基于miR-15a-5p/P53信号通路探讨EMT与肺癌细胞阿霉素耐药的关系  

Study on the relationship between EMT and lung cancer cell adriamycin resistance based on miR-15a-5p/P53 signaling pathway

作　　者：魏东[1] 辛运超 刘博[3] 容宇[1] 李彦明[1] 郝雁冰[1] Wei Dong;Xin Yunchao;Liu Bo;Rong Yu;Li Yanming;Hao Yanbing(Dept of thoracic surgery,the First Affiliated Hospital of Hebei North University,Zhangjiakou 075000;Dept of otolaryngology head and neck surgery,the First Affiliated Hospital of Hebei North University,Zhangjiakou 075000;Dept of pathology,the First Affiliated Hospital of Hebei North University,Zhangjiakou 075000)

机构地区：[1]河北北方学院附属第一医院胸外科,张家口075000 [2]河北北方学院附属第一医院耳鼻咽喉头颈外科,张家口075000 [3]河北北方学院附属第一医院病理科,张家口075000

出　　处：《安徽医科大学学报》2022年第7期1127-1133,共7页Acta Universitatis Medicinalis Anhui

基　　金：河北省卫建委重点科技研究计划(编号:20200552)。

摘　　要：目的探讨miR-15a-5p在肺癌细胞对阿霉素(DOX)耐药中的作用,并阐明其与DOX耐药之间的功能和机制联系。方法分别采用si-miR-15a-5p、miR-15a-5p模拟物转染A549、A549/DOX抗性细胞(A549/D)。采用MTT法测定细胞活力,流式细胞术检测细胞凋亡,Western blots检测上皮间充质转化(EMT)相关蛋白及P53蛋白表达,qRT-PCR检测miR-15a-5p表达。通过生物信息学预测与双荧光素酶报告子分析miR-15a-5p潜在靶基因。采用A549/D细胞构建裸鼠移植瘤模型,分析miR-15a-5p过表达促进DOX的体内抗肿瘤作用。结果MTT分析结果显示,miR-15a-5p的敲低提高了A549细胞的细胞活力(IC 50值:8.86±0.32μmol/L),miR-15a-5p的过表达降低了A549/D细胞的细胞活力(IC 50值:1.92±0.11μmol/L)。并且在A549细胞中阻断miR-15a-5p减少凋亡(P<0.001),在A549/D细胞中增加miR-15a-5p表达促进凋亡(P<0.001)。Western blot分析显示转染si-miR-15a-5p逆转了DOX调节EMT的作用。生物信息学预测证明P53和miR-15a-5p之间存在特异性结合位点。在A549细胞中阻断miR-15a-5p减少P53蛋白表达(P<0.001),在A549/D细胞中增加miR-15a-5p表达增加P53蛋白表达(P<0.01)。体内实验显示,miR-15a-5p agomir联合DOX可降低肿瘤体积和N-cadherin的表达水平,同时增强了P53、E-cadherin蛋白的表达水平。结论miR-15a-5p过表达可能通过靶向P53抑制EMT过程,增强肺癌细胞对DOX治疗的敏感性。Objective To investigate the role of miR-15 a-5 p in DOX resistance of lung cancer cells, and to elucidate the relationship between miR-15 a-5 p and DOX resistance. Methods miR-15 a-5 p inhibitor and miR-15 a-5 p mimics were used to transfect A549 and A549/DOX resistant cells(A549/D). MTT assay was used to detect cell viability, flow cytometry was used to detect cell apoptosis, Western blot was used to detect the expression of EMT related protein and P53 protein, and QRT PCR was used to detect the expression of miR-15 a-5 p. The potential target genes of miR-15 a-5 p were analyzed by bioinformatics prediction, dual luciferase reporter. A549/D cells were used to establish the xenograft tumor model in nude mice, and the effect of miR-15 a-5 p overexpression on DOX in vivo was analyzed. Results MTT analysis showed that the knockdown of miR-15 a-5 p increased the cell viability of A549 cells(IC_(50) value: 8.86±0.32 μmol/L), and the overexpression of miR-15 a-5 p decreased the cell viability of A549/D cells(IC_(50) value: 1.92±0.11 μmol/L). Blocking miR-15 a-5 p in A549 cells reduced apoptosis(P<0.001), and increasing the expression of miR-15 a-5 p in A549/D cells promoted apoptosis(P<0.001). The role of DOX in regulating the EMT was reversed by the transfection of miR-451 a inhibitor through Western blot. Bioinformatics prediction showed that there was a specific binding site between P53 and miR-15 a-5 p. miR-15 a-5 p inhibition reduced the expression of P53 protein in A549 cells(P<0.001), and miR-15 a-5 p over-expression increased the expression of P53 protein in A549/D cells(P<0.01). In vivo experiments showed that combination of agomir-miR-15 a-5 p and DOX could reduce the tumor volume and the expression level of N-cadherin, and enhance the expression levels of P53 and E-cadherin. Conclusion miR-15 a-5 p over-expression may inhibit EMT by targeting P53 and enhance the sensitivity of lung cancer cells to DOX therapy.

关 键 词：miR-15a-5p P53 上皮间充质转化 非小细胞性肺癌 耐药 

分 类 号：R734.2[医药卫生—肿瘤]