Hsa_circRNA_0006168在脑胶质瘤中的表达及其生物学意义

Expression of Hsa_circRNA_0006168 in gliomas and its biological significance

目的探讨胶质瘤细胞及脑胶质瘤组织中Hsa_circRNA_0006168的表达水平及其对胶质瘤细胞的增殖、迁移、侵袭能力,以及对替莫唑胺(TMZ)敏感性的影响,并探讨Hsa_circRNA_0006168的表达水平对脑胶质瘤患者生存期的影响.方法选取2018年1月至2021年5月儋州市人民医院神经外科采用手术治疗的86例脑胶质瘤患者的肿瘤组织标本和25例因颅脑外伤行内减压术的非肿瘤脑组织标本进行细胞培养.采用人脑正常胶质细胞株(HEB)及人胶质瘤细胞株(U251、A172、U87和SHG-4)进行相关细胞研究.采用实时荧光定量PCR(RT-qPCR)技术检测所有细胞Hsa_circRNA_0006168的表达水平.Kaplan-Meier法分析Hsa_circRNA_0006168表达水平与脑胶质瘤患者生存期的关系,单因素及多因素Cox回归分析法分析Hsa_circRNA_0006168表达水平对脑胶质瘤患者生存期的影响.选择胶质瘤细胞U251,分为siRNA-circ抑制组、siRNA-对照组和空白对照组.CCK-8法检测细胞的增殖能力和对TMZ的敏感性,Transwell小室实验检测细胞迁移和侵袭能力.结果与非肿瘤脑组织标本及人脑正常胶质细胞比较,脑胶质瘤组织及所有胶质瘤细胞中的Hsa_circRNA_0006168的表达水平均明显升高(均P<0.001).世界卫生组织(WHO)高级别脑胶质瘤患者肿瘤组织中Hsa_circRNA_0006168的表达水平高于低级别脑胶质瘤(分别为2.06±0.81、0.97±0.38,P<0.001).Kaplan-Meier分析结果显示,Hsa_circRNA_0006168高表达与脑胶质瘤患者生存期短有关(P<0.001).多因素Cox回归分析显示,除高级别脑胶质瘤、肿瘤远隔部位转移、TMZ耐药外(均P<0.05),Hsa_circRNA_0006168高表达是影响脑胶质瘤患者生存期的危险因素(HR=3.32,95%CI:2.92~7.31,P<0.001).siRNA-circ抑制组、siRNA-对照组及空白对照组Hsa_circRNA_0006168的表达水平分别为0.28±0.13、0.90±0.34、0.87±0.31,差异有统计学意义(P<0.001).siRNA-circ抑制组细胞增殖率低于siRNA-对照组(P<0.001).Transwell小室实验结果显示,U251细胞培养24 h,siRNA-circ抑制组、siRNA-对照组及空白对照组的细胞迁移数分别为(81.35±10.72)个、(173.26±28.40)个、(174.80±27.25)个;培养48h,3组的侵袭细胞数分别为(39.85±6.24)个、(106.48±14.73)个、(104.72±13.84)个,差异均有统计学意义(均P<0.001).给予不同浓度的TMZ处理U251细胞24 h,与siRNA-对照组比较,siRNA-circ抑制组细胞存活率均降低,差异均有统计学意义(P<0.001).结论Hsa_circRNA_0006168在脑胶质瘤组织及细胞中均呈高表达,其高表达与脑胶质瘤患者的预后差有关;下调Hsa_circRNA_0006168表达能抑制胶质瘤细胞的增殖、迁移、侵袭,并能促进对TMZ的敏感性.

Objective To investigate the relationship between expression level of Hsa_circRNA_0006168 and prognosis of patients with gliomas,the effect of Hsa_circRNA_0006168 on glioma cell proliferation,migration and invasion,and the influence on temozolomide(TMZ)sensitivity.Methods A total of 86 tissue samples from glioma patients and 25 normal brain tissue samples were collected,and the expression level of Hsa_circRNA_0006168 was detected by real-time quantitative PCR.Kaplan-Meier method was used to analyze the relationship between the expression of Hsa_circRNA_0006168 and the survival of patients with gliomas.Univariate and multivariate Cox regression analyses were used to analyze the factors affecting the prognosis of patients with gliomas.Glioma cells U251 were selected and randomly divided into siRNA-circ inhibition group,siRNA-NC group and NC group.CCK-8 method was used to detect the proliferation of cells and the sensitivity of temozolomide(TMZ).Transwell assay was used to detect cell migration and invasion abilities Results Compared with normal brain tissue and normal human glioma cells,Hsa_circRNA_0006168 expression level in glioma tissues and glioma cells was significantly increased(P<0.001).The expression level of Hsa_circRNA_0006168 in the tissues of high-grade glioma patients was significantly higher than that of low-grade glioma patients(2.06±0.81 vs.0.97±0.38,P<0.001).Kaplan-Meier analysis showed that high expression of Hsa_circRNA_0006168 was associated with shorter survival in glioma patients(P<0.001).Multivariate Cox regression analysis showed that in addition to high-grade glioma,distant tumor metastasis and TMZ resistance(all P<0.05),high expression of Hsa_circRNA_0006168 was also a risk factor affecting the survival of patients with gliomas(HR=3.32,95%CI:2.92-7.31,P<0.001).The expression level of Hsa_circRNA_0006168 in siRNA-circ inhibition group,siRNA-NC group and NC group was 0.28±0.13,0.90±0.34 and 0.87±0.31 respectively,and the differences were statistically significant(P<0.001).The cell proliferation rate of siRNA-circ inhibition group was lower than that of siRNA-NC group(P<0.001).The results of 24 h transwell cell culture showed that the number of migrated U251 cells in the siRNA-circ inhibition group,siRNA-NC group and NC group were 81.35±10.72,173.26±28.40 and 174.80±27.25 respectively.After 48 h of culture,the number of invaded U251 cells in the three groups were 39.85±6.24,106.48±14.73 and 104.72±13.84 respectively with statistical significance(all P<0.001).When cultured cells were treated with different concentrations of TMZ for 24 h,the survival rate of U251 cells in siRNA-circ inhibition group was decreased compared with that in siRNA-NC group with statistical significance(P<0.001).Conclusions Hsa_circRNA_0006168 is highly expressed in glioma tissues and cells,which is related to the poor prognosis of glioma patients.Downregulation of Hsa_circRNA_0006168 expression can inhibit the proliferation,migration and invasion of glioma cells and promote the sensitivity to TMZ chemotherapy.