circFAT1对胶质母细胞瘤细胞生物过程的机制研究

doi:10.11958/20230224

天津医药 ›› 2023, Vol. 51 ›› Issue (8): 797-802.doi: 10.11958/20230224

circFAT1对胶质母细胞瘤细胞生物过程的机制研究

张立群¹(), 乌日吉木斯¹, 郑晓明², 汪琳¹, 韩玉秀¹, 张薇¹, 阎涛¹^,^△()

1 天津市神经病学研究所，天津医科大学总医院（邮编300052）
2 天津市第一中心医院妇产科

收稿日期:2023-02-22 修回日期:2023-05-16 出版日期:2023-08-15 发布日期:2023-08-10
通讯作者: ^△E-mail：taoyan@tmu.edu.cn
作者简介:张立群（1964），男，副主任医师，主要从事中枢神经系统疾病方面研究。E-mail：13920120758@163.com

The mechanisms of circFAT1 on the biological process of GBM cells

ZHANG Liqun¹(), WURI Jimusi¹, ZHENG Xiaoming², WANG Lin¹, HAN Yuxiu¹, ZHANG Wei¹, YAN Tao¹^,^△()

1 Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin 300052, China
2 Department of Obstertrics and Gynecology, Tianjin First Central Hospital

Received:2023-02-22 Revised:2023-05-16 Published:2023-08-15 Online:2023-08-10
Contact: ^△E-mail: taoyan@tmu.edu.cn

张立群, 乌日吉木斯, 郑晓明, 汪琳, 韩玉秀, 张薇, 阎涛. circFAT1对胶质母细胞瘤细胞生物过程的机制研究[J]. 天津医药, 2023, 51(8): 797-802.

ZHANG Liqun, WURI Jimusi, ZHENG Xiaoming, WANG Lin, HAN Yuxiu, ZHANG Wei, YAN Tao. The mechanisms of circFAT1 on the biological process of GBM cells[J]. Tianjin Medical Journal, 2023, 51(8): 797-802.

摘要/Abstract

摘要：

目的探讨circFAT1吸附miR-1182调控转化生长因子（TGF）-β/Smad信号通路对胶质母细胞瘤（GBM）增殖、侵袭、上皮-间充质转化（EMT）的调控作用及机制。方法收集50例GBM样本和5例非瘤脑组织（NBT）样本，随机抽取3例GBM和3例NBT样本进行组织RNA测序。体外培养GBM细胞系U87、U251、T98G、LN229及星形胶质细胞系NHA，通过qRT-PCR检测组织和细胞系中circFAT1表达。通过RNase R选择性降解线性转录本以明确circFAT1成环特性。将GBM细胞分为sh-对照组和sh-circFAT1组，采用慢病毒进行转染。转染后集落形成实验、Transwell实验、Western blot实验分别检测细胞增殖、侵袭及EMT能力。荧光原位杂交（FISH）检测circFAT1在GBM细胞内定位，通过双萤光素酶报告基因和RNA免疫共沉淀（RIP）实验验证circFAT1和miR-1182的相互作用。Western blot实验验证敲低circFAT1后或抑制miR-1182表达对TGF-β/Smad信号通路的调控作用。结果 circFAT1在GBM组织和细胞中表达增加（P<0.05）。RNase R可减少线性FAT1表达（P<0.05），而不影响circFAT1。敲除circFAT1抑制了GBM细胞的增殖、侵袭和EMT（P<0.05）。双萤光素酶报告基因和RIP实验证实circFAT1靶向结合miR-1182。敲低circFAT1后TGFB2、p-SMAD2、p-SMAD3蛋白表达下降，抑制miR-1182表达后GFB2、p-SMAD2、p-SMAD3蛋白表达升高。结论敲除circFAT1可抑制GBM细胞增殖、侵袭和EMT，其作用机制可能与吸附miR-1182调控TGF-β/Smad信号通路有关。

关键词: 胶质母细胞瘤, 细胞增殖, 转化生长因子β, 微RNAs, circFAT1, 上皮间充质转化

Abstract:

Objective To investigate the regulation and mechanism of circFAT1 on proliferation, invasion and epithelial-mesenchymal transition (EMT) of glioblastoma (GBM). Methods Fifty GBM samples and 5 non-tumor brain tissue (NBT) samples were collected, and 3 GBM and 3 NBT samples were randomly selected for tissue RNA sequencing. The expression levels of circFAT1 in U87, U251, T98G, LN229 GBM cell lines and NHA astrocyte lines were detected by qRT-PCR. The cyclic properties of circFAT1 were determined by selective degradation of linear transcripts by RNase R. GBM cells were divided into the sh-control group and the sh-circFAT1 group. Cell proliferation, invasion and EMT were detected by colony formation assay, transwell assay and Western blot assay after lentivirus transfection. Fluorescence in situ hybridization (FISH) was used to detect the localization of circFAT1 in GBM cells, and the interaction between circFAT1 and miR-1182 was verified by dual-luciferase reporter and RNA immunocoprecipitation (RIP) assay. Finally, Western blot assay was performed to verify the regulatory effect of circFAT1 knockdown or inhibition of miR-1182 expression on TGF-β/Smad signaling pathway. Results The expression of circFAT1 was increased in GBM tissue and cells (P<0.05). RNase R reduced linear FAT1 expression without affecting circFAT1 (P<0.05). Knockout of circFAT1 inhibited the proliferation, invasion and EMT of GBM cells (P<0.05). Dual-luciferase reporter and RIP assay confirmed that circFAT1 targeting on miR-1182. The protein levels of TGFB2, p-SMAD2 and p-SMAD3 were decreased after circFAT1 knockdown, while protein expressions of TGFB2, p-SMAD2 and p-SMAD3 increased after inhibiting the expression of miR-1182 (all P<0.05). Conclusion Knockout of circFAT1 can inhibit proliferation, invasion and EMT of GBM cells, and its mechanism may be related to the regulation of TGF-β/Smad signaling pathway by sponge miR-1182.

Key words: glioblastoma, cell proliferation, transforming growth factor beta, microRNAs, circFAT1, epithelial-mesenchymal transition

中图分类号:

R739.41

图/表 8

表1 qRT-PCR引物序列

Tab.1 Primer sequence for qRT-PCR

基因名称	引物序列（5′→3′）	产物大小（bp）
circFAT1	上游：GATGAGGACGCCAGAAGAGA 下游：CAAATGTCTCCCCATTGCTT	121
linerFAT1	上游：GGGCCAAAGACAAGGGAAAG 下游：TGAAAACACCAACGCCAGAG	243
β-actin	上游：CCTCTATGCCAACACAGTGC 下游：CCTGCTTGCTGATCCACATC	243
β-actin	上游：CCTCTATGCCAACACAGTGC 下游：CCTGCTTGCTGATCCACATC	206

图1 GBM中差异表达的circRNA

Fig.1 Differentially expressed circRNA in GBM

图2 circFAT1的环状结构模式图 A：平板克隆集落显示细胞增殖情况；B：Transwell实验显示细胞侵袭情况（结晶紫染色，×200，比例尺=100 μm）；C：Western blot检测EMT相关蛋白表达情况。

Fig.2 Schematic diagram of the circular structure of circFAT1

图3 下调circFAT1表达抑制GBM细胞的增殖、侵袭和EMT

Fig.3 Knocking down circFAT1 expression inhibited proliferation, invasion and EMT of GBM cells

表2 2组U87细胞中细胞增殖、侵袭和EMT相关蛋白表达情况（n=3，$\bar{x}±s$）

Tab.2 Comparison of the colony number, invasion rate and protein level of U87 cells between two groups

组别		细胞集落数/（个/视野）		侵袭率/%
sh-对照组		499±46		1.01±0.02
sh-circFAT1组		182±65		0.33±0.06
t		6.894^＊＊		19.513^＊＊
组别	E-cadherin		N-cadherin		Vimentin
sh-对照组	0.59±0.11		1.47±0.13		1.53±0.09
sh-circFAT1组	1.04±0.03		0.35±0.07		0.27±0.08
t	9.233^＊＊		6.884^＊＊		10.582^＊＊

图4 circFAT1与miR-1182的相互作用 A：circFAT1（绿色荧光）与胶质瘤细胞核（蓝色荧光）的位置关系（免疫荧光染色，×400，比例尺=50 μm）；B：RIP实验结合qRT-PCR检测免疫共沉淀中circFAT1的表达；C：circFAT1与miR-1182的结合位点及序列突变模式图；D：双萤光素酶报告基因验证circFAT1与miR-1182的靶向关系。

Fig.4 Interaction of circFAT1 and miR-1182

图5 不同处理条件下TGFB2、p-SMAD2、p-SMAD3的表达水平

Fig.5 Expression levels of TGFB2, p-SMAD2 and p-SMAD3 under different conditions

表3 各组中TGFB2、p-SMAD2、p-SMAD3蛋白相对表达水平比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of TGFB2, p-SMAD2 and p-SMAD3 protein expression levels between four groups

组别	TGFB2	p-SMAD2	p-SMAD3
A组	0.98±0.04	0.69±0.05	0.92±0.04
B组	0.75±0.05^a	0.36±0.01^a	0.56±0.05^a
C组	1.24±0.05^a	1.16±0.03^a	1.13±0.09^a
D组	0.92±0.02^bc	0.81±0.05^bc	0.94±0.04^bc
F	76.842^＊＊	147.204^＊＊	43.651^＊＊

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circFAT1对胶质母细胞瘤细胞生物过程的机制研究

The mechanisms of circFAT1 on the biological process of GBM cells

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