Role and mechanism of miR-519d in brain metastasis of non-small cell lung cancer

SU Ying; MA Li-li; LIU Jiang; HAN Zhong-cheng

doi:10.11659/jjssx.09E023022

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Role and mechanism of miR-519d in brain metastasis of non-small cell lung cancer

Basic Research | 更新时间：2024-11-18

- Role and mechanism of miR-519d in brain metastasis of non-small cell lung cancer
- JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY Vol. 33, Issue 11, Pages: 974-979(2024)
- 作者机构：
  
  新疆维吾尔自治区人民医院肿瘤科，新疆乌鲁木齐 830000
- 作者简介：
- 基金信息：
- DOI：10.11659/jjssx.09E023022
  CLC： R734.2
- Published：25 November 2024，
  
  Received：05 September 2023，
- 稿件说明：
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苏莹,马丽丽,柳江等.miR-519d在非小细胞肺癌脑转移中的作用及机制[J].局解手术学杂志,2024,33(11):974-979.

SU Ying,MA Li-li,LIU Jiang,et al.Role and mechanism of miR-519d in brain metastasis of non-small cell lung cancer[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2024,33(11):974-979.
苏莹,马丽丽,柳江等.miR-519d在非小细胞肺癌脑转移中的作用及机制[J].局解手术学杂志,2024,33(11):974-979. DOI： 10.11659/jjssx.09E023022.

SU Ying,MA Li-li,LIU Jiang,et al.Role and mechanism of miR-519d in brain metastasis of non-small cell lung cancer[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2024,33(11):974-979. DOI： 10.11659/jjssx.09E023022.

摘要

目的

探究miR-519d在非小细胞肺癌（NSCLC）脑转移（BM）中的作用及其机制。

方法

采用RT-PCR检测miR-519d在伴有BM的NSCLC患者和无BM的患者癌旁正常肺组织、NSCLC肺组织、NSCLC脑组织，人NSCLC细胞系（SK-MES-1、NCI-H226、NCIH1299和NCL-H820）、正常人支气管上皮细胞（HBECs）、人脑微血管内皮细胞（HBMECs）及正常人星形胶质细胞（NHA）中的表达。将过表达miR-519d慢病毒（OE-miR-519d-LV）或空载体慢病毒（NC-LV）转染至NSCLC细胞系NCI-H1299中，作为OE-miR-519d-LV组和NC-LV组；联合转染OE-miR-519d-LV与pcDNA3.1-HER3的细胞作为OE-miR-519d-LV+pcDNA3.1-HER3组。将OE-miR-519d-LV组和NC-LV组细胞经心脏注射至裸鼠体内，以构建伴BM的NSCLC动物模型，并按注射细胞的不同将小鼠分为Ctrl组与miR-519d组。使用生物发光成像系统观察NCI-H1299细胞在小鼠大脑中的转移情况。CCK-8法检测OE-miR-519d-LV组和NC-LV组中NCI-H1299细胞的增殖情况。采用HBMECs和NHA包被的Transwell小室，以建立体外血脑屏障（BBB）细胞模型，并检测OE-miR-519d-LV组和NC-LV组NCI-H1299细胞跨内皮细胞的迁移能力。Western blot检测各组细胞中上皮间质转化（EMT）相关蛋白MMP-2、MMP-9、E-cadherin、N-cadherin和Vimentin的表达。生物信息学技术分析miR-519d与HER3的潜在结合位点，双荧光素酶基因报告实验验证两者的靶向调控关系。

结果

与癌旁正常肺组织相比，miR-519d在NSCLC肺组织和NSCLC脑组织中的表达降低（

0.05）；与NSCLC肺组织相比，NSCLC脑组织中miR-519d表达降低（

0.05）。与HBECs相比，miR-519d在NSCLC细胞系中的表达降低（

0.05），且miR-519d在NCI-H1299、NCI-H226和NCL-H820细胞中的表达水平低于SK-MES-1细胞（

0.05）。与Ctrl组裸鼠相比，miR-519d组裸鼠的BM发生率更低，生存时间更长，差异有统计学意义（

0.05）。与NC-LV组细胞相比，OE-miR-519d-LV组NCI-H1299细胞增殖活力、跨内皮细胞迁移能力及MMP-2、MMP-9、N-cadherin和Vimentin蛋白表达降低（

0.05），而E-cadherin蛋白表达升高（

0.05）。miR-519d可靶向调控HER3蛋白表达。与OE-miR-519d-LV组相比，OE-miR-519d-LV+pcDNA3.1-HRE3组细胞的迁移能力升高（

0.05），MMP-2、MMP-9、N-cadherin和Vimentin蛋白表升高（

0.05），而E-cadherin表达降低（

0.05）。

结论

miR-519d在伴有BM的NSCLC中表达水平降低，上调其表达可通过靶向调控HER3来抑制肿瘤的BM。

Abstract

Objective

To investigate the role and mechanism of miR-519d in brain metastasis （BM） of non-small cell lung cancer （NSCLC）.

Methods

RT-PCR was used to detect the expression of miR-519d in normal lung tissues adjacent to cancer， NSCLC lung tissues， NSCLC brain tissues in NSCLC patients with or without BM，human NSCLC cell lines （SK-MES-1，NCI-H226，NCI-H1299，and NCL-H820），normal human bronchial epithelial cells （HBECs），human brain microvascular endothelial cells （HBMECs），and normal human astrocytes （NHA）. NSCLC cell line NCI-H1299 was transfected with either miR-519d overexpression lentivirus （OE-miR-519d-LV） or empty vector lentivirus （NC-LV），and set as the OE-miR-519d-LV group and the NC-LV group；cells co-transfected with OE-miR-519d-LV and pcDNA3.1-HER3 were designated as the OE-miR-519d-LV+pcDNA3.1-HER3 group. The cells of the OE-miR-519d-LV group and the NC-LV group were injected intracardially into nude mice to establish an animal model of NSCLC with BM， and the mice were divided into the Ctrl group and the miR-519d group based on the injected cells.Bioluminescence imaging was used to observe NCI-H1299 cell metastasis in the brain of mice. The CCK-8 assay was used to determine the proliferation of NCI-H1299 cells in the OE-miR-519d-LV group and the NC-LV group. An in vitro blood-brain barrier （BBB） model was established using HBMECs and NHA-coated Transwell chambers， and the transendothelial migration ability of NCI-H1299 cells was detected. Western blot was used to detect the expression of epithelial-mesenchymal transition （EMT）-related proteins of MMP-2， MMP-9， E-cadherin， N-cadherin， and Vimentin in each group. Bioinformatics analysis was used to identify the potential binding sites between miR-519d and HER3，and their targeted regulatory relationship was validated by dual-luciferase gene reporter assays.

Results

Compared with the normal lung tissues adjacent to cancer， m

iR-519d expression was reduced in the NSCLC lung tissues and NSCLC brain tissues （

0.05）； miR-519d expression was lower in NSCLC brain tissues compared with the NSCLC lung tissues （

0.05）. Compared with HBECs， miR-519d expression was reduced in NSCLC cell lines （

0.05）， with lower levels in NCI-H1299， NCI-H226， and NCL-H820 cells compared with SK-MES-1 cells （

0.05）. Nude mice in the miR-519d group had a lower incidence of BM and longer survival compared with those in the Ctrl group， with statistically significant differences （

0.05）. Compared with the NC-LV group，NCI-H1299 cells in the OE-miR-519d-LV group showed decreases in the proliferation viability， transendothelial migration ability， and protein expression of MMP-2， MMP-9， N-cadherin， and Vimentin （

0.05），and an increase in the protein expression of E-cadherin （

0.05）. miR-519d had a targeted regulatory effect on HER3 protein expression. Compared with the OE-miR-519d-LV group， the migration ability of cells in the OE-miR-519d-LV+pcDNA3.1-HRE3 group was increased （

0.05）， the protein expression of MMP-2， MMP-9， N-cadherin and Vimentin was increased （

0.05）， and the expression of E-cadherin was decreased （

0.05）.

Conclusion

miR-519d expression is reduced in NSCLC with BM， and its upregulation can inhibit the BM of NSCLC by targeting HER3.

关键词

非小细胞肺癌miR-519d人表皮生长因子受体3脑转移

Keywords

non-small cell lung cancermiR-519dhuman epidermal growth factor receptor 3brain metastasis

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