lncRNA NEAT1通过miR-377-3p/Wnt通路影响ox-LDL诱导的人血管平滑肌细胞增殖、侵袭迁移

doi:10.3969/j.issn.1674-8115.2022.11.003

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (11): 1534-1541.doi: 10.3969/j.issn.1674-8115.2022.11.003

• 论著 · 基础研究 • 上一篇

lncRNA NEAT1通过miR-377-3p/Wnt通路影响ox-LDL诱导的人血管平滑肌细胞增殖、侵袭迁移

袁咏梅(), 程晓丹, 孙家安, 常东歌, 何莹莹, 刘畅()

郑州大学附属郑州中心医院急诊科，郑州 450007

收稿日期:2022-04-21 接受日期:2022-08-21 出版日期:2022-11-28 发布日期:2023-01-04
通讯作者: 刘畅 E-mail:yuanyongmei1977@163.com;lcjzk0@163.com
作者简介:袁咏梅（1977—），女，副主任医师；电子信箱：yuanyongmei1977@163.com。
基金资助:
2020年度河南省高等学校重点科研项目(20B320024)

lncRNA NEAT1 affects the proliferation, invasion and migration of ox-LDL-induced human vascular smooth muscle cells through miR-377-3p/Wnt pathway

YUAN Yongmei(), CHENG Xiaodan, SUN Jiaan, CHANG Dongge, HE Yingying, LIU Chang()

Department of Emergency, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, China

Received:2022-04-21 Accepted:2022-08-21 Online:2022-11-28 Published:2023-01-04
Contact: LIU Chang E-mail:yuanyongmei1977@163.com;lcjzk0@163.com
Supported by:
2020 Key Scientific Research Projects of Higher Education Institutions in Henan Province(20B320024)

摘要/Abstract

摘要：

目的·研究长链非编码RNA（long non-coding RNA，lncRNA）核旁斑组装转录本1（nuclear paraspeckle assembly transcript 1，NEAT1）通过微RNA-377-3p（microRNA-377-3p，miR-377-3p）调控Wnt通路对氧化低密度脂蛋白（oxidized low-density lipoprotein，ox-LDL）诱导人血管平滑肌细胞（human vascular smooth muscle cell，HVSMC）增殖、侵袭迁移的影响。方法·以100 μg/mL的ox-LDL处理体外培养的HVSMC。将HVSMC随机分为对照组、模型组、lncRNA NEAT1 siRNA组、miR-377-3p抑制组、转染+抑制组（转染NEAT1 siRNA和miR-377-3p抑制剂）、转染阴性对照组（NEAT1 siRNA阴性对照）。除对照组外，其余各组以100 μg/mL的ox-LDL诱导建立动脉粥样硬化细胞模型，分组转染处理后，通过细胞计数试剂盒8（cell counting kit-8，CCK-8）测定各组细胞活力；通过流式细胞实验测定各组细胞凋亡率；通过Transwell实验评测各组细胞迁移侵袭情况；通过免疫印迹实验评测各组细胞增值细胞标志蛋白［增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）］及上皮-间充质转化（epithelial-mesenchymal transition，EMT）标志蛋白［E-cadherin、vimentin］的表达；通过qRT-PCR实验测定各组细胞NEAT1、miR-377-3p及无翅型MMTV整合位点家族成员5A（wingless-related MMTV integration site 5A，WNT5A）mRNA表达。结果·与对照组相比，模型组NEAT1 mRNA表达升高，miR-377-3p mRNA表达降低，细胞凋亡率降低，细胞活力、迁移侵袭细胞数升高，PCNA、vimentin蛋白表达蛋白升高，E-cadherin表达降低，WNT5A mRNA表达升高（均P=0.000）。干扰NEAT1后，细胞凋亡率升高，细胞活力、迁移侵袭细胞数降低，PCNA、vimentin蛋白表达降低，E-cadherin蛋白表达升高，WNT5A mRNA表达降低（均P=0.000）；抑制miR-377-3p后上述各项指标均呈现相反的表达趋势（均P<0.05）。此外，抑制miR-377-3p可逆转NEAT1干扰对细胞增殖、侵袭迁移和凋亡的调控作用（均P=0.000）。结论·下调NEAT1表达，可能通过与miR-377-3p竞争性结合，升高miR-377-3p表达，降低Wnt通路蛋白表达，进而抑制ox-LDL诱导的HVSMC异常增殖及侵袭迁移，并诱导其凋亡。

关键词: 长链非编码RNA, 核旁斑组装转录本1, 微RNA-377-3p, 人血管平滑肌细胞, 增殖, 侵袭迁移

Abstract:

Objective ·To study the effects of long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) on the proliferation, invasion and migration of oxidized low-density lipoprotein (ox-LDL)-induced human vascular smooth muscle cells (HVSMCs) through regulating microRNA-377-3p (miR-377-3p) /Wnt pathway. Methods ·HVSMCs cultured in vitro were treated with 100 μg/mL ox-LDL. HVSMCs were randomly divided into control group, model group, NEAT1 siRNA group, miR-377-3p inhibitor group, transfection+inhibitor (NEAT1 siRNA+miR-377-3p inhibitor) group, and transfection-negative control (NEAT1 siRNA-negative control) group. Except for the control group, the atherosclerosis cell models were induced with 100 μg/mL ox-LDL in other groups. After grouping and transfection, the cell viability of each group was determined by using cell counting kit-8 (CCK-8) experiment; the apoptosis rate of each group was measured by flow cytometry; the migration and invasion of cells in each group were evaluated by the Transwell experiment; the expression of cell proliferation markers such as proliferating cell nuclear antigen (PCNA), and epithelial-mesenchymal transition (EMT) markers, such as E-cadherin and vimentin in each group, was determined by immunoblotting experiment; the expression of NEAT1, miR-377-3p and wingless-related MMTV integration site 5A (WNT5A) mRNA in each group was determined by qRT-PCR experiment. Results ·Compared with the control group, the mRNA expression of NEAT1 in the model group increased, the mRNA expression of miR-377-3p decreased, the apoptosis rate decreased, the cell viability, and the number of migrating and invasive cells increased, the protein expression of PCNA and Vimentin increased, the expression of E-cadherin decreased, and WNT5A mRNA expression increased (all P=0.000). After NEAT1 was interfered, the apoptosis rate increased, the cell viability, and the number of migrating and invasive cells decreased, the protein expression of PCNA and Vimentin decreased, the expression of E-cadherin increased, and WNT5A mRNA expression decreased (all P=0.000); after miR-377-3p was inhibited, all the above indicators showed opposite expression trends (P<0.05). In addition, inhibition of miR-377-3p reversed the regulation of NEAT1 interference on cell proliferation, invasion, migration and apoptosis (all P=0.000). Conclusion ·The down-regulating of NEAT1 expression may increase miR-377-3p expression by competitive binding to miR-377-3p, reduce protein expression associated with the Wnt pathway, inhibit the abnormal proliferation, invasion and migration of HVSMCs induced by ox-LDL, and induce apoptosis.

Key words: long non-coding RNA (lncRNA), nuclear paraspeckle assembly transcript 1 (NEAT1), microRNA-377-3p (miR-377-3p), human vascular smooth muscle cell (HVSMC), proliferation, invasion and migration

中图分类号:

R543.5

袁咏梅, 程晓丹, 孙家安, 常东歌, 何莹莹, 刘畅. lncRNA NEAT1通过miR-377-3p/Wnt通路影响ox-LDL诱导的人血管平滑肌细胞增殖、侵袭迁移[J]. 上海交通大学学报（医学版）, 2022, 42(11): 1534-1541.

YUAN Yongmei, CHENG Xiaodan, SUN Jiaan, CHANG Dongge, HE Yingying, LIU Chang. lncRNA NEAT1 affects the proliferation, invasion and migration of ox-LDL-induced human vascular smooth muscle cells through miR-377-3p/Wnt pathway[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(11): 1534-1541.

图/表 8

表1 各基因引物序列

Tab 1 Primer sequences of each gene

Gene	Forward sequence （5'→3'）	Reverse sequence （5'→3'）
NEAT1	CAATGACTTGGGGATGATGCAAACAATTACTG	GTACTCCCCACCTACACACCCAC
WNT5A	GCGGGACTTTCTCAAGGACA	CGGCTGCCTATTTGCATCAC
β-actin	AGTGCGACGTGGACATCCG	TGGCTCTAACAGTCCGCCTAG
miR-377-3p	GGGAATCACACAAAGGCAAC	GTGCGTGTCGTGGAGTCG
U6	GCTTCGGCAGCACATATACTAAAAT	CGCTTCAGAATTTGCGTGTCAT

图1 各组细胞 NEAT1 、miR-377-3p及 WNT5A mRNA相对表达(n=6)Note： A. Relative expressions of NEAT1 in each group. B. Relative expressions of WNT5A in each group. C. Relative expressions of miR-377-3p in each group. ①P=0.000, compared with the control group; ②P=0.000, compared with the model group; ③P=0.000, compared with the transfection+inhibition group; ④P=0.002, compared with the model group; ⑤P=0.001, compared with the transfection+inhibition group.

Fig 1 Relative expression of NEAT1, miR-377-3p and WNT5A mRNA in each group of cells (n=6)

图2 流式细胞实验检测各组细胞凋亡情况

Fig 2 Detection of apoptosis in each group by flow cytometry

图3 Transwell实验检测各组细胞侵袭情况

Fig 3 Cell invasion in each group was detected by Transwell migration test

图4 Transwell实验检测各组细胞迁移情况

Fig 4 Cell migration was detected by Transwell invasion test

图5 各组侵袭迁移细胞数Note： A. Comparison of numbers of invasive cells in each group. B. Comparison of numbers of migrating cells in each group. n=6. ①P=0.000, compared with the control group; ②P=0.000, compared with the model group; ③P=0.000, compared with the transfection+inhibition group.

Fig 5 Number of invasive and migrating cells in each group

图6 免疫印迹检测各组细胞增殖及EMT标志蛋白表达

Fig 6 Western blotting detection of cell proliferation and EMT marker proteins expression in each groupNote: A. Control group. B. Model group. C. NEAT1 siRNA group. D. miR-377-3p inhibitor group. E. Transfection negative control group. F. Transfection+inhibition group.

图7 各组细胞PCNA、E-cadherin、vimentin蛋白相对表达Note： A. Comparison of PCNA in each group. B. Comparison of E-cadherin in each group. C. Comparison of Vimentin in each group. n=6. ①P=0.000, compared with the control group; ②P=0.000, compared with the model group; ③P=0.000, compared with the transfection+inhibition group.

Fig 7 Relative expression of PCNA, E-cadherin and vimentin proteins in each group of cells

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lncRNA NEAT1通过miR-377-3p/Wnt通路影响ox-LDL诱导的人血管平滑肌细胞增殖、侵袭迁移

lncRNA NEAT1 affects the proliferation, invasion and migration of ox-LDL-induced human vascular smooth muscle cells through miR-377-3p/Wnt pathway

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