miR-1254 inhibits the proliferation and invasion of glioma cells by targeting CSF-1

doi:10.3760/cma.j.cn371439-20200416-00082

Abstract

Abstract:

Objective To investigate the expressions of miR-1254 and its target gene colony-stimulating factor-1 (CSF-1) in glioma tissues and glioma cells, and the effects of miR-1254 and CSF-1 on the proliferation and invasion of glioma cells. Methods The clinicopathological specimens and paracancerous tissues of 30 patients with glioma who underwent surgical treatment in Shiyan Taihe Hospital of Hubei Province from April 2017 to May 2019 were collected. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect the expression levels of miR-1254 and CSF-1 mRNA in glioma tissues, paracancerous tissues, U87 cells and human brain normal glial cells HEB. The glioma U87 cells were divided into blank control group, mimic NC group, miR-1254 mimic group, and siRNA NC group, CSF-1 siRNA group. The expression levels of miR-1254 and CSF-1 mRNA were detected by qRT-PCR. The protein expression levels of CSF-1 in each group were detected by Western blotting. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-1254 and CSF-1 gene. CCK-8 method and Transwell invasion experiment were used to detect the proliferation and invasion ability of cells. Results The relative expression of miR-1254 mRNA in glioma tissues was 0.44±0.16, that in adjacent tissues was 1.15±0.28, and there was a statistically significant difference (t=12.914, P<0.001). The relative expression of CSF-1 mRNA in glioma tissues was 1.96±0.27, that in adjacent tissues was 0.98±0.22, and there was a statistically significant difference (t=14.970, P<0.001). The relative expression of miR-1254 mRNA in glioma cells U87 was 0.39±0.11, that in human brain normal glial cells HEB was 1.03±0.02, and there was a statistically significant difference (t=10.113, P=0.008). The relative expression of CSF-1 mRNA in glioma cell U87 was 1.02±0.03, that in human brain normal glial cell HEB was 0.32±0.13, and there was a statistically significant difference (t=9.037, P=0.009). The expression levels of CSF-1 mRNA and protein decreased with the increase of miR-1254 after transfection of miR-1254. The results of dual luciferase reporter gene assay showed that compared with the mimic NC group (1.04±0.12), the fluorescence activity of CSF-1-WT cells in the miR-1254 mimic group (0.31±0.02) was significantly reduced (t=10.430, P<0.001). The difference in cell proliferation ability among the blank control group (0.71±0.01), mimic NC group (0.68±0.04) and miR-1254 mimic group (0.35±0.01) was statistically significant 48 h after transfection (F=252.651, P<0.001). The difference was statistically significant between miR-1254 mimic group and blank control group (P<0.001), and the difference was also statistically significant between miR-1254 mimic group and mimic NC group(P<0.001). The difference in cell proliferation ability among the blank control group (0.71±0.01), siRNA NC group (0.68±0.04) and CSF-1 siRNA group (0.25±0.01) was statistically significant (F=320.309, P<0.001). The difference was statistically significant between CSF-1 siRNA group and blank control group (P<0.001), and the difference was also statistically significant between CSF-1 siRNA group and siRNA NC group (P<0.001). Invasion experiments showed that the difference of transmembrane cells number among the blank control group (365±27), mimic NC group (388±24) and miR-1254 mimic group (83±15) was statistically significant (F=173.915, P<0.001). The blank control group (365±27), siRNA NC group (404±32) and CSF-1 siRNA group (87±14) had statistically significant difference in the number of transmembrane cells (F=141.294, P<0.001). Conclusion The expressions of miR-1254 in glioma tissues and glioma cells U87 are significantly decreased, and the expressions of CSF-1 in glioma tissues and glioma cells U87 are significantly increased. Overexpression of miR-1254 may inhibit the proliferation and invasion of glioma U87 cells by reducing the expression of CSF-1 targetedly.

Key words: Glioma, Macrophage colony-stimulating factor, Cell proliferation, Neoplasm invasiveness, miR-1254

Chen Liang, Qin Jun, Lei Junrong, Liu Jun, Wang Lu. miR-1254 inhibits the proliferation and invasion of glioma cells by targeting CSF-1[J]. Journal of International Oncology, 2020, 47(10): 577-584.

Figures/Tables 11

References 18

[1]	杨卓, 王新军, 付旭东, 等. 脑胶质瘤中Fbxw7蛋白的表达[J]. 中国神经精神疾病杂志, 2019,45(5):305-309. DOI: 10.3969/j.issn.1002-0152.2019.05.009.
[2]	Goode B, Mondal G, Hyun M, et al. A recurrent kinase domain mutation in PRKCA defines chordoid glioma of the third ventricle[J]. Nat Commun, 2018,9(1):810. DOI: 10.1038/s41467-018-02826-8. doi: 10.1038/s41467-018-02826-8 pmid: 29476136
[3]	Zhang M, Huang N, Yang X, et al. A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis[J]. Oncogene, 2018,37(13):1805-1814. DOI: 10.1038/s41388-017-0019-9. doi: 10.1038/s41388-017-0019-9 pmid: 29343848
[4]	Disney-Hogg L, Cornish AJ, Sud A, et al. Impact of atopy on risk of glioma: a mendelian randomisation study[J]. BMC Med, 2018,16(1):42. DOI: 10.1186/s12916-018-1027-5. pmid: 29540232
[5]	李京泰, 周恩相. 成纤维细胞生长因子受体1及miR-1254表达缺失在乳腺癌中的表达意义研究[J]. 中国妇幼健康研究, 2017, (S3):489-490.
[6]	Li B, Chen P, Wang J, et al. MicroRNA-1254 exerts oncogenic effects by directly targeting RASSF9 in human breast cancer[J]. Int J Oncol, 2018,53(5):2145-2156. DOI: 10.3892/ijo.2018.4530.
[7]	Li H, Yang T, Shang D, et al. MiR-1254 promotes lung cancer cell proliferation by targeting SFRP1[J]. Biomed Pharmacother, 2017,92:913-918. DOI: 10.1016/j.biopha.2017.05.116. doi: 10.1016/j.biopha.2017.05.116 pmid: 28605875
[8]	Zhou J, Liu X, Wang CH, et al. Decreased expression of miR-1254 is associated with cancer aggressiveness and predicts poor outcome in cervical cancer[J]. Eur Rev Med Pharmacol Sci, 2018,22(10):2997-3001. DOI: 10.26355/eurrev_201805_15056. pmid: 29863243
[9]	Li Q, Shen W, Li X, et al. The lncRNA n340790 accelerates carcinogenesis of thyroid cancer by regulating miR-1254[J]. Am J Transl Res, 2017,9(5):2181-2194.
[10]	Okugawa Y, Toiyama Y, Ichikawa T, et al. Colony-stimulating factor-1 and colony-stimulating factor-1 receptor co-expression is associated with disease progression in gastric cancer[J]. Int J Oncol, 2018,53(2):737-749. DOI: 10.3892/ijo.2018.4406. doi: 10.3892/ijo.2018.4406 pmid: 29767252
[11]	Colombo M, de Fraga Dias A, et al. Kaempferol-loaded mucoadhesive nanoemulsion for intranasal administration reduces glioma growth in vitro[J]. Int J Pharm, 2018,543(1-2):214-223. DOI: 10.1016/j.ijpharm.2018.03.055. doi: 10.1016/j.ijpharm.2018.03.055 pmid: 29605695
[12]	Shibao S, Ueda R, Saito K, et al. A pilot study of peptide vaccines for VEGF receptor 1 and 2 in patients with recurrent/progressive high grade glioma[J]. Oncotarget, 2018,9(30):21569-21579. DOI: 10.18632/oncotarget.25131. pmid: 29765561
[13]	Bester R, Burger JT, Maree HJ. Differential expression of miRNAs and associated gene targets in grapevine leafroll-associated virus 3-infected plants[J]. Arch Virol, 2017,162(4):987-996. DOI: 10.1007/s00705-016-3197-9. doi: 10.1007/s00705-016-3197-9 pmid: 28025711
[14]	Jiang M, Shi L, Yang C, et al. MiR-1254 inhibits cell proliferation, migration, and invasion by down-regulating Smurf1 in gastric cancer[J]. Cell Death Dis, 2019,10(1):32. DOI: 10.1038/s41419-018-1262-x.
[15]	Chen R, Zhang Y, Zhang X. MiR-1254 functions as a tumor suppressor in oral squamous cell carcinoma by targeting CD36[J]. Technol Cancer Res Treat, 2019,18:1533033819859447. DOI: 10.1177/1533033819859447. doi: 10.1177/1533033819859447 pmid: 31401948
[16]	Chu YM, Peng HX, Xu Y, et al. MicroRNA-1254 inhibits the migration of colon adenocarcinoma cells by targeting PSMD10[J]. J Dig Dis, 2017,18(3):169-178. DOI: 10.1111/1751-2980.12463. pmid: 28296190
[17]	Sapi E. The role of CSF-1 in normal physiology of mammary gland and breast cancer: an update[J]. Exp Biol Med (Maywood), 2004,229(1):1-11. DOI: 10.1177/153537020422900101.
[18]	Hua F, Tian Y, Gao Y, et al. Colony stimulating factor 1 receptor inhibition blocks macrophage infiltration and endometrial cancer cell proliferation[J]. Mol Med Rep, 2019,19(4):3139-3147. DOI: 10.3892/mmr.2019.9963.

基因名称	引物序列
miR-1254	正向引物:5'-AGCCTGGAAGCTGGAGCCTGCAGT-3'
	反向引物:5'-GCGAGCACAGAATTAATACGAC-3'
内参U6	正向引物:5'-CTCGCTTCGGCAGCACA-3'
	反向引物:5'-AACGCTTCACGAATTTGCGT-3'
CSF-1	正向引物:5'-ACCCCTCCACCCTCTCTG-3'
	反向引物:5'-CTGCCCCTTCACTTGCTG-3'
GAPDH	正向引物:5'-AACGGATTTGGTCGTATTG-3'
	反向引物:5'-GGAAGATGGTGATGGGATT-3'

组别	miR-1254 mRNA	CSF-1 mRNA
空白对照组	0.44±0.02	1.20±0.01
mimic NC组	0.45±0.01	1.18±0.02
miR-1254 mimic组	1.20±0.02^ab	0.50±0.02^ab
F值	4 561.419	4 503.095
P值	<0.001	<0.001

组别	A₄₅₀值
组别	0 h	48 h	72 h	96 h
空白对照组	0.20±0.03	0.71±0.01	0.87±0.04	0.98±0.05
mimic NC组	0.22±0.02	0.68±0.04	0.82±0.05	0.93±0.05
miR-1254 mimic组	0.19±0.03	0.35±0.01^ab	0.50±0.01^ab	0.66±0.01^ab
F值	0.875	252.651	87.500	55.657
P值	0.464	<0.001	<0.001	<0.001

组别	A₄₅₀值
组别	0 h	48 h	72 h	96 h
空白对照组	0.20±0.03	0.71±0.01	0.87±0.04	0.98±0.05
siRNA NC组	0.20±0.04	0.68±0.04	0.84±0.09	1.02±0.03
CSF-1 siRNA组	0.18±0.02	0.25±0.01^ab	0.40±0.01^ab	0.57±0.01^ab
F值	0.590	320.309	61.693	163.287
P值	0.583	<0.001	<0.001	<0.001