白花蛇舌草提取物调控微小RNA-340对肝癌细胞增殖、迁移及侵袭的影响  

Effect of Hedyotisdiffusa extract on the proliferation,migration,and invasion of hepatocellular carcinoma cells by regulating MiR-340

作　　者：彭亚楠 单丽芳 姜俊玲 唐晓冰 康国娇 罗克胜 周葵 赵强 PENG Yanan;SHAN Lifang;JIANG Junling;TANG Xiaobing;KANG Guojiao;LUO Kesheng;ZHOU Kui;ZHAO Qiang(Department of Pharmacy,Pu'er Hospital of Traditional Chinese Medicine,Pu'er,Yunnan 665000;Department of Pharmacy,Lancang Hospital of Traditional Chinese Medicine,Lancang,Yunnan 665600;Preparation Room,Pu'er Institute of Traditional Chinese Medicine,Pu'er,Yunnan 665000)

机构地区：[1]云南省普洱市中医医院药剂科,云南普洱665000 [2]云南省澜沧县中医医院药剂科,云南澜沧665600 [3]云南省普洱市民族传统医药研究所制剂室,云南普洱665000

出　　处：《河北中医》2024年第12期2035-2042,共8页Hebei Journal of Traditional Chinese Medicine

基　　金：2019年云南省科学技术厅—云南中医药大学应用基础研究联合专项项目[编号:2019FF002(-067)]。

摘　　要：目的观察白花蛇舌草提取物(HDE)对肝癌细胞增殖、迁移、侵袭的作用及机制。方法将肝癌细胞HCC-7721、LM3分为对照组(NC组)、HDE低剂量组(HDE-L组)和HDE高剂量组(HDE-H组),并予以相应的药物干预48 h,分别以CCK-8、EdU细胞增殖实验、克隆形成实验检测细胞活力、增殖及克隆情况,划痕实验、Trnaswell小室侵袭实验检测细胞迁移、侵袭情况,F-actin染色观察细胞骨架形态。通过RNA-seq分析高剂量HDE干预对微小RNA(miRNA)的影响,通过实时荧光定量聚合酶链式反应法(qRT-PCR)验证HDE对HCC-7721细胞中差异miRNA表达的影响。在高剂量HDE干预的同时过表达或低表达miR-340,观察肝癌细胞活力、增殖、克隆、迁移和侵袭能力的变化。结果HDE能呈剂量依赖地抑制肝癌细胞HCC-7721、LM3细胞活力、增殖、克隆、迁移和侵袭能力,比较差异均有统计学意义(P<0.05)。F-actin染色发现,NC组细胞的丝状伪足及片状伪足较多;HDE-L组和HDE-H组细胞形态出现皱缩,细胞间连接减少,丝状伪足及片状伪足也明显减少,同时2组F-actin荧光强度较对照组均降低(P<0.05),且HDE-H组低于HDE-L组(P<0.05)。RNA-seq显示,HDE干预HCC-7721细胞后,共检测到1769个差异表达基因,其中miR-340表达变化最显著,较对照细胞上调12.64倍,且qRT-PCR也证实,HDE能呈剂量依赖地上调HCC-7721细胞中miR-340的表达(P<0.05)。在高剂量HDE干预的同时过表达miR-340后,肝癌细胞HCC-7721、LM3的细胞活力、增殖、克隆、迁移和侵袭能力均下降(P<0.05),低表达miR-340后,肝癌细胞HCC-7721、LM3的细胞活力、增殖、克隆、迁移和侵袭能力均增强(P<0.05)。结论HDE通过上调miR-340的表达,进而抑制肝癌细胞HCC-7721、LM3增殖、迁移及侵袭能力。Objective To explore the effect of Hedyotisdiffusa extract(HDE)on the proliferation,migration,and invasion of liver cancer cells and the underlying mechanism.Methods Liver cancer cells HCC-7721 and LM3 induced with blank control,and low-dose and high-dose HDE for 48 h.Cell viability,proliferation and colony were assessed by cell counting kit-8(CCK-8)assay,EdU assay and colony formation,respectively.Cell migration and invasion were assessed by wound healing assay and Trasnwell assay,respectively.Cell cytoskeleton was observed by immunostaining of F-actin.RNA-seq was performed to identify differentially expressed genes in liver cancer cells induced with high-dose HDE,and verified in HCC-7721 cells by quantitative reverse-transcription polymerase chain reaction(qRT-PCR).Changes in the viability,proliferation,colony,migration and invasion of liver cancer cells induced with high-dose HDE and overexpression/knockdown of miR-340 were detected.Results HDE dose-dependently inhibited the viability,proliferation,colony,migration and invasion of HCC-7721 and LM3 cells(P<0.05).F-actin staining found that liver cancer cells induced with blank control had more filopodia and lamellipodia,while those induced with low-dose and high-dose HDE showed shrinkage,reduced intercellular connections,and filopodia and lamellipodia.The fluorescence intensity of F-actin was significantly lower in liver cancer cells induced with low-dose and high-dose HDE than those of controls,which was significantly lower in the high-dose HDE group than the low-dose HDE group(P<0.05).RNA-seq showed 1769 differentially expressed genes in HCC-7721 cells induced with high-dose HDE,among which miR-340 was the mostly upregulated for 12.64 times.qRT-PCR also confirmed that HDE dose-dependently upregulated miR-340 in HCC-7721 cells(P<0.05).High-dose HDE induction and overexpression of miR-340 significantly inhibited the viability,proliferation,colony,migration and invasion of HCC-7721 and LM3 cells(P<0.05).Knockdown of miR-340 significantly promoted the viability,p

关 键 词：肝癌 肝细胞 白花蛇舌草 miR-340 肿瘤转移 肿瘤浸润 细胞增殖 药理作用分子作用机制(中药) 

分 类 号：R735.705.31[医药卫生—肿瘤] R285.5[医药卫生—临床医学]