LncRNA FEZF1-AS1靶向调控miR-200c-3p对人肺成纤维细胞生物学行为的影响

doi:10.11958/20230719

天津医药 ›› 2024, Vol. 52 ›› Issue (3): 231-236.doi: 10.11958/20230719

LncRNA FEZF1-AS1靶向调控miR-200c-3p对人肺成纤维细胞生物学行为的影响

满君¹(), 高艳艳¹, 宋龙飞², 高福生¹^,^△()

1.潍坊医学院附属医院呼吸内科（邮编261035），2.康复医学科

收稿日期:2023-05-19 修回日期:2023-08-12 出版日期:2024-03-15 发布日期:2024-03-13
通讯作者: ^△E-mail：gaofs888@163.com
作者简介:满君（1988），女，主治医师，主要从事中医药治疗呼吸系统疾病方面的研究。E-mail：manjun0229@126.com
基金资助:
国家自然科学基金资助项目(82205079)

The effect of lncRNA FEZF1-AS1 targeting regulation of miR-200c-3p on biological behaviors of human lung fibroblasts

MAN Jun¹(), GAO Yanyan¹, SONG Longfei², GAO Fusheng¹^,^△()

1. Department of Respiratory Medicine, 2. Department of Rehabilitation Medicine, Affiliated Hospital of Weifang Medical University, Weifang 261035, China

Received:2023-05-19 Revised:2023-08-12 Published:2024-03-15 Online:2024-03-13
Contact: ^△E-mail: gaofs888@163.com

满君, 高艳艳, 宋龙飞, 高福生. LncRNA FEZF1-AS1靶向调控miR-200c-3p对人肺成纤维细胞生物学行为的影响[J]. 天津医药, 2024, 52(3): 231-236.

MAN Jun, GAO Yanyan, SONG Longfei, GAO Fusheng. The effect of lncRNA FEZF1-AS1 targeting regulation of miR-200c-3p on biological behaviors of human lung fibroblasts[J]. Tianjin Medical Journal, 2024, 52(3): 231-236.

摘要/Abstract

摘要：

目的探讨FEZ家族锌指1-反义RNA1（LncRNA FEZF1-AS1）靶向调控miR-200c-3p对人肺成纤维细胞HLF生物学行为的影响。方法采用转化生长因子β1（TGF-β1）诱导HLF向肌成纤维细胞转化，分为空白对照组（Blank组）和造模组（HLF+TGF-β1组），另根据转染质粒不同将细胞分为Blank组、TGF-β1+Si LncRNA FEZF1-AS1 NC组和TGF-β1+Si LncRNA FEZF1-AS1组。采用Western blot法检测α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白（CollagenⅠ）和波形蛋白（Vimentin）蛋白的表达。采用实时荧光定量PCR（qRT-PCR）检测LncRNA FEZF1-AS1和miR-200c-3p的表达。采用CCK-8法检测细胞增殖，细胞划痕实验检测迁移能力，Transwell实验检测侵袭能力；采用双萤光素酶实验检测FEZF1-AS1与miR-200c-3p的靶向作用关系。结果与Blank组比较，HLF+TGF-β1组α-SMA、CollagenⅠ、Vimentin蛋白表达及LncRNA FEZF1-AS1表达水平升高，miR-200c-3p表达水平降低（P<0.05）；与TGF-β1+Si LncRNA FEZF1-AS1 NC组比较，TGF-β1+Si LncRNA FEZF1-AS1组细胞增殖、迁移、侵袭能力下降，LncRNA FEZF1-AS1表达及α-SMA、CollagenⅠ、Vimentin蛋白表达水平降低，miR-200c-3p表达水平升高（P<0.05）；FEZF1-AS1与miR-200c-3p基因序列上存在结合位点。结论 LncRNA FEZF1-AS1通过抑制miR-200c-3p促进特发性肺间质纤维化的发生、发展。

关键词: 特发性肺间质纤维化, 肺成纤维细胞, 肌成纤维细胞, FEZ家族锌指1-反义RNA1, 微小RNA-200c-3p

Abstract:

Objective To investigate the effect of FEZ family zinc finger 1-antisense RNA 1 (LncRNA FEZF1-AS1) targeting regulation of miR-200c-3p expression on biological behaviors of human lung fibroblasts (HLF). Methods Transforming growth factor β1 (TGF-β1) was used to induce the transformation of HLF into myofibroblasts, which were divided into the Blank group and the model group (HLF+TGF-β1 group). According to different transfection plasmid, cells were divided into the Blank group, the TGF-β1+Si LncRNA FEZF1-AS1 NC group and the TGF-β1+Si LncRNA FEZF1-AS1 group. The protein expressions of α-SMA, Collagen Ⅰ and Vimentin were detected by Western blot assay. The expressions of LncRNA FEZF1-AS1 and miR-200c-3p were detected by quantitative real-time PCR (qRT-PCR). Cell proliferation ability was detected by CCK-8 method, migration ability was detected by cell scratch experiment and invasion ability was detected by Transwell assay. The targeting relationship between FEZF1-AS1 and miR-200c-3p was detected by dual-luciferase reporter assay. Results Compared with the Blank group, protein expressions of α-SMA, Collagen Ⅰ, Vimentin and the expression of LncRNA FEZF1-AS1 were increased in the HLF+TGF-β1 group (P<0.05), and the expression of miR-200c-3p was decreased (P<0.05). Compared with the TGF-β1+Si LncRNA FEZF1-AS1 NC group, cell proliferation, migration, invasion ability, LncRNA FEZF1-AS1 expression, protein expressions of α-SMA, Collagen Ⅰ and Vimentin were decreased in the TGF-β1+Si LncRNA FEZF1-AS1 group (P<0.05), and the expression of miR-200c-3p was increased (P<0.05). There were binding sites between miR-200c-3p and FEZF1-AS1 gene sequence. Conclusion LncRNA FEZF1-AS1 promotes the formation and progression of idiopathic pulmonary interstitial fibrosis by inhibiting miR-200c-3p.

Key words: idiopathic pulmonary interstitial fibrosis, lung fibroblasts, myofibroblasts, FEZ family zinc finger 1-antisense RNA1, microRNA-200c-3p

中图分类号:

R563.9

图/表 11

表1 引物序列

Tab.1 Primer sequence

基因名称	引物序列（5'→3'）	产物大小/bp
LncRNA FEZF1-AS1	上游：TTAGGAGGCTTGTTCTGTGT	239
LncRNA FEZF1-AS1	下游：GCGCAGGTACTTAAGAAAGA	239
miR-200c-3p	上游：UAAUACUGCCGGGUAAUGAUGGA	70
	下游：UCCAUCAUUACCCGGCAGUAUUA	70
GAPDH	上游：GCCTTCCGTGTCCCCACTGC	216
	下游：GGCTGGTGGTCCAGGGGTCT	216
U6	上游：CTCGCTTCGGCAGCACA	65
	下游：AACGCTTCACGAATTTGCGT	65

表2 2组细胞α-SMA、Collagen Ⅰ、Vimentin蛋白表达水平比较（n=3，$\bar{x} \pm s$）

Tab.2 Comparison of α-SMA, Collagen Ⅰ and Vimentin protein expressions between two groups of cells

组别	α-SMA	Collagen Ⅰ	Vimentin
Blank组	0.68±0.02	0.37±0.05	0.66±0.06
HLF+TGF-β1组	0.90±0.02	0.68±0.05	0.98±0.04
t	7.621^＊	7.431^＊	4.583^＊

图1 2组细胞α-SMA、Collagen Ⅰ、Vimentin蛋白表达水平

Fig.1 Protein expressions of α-SMA, Collagen Ⅰ and Vimentin in two groups of cells

表3 各组细胞增殖能力比较（n=3，OD450，$\bar{x} \pm s$）

Tab.3 Comparison of cell proliferation between three groups

组别	0 h	24 h	48 h	F
Blank组	0.49±0.00	0.65±0.01^A	1.08±0.02^AB	640.000^＊
TGF-β1+Si LncRNA FEZF1-AS1 NC组	0.48±0.01	1.08±0.03^aA	1.87±0.03^aAB	641.500^＊
TGF-β1+Si LncRNA FEZF1-AS1组	0.47±0.02	0.84±0.01^abA	1.53±0.07^abAB	185.300^＊
F	0.719	108.400^＊	79.310^＊

表4 各组细胞划痕愈合率、侵袭细胞数比较（n=3，$\bar{x} \pm s$）

Tab.4 Comparison of cell scratch healing rate and invasion number between three groups

组别	划痕愈合率/%	侵袭细胞数/（个/视野）
Blank组	61.98±1.56	236.00±2.08
TGF-β1+Si LncRNA FEZF1-AS1 NC组	89.16±0.78^a	413.30±16.23^a
TGF-β1+Si LncRNA FEZF1-AS1组	79.74±0.68^ab	329.30±25.22^ab
F	162.500^＊	26.120^＊

图2 沉默LncRNA FEZF1-AS1对HLF细胞划痕愈合的影响（×100） A：Blank组；B：TGF-β1+Si LncRNA FEZF1-AS1 NC组；C：TGF-β1+Si LncRNA FEZF1-AS1组。

Fig.2 Effects of silencing LncRNA FEZF1-AS1 on the scratch healing of HLF cells (×100)

图3 沉默LncRNA FEZF1-AS1对HLF细胞侵袭的影响（结晶紫染色，×100）

Fig.3 Effects of silencing LncRNA FEZF1-AS1 on the invasion of HLF cells (crystal violet staining, ×100)

图4 LncRNA FEZF1-AS1促进肺间质纤维化相关蛋白的表达 A：Blank组；B：TGF-β1+Si LncRNA FEZF1-AS1 NC组；C：TGF-β1+Si LncRNA FEZF1-AS1组。

Fig.4 LncRNA FEZF1-AS1 promoted the expression of interstitial fibrosis related proteins

表5 各组细胞α-SMA、CollagenⅠ、Vimentin蛋白表达（n=3，$\bar{x} \pm s$）

Tab.5 Expressions of α-SMA, CollagenⅠ and Vimentin in each group

组别	α-SMA	CollagenⅠ	Vimentin
Blank组	0.57±0.01	0.32±0.02	0.33±0.01
TGF-β1+Si LncRNA FEZF1-AS1 NC组	0.85±0.02^a	0.84±0.01^a	0.92±0.01^a
TGF-β1+Si LncRNA FEZF1-AS1 组	0.70±0.02^ab	0.67±0.03^ab	0.67±0.01^ab
F	65.610^＊	129.100^＊	1170.000^＊

表6 各组细胞LncRNA FEZF1-AS1、miR-200c-3p表达水平比较（n=3，$\bar{x} \pm s$）

Tab.6 Expressions of LncRNA FEZF1-AS1 and miR-200c-3p in each group

组别	LncRNA FEZF1-AS1	miR-200c-3p
Blank组	1.28±0.11	1.05±0.02
TGF-β1+Si LncRNA FEZF1-AS1 NC组	7.53±0.14^a	0.15±0.01^a
TGF-β1+Si LncRNA FEZF1-AS1组	2.62±0.04^ab	0.45±0.00^ab
F	952.100^＊	1 065.000^＊

图5 LncRNA FEZF1-AS1与miR-200c-3p的结合位点

Fig.5 Binding sites of LncRNA FEZF1-AS1 and miR-200c-3p

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LncRNA FEZF1-AS1靶向调控miR-200c-3p对人肺成纤维细胞生物学行为的影响

The effect of lncRNA FEZF1-AS1 targeting regulation of miR-200c-3p on biological behaviors of human lung fibroblasts

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