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

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的表达水平上调,NCadherin及Vimentin的表达水平下升(P<0.05),两组均在不同程度上抑制EMT的发生;上调TGFBR3和加入解毒复正汤作用能有效增加miR-223-3p对细胞迁移相关蛋白的负向调节作用(P<0.05)。结论解毒复正汤对肺癌A549细胞的侵袭迁移有抑制作用,其机制可能是通过调节miR-223-3p并靶向调控TGFBR3及其下游EMT相关指标的表达来实现的。

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-regulated,and the expression levels of N-cadherin and Vimentin were up-regulated The up regulation of TGFBR3 and the addition of JieduFuzheng Decoction can effectively increase the negative regulation of miR-223-3p on cell migration related proteins(P<0.05).Conclution JieduFuzheng Decoction can inhibit the invasion and migration of lung cancer A549 cells,and its mechanism may be realized by regulating miR-223-3p and targeting the expression of TGFBR3 and its downstream EMT related indicators.