作者：田凯兵，王亮，马骏鹏，王科，张俊廷，吴震

单位：

100070北京市，首都医科大学附属北京天坛医院神经外科

Units：

Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China

关键词：

脊索瘤；颅底；长链非编码RNA；FAM230B；细胞增殖；细胞凋亡

Keywords：

Chordoma; Skull base; Long chain noncoding RNA; FAM230B; Cell proliferation; Apoptosis

CLC：

R 730.269

DOI：

10.12114/j.issn.1008-5971.2022.00.262

Funds：

国家自然科学基金资助项目（81802683，82141113）；北京市医院管理中心创新梦工场-田凯兵青年职工创新工作室（2022-2023）；北京市优秀人才青年骨干个人项目（2018-田凯兵）

摘要：

目的　筛选颅底脊索瘤中的关键长链非编码RNA（lncRNA），并探讨其促肿瘤作用及机制。方法　选择在首都医科大学附属北京天坛医院临床医学研究中心存放的3份脊索瘤组织样本和3份脊索组织样本，进行lncRNA测序，筛选与肿瘤发生发展关系密切的lncRNA。采用笔者所在课题组自行培育的颅底脊索瘤细胞系HBC-2进行细胞培养及转染，其中FAM230B敲降组采用si-FAM230B进行细胞转染，对照1组采用si-FNC进行细胞转染，AC079630.2敲降组采用si-AC079630.2进行细胞转染，对照2组采用si-ANC进行细胞转染。采用CCK-8法检测细胞增殖能力，采用流式细胞仪检测细胞凋亡率。选择2005—2014年在首都医科大学附属北京天坛医院神经外科接受手术治疗的原发颅底脊索瘤患者83例，收集患者的临床资料。原发颅底脊索瘤患者术后肿瘤进展的影响因素分析采用单因素及多因素Cox回归分析。应用AnnoLnc数据库筛选与FAM230B相互作用的蛋白，采用RNA pull-down实验和Western blot法检测结合蛋白表达情况。结果　lncRNA测序结果显示，筛选后获取2 153个lncRNA集，包括差异表达lncRNA 169个，其中显著高表达lncRNA 11个、显著低表达lncRNA 4个。功能测序结果显示，共两个lncRNA（FAM230B和ACO79630.2）与肿瘤发生发展密切相关。FAM230B敲降组培养24、48、72 h细胞活性低于对照1组，细胞凋亡率高于对照1组（P ＜0.05）；AC079630.2敲降组和对照2组培养24、48、72 h细胞活性及细胞凋亡率比较，差异无统计学意义（P ＞0.05）。多因素Cox回归分析结果显示，FAM230B水平是原发颅底脊索瘤患者术后肿瘤进展的独立影响因素〔HR =0.749，95%CI（0.616，0.912），P ＜0.05〕。与FAM230B相关的PTEN蛋白得分较高（96.025 1分，P =1.14e-3）。RNA pull-down实验结果显示，FAM230B可与PTEN蛋白直接结合。Western blot法结果显示，干扰前后实验组PTEN蛋白条带无明显变化；与干扰前相比，干扰后实验组PTEN通路关键蛋白磷酸化蛋白激酶B（p-AKT）条带明显减弱，提示采用FAM230B干扰后细胞中p-AKT表达降低。结论　lncRNA FAM230B在颅底脊索瘤组织中呈高表达，其可与PTEN蛋白结合，导致AKT磷酸化水平升高，进而发挥促进肿瘤细胞增殖、抑制肿瘤细胞凋亡的作用。

Abstract：

Objective　To screen the key long chain noncoding RNA (lncRNA) in skull base chordoma and exploreits promote carcinogenesis and mechanism. Methods　Three chordoma tissue samples and three notochord tissue samplesstored in the Clinical Medical Research Center of Beijing Tiantan Hospital, Capital Medical University were selected for lncRNAsequencing, lncRNAs closely related to tumor development were screened. The skull base chordoma cell line HBC-2, bred by theauthors' group, was used for cell culture and transfection. si-FAM230B was used for cell transfection in FAM230B knockdowngroup, and control group 1 was transfected with si-FNC. si-AC079630.2 was used for cell transfection in AC079630.2 knockdowngroup, and control group 2 was transfected with si-ANC. CCK-8 assay was used to detect cell proliferation and flow cytometry was used to detect apoptosis. A total of 83 patients with primary skull base chordoma who underwent surgical treatment in Departmentof Neurosurgery, Beijing Tiantan Hospital, Capital Medical University from 2005 to 2014 were selected, the clinical data ofpatients were collected. Univariate and multivariate Cox regression analysis were used to analyze the risk factors of postoperativetumor progression in patients with primary skull base chordoma. The proteins interacting with FAM230B were screened byAnoLnc database, and the expression of binding protein was detected by RNA pull-down test and Western blot. Results　lncRNAsequencing results showed that 2 153 lncRNA sets were obtained after screening, and 169 differentially expressed lncRNA wereobtained, including 11 significantly over-expressed lncRNA and 4 significantly under-expressed lncRNA. Functional sequencingresults showed that a total of two lncRNA (FAM230B and ACO79630.2) were closely related to tumorigenesis and development.The cell viability at 24, 48, 72 h after cell culture and apoptosis rate of FAM230B knockdown group were lower than those ofcontrol group 1 (P < 0.05) , but there was no significant difference in cell viability at 24, 48, 72 h after cell culture and apoptosisrate between AC079630.2 knockdown group and control group 2 (P > 0.05) . Multivariate Cox regression analysis showed thatFAM230B expression level was an independent risk factor for postoperative tumor progression in patients with primary skull basechordoma [HR =0.749, 95%CI (0.616, 0.912) , P < 0.05] . The score of PTEN protein related to FAM230B was higher (96.025 1points，P =1.14e-3) . RNA pull-down assay showed that FAM230B could directly bind to PTEN protein. The results of Westernblot showed that the PTEN protein band had no significant change before and after interference in experimental group; comparedwith that before interference, the phosphorated protein kinase B (p-AKT) band was significantly weakened after interference inexperimental group, suggesting that the expression of p-AKT in cells was decreased after FAM230B interference. Conclusion　lncRNA FAM230B is highly expressed in skull base chordoma tissues, it can bind to PTEN protein and lead to increasedphosphorylation of AKT, which can promote the cell proliferation and inhibit the cell apoptosis.

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