摘要
脑胶质瘤是中枢神经系统最常见的恶性肿瘤之一,约占全部脑肿瘤的45～55%。目前,脑胶质瘤的治疗手段主要包括手术治疗、放射治疗、化学治疗和综合治疗等,尽管近年来上述治疗措施已取得长足进步,但由于胶质瘤呈浸润性生长,与周围脑组织无明显分界且易于复发,且疗效均不甚理想。新的治疗手段如基因治疗等虽有望成为治愈胶质瘤的途径之一,但是目前仍缺乏理想可靠的治疗靶标。因此寻找调控胶质瘤细胞恶性生物学行为的分子靶标,明确其在胶质瘤发病过程中的病理机制,已成为神经外科领域的研究热点之一。
泛素/蛋白酶体介导的蛋白质降解途径(Ubiquitin-proteasome proteolytic pathway,UPP途径)是蛋白质转录后降解的重要方式之一,此途径主要由泛素活化酶(E1)、泛素结合酶(E2)、泛素连接酶(E3)和26S蛋白酶体组成。目标底物蛋白通过上述酶的序贯作用后与泛素发生共价结合,继而被26S蛋白酶体识别并降解。UPP途径参与细胞周期进程、DNA转录/修复、细胞增殖分化和凋亡等诸多生物学过程,该途径的异常与肿瘤的发生发展关系密切。我科前期基因芯片以及ONCOMINE生物芯片研究结果证实,E2蛋白酶家族成员之一—泛素结合酶UbcH10在脑胶质瘤组织中表达异常上调,并在多种周围肿瘤组织中出现过度表达。作为在多种来源肿瘤组织中异常表达的“枢纽基因(hub gene)”,UbcH10过度表达可能与肿瘤发生发展过程密切相关。因此,探讨UbcH10参与脑胶质瘤发生发展以及恶变的分子病理机制,有助于明确胶质瘤的恶性生物学行为及机理,有望为胶质瘤的临床治疗提供新的分子靶标。
本研究拟通过观察UbcH10在不同病理级别脑胶质瘤以及胶质瘤细胞株中的确切表达情况,并通过敲减UbcH10在胶质瘤细胞株中的表达,研究UbcH10在胶质瘤发生发展过程中的功能角色。实验共分为三个部分:第一部分,观察UbcH10在脑胶质瘤组织中的mRNA和蛋白表达水平以及与胶质瘤病理级别的相关性;第二部分,观察UbcH10在U251和SHG-44脑胶质瘤细胞株中的mRNA和蛋白表达水平及其与胶质瘤细胞细胞周期的关系;第三部分,通过RNA干扰的方法敲减UbcH10的表达观察其对U251胶质瘤细胞株增殖、凋亡等细胞表型的影响。
第一部分泛素结合酶基因UbcH10在脑胶质瘤组织中的表达研究
目的:研究UbcH10在脑胶质瘤组织中的mRNA剪切形式、mRNA和蛋白表达水平及其与胶质瘤病理级别的相关性。
方法:根据UbcH10的cDNA序列设计相应引物,应用RT-PCR和直接测序的方法检测UbcH10 mRNA的不同剪切形式在32例不同级别脑星形胶质细胞瘤和6例正常脑组织中的表达。应用实时荧光定量PCR检测UbcH10 mRNA在上述组织标本中的相对表达水平,应用免疫组织化学染色检测UbcH10在55例脑星形胶质细胞瘤组织和7例正常脑组织中的蛋白表达水平,并检测UbcH10蛋白与增殖抗原Ki-67表达的相关性,最后应用免疫荧光双重染色技术获取UbcH10蛋白的细胞定位信息。
结果:32例脑星形胶质细胞瘤和6例正常脑组织mRNA反转录后进行PCR扩增获得450bp左右产物,经直接测序证实与UbcH10 splice vl序列一致(GenBankaccession nos.NM_007019)。实时荧光定量PCR结果提示UbcH10 mRNA表达水平在星形胶质瘤组织中明显上调,与正常脑组织相比差别显著(44.33±55.32 vs 1.00±1.57;P=0.000)。并且,UbcH10 mRNA水平在高级别胶质瘤组织中的表达水平明显高于低级别胶质瘤,二者相比差异显著(64.33±60.98 vs 8.36±8.15;p=0.000)。相似地,免疫组织化学染色提示UbcH10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(7.90±5.70%vs 0.0±0.0%;P=0.000),且高、低级别胶质瘤之间差别显著(10.53±5.79%vs 4.23±2.85%;P=0.000),UbcH10阳性细胞染色率与增殖抗原Ki-67蛋白表达呈明显的正相关关系(Spearman r=0.63,P＜0.001)。双重免疫荧光染色提示UbcH10蛋白与GFAP蛋白共定位于胶质瘤细胞胞浆。
结论:UbcH10以splice vl mRNA剪切形式表达于所有被检测的星形胶质细胞瘤和正常脑组织中,但其在星形胶质细胞瘤组织中的mRNA和蛋白表达水平明显高于正常脑组织,并和胶质瘤病理级别以及增殖抗原Ki-67表达呈正相关性,提示UbcH10可能在星形胶质瘤的发生发展恶变过程中具有重要作用。
第二部分UbcH10在U251,SHG-44胶质瘤细胞株中的表达及其与细胞周期的关系
目的:研究泛素结合酶UbcH10在胶质瘤细胞系U251和SHG-44中的mRNA、蛋白表达水平和细胞亚定位水平;研究UbcH10蛋白在不同细胞周期的表达及与细胞周期蛋白Cyclin A、Cyclin B1的关系。
方法:应用RT-PCR、实时荧光定量PCR等方法检测UbcH10在胶质瘤细胞系U251和SHG-44中的mRNA剪切形式和表达水平,应用细胞免疫组化、蛋白印迹等方法研究UbcH10在胶质瘤细胞系中的蛋白表达水平,应用免疫荧光染色检测UbcH10在胶质瘤细胞中的细胞亚定位水平及其与Ki67蛋白的定位关系。采用无血清饥饿、胸腺嘧啶阻断和Nocodazole等方法分别同步化细胞于G_0、G_1/S和G_2/M期,应用蛋白印迹实验检测UbcH10在上述不同细胞周期的表达情况及其与细胞周期相关蛋白Cyclin A、Cyclin B1的相互关系。
结果:经过PCR扩增和直接测序发现UbcH10在胶质瘤细胞系中的mRNA剪切形式为splice vl(GenBank accession nos.NM_007019)。荧光定量RT-PCR结果提示UbcH10 mRNA表达水平在胶质瘤细胞系U251和SHG-44中明显上调,与正常脑组织相比差别显著(33.77±21.54,30.20±20.13 vs 1.00±1.57;P=0.000)。免疫组织化学染色和免疫荧光染色提示UbcH10蛋白主要表达于胶质瘤细胞胞浆,并且部分表达UbcH10细胞亦表达增殖抗原Ki67,蛋白印迹实验证实其在U251和SHG-44细胞系中的表达水平明显高于正常脑组织(0.40±0.23 vs 0.0±0.0;P＜0.05)。UbcH10主要表达于G_2/M期,在G_0,G_1/S期表达甚少。自双胸腺嘧啶阻断释放后,UbcH10的表达逐渐升高,在G_2/M期达到高峰,并与细胞周期蛋白Cyclin A、Cyclin B1呈消长关系;自Nocodazole阻断释放后,UbcH10表达逐渐下降,细胞周期蛋白Cyclin A、Cyelin B1表达亦逐渐下降。
结论:UbcH10以splice vl mRNA剪切形式表达于所有被检测的星形胶质瘤细胞系中,其mRNA和蛋白表达水平明显高于正常脑组织,UbcH10在胶质瘤细胞系中的表达与细胞周期密切相关,在G_2/M期达到峰值。
第三部分靶向UbcH10的RNA干扰对U251胶质瘤细胞株增殖、凋亡和细胞周期改变的作用
目的:研究靶向UbcH10的RNA干扰对U251胶质瘤细胞株增殖、凋亡的作用。
方法:设计并合成靶向UbcH10的siRNA,脂质体法瞬时转染U251细胞株,优化转染条件。MTT法测定转染后24、48、72和96小时U251细胞增殖率,Annexin/PI双染测定干扰后24、48和72小时早期凋亡细胞比例和细胞周期改变,TUNEL原位检测UbcH10 siRNA 48h后凋亡细胞比例,蛋白印迹实验测定转染后48小时Cyclin A、Cyclin B1、PCNA、Bax、Bcl-2、P53等蛋白表达水平变化。
结果:UbcH10 siRNA可有效敲减U251细胞UbcH10的表达。MTT法检测UbcH10siRNA转染组24,48,72和96小时的细胞生长吸光值分别为0.060±0.009,0.082±0.010,0.139±0.028,0.235±0.045;较阴性对照组(0.115±0.026,0.217±0.038,0.381±0.040,0.646±0.077)和未转染组(0.101±0.017,0.215±0.048,0.390±0.047,0.747±0.126)降低;其中24、48、72和96小时细胞生长与对照组相比,差异均有统计学意义(P＜0.05)。经流式细胞仪检测,24,48,72小时靶向siRNA转染引起U251胶质瘤细胞的凋亡率分别为22.70±8.83%,12.37±2.14%和5.97±1.20%,阴性对照组为8.00±1.67%,4.63±1.45%和5.77±1.34%,未转染组为4.07±1.06%,4.60±1.01%和4.27±0.90%。在24、48小时,早期凋亡比例均高于对照组和未转染组,两者之间差异显著(P＜0.05)。靶向UbcH10 siRNA转染U251细胞48小时后G_2/M期细胞比例显著升高,较阴性对照组和未转染组差异显著(37.53±2.04%vs 18.01±1.60%,18.62±0.69%;P＜0.05)。TUNEL检测结果显示,在UbcH10 siRNA转染组中,有散在分布的阳性染色细胞,其比例明显高于阴性对照组和未转染组(26.75±7.56%vs 7.64±3.75%,4.33±2.10%,P＜0.05)。敲减UbcH10的表达后,Cyclin A的表达在干扰后出现升高,与对照组相比差异显著;Cyclin B1的表达在干扰后亦出现升高趋势;PCNA的表达在干扰后即下降,并且持续至干扰后72小时,与对照组相比差异显著;Bax的表达在干扰后出现升高,Bcl-2的表达在干扰后24至48小时出现降低,与对照组相比差异显著;P53的表达在干扰后出现升高,与对照组相比差异显著,p-P53的表达在干扰后亦出现升高趋势,48和72小时与对照组相比差异显著。然而,Akt、p-Akt、P38、p-P38、c-Jun和p-c-Jun等蛋白表达水平无明显改变。
结论:通过RNA干扰敲减UbeH10在U251胶质瘤细胞中的表达可有效抑制肿瘤细胞的增殖,其可能原因是通过减少cyclin A和cyclin B1的降解,由此导致细胞周期的阻滞;敲减UbcH10表达可促进P53、Bax蛋白的表达,并抑制Bcl-2的表达,由此促进胶质瘤细胞P53途径依赖的凋亡;敲减UbcH10对MAPKs、P13K/Akt和c-Jun介导的信号转导通路重要调控分子活性无明显影响。由此可见,敲减UbcH10的表达可以抑制胶质瘤细胞的增殖并且促进其凋亡,抑制UbcH10表达可能作为未来胶质瘤靶向治疗的选择之一。
Astrocytoma remains the most common primary neoplasm of the central nervous system and accounts for approximately 45～55%of all brain tumors.It represents a heterogenous group of diseases with different degree of malignancy from relatively indolent pilocytic astrocytomas to highly aggressive glioblastomas.Unfortunately,current therapeutic modalities including surgical resections,chemotherapy,radiotherapy or combinations can not ensure a cure and,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 astrocytoma tumorigenesis may offer important targets for treatment strategies.
Ubiquitin-proteasome proteolytic pathway is a major way for the post-transcriptional degradation of most proteins and is consisted of E1,E2,E3 protein and 26S proteasome. Ubiquitin-conjugating enzymes(E2) belong to a family of structurally related proteins that mediate the process of ubiquitin-dependent proteolysis,which pair with specific ubiquitin-ligase proteins(E3) and catalyze the ubiquitination of substrate proteins.As a member of the E2 family,ubiquitin-conjugating enzyme E2C(UbcH10) has drawn lots of attention lately owing to its involvement in the carcinogenesis of certain tumors.It is required for mitotic cyclins destruction and for cell cycle progression and,however,is significantly overexpressed in a variety of malignancies including breast,ovarian,thyroid, esophageal,and hepatocellular carcinomas.As a "hub gene" which has been involved in the carcinogenesis of various cancers,UbcH10 might play an important role in the malignant transformation of tumor cells.Hence,further investigation of the functional role of UbcH10 in the carcinogenesis of glioma cells may offer a better understanding of malignant behavior of glioma cells.
The aim of this study is to clarify the exact role for UbcH10 in the oncogenic process of glioma cells by examining its expression pattern in glioma tissues of different grades and glioma cell lines and knocking down its expression level in glioma cell lines.This study is consisted of three main parts:the first part is to investigate the splice pattern and expression level of UbcH10 gene in astrocytomas of different grades and normal brain tissues,the second is to investigate the expression pattern of UbcH10 in glioma cell lines and its relationship with cell cycle process and finally,the effects of siRNA targeting human UbcH10 on tumor proliferation and apoptosis are evaluated in U251 glioma cells.
PartⅠExpression of ubiquitin-conjugating enzyme E2C/UbcH10 in astrocytic tumors
Objective:To investigate the splice pattern and expression level of UbcH10 gene in astrocytomas of different grades and normal brain tissues.
Methods:The splice pattern of UbcH10 mRNA was investigated by RT-PCR and direct sequencing in 32 astrocytic tumors and 6 normal brain tissue samples.The expression levels of UbcH10 mRNA were evaluated by real-time quantitative PCR.By using immunohistochemistry,expression levels of UbcH10 protein were assessed in 55 astrocytic tumors of different pathological grades and 7 normal brain controls.Further,the correlation between UbcH10 and Ki-67 immunoreactivity was examined with the Spearman's correlation coefficient.Finally,immunofluorescene labelling assay was employed to investigate the celluar location of UbcH10 protein in astrocytomas.
Results:RT-PCR analysis revealed single bands corresponding to the calculated size of the amplified UbcH10 splice v1 in either astrocytic tumors or normal brain tissue samples using the sets of primers.Sequence analysis identified that each RT-PCR amplification product matched the published human UbcH10 v1 gene(GenBank accession nos. NM_007019).Quantitative real time PCR analysis demonstrated elevated expression levels of UbcH10/β-actin in high-grade astrocytomas versus low-grade(64.33±60.98 vs 8.36±8.15,respectively;P=0.000) or normal brain tissues(64.33±60.98 vs 1.00±1.57, respectively;P=0.000).Further,UbcH10 immunoreactivity was predominantly detected in the cytoplasm of tumor cells,whereas no positive staining for UbcH10 was observed in normal brain tissues.Statistical analysis showed increased UbcH10 labelling index in high-grade astrocytomas versus low-grade tumors(10.53±5.79%vs 4.23±2.85%, respectively;P=0.000) or normal controls(10.53±5.79%vs 0.0±0.0%,respectively;P =0.000).Importantly,UbcH10 staining positively correlated with the proliferative activity indicated by Ki-67 staining(P＜0.001,r=0.63,by Spearman's correlation coefficient). Double immunofluorescene labelling assay demonstrated a close co-localization of UbcH10 and GFAP in the cytoplasm of glioma cells.
Conclusion:In summary,we demonstrate that UbcH10 splice variant 1 is present in either astrocytomas or normal brain tissues examined,and its expression positively correlates with the degree of malignancy at both RNA and protein levels.These data suggest that overexpression of UbcH10 may serve as one important molecular mechanism that underlies the development and progression of astrocytic tumors.
PartⅡExpression of ubiquitin-conjugating enzyme E2C/UbcH10 in U251 and SHG-44 glioma cell lines
Objective:To investigate the expression pattern of UbcH10 in glioma cell lines and its relationship with cell cycle process.
Methods:The splice pattern of UbcH10 mRNA was investigated by RT-PCR and direct sequencing in U251 and SHG-44 glioma cell lines.The expression level of UbcH10 mRNA was also evaluated by real-time quantitative PCR in these two glioma cell lines. Immunofluorescene labelling assay was employed to investigate the celluar location of UbcH10 protein in U251 and SHG-44 glioma cell lines.Further,UbcH10 protein expression pattern and its relationship with cell cycle were investigated at G1,S and G2/M phase which were blocked by no serum starve,thymidine and nocodazole,respectively.
Results:RT-PCR analysis revealed single bands corresponding to the calculated size of the amplified UbcH10 splice variant 1(v1) in U251 and SHG-44 glioma cells.Sequence analysis identified that each RT-PCR amplification product matched the published human UbcH10 v1 gene(GenBank accession nos.NM_007019).Quantitative real time PCR analysis demonstrated elevated expression levels of UbcH10/β-actin in U251 and SHG-44 glioma cells versus normal brain tissues(33.77±21.54,30.20±20.13 vs 1.00±1.57; P=0.000).UbcH10 immunoreactivity was predominantly detected in the cytoplasm of tumor cells by immunohistochemistry and immunofluorescene labelling assay.Western blot analysis showed increased UbcH10 expression levels in glioma cells versus normal brain tissues(0.40±0.23 vs 0.0±0.0;P＜0.05).Expression of UbcH10 protein was mainly detected in G_2/M phase of cell cycle instead of G_0 and S phase.Expression levels of UbcH10 increased in a time-dependant manner after release from double thymidine arrest, whereas expression levels of cyclin A and cyclin B1 decreased in an opposite way.After release from nocodazole arrest,expression levels of UbcH10 as well as cyclin A and cyclin B1 decreased in a time-dependant manner.
Conclusion:UbcH10 splice variant 1 is present in glioma cell lines examined,and its expression levels significantly increase at both mRNA and protein levels versus normal brain tissues.The expression pattern of UbcH10 is regulated in a cell cycle-dependent manner,and its expression level reachs a peak value at G_2/M phase transition.
PartⅢRNA interference targeting UbcH10 inhibits glioma cell proliferation,enhance cell apotosis in vitro
Objective:To investigate the effects of siRNA targeting human UbcH10 on tumor proliferation and apoptosis of U251 glioma cells.
Methods:Three siRNA targeting human UbcH10 were transfected into U251 glioma cells. Cell proliferative activity was assessed by MTT assay and,cell apoptosis was analyzed by flow cytometry and in situ TUNEL assay.The expression levels of cyclin A,cyclin B1, PCNA,Bax,Bcl-2 and P53 Protein were measured by western blot.
Results:The expression level of UbcH10 protein was knocked down by UbcH10 siRNA as indicated by western blotting analysis.Proliferation of U251 glioma cells was significantly reduced compared with that of control at 24,48,72 and 96h post transfection.The flow cytometric analysis showed increased proportion of annexinⅤpositive cells in UbcH10 siRNA group at 24 and 48h post transfection versus si-scrambled and untransfected groups(22.70±8.83%vs 8.00±1.67%,4.07±1.06%at 24h;12.37±2.14%vs 4.63±1.45%,4.60±1.01%at 48h post transfection).Similar results were obtained with TUNEL staining(26.75±7.56%vs 7.64±3.75%,4.33±2.10%;P＜0.05).Expression levels of cyclin A siginificantly increased at 24 and 48h after knocking down UbcH10 expression as well as cyclin B1.Expression levels of PCNA decreased significantly post transfection of UbcH10 siRNA.Expression levels of Bax siginificantly increased after knocking down UbcH10 expression,whereas expression levels of Bcl-2 decreased post transfection. Expression levels of P53 increased significantly post transfection of UbcH10 siRNA as well as the expression levels of p-P53.However,no significant difference for the expression levels of Akt、p-Akt、P38、p-P38、c-Jun and p-c-Jun was noted.
Conclusion:Overall,our results indicate that siRNA transfection targeting UbcH10 can inhibit cell proliferation and induce apoptosis through a P53 dependant way.Given the strong upregulation of UbcH10 expression in malignant gliomas,knock-down of UbcH10 expression may therefore represent a potentially treatment strategy against glioma.
引文
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