多发性骨髓瘤预后相关基因的鉴定及核心基因CLDN1的机制探讨  

Identification of prognosis-related genes in multiple myeloma and mechanism of core gene CLDN1

作　　者：龚高明 陈博 舒鹏 GONG Gaoming;CHEN Bo;SHU Peng(Department of Orthopedics,Beilun People's Hospital,Ningbo 315800,China)

机构地区：[1]宁波市北仑区人民医院骨科,315800

出　　处：《浙江医学》2021年第5期475-481,共7页Zhejiang Medical Journal

基　　金：浙江省基础公益研究计划项目(LGF19H200001)。

摘　　要：目的明确与多发性骨髓瘤(MM)患者预后相关的基因,并探讨相应的机制。方法使用随机森林算法与Cox回归分析相结合的方法得到预后基因列表,并选定紧密连接蛋白1(CLDN1),通过构建CLDN1敲除载体,采用qRT-PCR法检测健康志愿者与MM患者中CLDN1 mRNA表达水平,Western blot检测CLDN1、转化生长因子-β3(TGF-β3)蛋白表达水平,Annexin V FITC/PI双染法检测细胞凋亡的变化,Transwell法检测细胞迁移的变化,CCK-8法检测细胞增殖的变化。论证CLDN1表达的变化对MM细胞系凋亡、增殖和迁移的影响。同时通过基因富集分析、相关性分析、Western blot、Transwell法证实CLDN1通过TGF-β3通路调控MM细胞系的迁移。结果通过随机森林算法与Cox回归分析,得到25个与MM预后相关的基因,通过这些基因的表达能很好地预测患者的总体生存率,其中CLDN1不仅在25个预后相关基因中高表达,且在宁波市北仑区人民医院MM样本中始终高表达,在MM细胞系RPMP8226中CLDN1表达的缺失诱导了RPMP8226的凋亡,抑制了RPMI8226的增殖与迁移。与此同时,CLDN1高表达的样本中,细胞外基质相关通路高度富集,其中TGF-β3的表达与CLDN1的表达呈正相关,TGF-β3的表达不仅能够改变CLDN1的表达,且CLDN1对迁移的调控依赖于TGF-β3的表达。结论25个预后相关基因能够预测患者生存,其中CLDN1基因与肿瘤发生、发展密切相关,而CLDN1的功能依赖于TGF-β3的表达。Objective To identify the prognosis-related genes in multiple myeloma(MM) patients and explore the corresponding mechanism. Methods The random forest algorithm and Cox regression analysis were used to get the gene signature of prognosis-related genes, and CLDN1 was selected as the candidate gene. The CLDN1 was knocked-down in MM RPMP8226 cells. The expression of CLDN1 mRNA was detected with qRT-PCR in healthy donor and MM patients. The expression of CLDN1, TGF-β3 proteins were detected with Western blot. The cell apoptosis was detected with Annexin V FITC/PI double staining. The cell migration was detected with Transwell method, and the cell proliferation was detected with CCK-8. The effect of CLDN1 in regulation of migration of MM cells was verified through gene enrichment analysis, correlation analysis,Western blot and Transwell method. Results Through random forest algorithm and Cox regression analysis, we obtained 25 genes related to the prognosis of MM. Among 25 gene signatures CLDN1 was highly expressed in myeloma samples. In RPMI8226 cells, the loss of CLDN1 expression induced the cell apoptosis, inhibited cell proliferation and migration. At the same time,in CLDN1 high expression samples, the extracellular matrix pathway was highly enriched, and the expression of TGF-β3 was correlated with the expression of CLDN1, which regulated the migration through the expression of TGF-β3. Conclusion A total of 25 genes may predict the survival of MM patients, among which CLDN1 gene is closely related to tumor development, and the function of CLDN1 depends on the expression of TGF-β3.

关 键 词：预后相关基因 紧密连接蛋白1 转化生长因子-Β3 多发性骨髓瘤 

分 类 号：R733.3[医药卫生—肿瘤] Q811.4[医药卫生—临床医学]