miR-27a-3p_RC...毒方抑制上皮间质转化的机制_吴存恩.pdf

中华中医药杂志(原中国医药学报)2023年2月第38卷第2期 CJTCMP,February 2023,Vol.38,No.2 819 miR-27a-3p/RCBTB1/-catenin轴与胃癌临床 特征的关联性及益气化瘀解毒方 抑制上皮间质转化的机制吴存恩，壮雨雯，周锦勇，王琼，钱军，刘沈林（南京中医药大学附属医院，南京 210029）摘要：目的：明确miR-27a-3p/RCBTB1/-catenin与胃癌临床特征的关系，探讨益气化瘀解毒方抑制胃癌上皮间质转化（EMT）的作用机制。方法：生物信息学分析miR-27a-3p、RCBTB1在胃癌中的表达与临床意义；高通量测序检测小鼠肝转移瘤组织内mRNA表达差异；质粒转染构建沉默或过表达miR-27a-3p、RCBTB1的稳定细胞株；Transwell、划痕实验检测胃癌细胞侵袭迁移能力的改变；脾脏注射MFC细胞建立小鼠胃癌肝转移模型并以益气化瘀解毒方干预，检测肝转移瘤数量，苏木精-伊红染色（HE）染色法检测胃癌细胞肝脏浸润情况；RT-PCR、Western Blot检测miR-27a-3p、RCBTB1、-catenin、E-cadherin、Snail的表达。结果：miR-27a-3p在胃癌组织中较正常组织升高（P0.01），且期胃癌组织中miR-27a-3p表达较、期增高（P0.05）。与对照组比较，miR-27a-3p过表达组胃癌细胞侵袭迁移能力增强，miR-27a-3p沉默组细胞侵袭性减弱（P0.01）。高通量测序显示，益气化瘀解毒方组小鼠肝转移瘤组织内RCBTB1较对照组升高，且TargetScan预测RCBTB1为miR-27a-3p的潜在靶基因。与对照组比较，过表达miR-27a-3p能抑制RCBTB1（P0.01），沉默miR-27a-3p能上调RCBTB1表达（P0.01）。生信分析提示，RCBTB1低表达组胃癌患者PFI、DSS较高表达组降低（P0.05）。细胞实验提示，沉默RCBTB1可增强胃癌细胞侵袭性（P0.01），过表达RCBTB1能抑制胃癌细胞侵袭性（P0.01，P0.05）。与对照组比较，益气化瘀解毒方含药血清组E-cadherin表达升高（P0.01），Snail、-catenin表达降低（P0.01，P0.05）。体内实验显示与对照组比较，益气化瘀解毒方组胃癌细胞在肝组织中的浸润减少，肿瘤组织内 miR-27a-3p、-catenin、Snail表达下调（P0.01），RCBTB1、E-cadherin表达上调（P0.01，P0.05）。结论：miR-27a-3p/RCBTB1/-cateni是促进胃癌EMT的重要信号轴，益气化瘀解毒方能调控miR-27a-3p/RCBTB1/-catenin信号以抑制EMT，进而发挥抗胃癌转移的作用。关键词：益气化瘀解毒方；胃癌；临床特征；上皮间质转化；miR-27a-3p/RCBTB1/-catenin基金资助：国家自然科学基金项目（No.82104950），2019国家中医药管理局中医药循证能力建设项目（No.2019XZZX-ZL003），重大疑难疾病中西医临床协作试点，国家中医临床研究基地开放课题（No.JD2019SZXYB01），江苏省卫生健康委医学科研项目（No.H2019094），国家中医药管理局全国名中医传承工作室建设项目（No.2018-119），中医药传承创新平台建设项目，江苏省中医院院级课题（No.Y2020CX57，No.Y2020CX60，No.Y2020CX67，No.Y2021CX06）Relationship between miR-27a-3p/RCBTB1/-catenin axis and clinical features of gastric cancer and the mechanism of Yiqi Huayu Jiedu Decoction inhibiting epithelial mesenchymal trasformationWU Cun-en，ZHUANG Yu-wen，ZHOU Jin-yong，WANG Qiong，QIAN Jun，LIU Shen-lin(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China)Abstract:Objective:To investigate the relationship between miR-27a-3p/RCBTB1/-catenin and the clinical characteristics of gastric cancer,and to explore the mechanism of Yiqi Huayu Jidu Decoction inhibiting epithelial mesenchymal trasformation(EMT).Methods:The expression and clinical significance of miR-27a-3p and RCBTB1 in gastric cancer was 研究报告通信作者：刘沈林，江苏省南京市汉中路155号南京中医药大学附属医院，邮编：210029，电话：025-86617141E-mail：内文2.indd 8192023/2/28 10:36:10中华中医药杂志(原中国医药学报)2023年2月第38卷第2期 CJTCMP,February 2023,Vol.38,No.2 820 analyzed by bioinformatics.High-throughput sequencing was used to detect the difference of mRNA expression in liver metastatic tumor tissues of mice.Stable cell lines with silenced or overexpressed miR-27a-3p or RCBTB1 were constructed by plasmid transfection.The invasion and migration ability of gastric cancer cells was Detected by Transwell and Wound-healing assays.MFC cells were injected into the spleen to establish liver metastasis model of gastric cancer in mice,and Yiqi Huayu Jiedu Decoction was used for intervention.The number of liver metastases was detected,and the liver infiltration of gastric cancer cells was detected by HE staining.Expression of miR-27a-3p,RCBTB1,-catenin,E-cadherin and Snail were detected by RT-PCR and Western Blot.Results:The expression of miR-27a-3p was higher in gastric cancer than normal tissues(P0.01),mir-27a-3p expression in stage gastric cancer was higher than that in stage,and (P0.05).Compared with the control group,the invasion and migration of gastric cancer cells in miR-27a-3p overexpression group was enhanced,while it got weakened in miR-27a-3p silencing group(P0.01).High-throughput sequencing showed that RCBTB1 in the liver metastatic tumor tissues of mice in Yiqi Huayu Jiedu Decoction group was higher than that in the control group,and TargetScan predicted that RCBTB1 was a potential target gene of miR-27a-3p.Compared with control group,overexpression of miR-27a-3p inhibited RCBTB1(P0.01),while silencing of miR-27a-3p up-regulated the expression of RCBTB1(P0.01).Bioinformatics analysis suggested that PFI and DSS in gastric cancer patients with low expression of RCBTB1 were decreased than those of patients with high expression of RCBTB1(P0.05).In vitro experiments suggested that silencing RCBTB1 can enhance the aggressiveness of gastric cancer cells(P0.01),while overexpression of RCBTB1 can inhibit it(P0.01,P0.05).Compared with control group,the expression of E-cadherin was increased(P0.01),the expression of Snail and-catenin were decreased in Yiqi Huayu Jiedu Decoction containing serum group(P0.01,P0.05).In vivo experiments showed that compared with the control group,the infiltration of gastric cancer cells in the liver tissues of Yiqi Huayu Jiedu Decoction group was reduced,the expression of miR-27a-3p,-catenin and Snail in tumor tissues were down-regulated(P0.01),and the expression of RCBTB1 and E-cadherin were up-regulated (P0.01,P2倍或FC0.5倍且P0.05的基因定义为差异基因。5.Transwell实验检测细胞侵袭能力 将Matrigel基质胶包被好的小室至于24孔板中，向上室加入无血清培养液，室温下水化。将对照组正常AGS细胞，miR-27a-3p过表达对照组、miR-27a-3p过表达组、miR-27a-3p沉默对照组、miR-27a-3p沉默组、RCBTB1沉默对照组、RCBTB1沉默组、RCBTB1过表达对照组、RCBTB1过表达组各组细胞饥饿24 h后，无血清培养液