miR-95-3p在胶质瘤细胞中的表达及对其生物学功能的影响

发布时间：2018-04-05 02:03

本文选题：胶质瘤　切入点：细胞增殖　出处：《河北医科大学》2016年博士论文

【摘要】：胶质瘤是人类中枢神经系统中最为常见的原发性恶性肿瘤,约占颅内肿瘤的40%~60%。目前对胶质瘤患者普遍采用手术切除联合术后放化疗等综合治疗方法。虽然近年来,神经影像技术、显微外科技术以及放化疗治疗方案等多方面均取得了突飞猛进的发展,但对于胶质瘤患者,尤其是高级别的胶质瘤患者,预后仍然不佳。胶质母细胞瘤(WHOⅣ级)患者术后复发率仍接近100%,中位生存时间少于1年。文献报道,低级别胶质瘤患者平均生存时间为3~5年,而高级别患者则是1~2年。治疗效果的不佳主要是由于胶质瘤细胞具有高度侵袭、增殖、迁移能力以及凋亡抑制的特性,决定了胶质瘤不可能单纯通过外科方法得到治愈,而目前的放化疗治疗也无法满意控制胶质瘤的发展。因此,深入了解胶质瘤发生发展机制,研究靶向药物以特异性地抑制甚至灭活残存瘤细胞,是未来胶质瘤治疗的方向。目前已经发现许多与肿瘤相关的基因及蛋白因子,有些已经研究的比较深入,一部分新型抗肿瘤药物已应用临床并显著改善预后。但是针对胶质瘤的药物研究大多仍处于实验的初级阶段,仍需进一步研究探索。mi RNA即微小RNA(microRNA),是一类新近发现的、内源性、非蛋白质编码RNA,因其在各种病理生理过程中的强大作用逐渐受到重视。文献指出,miR-95-3p在直肠癌、胰腺癌、乳腺癌及前列腺癌等癌症中具有致癌作用。考虑到miR-95-3p尚未在胶质瘤细胞中进行过研究,故在本实验中我们选取miR-95-3p作为对象,并通过MTT实验,流式细胞技术以及Transwell实验探讨其对于胶质瘤的作用。CELF2(CUGBP-and ETR-3-like family)为一组RNA连接蛋白,包含众多与基因转录后调控相关的蛋白。研究表明,CELF2编码基因位于10号染色体上,该染色体在60%的低级别胶质瘤中呈单体型,并在80%的高级别胶质瘤中部分缺失,我们因此猜测CELF2与胶质瘤的发生发展可能相关。由于CELF2出现在Targetscan的预测结果中,我们进一步研究其与miR-95-3p的关系。实验中我们通过荧光素酶报告基因实验确定mir-95-3p与celf2的靶向作用关系,并分析两者表达量的相关性进一步证实两者相关性。最后通过复活实验证实mir-95-3p对胶质瘤的作用是通过靶向调控celf2引起的。第一部分mir-95-3p在人脑胶质瘤组织中的表达及其与胶质瘤病理级别的相关性目的:研究mir-95-3p在人脑胶质瘤组织中的表达,并分析mir-95-3p的表达与胶质瘤病理级别的关系。方法:1组织标本的采集:35例胶质瘤样本及15例非肿瘤脑组织样本均于2014年采集自河北医科大学第二医院神经外科。包括19名女性,31名男性,年龄从18至68岁,共收集胶质瘤样本35例,其中Ⅰ级胶质瘤4例、ii级5例、iii级17例、iv级9例,均由术中切除获得。15例非肿瘤脑组织样本来自于对严重的脑外伤患者进行的颅内减压术。诊断结果均由2位病理医师根据who分级指导得出。所有样本在采集后立即保存至-80℃液氮中。2样本mir-95-3p表达水平的测定:反转录定量聚合酶链反应(qrt-pcr)方法测定35例胶质瘤样本以及15例非肿瘤脑组织样本中的mir-95-3p含量。3相关性分析:应用t检验分析胶质瘤中mir-95-3p含量与胶质瘤级别之间的关系。结果:1qrt-pcr结果:胶质瘤组织与非肿瘤脑组织中mir-95-3p表达量具有显著性差异。与对照非肿瘤脑组织相比,胶质瘤组织中mir-95-3p含量显著升高,差别具有统计学意义(p0.001)。2t检验结果:低级别胶质瘤(whoⅠ、Ⅱ级)、高级别胶质瘤(whoⅢ、Ⅳ级)及非肿瘤脑组织中mir-95-3p表达量具有显著性差异。在高级别胶质瘤组织中mir-95-3p的含量较低级别更高。结论:1mir-95-3p在人脑胶质瘤组织中高表达,且其表达量随着胶质瘤级别的升高而升高。高级别胶质瘤mir-95-3p的表达量要高于低级别胶质瘤,差别具有统计学意义。2mir-95-3p的表达水平与胶质瘤的级别呈显著的正相关,提示mir-95-3p可能成为胶质瘤临床分级的分子标志物。第二部分mir-95-3p对胶质瘤细胞生物学活性的影响目的:研究mir-95-3p对u87和u251细胞的增殖、侵袭、凋亡及细胞周期等方面的影响。方法:1培养u87和u251细胞系用于体外功能学实验。向实验组中转染mir-95-3paso以降低细胞内mir-95-3p的表达。对照组转染ctrlaso。2进行mtt实验研究降低mir-95-3p表达后胶质瘤细胞增殖能力的变化。3进行流式细胞实验研究降低mir-95-3p表达后胶质瘤细胞周期及凋亡能力的变化。4进行transwell实验研究降低mir-95-3p表达后胶质瘤细胞侵袭能力的变化。结果:1qrt-pcr结果显示,转染mir-95-3paso后,u87及u251细胞内mir-95-3p的表达量显著降低(p0.05)。2mtt实验结果显示,降低mir-95-3p表达后,u87及u251细胞增殖能力显著降低(p0.05)。3流式细胞实验结果显示,降低mir-95-3p表达后,u87及u251细胞周期无明显改变,凋亡增殖能力显著增高(p0.05)。4transwell实验结果显示,降低mir-95-3p表达后,u87及u251细胞侵袭能力显著降低(p0.05)。结论:降低mir-95-3p表达,可以使u87及u251细胞增殖及侵袭能力显著降低,凋亡能力显著增高,细胞周期无明显差别。mir-95-3p可以影响胶质瘤细胞的增殖、侵袭及凋亡水平。第三部分celf2为mir-95-3p的下游靶基因目的:寻找mir-95-3p的下游作用靶点,进一步了解mir-95-3p对胶质瘤生物学影响的作用机制。方法:1使用在线工具targetscan预测mir-95-3p的下游作用靶点,并根据文献资料筛选结果。2双荧光素酶报告基因实验验证mir-95-3p与celf2的靶向作用关系。3应用反转录定量聚合酶链反应(qrt-pcr)及蛋白质免疫印迹(westernblot)方法检测mir-95-3p与celf2在u87及u251细胞中的表达量,并分析两者的相关性。结果:1根据targetscan的预测结果,并结合相关文献资料,猜测celf2上可能存在mir-95-3p的靶向作用位点。2相对于mir-95-3p反义寡核苷酸(mir-95-3paso),对照组中共转染mir-95-3p与ctrlaso可以显著抑制荧光素酶活性,而突变靶位点后此抑制作用消失。3mir-95-3p与celf2在u87及u251中的表达量呈负相关。结论:celf2的mrna中存在mir-95-3p在胶质瘤中的靶向作用位点。第四部分mir-95-3p通过调控celf2影响胶质瘤细胞增殖、侵袭、凋亡等的生物学活性目的:证实mir-95-3p对胶质瘤的生物学活性影响是通过调控celf2而完成的。方法:1向u251细胞内转染mir-95-3paso后,同时转染小干扰rna(smallinterferingrna,sirna),以降低细胞中celf2的含量。2进行mtt实验,实验组共转染mir-95-3paso与celf2sirna,对照组共转染mir-95-3p与ctrlsirna,检测u251细胞增殖能力的改变。3进行流式细胞实验,实验组共转染mir-95-3paso与celf2sirna,对照组共转染mir-95-3p与ctrlsirna,检测u251细胞凋亡能力的改变。4进行transwell实验,实验组共转染mir-95-3paso与celf2sirna,对照组共转染mir-95-3p与ctrlsirna,检测u251细胞侵袭能力的改变。1 Western blot实验证实转染CELF2 siRNA后,U251细胞内CELF2含量显著降低(P0.05)。2 MTT实验结果显示,相对于对照组,实验组中U251细胞增殖能力明显降低(P0.05)。3流式细胞实验结果显示,相对于对照组,实验组中U251细胞凋亡能力明显增高(P0.05)。4 Transwell实验结果显示,相对于对照组,实验组中U251细胞侵袭能力明显降低(P0.05)。结论:miR-95-3p是通过调控CELF2的表达而影响胶质瘤增殖、凋亡、侵袭能力改变的。
[Abstract]:Glioma is the human central nervous system is the most common primary malignant tumor, accounnting 40%~60%. of glioma patients commonly used surgery chemotherapy and other comprehensive treatment method of resection. Although in recent years, neuroimaging techniques, microsurgical technique and chemoradiotherapy achieved etc. the rapid development, but for glioma patients, especially in patients with high grade gliomas, the prognosis is still poor. Glioblastoma (WHO IV) recurrence rate is close to 100%, the median survival time of less than 1 years. The literature reported that patients with low grade gliomas the average survival time was 3~5 however, patients with high grade is 1~2 years. The poor treatment effect is mainly due to glioma cells with high invasion, proliferation, migration and apoptosis characteristics, determines the glioma may not only by Methods surgical cure and chemotherapy in the treatment of current cannot satisfy the development control of glioma. Therefore, in-depth understanding of the occurrence and development mechanism of glioma research, targeted drugs to specifically inhibit or inactivate the residual tumor cells, glioma is the future direction of the treatment. It was found that many tumor related genes and some of the proteins, has been more in-depth, part of a new antitumor drug has clinical application and improve the prognosis. But research on drugs of glioma are still in the primary stage of the experiment, further research is needed to explore the.Mi RNA micro RNA (microRNA), is a newly discovered endogenous non protein. RNA encoding, because of its powerful role in various pathophysiological processes has attracted much attention. The literature pointed out, miR-95-3p in rectal cancer, pancreatic cancer, breast cancer and prostate cancer and other cancers. Taking into account the carcinogenic effect. MiR-95-3p has not been in glioma cells studied in this experiment, we select miR-95-3p as the object, and through the MTT experiment to investigate the effect of.CELF2 glioma cells and Transwell (CUGBP-and ETR-3-like family) flow experiment for a group of RNA binding protein related protein regulation, including many of the post transcriptional gene. The results show that the CELF2 encoding gene is located on chromosome 10, the chromosome haplotype was in low grade glioma in 60%, and 80% in the high grade gliomas excalation, so I guess the occurrence and development of CELF2 and glioma may be related. Because CELF2 appear in the prediction results in Targetscan, we further study the relationship with miR-95-3p. In our experiment by luciferase reporter assay and determine mir-95-3p targeting celf2, and analysis The expression of correlation further confirmed the relationship between them. Finally, the resurrection experiments confirmed that the effect of mir-95-3p on glioma is caused by celf2 to control the target. Part 1 expression of mir-95-3p in human glioma tissues and its correlation with the pathological grade of glioma Objective: To study the expression of mir-95-3p in human glioma tissues, and analysis the relationship between mir-95-3p expression and pathological grade of glioma. Methods: 1 tissue specimens collected: 35 cases of glioma samples and 15 cases of non tumor brain tissue samples were collected in 2014 from the second hospital of Hebei Medical University, Department of neurosurgery. Including 19 women, 31 men, aged from 18 to 68 years, a total of 35 cases of glioma samples, including 4 cases of glioma, 5 cases of grade II, III grade 17 cases, 9 cases of grade IV, were obtained by resection of.15 cases of non tumor brain tissue samples from the patients with severe traumatic brain injury Intracranial decompression performed. According to the WHO grading diagnosis results by 2 pathologists. All the samples that guide preservation to the level of mir-95-3p expression were detected by.2 -80 C in liquid nitrogen immediately after collection: reverse transcription polymerase chain reaction (qRT-PCR) analysis of.3 correlation of mir-95-3p content in 35 cases of glioma and 15 cases of non tumor samples brain tissue samples: application of t test method to analyze the relationship between the content of mir-95-3p in glioma and glioma grade. Results: 1qrt-pcr results: a significant difference of mir-95-3p expression of glioma and non tumor brain tissues. Compared with non tumor brain tissue, the content of mir-95-3p in glioma tissues increased significantly. The difference was statistically significant (p0.001).2t test results: low grade gliomas (who I, II) and high grade gliomas (who III, IV) and non tumor brain tissue of mir-95-3p expression The difference is significant. The content of mir-95-3p in high grade glioma tissues of the lower level higher. Conclusion: high expression of 1mir-95-3p in human glioma tissues, and its expression increased with the glioma grade. The expression of mir-95-3p in high grade gliomas than in low grade gliomas, a significant positive expression was statistically significant difference.2mir-95-3p and glioma grade, suggesting that mir-95-3p may become a molecular marker for glioma clinical classification. The second part: the effect of mir-95-3p on glioma cells Objective: To study the biological activity of mir-95-3p and U251 on U87 cell proliferation, invasion, apoptosis and cell cycle and other aspects of the method. 1: the culture of U87 and U251 cells for in vitro functional experiments. The experimental group was transfected with mir-95-3paso to reduce the expression of mir-95-3p in cells transfected with ctrlaso.2 in the control group MTT experimental study on reducing the change of.3 expression of mir-95-3p glioma cell proliferation by flow cytometry experiments of experimental study of Transwell decreased after glioma cell invasion of the expression of mir-95-3p decreased.4 mir-95-3p expression in glioma cell cycle and apoptosis ability. Results: 1qrt-pcr results showed that after mir-95-3paso transfection, expression U87 and U251 cells significantly decreased mir-95-3p (P0.05).2mtt experimental results show that the decreased expression of mir-95-3p, U87 and U251 Cell proliferation ability decreased significantly (P0.05).3 flow cytometry results showed that decreased expression of mir-95-3p, U87 and U251 no significant changes in the cell cycle, proliferation apoptosis significantly increased (P0.05).4transwell the experimental results show that the reduced expression of mir-95-3p, U87 and the invasion ability of U251 cells was significantly decreased (P0.05). Conclusion: the decreased expression of mir-95-3p, can make u 87 and U251 Cell Proliferation and invasion ability decreased significantly, the apoptosis increased significantly, there was no significant difference in cell cycle of.Mir-95-3p can affect the glioma cell proliferation, invasion and apoptosis level. The third part celf2 is the downstream target genes of mir-95-3p Objective: to find out the mir-95-3p downstream targets, to further understand the mechanism of mir-95-3p effect on glioma biology. Methods: 1 using the online tool targetscan to predict downstream effect of mir-95-3p target, and screening results of mir-95-3p and celf2 to verify the.2 dual luciferase assay targeting.3 by reverse transcription polymerase chain reaction (qRT-PCR) based on the literature and Western blot (Westernblot) method to detect mir-95-3p and celf2 expression in U87 and U251 cells, and to analyze the relationship between them. Results: 1 according to the prediction results of targetscan, and combined with the relevant Literature, guess mir-95-3p might exist on celf2 targeting sites of.2 compared with mir-95-3p antisense oligonucleotide (mir-95-3paso), the control group transfected with mir-95-3p and ctrlaso can significantly inhibit the luciferase activity, and mutation of target sites of this inhibition of the expression of.3mir-95-3p and celf2 disappeared in U87 and in U251 was negatively correlated. Conclusion: mir-95-3p in glioma targeting sites of celf2 mRNA in mir-95-3p. The fourth part through the regulation of celf2 proliferation effect of glioma cell invasion, apoptosis and biological activity Objective: To study the effects of biological activity of mir-95-3p on glioma is completed by the regulation of celf2. Methods: 1 to U251 cells after transfection of mir-95-3paso. At the same time, transfection of small interfering RNA (smallinterferingrna, siRNA), in order to reduce the content of.2 in celf2 cells by MTT experiment, the experimental group were transfected into mir-95-3paso and CE Lf2sirna, the control group were transfected into mir-95-3p and ctrlsirna, U251 to detect the changes of cell proliferation..3 flow cytometry experiment, the experimental group were transfected into mir-95-3paso and celf2sirna, the control group were transfected into mir-95-3p and ctrlsirna, U251 apoptosis detection ability change.4 Transwell experiment, the experimental group were transfected into mir-95-3paso and celf2sirna, CO transfected control group mir-95-3p ctrlsirna and.1 Western blot, change the experimental invasion ability of U251 cells were detected by CELF2 siRNA after transfection confirmed, CELF2 in U251 cells was significantly reduced (P0.05).2 MTT experimental results show that compared with the control group, U251 cell proliferation in experimental group was significantly lower (P0.05).3 flow cytometry results showed that compared with the control U251 group, apoptosis in experimental group was significantly increased (P0.05).4 Transwell experimental results show that compared with the control group, U251 cell infiltration in the experimental group The attack ability was significantly reduced (P0.05). Conclusion: miR-95-3p affects the proliferation, apoptosis, and invasion of glioma by regulating the expression of CELF2.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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