中华肿瘤防治杂志2024，Vol.31Issue(1) ：1-10.DOI:10.16073/j.cnki.cjcpt.2024.01.01

miRNA-423-5p和干扰素调节因子1对肺腺癌细胞影响的研究

miRNA-423-5p can promote the malignant phenotype of lung adenocarcinoma cells by regulating IRF1

崔逸爽 赵阅 吴亚男 郑璇 洪紫谦 潘可心 孙国贵
中华肿瘤防治杂志2024，Vol.31Issue(1) ：1-10.DOI:10.16073/j.cnki.cjcpt.2024.01.01
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miRNA-423-5p和干扰素调节因子1对肺腺癌细胞影响的研究

miRNA-423-5p can promote the malignant phenotype of lung adenocarcinoma cells by regulating IRF1

崔逸爽 1赵阅 2吴亚男 3郑璇 3洪紫谦 3潘可心 1孙国贵3
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作者信息

  • 1. 华北理工大学公共卫生学院,河北唐山 063210
  • 2. 中国科学院基础医学与癌症研究所,浙江杭州 310000
  • 3. 河北省医工融合精准医疗重点实验室,河北唐山 063000
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摘要

目的 探讨miRNA-423-5p与干扰素调节因子1(IRF1)在肺腺癌中的作用及机制.方法 体外培养肺腺癌细胞A549、H1299和H1975.实验分组:(1)在过表达miRNA-423-5p实验中,mimics NC作为对照组,miRNA-423-5p作为过表达组;(2)在敲降miRNA-423-5p实验中,ASO-NC作为对照组,ASO-miRNA-423-5p作为敲降组;(3)过表达IRF1实验中,pcDNA3.1作为对照组,pcDNA3.1-IRF1作为过表达组;(4)同时过表达miRNA-423-5p和IRF1实验中,mimics NC+pcDNA3.1 作为对照组,miRNA-423-5p+pcDNA3.1 作为只过表达 miRNA-423-5p 组,miRNA-423-5p+pcD-NA3.1-IRF1作为同时过表达miRNA-423-5p和IRF1组.采用CCK-8、克隆形成、5-乙炔基-2'-脱氧尿嘧啶核苷(EDU)、蛋白质印迹法、Transwell和划痕实验分别检测各组细胞增殖凋亡、侵袭和迁移能力的影响.通过Star Base数据库预测miRNA-423-5p与IRF1的结合位点,双荧光素酶实验验证miRNA-423-5p与IRF1之间的结合作用.裸鼠皮下成瘤实验检测稳定敲降miRNA-423-5p和稳定过表达IRF1细胞对移植瘤生长的影响.多组比较采用单因素方差分析,两组间比较采用独立样本t检验.结果 CCK-8、克隆形成、EDU、蛋白质印迹法、划痕和Transwell实验结果示,过表达miRNA-423-5p会促进H1299细胞的增殖、迁移和侵袭能力,抑制H1299细胞的凋亡能力,均P＜0.001.敲低miRNA-423-5p会抑制H1299细胞的增殖、迁移和侵袭能力,促进H1299细胞的凋亡能力,均P＜0.001.过表达IRF1会抑制H1299细胞的增殖、迁移和侵袭能力,促进H1299细胞的凋亡能力,均P＜0.001.过表达IRF1可以挽救过表达miRNA-423-5p对H1299细胞增殖、凋亡、迁移和侵袭能力的作用,均P＜0.001.Star Base数据库预测显示,miR-NA-423-5p与IRF1之间存在结合位点.双荧光素酶实验证明miRNA-423-5p与IRF1可以结合,t=26.470,P＜0.001.qRT-PCR(t=7.085,P=0.001)和蛋白质印迹法(t=28.660,P＜0.001)实验表明,miRNA-423-5p 可以调节IRF1的表达.裸鼠皮下成瘤实验结果提示,稳定敲降miRNA-423-5p的H1299细胞瘤体体积[(491.40±94.34)mm3 vs(382.20±16.04)mm3,t=2.825,P=0.037]、瘤组织 Ki-67(0.40±0.10 vs 0.24±0.02,t=3.843,P=0.012)和PCNA表达(0.45±0.09 vs 0.25±0.04,t=4.974,P=0.004)均显著降低,稳定过表达IRF1的H1299细胞瘤体体积[(291.50±109.1)mm3 vs(125.46±98.52)mm3,t=2.766,P=0.040]、瘤组织 Ki-67(0.44±0.04 vs 0.25±0.02,t=10.407,P＜0.001)和 PCNA 表达(0.39±0.03 vs 0.15±0.02,t=16.305,P＜0.001)均显著降低.结论 miRNA-423-5p可能通过调控IRF1促进肺腺癌的恶性表型.

Abstract

Objective To explore the role and mechanism of miRNA-423-5p and interferon regulatory factor 1(IRF1)in lung adenocarcinoma(LU AD).Methods LU AD cells A549,H1299 and H1975 were cultured in vitro.The experiment was divided into four parts:(1)In the overexpression of miRNA-423-5p experiment,mimics NC was used as the control group,and miRNA-423-5p was used as the overexpression group;(2)In the knockdown miRNA-423-5p experiment,ASO-NC was used as the control group and ASO-miRNA-423-5p was used as the knockdown group;(3)In the overex-pression IRF1 experiment,pcDNA3.1 was used as the control group and pcDNA3.1-IRF1 was used as the overexpres-sion group;(4)In the simultaneous overexpression of miRNA-423-5p and IRF1 experiments,mimics NC+pcDNA3.1 was used as the control group,miRNA-423-5p+pcDNA3.1 was used as the only overexpression of miRNA-423-5p group,and miRNA-423-5p+pcDNA3.1-IRF1 was used as the simultaneous overexpression of miRNA-423-5p and IRF1 group.CCK-8,cloning,5-Acetynyl-2'-deoxyuridine nucleoside(EDU),Western blot,trans well and scratch test were used to detect the effects of the above groups on the proliferation,apoptosis,invasion and migration of LU AD cells.The binding sites of miRNA-423-5p and IRF1 were predicted by Star Base database,and the binding effect between miRNA-423-5p and IRF1 was verified by double luciferase assay.The effect of stably knockdown miRNA-423-5p and stably over-expression of IRF1 on the growth of the tumorigenicity was detected by subcutaneous tumorigenesis test in nude mice.Multiple group comparisons were conducted using one-way ANOVA,and independent sample t-tests were used for com-parison between the two groups.Results The results of CCK-8,clone formation,EDU,Western blot,scratch,and Tr-answell experiments showed that overexpression of miRNA-423-5p promoted the proliferation of H1299 cells,migration and invasion.The ability to inhibit apoptosis of H1299 cells.Knocking down miRNA-423-5p inhibited the proliferation of H1299 cells,migration and invasion,and promoted the apoptosis of H1299 cells.Overexpression of IRF1 inhibited the proliferation of H1299 cells,migration and invasion,and promoted the apoptosis of H1299 cells.Overexpression of IRF1 could salvage the effects of overexpression of miRNA-423-5p on H1299 cell proliferation,apoptosis,and the role of mi-gration and invasion ability.The binding sites of miRNA-423-5p and IRF1 were predicted by Star Base database,and the binding effect between miRNA-423-5p and IRF1 was verified by double luciferase assay(t=26.470,P＜0.001).QRT PCR(t=7.085,P=0.001)and Western blot(t=28.660,P＜0.001)experiments showed that miRNA-423-5p could regulate the expression of IRF1.The results of the subcutaneous tumorigenesis experiment in nude mice suggested that the tumour volume of H1299 cells stably knockdown miRNA-423-5p was significantly reduced[(491.40±94.34)mm3 vs(382.20±16.04)mm3,t=2.825,P=0.037],tumour tissue Ki-67(0.40±0.10 vs 0.24±0.02,t=3.843,P=0.012),and PCNA expression was significantly reduced(0.45±0.09 vs 0.25±0.04,t=4.974,P=0.004),the tumour volume of H1299 cells stably overexpressing IRF1 was significantly reduced[(291.50±109.1)mm3 vs(125.46± 98.52)mm3,t=2.766,P=0.040)],tumour tissue Ki-67(0.44±0.04 vs 0.25±0.02,t=10.407,P＜0.001),and PCNA expression was significantly reduced(0.39±0.03 vs 0.15±0.02,t=16.305,P＜0.001).Conclusion miRNA-423-5p can promote the malignant phenotype of lung adenocarcinoma cells by regulating IRF1.

关键词

miRNA-423-5p/干扰素调节因子1/肺腺癌/增殖凋亡/侵袭迁移

Key words

miRNA-423-5p/interferon regulatory factor 1/lung adenocarcinoma/proliferation and apoptosis/invasive and migration

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基金项目

国家自然科学基金(82172658)

唐山市小细胞肺癌医工融合精准诊疗基础创新团队(21130203D)

华北理工大学公共卫生学院高水平科研创新团队建设计划(KYTD202309)

出版年

2024
中华肿瘤防治杂志
中华预防医学会 山东省肿瘤防治研究院

中华肿瘤防治杂志

CSTPCD北大核心
影响因子：1.292
ISSN：1673-5269
参考文献量35
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