miR-218靶向调控HMGB1-RAGE通路抑制胶质瘤发生发展机制研究

发布时间：2018-07-15 14:09

【摘要】：背景:脑胶质瘤是脑中最常见的恶性肿瘤,具有快速浸润性生长和术后高复发率的特点。低级别胶质瘤患者(世界卫生组织WHO I级和II级)的5年生存率约为30%至70%,而高级别胶质母细胞瘤(IV级)的中位生存期为9至12个月。因此,有必要确定这种恶性疾病更多的生物标志物和潜在的治疗靶点。微小RNA(microRNA,miRNA)涉及一系列重要的生物过程,如细胞发育,分化,凋亡和增殖。异常mi RNA表达与各种肿瘤的发生和进展相关。此外,表达失调的mi RNA可作为诊断和评估预后的生物标志物。目的:研究miRNA在不同级别胶质瘤中的差异表达情况,探讨miRNA通过靶基因调控胶质发生发展的分子机制。方法:(1)利用生物信息学对NCBI GEO数据库内胶质瘤数据分析得出差异表达mi RNA,对22例胶质瘤组织标本通过PCR Array(内含184miRNA引物预设计的PCR芯片)方法筛选胶质瘤内差异表达mi RNA,选取在胶质瘤内表达差异倍数高的mi RNA探讨其在胶质瘤内的调节机制,并通过生物信息学软件预测所选取mi RNA的靶基因进行下一步研究。(2)对66例胶质瘤组织标本及26例正常脑组织标本中,通过荧光定量PCR检测mi R-218在正常脑组织及胶质瘤内表达量,分析miR-218在不同级别胶质瘤内表达情况及表达差异。(3)应用半定量PCR初步检测HMGB1和RAGE基因水平在不同级别胶质瘤内表达情况,在此基础上通过定量PCR对HMGB1和RAGE基因水平在正常脑组织及胶质瘤内表达情况进行验证,分析HMGB1和RAGE在正常脑组织及不同级别胶质瘤内表达情况及表达差异。(4)利用免疫组织化学方法对HMGB1和RAGE在蛋白水平表达量进行检测,分析其在正常脑组织及胶质瘤中阳性细胞表达情况及表达差异,通过蛋白印迹实验检测HMGB1和RAGE的在正常脑组织及胶质瘤中表达量。(5)收集纳入研究患者临床资料并进行随访。通过Kaplan-Meier方法和Cox回归分析患者的生存曲线,进行单变量及多变量Cox回归分析以检查每个临床协变量及mi R-218、HMGB1、RAGE在胶质瘤内表达强度对患者存活的影响。结果:(1)通过对22例胶质瘤组织标本进行PCR Array分析,筛选出明显具有表达差异(Fold Difference2)的miRNA 11个,(miR-22-3p、miR-363-3p、mi R-451a、mi R-20b、miR-450、mi R-149-5p、mi R-15b、miR-106b、miR-148a、mi R-93、miR-218-5p)。通过对NCBI GEO数据分析,获取在胶质瘤明显具有表达差异(Fold Difference4)的miRNA 7个,(miR-7、miR-10b、miR-20b、mi R-551b、miR-455、mi R-218-5p、miR-137)。依据其表达差异选取miR-218为研究对像并利用生物信息学预测mi R-218靶基因HMGB1、RAGE。(2)miR-218在正常脑组织及胶质瘤内均有表达,在不同级别胶质瘤内mi R-218的表达量各不相同。与正常脑组织内miR-218表达量相比较,miR-218表达量与肿瘤等级呈现逆相关。miR-218在高级别(Ⅲ级和Ⅳ级)胶质瘤组织中的表达量明显低于低级别(Ⅰ级和Ⅱ级)(p0.01)。(3)PCR、免疫组织化学染色、蛋白印迹实验检测表明在正常脑组织标本中和不同级别胶质瘤组织标本内均能检测到HMGB1、RAGE表达,HMGB1、RAGE表达量与肿瘤级别正相关,HMGB1、RAGE在高级别(Ⅲ级和Ⅳ级)胶质瘤组织中的表达量明显高于低级别(Ⅰ级和Ⅱ级)(p0.05)。(4)Kaplan-Meier曲线显示低mi R-218表达患者的生存时间显著短于mi R-218高表达的患者,Kaplan-Meier曲线显示HMGB1、RAGE低表达组生存时间长于高表达组。(5)单因素分析显示miR-218低表达与性别(p=0.013),肿瘤大小(p=0.020),WHO分级(p0.001)和KPS评分(p0.001)显着相关,但与患者年龄以及切除程度无关。(6)Cox回归单变量分析显示肿瘤大小(p=0.018),WHO分级(p0.001),mi R-218表达水平(p0.001)为总生存时间(OS)的独立相关因素;肿瘤大小(p=0.045),切除范围(p=0.028),WHO分级(p0.001),miR-218的表达(p0.001)为无进展生存时间(PFS)的独立预后因子。Cox回归多变量分析显示mi R-218的表达(HR=0.32;95%CI 0.11,0.89;p=0.029),切除范围(HR=0.11;95%CI 0.03,0.37;p0.001),肿瘤大小(HR=0.39;95%CI 0.14,1.11;p=0.029),和WHO分级(HR=8.94;95%CI 0.11,1.94;p0.001)是总生存时间(OS)的独立预后因子;miR-218的表达(HR=0.46;95%CI 0.18,1.21;p=0.011),切除范围(HR=0.09;95%CI 0.02,0.30;p0.001),肿瘤大小(HR=0.53;95%CI 0.19,1.45;p=0.031),和WHO分级(HR=2.10;95%CI 1.52,2.91;p0.001)是无进展生存时间(PFS)的独立相关因素。结论:PCR Array及生物信息学筛选出miRNA-218在胶质瘤差异表达,在胶质瘤内表达呈下调趋势。生物信息学预测胶质瘤内HMGB1、RAGE为mi R-218潜在的靶基因。实验检测mi R-218、HMGB1及RAGE结果表明:miR-218可作为胶质瘤中的肿瘤抑制miRNA;miR-218在胶质瘤中下调表达并与HMGB1和RAGE表达呈负相关;mi R-218低表达与HMGB1、RAGE高表达预测胶质瘤患者的预后不良;miR-218能通过调节HMGB1-RAGE的表达抑制胶质瘤的进展。
[Abstract]:Background: glioma is the most common malignant tumor in the brain, characterized by rapid invasive growth and high postoperative recurrence. The 5 year survival rate of patients with low grade glioma (WHO WHO I and II) is about 30% to 70%, and the median survival period of high grade glioblastoma (IV grade) is 9 to 12 months. Therefore, it is necessary to determine this More biomarkers and potential therapeutic targets for malignant diseases. Small RNA (microRNA, miRNA) involves a series of important biological processes, such as cell development, differentiation, apoptosis and proliferation. Abnormal mi RNA expression is related to the occurrence and progression of various tumors. In addition, the expression of the misaligned mi RNA can be used as a biomarker for the diagnosis and evaluation of the prognosis. Objective: To study the differential expression of miRNA in different grade gliomas and to explore the molecular mechanism of miRNA regulation of glial development through target gene. Methods: (1) the differential expression of MI RNA was obtained by bioinformatics on the analysis of glioma data in NCBI GEO database, and 22 cases of glioma tissue were expressed through PCR Array (containing 184miRNA primers). The pre designed PCR chip was used to screen the differential expression of MI RNA in glioma, and to select the regulatory mechanism of MI RNA in glioma, and to predict the target gene of MI RNA by bioinformatics software. (2) 66 cases of glioma tissue specimens and 26 normal brain tissues were labeled. In this study, the expression of MI R-218 in normal brain tissue and glioma was detected by fluorescence quantitative PCR, and the expression and expression difference of miR-218 in different gliomas were analyzed. (3) a semi quantitative PCR was used to detect the level of HMGB1 and RAGE in different levels of glioma, and on this basis, the quantitative PCR to HMGB1 and RAGE based on the quantitative PCR The expression and expression of HMGB1 and RAGE in normal brain tissue and different grade gliomas were verified by the expression of the level in normal brain tissue and glioma. (4) the expression of HMGB1 and RAGE in the protein level was detected by immunohistochemical method, and the positive cells in normal brain tissue and glioma were analyzed. Expression and expression difference, the expression of HMGB1 and RAGE in normal brain tissue and glioma was detected by Western blot. (5) the clinical data of the patients were collected and followed up. The survival curves of the patients were analyzed by Kaplan-Meier and Cox regression, and the single variable and multivariable Cox regression analysis were used to examine each clinical trial. The effect of covariate and MI R-218, HMGB1, RAGE on the survival of patients with glioma. Results: (1) 11 miRNA (miR-22-3p, miR-363-3p, MI) were screened by PCR Array analysis of 22 glioma tissue specimens. 148A, MI R-93, miR-218-5p). Through the analysis of NCBI GEO data, we obtain miRNA 7 miRNA (miR-7, miR-10b, miR-20b, Fold Difference4) in glioma. 2) miR-218 was expressed in normal brain tissue and glioma, and the expression of MI R-218 in different gliomas was different. Compared with miR-218 expression in normal brain tissue, the expression of miR-218 expression and tumor grade presented inverse.MiR-218 in high grade (grade III and IV grade) glioma tissue, which was significantly lower than that of low grade (grade III and IV) glioma (grade III and IV). Grade I and II) (P0.01). (3) PCR, immunohistochemical staining and Western blot test showed that HMGB1, RAGE expression, HMGB1, RAGE expression were positively correlated with tumor grade in normal brain tissue specimens and different grade glioma tissue specimens. The expression of HMGB1, RAGE in high grade (grade III and IV grade) glioma tissues was expressed. Significantly higher than the low level (grade I and grade II) (P0.05). (4) the Kaplan-Meier curve showed that the survival time of the patients with low MI R-218 expression was significantly shorter than the high expression of MI R-218, the Kaplan-Meier curve showed HMGB1, and the survival time of the RAGE low expression group was longer than that of the high expression group. (5) the single factor analysis showed that the low expression of miR-218 and sex (p=0.013), the size of the tumor ( P=0.020), WHO grading (p0.001) and KPS score (p0.001) were significantly related, but not related to patient's age and degree of resection. (6) a single variable analysis of Cox regression showed the tumor size (p=0.018), WHO classification (p0.001), MI R-218 expression level (p0.001) as an independent correlation factor for the total survival time; tumor size, resection range, etc. P0.001, miR-218 expression (p0.001) is an independent prognostic factor of progression free survival time (PFS),.Cox regression multivariable analysis shows the expression of MI R-218 (HR=0.32; 95%CI 0.11,0.89; p=0.029). 001) is an independent prognostic factor of the total survival time (OS); the expression of miR-218 (HR=0.46; 95%CI 0.18,1.21; p=0.011), the range of the resection (HR=0.09; 95%CI 0.02,0.30; p0.001), the size of the tumor (HR=0.53; 95%CI), is an independent correlation factor of the progression free survival time. Y and bioinformatics screened the differential expression of miRNA-218 in glioma. The expression in glioma was down downward. Bioinformatics predicted HMGB1 in glioma and RAGE as a potential target gene for MI R-218. Experimental detection of MI R-218, HMGB1 and RAGE results showed that miR-218 could be used as a tumor suppressor in glioma; There is a negative correlation with the expression of HMGB1 and RAGE, and the low expression of MI R-218 and the high expression of HMGB1 and RAGE predict the poor prognosis of the patients with glioma, and miR-218 can inhibit the progression of glioma by regulating the expression of HMGB1-RAGE.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

【相似文献】