Med19在星形细胞瘤中的表达及对其恶性生物学行为调控的实验研究
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摘要
2010年,美国新增22,020例颅脑肿瘤病例,13,140例颅脑肿瘤患者死亡(源自www.cancer.org,Cancer Facts & Figures 2010)。脑星形细胞瘤是颅内最常见的起源于神经上皮的原发肿瘤之,约占成人颅内肿瘤的73%～86%,其中恶性星形细胞瘤约占90%,以间变型星形细胞瘤和胶质母细胞瘤最为多见。恶性星形细胞瘤预后差,病死率高,即使手术全部切除后其复发率仍高达60%以上,5年生存率极低。近5年来,随着基础和临床研究的深入探索,发现:胶质母细胞瘤患者接受手术、放疗和化疗后,平均生存期可由10个月增加至14个月。但是,由于星形细胞瘤具有血供丰富,呈浸润性生长的生物学特点,并且与周围脑组织无明显分界,易于复发,则其疗效均不甚理想。
既往研究表明:星形细胞瘤的恶性进展及其侵袭复发是个涉及多基因、多阶段、多步骤的复杂过程,是受多种基因、蛋白以及细胞因子等共同调控的结果。参与星形细胞瘤发生、发展过程中的基因,在其他肿瘤的发生发展过程中几乎均发挥作用,如:上皮生长因子、内皮生长因子、转化生长因子、肿瘤原癌基因和抑癌基因等;此外星形细胞瘤侵袭复发的过程可能涉及到肿瘤细胞粘附、基质降解、迁移、细胞增殖等诸多环节,但其具体机制仍不清楚。近年来,基于对星形细胞瘤分子机制的逐步了解,靶向性分子疗法为星形细胞瘤治疗提供了新思路,寻找星形细胞瘤发生发展过程中的重要通路和靶标分子以及安全高效的靶向性载体系统显得尤为重要,但尚未将任何种靶标分子用于星形细胞瘤的治疗具有明确疗效。因此寻找调控星形细胞瘤细胞恶性生物学行为的分子靶标,明确其在星形细胞瘤发病过程中的病理机制,已成为目前星形细胞瘤治疗研究领域的的热点之。
Med19基因,与RNA聚合酶Ⅱ相互作用介导子基因,是2002年应用差异显示PCR方法从两种不同高低转移潜能的人肺癌细胞系中克隆到的人类新基因,目前尚无其功能学的报道。既往研究显示,Med19基因在多种正常组织以及肿瘤中有所表达,推测其可能与细胞的增殖、凋亡以及细胞分化密切相关。而在肺癌组织中发现,Med19基因的表达与肿瘤的恶性程度密切相关。在乳腺癌组织中发现,Med19蛋白的异常高表达,且运用慢病毒感染干扰Med19基因表达水平后,乳腺癌细胞的增殖能力和细胞周期受到显著影响;Medl9基因沉默后胃癌细胞的增殖能力和细胞周期均受到显著影响,因此推测其可能参与胃癌的发生发展以及促进恶性肿瘤细胞的增殖和浸润性生长。而在星形细胞瘤中Med19基因对肿瘤细胞的增殖,凋亡,侵袭以及血管生成等方面的研究在国内外,尚未见诸报道。因此,探讨MED19参与脑星形细胞瘤发生发展以及恶变的分子病理机制,有助于明确星形细胞瘤的恶性生物学行为及机理,有望为星形细胞瘤的临床治疗提供新的分子靶标。
本研究拟通过观察Med19在不同病理级别脑星形细胞瘤以及星形细胞瘤细胞株中的确切表达情况,并通过研究Med19在星形细胞瘤细胞株中的异常表达,研究Med19在星形细胞瘤发生发展过程中的功能角色。实验共分为四个部分:第部分,观察Med19在脑星形细胞瘤组织中的表达水平以及与星形细胞瘤病理级别的相关性,探讨Med19的表达水平与星形细胞瘤患者生存周期之间的关系;第二部分,观察Med19在U251、U373和U87脑星形细胞瘤细胞株中的表达情况;第三部分,靶向Med19的RNA干扰和过表达慢病毒载体的构建和鉴定;第四部分,通过基因操作的方法上调或抑制Med19基因在星形细胞瘤细胞内表达,观察其对U251星形细胞瘤细胞株生长、增殖、凋亡、侵袭等细胞表型以及裸鼠皮下成瘤的体内外试验的影响。
第一部分Med19在不同级别脑星形细胞瘤组织中的表达和临床病理相关性分析的实验研究
目的:研究Med19在脑星形细胞瘤组织中表达水平及其与星形细胞瘤病理WHO级别的相关性,Med19表达水平同星形细胞瘤患者生存时间的关系。
方法:首先,运用实时定量RT-PCR和Western blot杂交分别检测星形细胞瘤组织中Med19的mRNA及蛋白的表达水平并分析Med19表达水平同星形细胞瘤组织级别的相关性;随后采用免疫组化染色法检测5例正常脑组织和79例不同级别星形细胞瘤组织中Med19的表达情况;最后,临床随访星形细胞瘤患者,运用生存分析检测Med19表达水平同患者生存时间的相关性。
结果:(1)应用实时定量RT-PCR检测Med19 mRNA在21例不同级别脑星形胶质细胞瘤和5例正常脑组织中的表达显示:Med19 mRNA表达水平在星形细胞瘤组织中明显上调,与正常脑组织相比差别显著(P<0.01);并且,Med19 mRNA水平在高级别星形细胞瘤组织中的表达水平明显高于低级别星形细胞瘤,二者相比差异显著(P<0.01)。(2)运用Western blot技术测定Med19蛋白在上述星形细胞瘤组织的表达水平,结果显示:Med19蛋白在星形胶质细胞瘤组织中表达明显上调,与正常脑组织相比差别显著(P<0.01)。Med19在III-IV级(高级别)星形细胞瘤中高表达,而在I-II级(低级别)星形细胞瘤中表达量较低,且有显著差异(P<0.001)。(3)采用免疫组化法检测了5例正常脑组织及79例人脑星形细胞瘤石蜡标本中Med19蛋白的表达情况。结果显示,正常脑星形胶质细胞中Med19蛋白染色为阴性,绝大部分星形细胞瘤标本中可见Med19蛋白的阳性表达。星形细胞瘤的临床病理级别与Med19的表达强度与肿瘤恶性程度呈正相关关系(P<0.01);而性别和年龄则无统计学上的差异(P>0.05)。生存分析表明,Med19免疫组化染色表达水平同患者生存时间呈负相关(p<0.05)。
结论:Med19在脑星形细胞瘤中呈阳性表达,其表达水平同脑星形细胞瘤的级别呈明显正相关,同星形细胞瘤患者的生存时间呈负相关。
第二部分Med19在脑星形细胞瘤细胞株中的表达
目的:研究Med19在胶质母细胞瘤(GBM)细胞株U87、U373和U251中的表达情况。
方法:应用细胞免疫荧光法检测Med19在胶质母细胞瘤细胞株U87、U373和U251中的定位;运用实时定量RT-PCR法和Western blot技术检测Med19在胶质母细胞瘤细胞株U87、U373和U251中的mRNA和蛋白表达情况。
结果: (1)细胞免疫荧光结果提示:Med19在胶质母细胞瘤细胞株U87、U373和U251中的定位于细胞浆;(2)实时定量RT-PCR结果提示:Med19 mRNA在胶质母细胞瘤细胞株U87、U373和U251中均表达;(3)Western blot结果表明:Med19蛋白在胶质母细胞瘤细胞株U87、U373和U251均表达。
结论:Med19在胶质母细胞瘤细胞株中均有表达。
第三部分:靶向Med19的RNA干扰和过表达慢病毒载体的构建和鉴定
目的:Med19在星形细胞瘤组织和细胞中均有表达,其表达量与星形细胞瘤的病理级别呈正相关,提示Med19可能与星形细胞瘤的恶性生物学行为密切相关。本部分拟构建靶向Med19的慢病毒RNA干扰和过表达载体,为进步研究Med19的生物学功能提供载体工具。
方法:1)设计siRNA靶点,利用外源筛靶实验挑选高效干扰序列,与慢病毒载体连接,并利用荧光显微镜,实时定量RT-PCR、Western blot在U373、U251和U87细胞中验证RNA干扰效果。2)设计Med19过表达质粒并经过测序后,连接慢病毒载体,并利用荧光显微镜、实时定量RT-PCR、Western blot在U373、U251和U87细胞中验证过表达效果。
结果:设计的siRNA干扰序列,其中两条干扰效率较高,连接入慢病毒载体后,转染效率超过75%。在U373和U251细胞中,RNAi能够在mRNA水平和蛋白水平显著降低内源性Med19的表达(p<0.01);设计的过表达质粒经过测序证实与原基因序列完全致,慢病毒过表达载体能够在使U373和U251细胞中内源性Med19的mRNA和(或)蛋白水平表达量增加(p<0.05)。
结论:本研究成功构建了能够稳定改变内源性Med19表达水平的RNA干扰和过表达慢病毒载体,可为后续细胞功能学实验提供实验依据。
第四部分:靶向Med19调控胶质母细胞瘤细胞恶性生物学行为的体内外实验研究
目的:利用前期构建的稳定改变内源性Med19表达水平的RNA干扰和过表达慢病毒载体感染胶质母细胞瘤细胞株U251和U373细胞后,从体外和体内两个角度来研究Med19对胶质母细胞瘤恶性生物学行为的作用。
方法:内源性Med19表达水平改变后,运用MTT法检测细胞增殖曲线变化、克隆形成实验检测肿瘤细胞克隆形成能力变化、流式细胞仪测肿瘤细胞周期与凋亡比例变化、transwell细胞侵袭试剂盒检测细胞侵袭能力、运用荷瘤裸鼠模型检测Med19表达量改变对皮下种植瘤生长速率的影响。
结果:(1)Med19表达量降低后,胶质母细胞瘤细胞增殖能力减弱,克隆形成能力减弱;上调胶质母细胞瘤细胞内源性Med19的表达量后,其增殖能力增强,克隆形成能力增强;(2)U251MG细胞中,Med19表达量改变对细胞凋亡和细胞周期可产生影响;(3)在胶质母细胞瘤细胞株中,过表达内源性Med19表达水平后的细胞的侵袭能力明显增加;(4)利用裸鼠皮下成瘤模型,发现干扰Med19的慢病毒可以减缓肿瘤的增长速度,抑制肿瘤的致瘤性。结论:Med19参与调控胶质母细胞瘤细胞的恶性生物学行为。
In 2010 , U.S.A added 22,020 new cases of brain tumor patients , in addition to 13,140 cases of brain tumor patients died ( www.cancer.org, Cancer Facts & Figures 2010). Astrocytomas are the most common primary intracranial tumors, which origin from neuroepithelial. Astrocytomas account for approximately 73%-86% of all adult intracranial tumors, among them, nearly 90% are malignant astrocytomas , which are prevalent of anaplastic astrocytoma and glioblastoma. After completly surgery, the recurrence rate of malignant astrocytomas is nearly 60%, with poor prognosis and low survival time. Nearly 5 years, with the development of basic and clinical research, it found that the average survival time of glioblastoma patients who receiving surgery, radiotherapy and chemotherapy, is increased from 10 months to 14 months. However, astrocytomas with the biological characteristics of a rich blood supply and infiltrating growth are easy to relapse.
Previous studies showed that the malignant progression, invasion and recurrence of astrocytomas are are a matter of multi-gene, multi-stage and multi-step complex processes, which are the results of a variety of genes, proteins and cytokines. The genes involved in the development of astrocytomas are epithelial growth factor, endothelial growth factor, transforming growth factor, tumor oncogenes and tumor suppressor genes. However, the molecular mechanisms underlying the initiation, maintenance and progression of astrocytomas still remain largely unclarified. Hence, identification and characterization of the regulatory molecules that involved in the astrocytomas tumorigenesis may offer important targets for treatment strategies.
Med19 gene, interacting with the RNA polymeraseⅡ, was found by means of differential display from two different metastatic potential of human lung carcinoma cell lines(95C and 95D ). In previous reports, Med19 was higher expressed in human breast cancer. Med19 played an important role in the proliferation of human breast cancer cells, which suggested that the lentiviruses delivering shRNA against Med19 could be a promising tool for breast cancer therapy. However, little was known about its role in malignant progression of astrocytomas tumorigenesis. Hence, further investigation of the functional role of Med19 in the carcinogenesis of astrocytomas may offer a better understanding of malignant behavior of astrocytomas. The aim of this study is to clarify the exactly role of Med19 in the oncogenic process of astrocytomas by examining its expression level in astrocytoma tissues of different grades and astrocytoma cell lines and knocking down or overexpressing its expression level in astrocytoma cell lines. This study is consisted of four main parts: the first part is to investigate the expression pattern of Med19 in astrocytomas of different grades and survival rate of the patients analysis; the second part is to study expression level of Med19 in different astrocytoma cell lines, including U87 , U251, U373 glioblastoma cell lines; the third part is to construct lentiviral-Mediated RNAi and overexpression system of Med19 gene. Finally, the effects of siRNA or overexpression targeting Med19 on tumor proliferation, colony formation, cell cycle and apoptosis are evaluated in vitro and in vivo.
PartⅠExpression of Med19 in Astrocytic Tumors and Survival Analysis of the Patients
Objective: The purpose of this study is to investigate the expression level of Med19 gene in astrocytomas of different grades and normal brain tissues and to analysis the direct relationship between the expression level of Med19 and patients’life span.
Methods: The expression levels of Med19 mRNA were evaluated by real-time quantitative PCR, and the protein levels of Med19 were assessed by using immunohistochemistry and western blot. Further, the direct relationship between the expression level of Med19 and patients’life span was evaluated by the product-limit estimate of the survival function (Kaplan-Meier method).
Results: First of all, quantitative real time PCR analysis demonstrated elevated expression levels of Med19/β-actin in high-grade astrocytomas versus low-grade (p<0.01) or normal brain tissues (p<0.01). And secondly, statistical analysis showed increased Med19 protein levels in high-grade astrocytomas versus low-grade tumors (p<0.001) or normal controls (p<0.01). Finally, Kaplan-Meier survival curves indicated that increased expression of Med19 was significantly associated with poor overall survival of astrocytoma patients (P<0.05).
Conclusion: In summary, we demonstrate that Med19 plays an important role in the astrocytic tumors. Med19 is positive in brain astrocytomas. The Med19 expression levels of astrocytomas were significantly correlated with pathological grade and were negatively correlated with patients’life span.
PartⅡExpression of Med19 in Astrocytic Tumors cell lines
Objective: To investigate the expression pattern of Med19 in glioblastoma cell lines, U87, U251, and U373.
Methods: Immunofluorescene labelling assay was employed to investigate the celluar location of Med19 protein in U251, U373, and U87 glioblastoma cell lines. The expression level of Med19 mRNA and protein were evaluated by real-time quantitative PCR and Western blotting respectively in U251, U373, and U87 glioblastoma cell lines.
Results: (1) Med19 protein was predominantly detected in the cytoplasm of astrocytoma cells by immunofluorescene labelling assay. (2) Quantitative real time PCR analysis demonstrated similar expression levels of Med19 /β-actin in U251, U373, and U87 glioblastoma cell lines. (3) Western blot analysis showed Med19 protein expressed in U251, U373, and U87 glioblastoma cell lines.
Conclusion: Med19 is present in glioblastoma cell lines, and its expression levels were examined at both mRNA and protein levels.
PartⅢConstruction and identification of lentiviral-mediated RNA interference and overexpression system targeting Med19
Objective: Med19 was detected in both astrocytoma tissues and glioblastoma cell lines. Its expression in astrocytoma samples was related to astrocytoma tumor grades. In this study, we constructed and identified the lentiviral-mediated RNA interference and overexpression system targeting Med19 for further study of its molecular function on the tumorgenesis of glioblastoma.
Method: 1) siRNA targeting sequences were constructed, after screening, they were connected with lentivirus, fluorescence microscope, real-time PCR, Western blot were then applied in U373, U251 and U87 cells to confirm the effects of RNA interference 2) Plasmid overexpressing Med19 was synthesed, after sequencing, it was connected with lentivirus, fluorescence microscope, real-time PCR, Western blot were then applied in U373, U251 and U87 cells to confirm the effects of overexpression.
Results: siRNA targeting sequences were constructed to knock-down Med19 expression, two of them were selected because of high interference efficacy of more than 75%. Med19 expression was significantly inhibited by these two siRNA sequences in U373, U87 and U251 cell lines at both mRNA and protein levels. The overexpression plasmid showed the same sequence with Med19, while Med19 expression was significantly increased by lentiviral-mediated overexpression plasmid in U373, U87 and U251 cell lines at mRNA and (or) protein levels.
Conclusion: We successfully constructed lentivirus based RNAi and overexpression system, it was highly effective to change the expression of Med19 in glioblastoma cells.
PartⅣLentiviral-mediated RNAi and overexpression targeting Med19 on oncogenic behavior of glioblastoma cells, in vitro and in vivo.
Objective: Using lentiviral-mediated RNAi and overexpression targeting Med19, we investigated the role of Med19 on oncogenic behavior of gliomblastoma cells, both in vitro and in vivo.
Method: MTT, colony formation, flow cytometry and nude mouse xenograft model were applied to assess the oncogenic behavior of gliomblastoma cell lines, U373 and U251. The fouces were on the effect of Med19 RNAi and overexpression on gliomblastoma cells growth both in vitro and in vivo.
Results: (1)In cell-based experiments, knocking down of Med19 can suppress the proliferation, colony formation of U251 and U87 glioblastoma cell lines, while overexpressing Med19 can enhance the proliferation, colony formation of glioblastoma cell lines. (2) In Med19 RNAi and overexpression U251 glioblastoma cells, the cell cycle and apoptosis change. (3)The capability of invasion in Med19 overexpression glioblastoma cells was enhanced. (4) In a nude mouse xenograft model, Med19 RNAi lentivirus significantly delayed tumor growth and extended mice’life span. Conclusion: Med19 played an important role in tumorigenesis of glioblastoma.
既往研究表明:星形细胞瘤的恶性进展及其侵袭复发是个涉及多基因、多阶段、多步骤的复杂过程,是受多种基因、蛋白以及细胞因子等共同调控的结果。参与星形细胞瘤发生、发展过程中的基因,在其他肿瘤的发生发展过程中几乎均发挥作用,如:上皮生长因子、内皮生长因子、转化生长因子、肿瘤原癌基因和抑癌基因等;此外星形细胞瘤侵袭复发的过程可能涉及到肿瘤细胞粘附、基质降解、迁移、细胞增殖等诸多环节,但其具体机制仍不清楚。近年来,基于对星形细胞瘤分子机制的逐步了解,靶向性分子疗法为星形细胞瘤治疗提供了新思路,寻找星形细胞瘤发生发展过程中的重要通路和靶标分子以及安全高效的靶向性载体系统显得尤为重要,但尚未将任何种靶标分子用于星形细胞瘤的治疗具有明确疗效。因此寻找调控星形细胞瘤细胞恶性生物学行为的分子靶标,明确其在星形细胞瘤发病过程中的病理机制,已成为目前星形细胞瘤治疗研究领域的的热点之。
Med19基因,与RNA聚合酶Ⅱ相互作用介导子基因,是2002年应用差异显示PCR方法从两种不同高低转移潜能的人肺癌细胞系中克隆到的人类新基因,目前尚无其功能学的报道。既往研究显示,Med19基因在多种正常组织以及肿瘤中有所表达,推测其可能与细胞的增殖、凋亡以及细胞分化密切相关。而在肺癌组织中发现,Med19基因的表达与肿瘤的恶性程度密切相关。在乳腺癌组织中发现,Med19蛋白的异常高表达,且运用慢病毒感染干扰Med19基因表达水平后,乳腺癌细胞的增殖能力和细胞周期受到显著影响;Medl9基因沉默后胃癌细胞的增殖能力和细胞周期均受到显著影响,因此推测其可能参与胃癌的发生发展以及促进恶性肿瘤细胞的增殖和浸润性生长。而在星形细胞瘤中Med19基因对肿瘤细胞的增殖,凋亡,侵袭以及血管生成等方面的研究在国内外,尚未见诸报道。因此,探讨MED19参与脑星形细胞瘤发生发展以及恶变的分子病理机制,有助于明确星形细胞瘤的恶性生物学行为及机理,有望为星形细胞瘤的临床治疗提供新的分子靶标。
本研究拟通过观察Med19在不同病理级别脑星形细胞瘤以及星形细胞瘤细胞株中的确切表达情况,并通过研究Med19在星形细胞瘤细胞株中的异常表达,研究Med19在星形细胞瘤发生发展过程中的功能角色。实验共分为四个部分:第部分,观察Med19在脑星形细胞瘤组织中的表达水平以及与星形细胞瘤病理级别的相关性,探讨Med19的表达水平与星形细胞瘤患者生存周期之间的关系;第二部分,观察Med19在U251、U373和U87脑星形细胞瘤细胞株中的表达情况;第三部分,靶向Med19的RNA干扰和过表达慢病毒载体的构建和鉴定;第四部分,通过基因操作的方法上调或抑制Med19基因在星形细胞瘤细胞内表达,观察其对U251星形细胞瘤细胞株生长、增殖、凋亡、侵袭等细胞表型以及裸鼠皮下成瘤的体内外试验的影响。
第一部分Med19在不同级别脑星形细胞瘤组织中的表达和临床病理相关性分析的实验研究
目的:研究Med19在脑星形细胞瘤组织中表达水平及其与星形细胞瘤病理WHO级别的相关性,Med19表达水平同星形细胞瘤患者生存时间的关系。
方法:首先,运用实时定量RT-PCR和Western blot杂交分别检测星形细胞瘤组织中Med19的mRNA及蛋白的表达水平并分析Med19表达水平同星形细胞瘤组织级别的相关性;随后采用免疫组化染色法检测5例正常脑组织和79例不同级别星形细胞瘤组织中Med19的表达情况;最后,临床随访星形细胞瘤患者,运用生存分析检测Med19表达水平同患者生存时间的相关性。
结果:(1)应用实时定量RT-PCR检测Med19 mRNA在21例不同级别脑星形胶质细胞瘤和5例正常脑组织中的表达显示:Med19 mRNA表达水平在星形细胞瘤组织中明显上调,与正常脑组织相比差别显著(P<0.01);并且,Med19 mRNA水平在高级别星形细胞瘤组织中的表达水平明显高于低级别星形细胞瘤,二者相比差异显著(P<0.01)。(2)运用Western blot技术测定Med19蛋白在上述星形细胞瘤组织的表达水平,结果显示:Med19蛋白在星形胶质细胞瘤组织中表达明显上调,与正常脑组织相比差别显著(P<0.01)。Med19在III-IV级(高级别)星形细胞瘤中高表达,而在I-II级(低级别)星形细胞瘤中表达量较低,且有显著差异(P<0.001)。(3)采用免疫组化法检测了5例正常脑组织及79例人脑星形细胞瘤石蜡标本中Med19蛋白的表达情况。结果显示,正常脑星形胶质细胞中Med19蛋白染色为阴性,绝大部分星形细胞瘤标本中可见Med19蛋白的阳性表达。星形细胞瘤的临床病理级别与Med19的表达强度与肿瘤恶性程度呈正相关关系(P<0.01);而性别和年龄则无统计学上的差异(P>0.05)。生存分析表明,Med19免疫组化染色表达水平同患者生存时间呈负相关(p<0.05)。
结论:Med19在脑星形细胞瘤中呈阳性表达,其表达水平同脑星形细胞瘤的级别呈明显正相关,同星形细胞瘤患者的生存时间呈负相关。
第二部分Med19在脑星形细胞瘤细胞株中的表达
目的:研究Med19在胶质母细胞瘤(GBM)细胞株U87、U373和U251中的表达情况。
方法:应用细胞免疫荧光法检测Med19在胶质母细胞瘤细胞株U87、U373和U251中的定位;运用实时定量RT-PCR法和Western blot技术检测Med19在胶质母细胞瘤细胞株U87、U373和U251中的mRNA和蛋白表达情况。
结果: (1)细胞免疫荧光结果提示:Med19在胶质母细胞瘤细胞株U87、U373和U251中的定位于细胞浆;(2)实时定量RT-PCR结果提示:Med19 mRNA在胶质母细胞瘤细胞株U87、U373和U251中均表达;(3)Western blot结果表明:Med19蛋白在胶质母细胞瘤细胞株U87、U373和U251均表达。
结论:Med19在胶质母细胞瘤细胞株中均有表达。
第三部分:靶向Med19的RNA干扰和过表达慢病毒载体的构建和鉴定
目的:Med19在星形细胞瘤组织和细胞中均有表达,其表达量与星形细胞瘤的病理级别呈正相关,提示Med19可能与星形细胞瘤的恶性生物学行为密切相关。本部分拟构建靶向Med19的慢病毒RNA干扰和过表达载体,为进步研究Med19的生物学功能提供载体工具。
方法:1)设计siRNA靶点,利用外源筛靶实验挑选高效干扰序列,与慢病毒载体连接,并利用荧光显微镜,实时定量RT-PCR、Western blot在U373、U251和U87细胞中验证RNA干扰效果。2)设计Med19过表达质粒并经过测序后,连接慢病毒载体,并利用荧光显微镜、实时定量RT-PCR、Western blot在U373、U251和U87细胞中验证过表达效果。
结果:设计的siRNA干扰序列,其中两条干扰效率较高,连接入慢病毒载体后,转染效率超过75%。在U373和U251细胞中,RNAi能够在mRNA水平和蛋白水平显著降低内源性Med19的表达(p<0.01);设计的过表达质粒经过测序证实与原基因序列完全致,慢病毒过表达载体能够在使U373和U251细胞中内源性Med19的mRNA和(或)蛋白水平表达量增加(p<0.05)。
结论:本研究成功构建了能够稳定改变内源性Med19表达水平的RNA干扰和过表达慢病毒载体,可为后续细胞功能学实验提供实验依据。
第四部分:靶向Med19调控胶质母细胞瘤细胞恶性生物学行为的体内外实验研究
目的:利用前期构建的稳定改变内源性Med19表达水平的RNA干扰和过表达慢病毒载体感染胶质母细胞瘤细胞株U251和U373细胞后,从体外和体内两个角度来研究Med19对胶质母细胞瘤恶性生物学行为的作用。
方法:内源性Med19表达水平改变后,运用MTT法检测细胞增殖曲线变化、克隆形成实验检测肿瘤细胞克隆形成能力变化、流式细胞仪测肿瘤细胞周期与凋亡比例变化、transwell细胞侵袭试剂盒检测细胞侵袭能力、运用荷瘤裸鼠模型检测Med19表达量改变对皮下种植瘤生长速率的影响。
结果:(1)Med19表达量降低后,胶质母细胞瘤细胞增殖能力减弱,克隆形成能力减弱;上调胶质母细胞瘤细胞内源性Med19的表达量后,其增殖能力增强,克隆形成能力增强;(2)U251MG细胞中,Med19表达量改变对细胞凋亡和细胞周期可产生影响;(3)在胶质母细胞瘤细胞株中,过表达内源性Med19表达水平后的细胞的侵袭能力明显增加;(4)利用裸鼠皮下成瘤模型,发现干扰Med19的慢病毒可以减缓肿瘤的增长速度,抑制肿瘤的致瘤性。结论:Med19参与调控胶质母细胞瘤细胞的恶性生物学行为。
In 2010 , U.S.A added 22,020 new cases of brain tumor patients , in addition to 13,140 cases of brain tumor patients died ( www.cancer.org, Cancer Facts & Figures 2010). Astrocytomas are the most common primary intracranial tumors, which origin from neuroepithelial. Astrocytomas account for approximately 73%-86% of all adult intracranial tumors, among them, nearly 90% are malignant astrocytomas , which are prevalent of anaplastic astrocytoma and glioblastoma. After completly surgery, the recurrence rate of malignant astrocytomas is nearly 60%, with poor prognosis and low survival time. Nearly 5 years, with the development of basic and clinical research, it found that the average survival time of glioblastoma patients who receiving surgery, radiotherapy and chemotherapy, is increased from 10 months to 14 months. However, astrocytomas with the biological characteristics of a rich blood supply and infiltrating growth are easy to relapse.
Previous studies showed that the malignant progression, invasion and recurrence of astrocytomas are are a matter of multi-gene, multi-stage and multi-step complex processes, which are the results of a variety of genes, proteins and cytokines. The genes involved in the development of astrocytomas are epithelial growth factor, endothelial growth factor, transforming growth factor, tumor oncogenes and tumor suppressor genes. However, the molecular mechanisms underlying the initiation, maintenance and progression of astrocytomas still remain largely unclarified. Hence, identification and characterization of the regulatory molecules that involved in the astrocytomas tumorigenesis may offer important targets for treatment strategies.
Med19 gene, interacting with the RNA polymeraseⅡ, was found by means of differential display from two different metastatic potential of human lung carcinoma cell lines(95C and 95D ). In previous reports, Med19 was higher expressed in human breast cancer. Med19 played an important role in the proliferation of human breast cancer cells, which suggested that the lentiviruses delivering shRNA against Med19 could be a promising tool for breast cancer therapy. However, little was known about its role in malignant progression of astrocytomas tumorigenesis. Hence, further investigation of the functional role of Med19 in the carcinogenesis of astrocytomas may offer a better understanding of malignant behavior of astrocytomas. The aim of this study is to clarify the exactly role of Med19 in the oncogenic process of astrocytomas by examining its expression level in astrocytoma tissues of different grades and astrocytoma cell lines and knocking down or overexpressing its expression level in astrocytoma cell lines. This study is consisted of four main parts: the first part is to investigate the expression pattern of Med19 in astrocytomas of different grades and survival rate of the patients analysis; the second part is to study expression level of Med19 in different astrocytoma cell lines, including U87 , U251, U373 glioblastoma cell lines; the third part is to construct lentiviral-Mediated RNAi and overexpression system of Med19 gene. Finally, the effects of siRNA or overexpression targeting Med19 on tumor proliferation, colony formation, cell cycle and apoptosis are evaluated in vitro and in vivo.
PartⅠExpression of Med19 in Astrocytic Tumors and Survival Analysis of the Patients
Objective: The purpose of this study is to investigate the expression level of Med19 gene in astrocytomas of different grades and normal brain tissues and to analysis the direct relationship between the expression level of Med19 and patients’life span.
Methods: The expression levels of Med19 mRNA were evaluated by real-time quantitative PCR, and the protein levels of Med19 were assessed by using immunohistochemistry and western blot. Further, the direct relationship between the expression level of Med19 and patients’life span was evaluated by the product-limit estimate of the survival function (Kaplan-Meier method).
Results: First of all, quantitative real time PCR analysis demonstrated elevated expression levels of Med19/β-actin in high-grade astrocytomas versus low-grade (p<0.01) or normal brain tissues (p<0.01). And secondly, statistical analysis showed increased Med19 protein levels in high-grade astrocytomas versus low-grade tumors (p<0.001) or normal controls (p<0.01). Finally, Kaplan-Meier survival curves indicated that increased expression of Med19 was significantly associated with poor overall survival of astrocytoma patients (P<0.05).
Conclusion: In summary, we demonstrate that Med19 plays an important role in the astrocytic tumors. Med19 is positive in brain astrocytomas. The Med19 expression levels of astrocytomas were significantly correlated with pathological grade and were negatively correlated with patients’life span.
PartⅡExpression of Med19 in Astrocytic Tumors cell lines
Objective: To investigate the expression pattern of Med19 in glioblastoma cell lines, U87, U251, and U373.
Methods: Immunofluorescene labelling assay was employed to investigate the celluar location of Med19 protein in U251, U373, and U87 glioblastoma cell lines. The expression level of Med19 mRNA and protein were evaluated by real-time quantitative PCR and Western blotting respectively in U251, U373, and U87 glioblastoma cell lines.
Results: (1) Med19 protein was predominantly detected in the cytoplasm of astrocytoma cells by immunofluorescene labelling assay. (2) Quantitative real time PCR analysis demonstrated similar expression levels of Med19 /β-actin in U251, U373, and U87 glioblastoma cell lines. (3) Western blot analysis showed Med19 protein expressed in U251, U373, and U87 glioblastoma cell lines.
Conclusion: Med19 is present in glioblastoma cell lines, and its expression levels were examined at both mRNA and protein levels.
PartⅢConstruction and identification of lentiviral-mediated RNA interference and overexpression system targeting Med19
Objective: Med19 was detected in both astrocytoma tissues and glioblastoma cell lines. Its expression in astrocytoma samples was related to astrocytoma tumor grades. In this study, we constructed and identified the lentiviral-mediated RNA interference and overexpression system targeting Med19 for further study of its molecular function on the tumorgenesis of glioblastoma.
Method: 1) siRNA targeting sequences were constructed, after screening, they were connected with lentivirus, fluorescence microscope, real-time PCR, Western blot were then applied in U373, U251 and U87 cells to confirm the effects of RNA interference 2) Plasmid overexpressing Med19 was synthesed, after sequencing, it was connected with lentivirus, fluorescence microscope, real-time PCR, Western blot were then applied in U373, U251 and U87 cells to confirm the effects of overexpression.
Results: siRNA targeting sequences were constructed to knock-down Med19 expression, two of them were selected because of high interference efficacy of more than 75%. Med19 expression was significantly inhibited by these two siRNA sequences in U373, U87 and U251 cell lines at both mRNA and protein levels. The overexpression plasmid showed the same sequence with Med19, while Med19 expression was significantly increased by lentiviral-mediated overexpression plasmid in U373, U87 and U251 cell lines at mRNA and (or) protein levels.
Conclusion: We successfully constructed lentivirus based RNAi and overexpression system, it was highly effective to change the expression of Med19 in glioblastoma cells.
PartⅣLentiviral-mediated RNAi and overexpression targeting Med19 on oncogenic behavior of glioblastoma cells, in vitro and in vivo.
Objective: Using lentiviral-mediated RNAi and overexpression targeting Med19, we investigated the role of Med19 on oncogenic behavior of gliomblastoma cells, both in vitro and in vivo.
Method: MTT, colony formation, flow cytometry and nude mouse xenograft model were applied to assess the oncogenic behavior of gliomblastoma cell lines, U373 and U251. The fouces were on the effect of Med19 RNAi and overexpression on gliomblastoma cells growth both in vitro and in vivo.
Results: (1)In cell-based experiments, knocking down of Med19 can suppress the proliferation, colony formation of U251 and U87 glioblastoma cell lines, while overexpressing Med19 can enhance the proliferation, colony formation of glioblastoma cell lines. (2) In Med19 RNAi and overexpression U251 glioblastoma cells, the cell cycle and apoptosis change. (3)The capability of invasion in Med19 overexpression glioblastoma cells was enhanced. (4) In a nude mouse xenograft model, Med19 RNAi lentivirus significantly delayed tumor growth and extended mice’life span. Conclusion: Med19 played an important role in tumorigenesis of glioblastoma.
引文
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