR-654-5p in lung cancer tissues andcells were significantly decreased (all P<0.01). Biological online software and luciferase detection verified that NORADhad 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 expressionlevels of miR-26b-5p (r=-0.435, P<0.05) and miR-654-5p (r=-0.395, P<0.05) were negatively correlated withNORAD. Compared with the Control group, si-NORAD significantly inhibited the proliferation of A549 cells (P<0.01)and promoted apoptosis (P<0.01), significantly increased the expression of Bad and Cleaved caspase-3 (all P<0.01),and significantly decreased the expression of Bcl-2 (P<0.05). Compared with si-NORAD group, si-NORAD combinedwith miR-26b-5p mimics group and si-NORAD combined with mi-654-5p mimics group could further inhibit cell pro-liferation (P<0.01) and promote cell apoptosis (P<0.01). Conclusion The expression of NORAD is significantlyup-regulated in lung cancer, which may promote the proliferation of lung cancer cells by inhibiting the expression ofmiR-26b-5p and miR-654-5p. NORAD may be used as a diagnostic marker of lung cancer.
      【Key words】 Lung cancer; Long non-coding RNA; NORAD; Cell proliferation; microRNA