肺癌放疗抵抗相关长链非编码RNA差异表达谱的筛选及功能研究  

Screening and functional analysis of differential expression profiles of long non-coding RNA related to radiotherapy resistance in lung cancer

作　　者：刘玉凤 凌春华[2] 殷红[3] 黄锦宏 Liu Yufeng;Ling Chunhua;Yin Hong;Huang Jinhong(Department of Pulmonary and Critical Care Medicine,Suzhou Ninth People’s Hospital,Suzhou 215200,China;Department of Pulmonary and Critical Care Medicine,the First Affiliated Hospital of Soochow University,Suzhou 215006,China;Department of Oncology,the First Affiliated Hospital of Soochow University,Suzhou 215006,China;Department of Respiratory Diseases,the Affiliated Changshu Hospital of Xuzhou Medical University,Suzhou 215500,China)

机构地区：[1]苏州市第九人民医院呼吸与危重症医学科,苏州215200 [2]苏州大学附属第一人民医院呼吸与危重症医学科,苏州215006 [3]苏州大学附属第一人民医院肿瘤内科,苏州215006 [4]徐州医科大学附属常熟市第二人民医院呼吸与危重症医学科,苏州215500

出　　处：《中华实验外科杂志》2023年第8期1484-1487,共4页Chinese Journal of Experimental Surgery

基　　金：江苏省高等学校自然科学研究重大项目(20KJA310004);常熟市科技发展计划项目(CS201502);苏州市吴江区科教兴卫项目(WWK201907)。

摘　　要：目的探讨长链非编码RNA(lncRNA)在肺癌放疗抵抗中的作用, 并观察lncRNA微小RNA(miR)-4453对肺癌放疗抵抗的影响。方法 H460细胞(大细胞肺癌细胞)购自上海中国科学院细胞研究所。应用梯度增加X射线剂量法对H460细胞反复照射, 剂量分别为前3次4 Gy, 中4次6 Gy, 后3次8 Gy, 累积剂量60 Gy。照射完成后放疗抵抗株命名为H460R细胞。通过高通量测序, 筛选出亲本株H460细胞与抵抗株H460R细胞差异表达的lncRNAs。利用实时荧光定量聚合酶链反应(qRT-PCR)技术, 对H460细胞与H460R细胞差异表达的lncRNAs和mRNAs进行验证。对放疗抵抗细胞株H460R细胞分别转染干扰序列[小干扰RNA(siRNA)-miR-4453]和对照序列(siRNA-NC)。采用Transwell法分析H460细胞、H460R细胞、siRNA-NC细胞和siRNA-miR-4453细胞的迁移能力。采用流式细胞术分析siRNA-NC细胞和siRNA-miR-4453细胞凋亡比例。利用细胞计数试剂盒(CCK-8)实验检测H460细胞、H460R细胞、siRNA-NC细胞和siRNA-miR-4453细胞放疗剂量的半数抑制浓度(IC50)值。组间比较采用t检验。结果放疗抵抗株H460R细胞的IC50值高于对照组H460细胞的IC50值[(28.580±4.181) Gy比(7.788±0.352) Gy, t=8.58, P<0.05]。H460R细胞株穿过Transwell膜的细胞数量高于对照组H460细胞的数量[(532.700±21.060)个比(220.000±14.150)个, t=11.09, P<0.05]。高通量测序结果表明, 在亲本H460和放疗抵抗株460R细胞中共检测到71个差异表达的lncRNAs和1461个差异表达的mRNAs。对H460R细胞敲除miR-4453后, 检测miR-4453在siRNA-NC对照组中的表达水平明显高于siRNA-miR-4453组[(0.032±0.008)比(0.001±0.000), t=6.860, P<0.05]。对照组siRNA-NC细胞的IC50值高于干扰组siRNA-miR-4453细胞的IC50值[(29.240±1.640) Gy比(16.503±1.713) Gy, t=9.30, P<0.05]。对照组siRNA-NC细胞的迁移能力高于干扰组siRNA-miR-4453细胞的迁移能力[(611.700±26.850)个比(317.700±11.860)个, t=19.54, P<0.05]。对照组siRNA-NC细胞的凋亡低Objective To investigate the role of long non-coding RNA(lncRNA)in radiation resistance of lung cancer,and observe the effect of lncRNA microRNA(miR)-4453 on radiotherapy resistance of lung cancer.Methods H460(large cell lung cancer cell)was purchased from the Shanghai Institute of Chinese Academy of Sciences Research.Establishment of radioresistant cells H460R.H460 cells were irradiated 3 times with the dose of 4 Gy,then 4 times with the dose of 6 Gy and 3 times with the dose of 8 Gy,and the cumulative dose was 60 Gy.The irradiated strains were named H460R.We performed high-throughput RNA sequencing to find the differences lncRNAs and mRNAs expression transwell assayprofiles between parent H460 and H460R cells.Quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to validate the differential lncRNAs and mRNAs expression.The cell line H460R was transfected with interference sequence[small interfering RNA(siRNA)-miR-4453]and control sequence(siRNA-NC),respectively.The migration ability of H460 cells,H460R cells,siRNA-NC cells and siRNA-miR-4453 cells was analyzed by Transwell method.The apoptosis rates of siRNA-NC cells and siRNA-miR-4453 cells were analyzed by flow cytometry analysis.Cell counting kit-8(CCK-8)assay was used to decete 50%inhibiting concentraion(IC50)of radiation dose of H460 cells,H460R cells,siRNA-NC cells and siRNA-miR-4453 cells.T test was used to compare the data between groups.Results The IC50 of radiation dose in radioresistant cell sublines H460R was higher than in parent cell sublines H460[(28.580±4.181)Gy vs.(7.788±0.352)Gy,t=8.59,P<0.05].Transwell assay showed that the number of cells penetrating the membrance in radioresistant cell sublines H460R was more than that in parent cell sublines H460[(532.700±21.060)vs.(220.000±14.150),t=11.09,P<0.05].The high-throughput RNA sequencing results showed that a total number of 71 differentially expressed lncRNAs and 1461 differentially expressed mRNAs were identified between parent H460 and 460R cells.After knocked out miR-445

关 键 词：肺癌 放疗抵抗 高通量测序 长链非编码RNA 微小RNA-4453 

分 类 号：R734.2[医药卫生—肿瘤]