LncRNA H19的表达异常影响垂体腺瘤的细胞表型.pdf

山东第一医科大学（山东省医学科学院）学报 2023年10月第44卷第10期Journal of Shandong First Medical University&Shandong Academy of Medical Sciences，October 2023，Vol.44 No.10LncRNA H19的表达异常影响垂体腺瘤的细胞表型王洋洋1 宋兆鹏1 曾成龙1 宋涛21.山东第一医科大学（山东省医学科学院）研究生部，山东 济南 250117；2.山东第一医科大学附属山东省立医院神经外科，山东 济南 250021摘要：目的目的以长链非编码 RNA（long noncoding RNA，LncRNA）H19 为着力点，通过研究其对垂体腺瘤（pituitary adenoma，PA）细胞表型的影响，寻求解决临床中因PA侵袭性生长，导致其治疗困难的新途径。方法方法采用实时荧光定量聚合酶链式反应（quantitative realtime PCR，qRTPCR）检测H19在PA组织中的表达水平。对HP75细胞系进行细胞培养，并构建高表达H19的HP75细胞模型，采用qRT-PCR检测其转染效率。然后分别采用细胞计数试剂盒8（CCK8）法、流式细胞术和Transwell法，检测H19对HP75细胞增殖、凋亡、迁移、侵袭等功能的影响。结果结果与正常组织相比，lncRNA H19在PA组织中表达下调，差异具有统计学意义（P 0.001）。oeH19组（转染H19质粒的HP75细胞）lncRNA H19表达量大于oenc组（未进行转染处理的HP75细胞），差异具有统计学意义（P 0.001），细胞模型构筑成功。CCK-8法中，oenc组具有更高的细胞增殖活性，差异具有统计学意义（P 0.001）。流式细胞术检测，oeH19组凋亡细胞率高于oenc组，差异具有统计学意义（P 0.001）。Transwell小室实验中，oeH19组Transwell小室上室底膜下表面细胞数，在迁移实验及侵袭实验中均低于oenc组，差异具有统计学意义（P 0.001）。结论结论H19在垂体腺瘤组织中表达下调，高表达的H19可抑制垂体腺瘤细胞的增殖、迁移和侵袭，促进其凋亡。关键词：Lnc RNA H19；垂体腺瘤；细胞表型doi：10.3969/j.issn.2097-0005.2023.10.004Effect of aberrant expression of lncrna H19 on the cellular phenotype of pituitary adenomasWANG Yangyang1，SONG Zhaopeng1，ZENG Chenglong1，SONG Tao21.School of Graduate studies，Shandong First Medical University&Shandong Academy of Medical Science，Jinan 250117，China；2.Department of Neurosurgery，Shandong Provincial Hospital Affiliated to Shandong First Medical University，Jinan 250021，ChinaAbstract：Objective：To seek a new way to solve the treatment difficulties caused by aggressive growth of PA in the clinic by studying its impact on the cell phenotype in Pituitary Adcnoma（PA），using lncRNA H19 as the focus in the study.Methods：In this study，Quantitative Real-time PCR（qRT-PCR）was used to detect the down-regulated expression of H19 in PA tissues.HP75 cell lines were purchased for cell culture，and HP75 cell models with high expression of H19 were constructed，and the transfection efficiency was detected by qRT-PCR.Then，the effects of H19 on the proliferation，apoptosis，migration and invasion of HP75 cells were detected by cell counting Kit 8（CCK-8），flow cytometry and Transwell method respectively.Results：The results showed that lncRNA H19 was down-regulated in PA tissues（P 0.001）and the difference was statistically significant.The expression level of lncRNA H19 in oe-H19 group（HP75 cells transfected with H19 plasmid）was higher than that in oe-nc group（HP75 cells not transfected with H19 plasmid），and the difference was statistically significant（P 0.001），indicating that the cell model was successfully constructed.By CCK-8 method，the oe-nc group showed higher cell proliferation activity（P 0.001），and the difference was statistically significant.Flow cytometry showed that the rate of apoptotic cells in the oe-H19 group was higher than that in the oe-nc group（P 0.001）.In the Transwell chamber test，the number of cells on the lower surface of the upper compartment membrane of the Transwell chamber in the oe-H19 group was lower than that in the oe-nc group in both the migration test and invasion test（P 0.05。正态分布数据用均数标准差表示。两组间比较采用独立样本t检验，方差不齐时采用t检验。检验水准=0.05。2结果2.1PA中H19的表达异常qRT-PCR检测结果显示，PA组织中H19的表达量低于正常垂体组织（表1），差异具有统计学意义（P 0.001）。2.2H19过表达影响体外PA细胞的细胞表型采用qRT-PCR检测高表达H19的PA细胞系的转染效率（表 2），oe-H19 组 H19 表达量高于 oe-nc组，差异具有统计学意义（P 0.001）。用CCK-8来检测高表达H19对PA细胞增殖的影响，转染后的HP75细胞活力明显降低，取培养96 h的2组数据，oe-nc组吸光值高于oe-H19组，差异具有统计学意义（P 0.001）。详见表3。采用流式细胞检测评价高表达H19对PA细胞凋亡的影响，oe-H19组凋亡细胞率明显高于oe-nc组，差异具有统计学意义（P 0.001）。详见表3。通过transwell小室研究H19过表达对PA细胞迁移和侵袭的影响。实验发现oe-nc组transwell小室膜下表面细胞数明显多于oe-H19组，差异具有统计学意义（P 0.001）。详见表3。3讨论PA是常见的中枢神经系统肿瘤之一，其发病率和预后不良程度逐年增加12。中枢神经系统疾病往往受其精密复杂的结构和功能影响，其诊断和治疗一直以来都是临床难点 13。靶向分子治疗作为近表1H19在PA组织与正常组织中的表达组别PA（n=47）正常组织（n=25）H19表达量0.260 0.1441.000 0.273t12.408P 0.001注：PA为垂体腺瘤。表2H19在转染的H75细胞中表达上调组别oe-H19（n=9）oe-nc（n=9）H19表达量2.795 0.2171.000 0.065t22.778P 0.001注：oe-H19为转染H19质粒的HP75细胞；oe-nc为未转染质粒的HP75细胞。表3高表达H19对PA细胞增殖、凋亡、迁移及侵袭能力的影响组别oe-H19（n=9）oe-nc（n=9）t/tPCCK-8（96 h）0.639 0.0310.876 0.02717.2990.001流式细胞测定（%）24.57 1.997.47 1.1622.2740.001迁移细胞数52.43 10.68251.27 27.7120.0870.001侵袭细胞数37.34 3.78175.24 8.8942.8270.001注：PA为垂体腺瘤。oe-H19为转染H19质粒的HP75细胞；oe-nc为未转染质粒的HP75细胞。741山东第一医科大学（山东省医学科学院）学报 2023年10月第44卷第10期Journal of Shandong First Medical University&Shandong Academy of Medical Sciences，October 2023，Vol.44 No.10年来新兴的治疗方法，在多种疾病（包括肿瘤）中发挥了重要作用14。LncRNA对PA的调控作用近年来已被大量报道。例如，LncRNA LINC01116通过调节miR-744-5p/HOXB8通路来促进PA的进展15；LncRNA LINC00473 通过介导 miR-502-3p/KMT5A轴，参与PA细胞的发育16。研究表明，LncRNA H19的异常表达在神经胶质瘤17、颅内动脉瘤18、神经母细胞瘤19等肿瘤的发生发展中起着重要作用。同时，LncRNA H19的异常表达也通过调控通路的关键酶基因，参与中枢神经系统疾病的发病机制20。这表明LncRNA H19可以成为中枢神经系统肿瘤的一个很有前途的治疗靶点和生物标志物。本研究发现，LncRNA H19在PA中的表达水平较低，这与之前的报道相同10。通过细胞转染、流式细胞测定、CCK-8等方法，证实H19的过表达影响了PA细胞的增殖、迁移、侵袭和凋亡等细胞表型，这揭示了H19在 PA 的发生发展中的作用。本研究探索了LncRNA H19作为PA靶向治疗部位的可能性，为PA的治疗提供了新的方向。利益冲突 所有作者均声明不存在利益冲突参考文献：1Beylerli OA.Diagnosis and treatment of pituitary adenomas J.Creative surgery and oncology，2020，9（4）：311.2Zhang R，Yang F，Fan H，et al.Long non-coding RNA 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