解毒复正汤通过干扰miR-223-3p并靶向调控TGFBR3影响肺癌侵袭转移的实验研究  

Experimental Study on the Effect of JieduFuzheng Decoction on the Invasion and Metastasis of Lung Cancer by Interfering with miR-223-3p and Targeting TGFBR3

作　　者：姚菲[1] 石玮[1] 吕梦玉 吴姗燕 王家晓[1] 李春姗[1] Yao Fei;Shi Wei;Lyu Mengyu;Wu Shanyan;Wang Jiaxiao;Li Chunshan(The First Affiliated Hospital of Guangxi University of Chinese Medical,Nanning 530200,China)

机构地区：[1]广西中医药大学第一附属医院,南宁530200

出　　处：《世界科学技术-中医药现代化》2023年第4期1358-1366,共9页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金会地区基金项目(82260956):解毒复正汤干预携带miR-223-3p的肿瘤成纤维细胞相关外泌体抑制肺癌转移的机制研究,负责人:姚菲;广西壮族自治区自然科学基金会面上基金项目(2020GXNSFAA297294):解毒复正汤通过干扰CAFs外泌体转运miR-223-3p影响肺癌侵袭的应用基础研究,负责人:姚菲;广西中医药适宜技术开发与推广项目(GZSY20-22):解毒扶正颗粒联合EGFR-TKIs治疗晚期非小细胞肺癌的临床研究,负责人:李春姗

摘　　要：目的探讨解毒复正汤(JieduFuzhengdecoction,JDFZ)通过影响微小核糖核酸-223-3p(Mircro ribonucleic acid-223-3p,miR-223-3p)靶向调控转化生长因子β受体3(Transforming growth factorβReceptor3,TGFBR3)的表达及其对肺癌细胞迁移的影响。方法利用RT-qPCR技术检测肺腺癌患者癌组织及癌旁组织、肺腺癌患者及健康人外周血清中miR-223-3p表达水平的差异;并检测不同种类的NSCLC细胞系肺癌细胞(95D、LTEP-α-2、A549及NCI-H460)以及人肺上皮细胞BEAS-2B中miR-223-3p的本底表达量,从组织、血浆及细胞3个维度验证miR-223-3p的差异性。利用生物信息学网站重叠分析,初步预测到miR-223-3p与TGFBR33’UTR具有潜在的结合位点序列。接着运用双荧光素酶报告基因实验验证miR-223-3p与TGFBR3的靶向关系。体外培养肺癌A549细胞,采用脂质体法分别将miR-223-3p-过表达序列(miR-223-3p-mimics)及携带TGFBR3全基因的重组载体(Over-expression-TGFBR3,OE-TGFBR3)分别转染至肺癌细胞,以转入无关序列的模拟剂(Negative control-mimics,NC)作为对照组,收集各组稳转细胞,Transwell小室检测转染处理后及JDFZ作用后的细胞迁移能力;收集各组细胞蛋白,蛋白免疫印迹法(Westernblot)检测细胞中EMT相关蛋白的表达。结果肺癌组织中miR-223-3p的表达量显著低于癌旁组织;肺癌患者血浆miR-223-3p表达水平明显低于健康正常人。不同种类的NSCLC细胞系肺癌细胞(95D、LTEP-α-2、A549及NCI-H460)中的miR-223-3p表达量亦低于正常人肺上皮细胞BEAS-2B。利用生物信息学网站及荧光素酶报告基因实验证实了miR-223-3p与TGFBR3的靶向调控关系。与miR-223-3p NC组对比,miR-223-3p mimics组和miR-223-3p mimics+OE-TGFBR3组的细胞迁移能力均下降(P<0.05),上调TGFBR3和加入解毒复正汤均能有效增强miR-223-3p对A549细胞迁移的负向调节作用;Westenblot结果发现,与miR-223-3p NC组对比,miR-223-3p mimics组和miR-223-3p mimics+OE-TGFBR3组E-Cadherin的�Objective To explore that JieduFuzhengDecotion(JDFZ)can regulate TGF by affecting mircro ribonucleic acid 223-3p(miR-223-3p)βReceptor 3(transforming growth factorβThe expression of Receptor 3,TGFBR3)and its effect on lung cancer cell migration.Methods RT-qPCR was used to detect the difference of miR-223-3p expression level in cancer tissues and adjacent tissues of lung adenocarcinoma patients,in peripheral serum of lung adenocarcinoma patients and healthy people;And detect different kinds of NSCLC cell line lung cancer cells(95D,LTEP-α-2.A549 and NCI-H460)and human pulmonary epithelial cell BEAS-2B.By overlapping analysis of bioinformatics websites,it was preliminarily predicted that miR-223-3p and TGFBR33'UTR had potential binding site sequences.Then,double luciferase reporter gene experiment was used to verify the targeting relationship between miR-223-3p and TGFBR3.In vitro culture of lung cancer A549 cells,miR-223-3p overexpression sequences(miR-223-3p mimics),Transwell chamber was used to detect the cell migration ability after transfection and JDFZ treatment;Collect the cell proteins of each group,and detect the expression of EMT related proteins in cells by Western blot.Results The expression of miR-223-3p in lung cancer tissues was significantly lower than that in adjacent tissues;The expression level of miR-223-3p in plasma of lung cancer patients was significantly lower than that of healthy controls.Different NSCLC cell lines lung cancer cells(95D,LTEP-α-2.The expression of miR-223-3p in A549 and NCI-H460)was also lower than that in BEAS-2B.The targeted regulation relationship between miR-223-3p and TGFBR3 was confirmed by bioinformatics website and luciferase reporter gene experiment.Compared with miR-223-3p NC group,the migration ability of cells in miR-223-3p mimics group and miR-223-3p mimics+OE-TGFBR3 group decreased Western blot results showed that,compared with miR-223-3p NC group,the expression level of E-cadherin in miR-223-3p mimics group and miR-223-3p mimics+OE-TGFBR3 group was up-regula

关 键 词：微小核糖核酸-223-3p(miR-223-3p) 人转化生长因子β受体3(TGFBR3) 解毒复正汤 侵袭转移 肺癌 

分 类 号：R285.5[医药卫生—中药学]