miR-1254通过靶向CSF-1抑制胶质瘤细胞的增殖和侵袭能力

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

目的探讨miR-1254及其靶基因巨噬细胞集落剌激因子-1(CSF-1)在胶质瘤组织、胶质瘤细胞中的表达,以及miR-1254和CSF-1对胶质瘤细胞增殖和侵袭能力的影响。方法收集2017年4月至2019年5月在湖北省十堰市太和医院接受手术治疗的30例胶质瘤患者的临床病理标本以及癌旁组织,采用实时荧光定量PCR(qRT-PCR)检测胶质瘤组织、癌旁组织以及细胞株U87和人脑正常胶质细胞HEB中miR-1254和CSF-1 mRNA的表达水平。将胶质瘤U87细胞分为空白对照组、mimic NC组、miR-1254 mimic组以及siRNA NC组、CSF-1 siRNA组。采用qRT-PCR检测各组细胞miR-1254及CSF-1的mRNA表达水平;采用Western blotting检测各组细胞CSF-1的蛋白表达水平;采用双荧光素酶报告基因实验验证miR-1254和CSF-1基因的靶向关系;采用CCK-8法和Transwell侵袭实验检测细胞的增殖和侵袭能力。结果胶质瘤组织中miR-1254 mRNA的相对表达量为0.44±0.16,癌旁组织为1.15±0.28,差异具有统计学意义(t=12.914,P<0.001);胶质瘤组织中CSF-1 mRNA的相对表达量为1.96±0.27,癌旁组织为0.98±0.22,差异具有统计学意义(t=14.970,P<0.001)。胶质瘤细胞U87中miR-1254 mRNA的相对表达量为0.39±0.11,人脑正常胶质细胞HEB中为1.03±0.02,差异具有统计学意义(t=10.113,P=0.008);胶质瘤细胞U87中CSF-1 mRNA相对表达量为1.02±0.03,人脑正常胶质细胞HEB为0.32±0.13,差异具有统计学意义(t=9.037,P=0.009)。转染miR-1254后,CSF-1 mRNA、蛋白的表达水平均随miR-1254表达水平的升高而降低。双荧光素酶报告基因实验结果显示,与mimic NC组(1.04±0.12)比较,miR-1254 mimic组(0.31±0.02)CSF-1-WT细胞中荧光活性显著降低(t=10.430,P<0.001)。转染48 h后,空白对照组(0.71±0.01)、mimic NC组(0.68±0.04)、miR-1254 mimic组(0.35±0.01)细胞增殖能力差异具有统计学意义(F=252.651,P<0.001);miR-1254 mimic组与空白对照组相比,差异具有统计学意义(P<0.001);miR-1254 mimic组与mimic NC组相比,差异具有统计学意义(P<0.001)。空白对照组(0.71±0.01)、siRNA NC组(0.68±0.04)、CSF-1 siRNA组(0.25±0.01)细胞增殖能力差异具有统计学意义(F=320.309,P<0.001);CSF-1 siRNA组与空白对照组相比,差异具有统计学意义(P<0.001);CSF-1 siRNA组与siRNA NC组相比,差异具有统计学意义(P<0.001)。侵袭实验结果显示空白对照组(365±27)、mimic NC组(388±24)、miR-1254 mimic组(83±15)细胞穿膜数差异具有统计学意义(F=173.915,P<0.001);空白对照组(365±27)、siRNA NC组(404±32)、CSF-1 siRNA组(87±14)细胞穿膜数差异具有统计学意义(F=141.294,P<0.001)。结论miR-1254在胶质瘤组织和胶质瘤细胞株U87中的表达均显著下降,CSF-1在胶质瘤组织和胶质瘤细胞株U87中的表达均显著升高。过表达miR-1254可能通过靶向降低CSF-1的表达来抑制胶质瘤U87细胞的增殖和侵袭能力。

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.