lncRNA OIP5-AS1调节miR-942-5p/CHEK1轴对脑胶质瘤细胞生物学行为的影响

doi:10.11958/20220867

摘要/Abstract

摘要：

目的探讨长链非编码RNA（lncRNA）OPA相互作用蛋白5反义转录本1（OIP5-AS1）对脑胶质瘤细胞增殖、凋亡、迁移和侵袭的影响机制。方法收集33例胶质瘤患者（低级别胶质瘤14例、高级别胶质瘤19例）和33例颅脑损伤患者的组织标本。实时荧光定量PCR（qPCR）检测组织和细胞中OIP5-AS1、微小RNA-942-5p（miR-942-5p）和检查点激酶1（CHEK1）mRNA表达，分析胶质瘤组织中OIP5-AS1、miR-942-5p和CHEK1 mRNA表达水平的相关性。体外培养人脑胶质瘤细胞系U87、SHG-44、U251、H4和正常人星形胶质细胞NHA，Western blot检测细胞中CHEK1蛋白表达。将U87细胞分为对照（NC）组、siRNA阴性对照（si-NC）组、OIP5-AS1 siRNA（si-OIP5-AS1）组、si-OIP5-AS1+inhibitor阴性对照（si-OIP5-AS1+anti-NC）组、si-OIP5-AS1+miR-942-5p抑制剂（si-OIP5-AS1+anti-miR-942-5p）组，采用Lipofectamine 3000试剂进行转染。转染后，qPCR和Western blot检测细胞中OIP5-AS1、miR-942-5p和CHEK1 mRNA和蛋白表达水平；MTT法测定细胞增殖活性；流式细胞术检测细胞凋亡；Transwell实验检测细胞迁移和侵袭能力。最后通过双荧光素酶和RNA免疫沉淀（RIP）实验验证OIP5-AS1和miR-942-5p以及CHEK1和miR-942-5p的相互作用。结果 OIP5-AS1和CHEK1在脑胶质瘤组织和细胞中过表达，miR-942-5p呈低表达（均P<0.05）；相关分析显示，脑胶质瘤组织中OIP5-AS1与miR-942-5p的表达水平呈负相关，miR-942-5p与CHEK1 mRNA的表达水平呈负相关，CHEK1与OIP5-AS1 mRNA的表达水平呈正相关；且OIP5-AS1、CHEK1 mRNA在高级别胶质瘤组织中的表达明显高于低级别组织，而高级别胶质瘤中的miR-942-5p水平明显低于低级别组织（P<0.01）。沉默OIP5-AS1可显著上调miR-942-5p，抑制CHEK1的mRNA和蛋白表达（均P<0.05）；沉默OIP5-AS1可显著抑制U87细胞增殖、迁移和侵袭，促进U87细胞凋亡（均P<0.05）；下调miR-942-5p可上调CHEK1表达，阻断OIP5-AS1沉默对脑胶质瘤细胞生物学行为的影响（均P<0.05）。双荧光素酶和RIP实验证实miR-942-5p是OIP5-AS1的靶基因，CHEK1是miR-942-5p的下游靶基因。结论沉默OIP5-AS1可能通过上调miR-942-5p抑制CHEK1表达，抑制脑胶质瘤细胞的侵袭和迁移，并促进细胞凋亡。

关键词: 神经胶质瘤, RNA，长链非编码, 微RNAs, 细胞增殖, 细胞凋亡, 细胞运动, 肿瘤侵润, OPA相互作用蛋白5反义转录本1, 微小RNA-942-5p, 检查点激酶1

Abstract:

Objective To investigate the influence of long non-coding RNA (lncRNA) OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) on the proliferation, apoptosis, migration and invasion of glioma cells. Methods Tissue specimens from 33 glioma patients (14 low-grade gliomas and 19 high-grade gliomas) and 33 patients with craniocerebral injury were collected. Real-time quantitative PCR (qPCR) was used to detect the mRNA expression levels of OIP5-AS1, microRNA-942-5p (miR-942-5p) and checkpoint kinase 1 (CHEK1) in tissue samples and cells, and analyze the correlation between OIP5-AS1, miR-942-5p and CHEK1 mRNA expression level in glioma tissues. Human glioma cell lines U87, SHG-44, U251 and H4 and normal human astrocytes NHA were cultured in vitro. The expression of CHEK1 protein in cells were detected by Western blot assay. U87 cells were divided into the control (NC) group, the siRNA negative control (si-NC) group, the OIP5-AS1 siRNA (si-OIP5-AS1) group, the si-OIP5-AS1+inhibitor negative control (si-OIP5-AS1+anti-NC) group and the si-OIP5-AS1+miR-942-5p inhibitor (si-OIP5-AS1+anti-miR-942-5p) group. Cells were transfected with Lipofectamine 3000 reagent. After transfection, the expression levels of OIP5-AS1, miR-942-5p and CHEK1 mRNA and protein in cells were detected by qPCR and Western blot assay. MTT assay was used to detect the cell proliferation activity. Flow cytometry was used to detect cell apoptosis. Transwell assay was used to detect cell migration and invasion abilities. Finally, the interaction of OIP5-AS1 and miR-942-5p and CHEK1 and miR-942-5p were verified by dual luciferase and RNA immunoprecipitation (RIP) experiments. Results OIP5-AS1 and CHEK1 were overexpressed in glioma tissue and cells, while miR-942-5p was underexpressed (all P<0.05). Correlation analysis showed that OIP5-AS1 was negatively correlated with miR-942-5p, and miR-942-5p and CHEK1 mRNA was negatively correlated in glioma tissue. The expression level of CHEK1 mRNA was positively correlated with OIP5-AS1. The expression levels of OIP5-AS1 and CHEK1 mRNA were significantly higher in high-grade glioma tissue than those in low-grade glioma tissue, while the expression level of miR-942-5p was significantly lower in high-grade glioma tissue than that in low-grade glioma tissue (P<0.01). Silencing OIP5-AS1 significantly up-regulated miR-942-5p and inhibited the mRNA and protein expression of CHEK1 (P<0.05). Silencing OIP5-AS1 significantly inhibited the proliferation, migration and invasion of U87 cells, promoting apoptosis of U87 cells (P<0.05). Down-regulation of miR-942-5p could up-regulate the expression of CHEK1 and block the effects of OIP5-AS1 silencing on glioma cell proliferation, migration, invasion and apoptosis (P<0.05). Dual-luciferase and RIP experiments confirmed that miR-942-5p was a target of OIP5-AS1, and CHEK1 was a downstream target gene of miR-942-5p. Conclusion Silencing OIP5-AS1 may inhibit the expression of CHEK1 by up-regulating miR-942-5p, inhibit the invasion and migration of glioma cells, and promote cell apoptosis.

Key words: glioma, RNA, long noncoding, microRNAs, cell proliferation, apoptosis, cell movement, neoplasm invasiveness, OPA-interacting protein 5 antisense transcript 1, miRNA-942-5p, checkpoint kinase 1

中图分类号:

R739.41

图/表 13

表1 qPCR引物序列

Tab.1 Primer sequences for qPCR

基因名称	引物序列（5′→3′）	产物大小（bp）
OIP5-AS1	上游：TGCGAAGATGGCGGAGTAAG	325
	下游：TAGTTCCTCTCCTCTGGCCG	325
miR-942-5p	上游：GCCAGATCTTGATTGACTTACAGCCCAGTT	129
	下游：GCCGAATTCCACCTGTCTTTATTCCACCC	129
CHEK1	上游：TTCCATCAACTCATGGCAGG	214
	下游：ACGCTCACGATTATTATACCGAA	214
U6	上游：GTGCTCGCTTCGGCAGCACATATAC	89
	下游：AAAAATATGGAACGCTCACGAATTTG	89
GAPDH	上游：CATGAGAAGTATGACAACAGCT	197
	下游：AGTCCTTCCACGATACCAAAGT	197

表2 2组研究对象中OIP5-AS1、miR-942-5p和CHEK1 mRNA表达水平比较（n=33，$\bar{x}\pm s$）

Tab.2 Comparison of expression levels of OIP5-AS1, miR-942-5p and CHEK1 mRNA between the two groups

组织	OIP5-AS1	miR-942-5p	CHEK1
正常组织	1.02±0.13	0.99±0.10	1.00±0.12
脑胶质瘤组织	2.35±0.48	0.55±0.09	2.78±0.45
t	15.364^＊＊	18.788^＊＊	21.956^＊＊

表3 不同病理级别胶质瘤中OIP5-AS1、miR-942-5p和CHEK1 mRNA表达水平比较（$\bar{x}\pm s$）

Tab.3 Comparison of expression levels of OIP5-AS1, miR-942-5p and CHEK1 mRNA between gliomas of different pathological grades

肿瘤级别	n	OIP5-AS1	miR-942-5p	CHEK1
低级别胶质瘤	14	1.83±0.20	0.71±0.10	2.05±0.29
高级别胶质瘤	19	2.73±0.36	0.43±0.08	3.32±0.56
t		8.423^＊＊	8.938^＊＊	7.734^＊＊

图1 不同脑胶质瘤细胞中CHEK1蛋白表达

Fig.1 Expression of CHEK1 protein in different glioma cells

表4 不同脑胶质瘤细胞中OIP5-AS1、miR-942-5p和CHEK1水平比较（n=6，$\bar{x}\pm s$）

Tab.4 Comparison of OIP5-AS1, miR-942-5p and CHEK1 levels between different glioma cells

细胞	OIP5-AS1mRNA	miR-942-5pmRNA	CHEK1mRNA	CHEK1蛋白
NHA	1.01±0.11	1.02±0.09	0.99±0.10	0.24±0.04
U87	3.26±0.40^a	0.29±0.05^a	3.65±0.41^a	0.87±0.09^a
SHG-44	1.65±0.23^ab	0.73±0.08^ab	2.30±0.27^ab	0.55±0.07^ab
U251	2.70±0.35^abc	0.45±0.06^abc	1.49±0.23^abc	0.36±0.05^abc
H4	1.52±0.21^abd	0.81±0.10^abd	1.81±0.25^abc	0.44±0.06^abc
F	65.245^＊＊	82.843^＊＊	84.278^＊＊	83.290^＊＊

图2 各组U87细胞中CHEK1蛋白表达 A：NC组；B：si-NC组；C：si-OIP5-AS1组；D：si-OIP5-AS1+anti-NC组；E：si-OIP5-AS1+anti-miR-942-5p组。

Fig.2 CHEK1 protein expression in U87 cells in each group

表5 各组U87细胞中OIP5-AS1、miR-942-5p和CHEK1水平比较（n=6，$\bar{x}\pm s$）

Tab.5 Comparison of OIP5-AS1, miR-942-5p and CHEK1 levels between the five groups of U87 cells

组别		OIP5-AS1 mRNA	miR-942-5p mRNA
NC组		0.99±0.10	1.00±0.12
si-NC组		1.02±0.11	1.01±0.10
si-OIP5-AS1组		0.28±0.05^ab	3.54±0.39^ab
si-OIP5-AS1+anti-NC组		0.26±0.04^ab	3.60±0.41^ab
si-OIP5-AS1+anti-miR-942-5p组		0.30±0.05^ab	1.32±0.20^cd
F		165.157^＊＊	142.939^＊＊
组别	CHEK1 mRNA			CHEK1蛋白
NC组	1.00±0.11			0.82±0.09
si-NC组	0.98±0.09			0.84±0.10
si-OIP5-AS1组	0.39±0.05^ab			0.32±0.05^ab
si-OIP5-AS1+anti-NC组	0.36±0.06^ab			0.30±0.04^ab
si-OIP5-AS1+anti-miR-942-5p组	0.87±0.09^cd			0.72±0.08^cd
F	88.736^＊＊			75.734^＊＊

表6 各组U87细胞活力比较（n=6，$\bar{x}\pm s$）

Tab.6 Comparison of the viability of U87 cells between the five groups

组别	细胞活力（OD₅₇₀）
组别	24 h	48 h	72 h
NC组	0.36±0.05	0.58±0.07	0.95±0.11
si-NC组	0.38±0.06	0.60±0.07	0.94±0.12
si-OIP5-AS1组	0.23±0.04^ab	0.37±0.05^ab	0.56±0.07^ab
si-OIP5-AS1+anti-NC组	0.21±0.03^ab	0.36±0.05^ab	0.58±0.06^ab
si-OIP5-AS1+anti-miR-942-5p组	0.34±0.05^cd	0.55±0.06^cd	0.89±0.10^cd
F	16.568^＊＊	17.730^＊＊	25.820^＊＊

图3 各组U87细胞凋亡水平比较 A：NC组；B：si-NC组；C：si-OIP5-AS1组；D：si-OIP5-AS1+anti-NC组；E：si-OIP5-AS1+anti-miR-942-5p组；a与NC比较，b与si-NC组比较，c与si-OIP5-AS1组比较，d与si-OIP5-AS1+anti-NC组比较，P<0.05。

Fig.3 Comparison of apoptosis levels of U87 cells in each group

表7 各组U87细胞迁移、侵袭的数量比较（n=6，个/视野，$\bar{x}\pm s$）

Tab.7 Comparison of migration and invasion numbers of U87 cells between the five groups

组别	迁移细胞数	侵袭细胞数
NC组	143.20±18.57	86.72±10.31
si-NC组	150.36±20.14	91.06±12.15
si-OIP5-AS1组	62.81±9.56^ab	45.54±6.78^ab
si-OIP5-AS1+anti-NC组	59.44±7.12^ab	42.93±7.12^ab
si-OIP5-AS1+anti-miR-942-5p组	127.13±15.08^cd	83.60±9.45^cd
F	52.220^＊＊	38.177^＊＊

图4 各组U87细胞迁移和侵袭情况（×200） A：NC组；B：si-NC组；C：si-OIP5-AS1组；D：si-OIP5-AS1+anti-NC组；E：si-OIP5-AS1+anti-miR-942-5p组。

Fig.4 Migration and invasion of U87 cells in each group (×200)

图5 OIP5-AS1与脑胶质瘤细胞中miR-942-5p的相互作用 A：生物信息学分析预测miR-942-5p与OIP5-AS1的结合位点；B：双荧光素酶报告基因测定法测定miR-942-5p与OIP5-AS1结合的荧光素酶活性；C：RIP实验结合qPCR检测免疫沉淀物中Ago2与miR-942-5p和OIP5-AS1的结合。

Fig.5 Interaction of OIP5-AS1 with miR-942-5p in glioma cells

图6 miR-942-5p直接靶向癌基因CHEK1 A：生物信息学分析预测miR-942-5p与CHEK1的结合位点；B：双荧光素酶报告基因测定法测定miR-942-5p与CHEK1结合的荧光素酶活性；C：qPCR检测miR-942-5p过表达和敲低对CHEK1 mRNA水平的影响；D、E：Western blot检测miR-942-5p过表达和敲低对CHEK1蛋白表达的影响。

Fig.6 miR-942-5p directly targets the oncogene CHEK1

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