lncRNA OIP5-AS1 regulates the effect of miR-942-5p/CHEK1 axis on the biological behavior of glioma cells

doi:10.11958/20220867

Abstract

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

CLC Number:

R739.41

Figures/Tables 13

References 17

[1]	MITCHELL D, SHIREMAN J M, DEY M. Surgical neuro-oncology:management of glioma[J]. Neurol Clin, 2022, 40(2):437-453. doi:10.1016/j.ncl.2021.11.003.
[2]	BAUSART M, PRÉAT V, MALFANTI A. Immunotherapy for glioblastoma:the promise of combination strategies[J]. J Exp Clin Cancer Res, 2022, 41(1):35-56. doi:10.1186/s13046-022-02251-2.
[3]	CHAE Y, ROH J, KIM W. The roles played by long non-coding RNAs in glioma resistance[J]. Int J Mol Sci, 2021, 22(13):6834. doi:10.3390/ijms22136834.
[4]	ZHENG C, CHU M, CHEN Q, et al. The role of lncRNA OIP5-AS1 in cancer development and progression[J]. Apoptosis, 2022, 27(5/6):311-321. doi:10.1007/s10495-022-01722-3.
[5]	LI H, WANG D, YI B, et al. SUMOylation of IGF2BP2 promotes vasculogenic mimicry of glioma via regulating OIP5-AS1/miR-495-3p axis[J]. Int J Biol Sci, 2021, 17(11):2912-2930. doi:10.7150/ijbs.58035.
[6]	SUN W L, KANG T, WANG Y Y, et al. Long noncoding RNA OIP5-AS1 targets Wnt-7b to affect glioma progression via modulation of miR-410[J]. Biosci Rep, 2019, 39(1):BSR20180395. doi:10.1042/BSR20180395.
[7]	ALKAN A H, AKGÜL B. Endogenous miRNA sponges[J]. Methods Mol Biol, 2022, 2257:91-104. doi:10.1007/978-1-0716-1170-8_5.
[8]	ZHAO W, XIN L, TANG L, et al. A positive feedback loop between LINC01605 and NF-κB pathway promotes tumor growth in nasopharyngeal carcinoma[J]. RNA Biol, 2022, 19(1):482-495. doi:10.1080/15476286.2022.2027149.
[9]	ZHANG W, MAO K, LIU S, et al. miR-942-5p promotes the proliferation and invasion of human melanoma cells by targeting DKK3[J]. J Recept Signal Transduct Res, 2021, 41(2):180-187. doi:10.1080/10799893.2020.1804280.
[10]	LIU N Z, LI T, LIU C M, et al. Hsa_circ_0000337 promotes proliferation,migration and invasion in glioma by competitively binding miRNA-942-5p and thus upregulates MAT2A[J]. Eur Rev Med Pharmacol Sci, 2020, 24(23):12251-12257. doi:10.26355/eurrev_202012_24017.
[11]	KRELL A, WOLTER M, STOJCHEVA N, et al. MiR-16-5p is frequently down-regulated in astrocytic gliomas and modulates glioma cell proliferation,apoptosis and response to cytotoxic therapy[J]. Neuropathol Appl Neurobiol, 2019, 45(5):441-458. doi:10.1111/nan.12532.
[12]	KHANNA A, THOMS J A I, STRINGER B W, et al. Constitutive CHK1 expression drives a pSTAT3-CIP2A circuit that promotes glioblastoma cell survival and growth[J]. Mol Cancer Res, 2020, 18(5):709-722. doi:10.1158/1541-7786.MCR-19-0934.
[13]	LI F, SHEN Z Z, XIAO C M, et al. YY1-mediated up-regulation of lncRNA LINC00466 facilitates glioma progression via miR-508/CHEK1[J]. J Gene Med, 2021, 23(1):e3287. doi:10.1002/jgm. 3287.
[14]	LIANG J, LIU C, XU D, et al. LncRNA NEAT1 facilitates glioma progression via stabilizing PGK1[J]. J Transl Med, 2022, 20(1):80. doi:10.1186/s12967-022-03273-2.
[15]	CHENG G, ZHENG J, WANG L. LncRNA SNHG7 promotes glioma cells viability,migration and invasion by regulating miR-342-3p/AKT2 axis[J]. Int J Neurosci, 2021, 131(12):1190-1202. doi:10.1080/00207454.2020.1790556.
[16]	LI Z, LI M, XIA P, et al. Targeting long non-coding RNA PVT1/TGF-β/Smad by p53 prevents glioma progression[J]. Cancer Biol Ther, 2022, 23(1):225-233. doi:10.1080/15384047.2022.2042160.
[17]	ZHONG J, CHEN J, WANG B, et al. OIP5-AS1:A fascinating long noncoding RNA in carcinoma[J]. Curr Pharm Des, 2021, 27(46):4699-4706. doi:10.2174/1381612827666210927105558.

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

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

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^＊＊

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^＊＊

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^＊＊