摘要
泛素结合酶UbcH10 (Ubiquitin-conjugating enzymes UbcH10)又称周期蛋白选择性泛素载体蛋白E2-C,属于泛素结合酶E2家族的成员。在细胞周期调控过程中,UbcH10通过与具有E3连接酶活性的细胞后期促进复合物APC相互作用,影响有丝分裂纺锤体装配检测点驱动细胞周期的进展。在泛素介导的蛋白质降解途径中,E2蛋白的作用是通过其活性位点的半胱氨酸与来自E1活化酶的泛素以硫酯键相连,形成Ub-E2中间体,然后直接转移或通过E3连接酶转移泛素至靶蛋白,泛素化的靶蛋白被26s蛋白酶体识别而降解。泛素蛋白酶体系统(UP-S)具有多种生理调控功能,该系统功能失常能引起包括肿瘤在内的多种病理损害。Yoshiaki Okamoto等在研究泛素结合酶E2家族成员对肿瘤发生的潜在作用时,指出UbcH10是肿瘤相关性泛素结合酶。随着一些与泛素蛋白酶体途径相关致病蛋白的发现以及蛋白酶抑制剂Bortezomib (Velcade)对多发性骨髓瘤治疗调控的认可,特异性干涉UP-S的某一环节已被认为是一种很有前途的创新性抗肿瘤治疗方法。
大肠癌(Colorectal cancer, CRC)包括直肠癌和结肠癌,是常见的恶性肿瘤之一,在发达国家大肠癌已成为死亡率位居第2位的恶性肿瘤。近日,由中国抗癌协会临床肿瘤学协作专业委员会公布的最新数据表明大肠癌在我国目前总人群的发病率为十万分之二十,发病率占常见肿瘤的第四位,2008年上海市大肠癌的发病率占恶性肿瘤的第二位。而且,近年来发病率上升趋势十分明显。目前治疗大肠癌首选手术以及术后常规化疗,手术后5年生存率不到40%。化疗和放疗的联合应用取得一定疗效,也不十分令人满意。因此寻找一种新型非手术干预的治疗方法对于大肠癌的预防和治疗具有非常重要意义。
本研究中,我们首先检测了UbcH10在大肠癌肿瘤组织及癌旁正常组织的表达,统计分析了UbcH10与临床病理学特征的相关性。结果表明UbcH10在大肠癌患者肿瘤组织中过度表达,并与肿瘤的分化程度及淋巴结转移相关。随后我们构建了UbcH10及其siRNA真核表达载体,在细胞学水平上证实了UbcH10过表达能促进大肠癌细胞的增殖和侵袭能力,基因沉默UbcH10则能抑制大肠癌细胞的增殖和侵袭能力。我们对细胞周期检测的结果表明UbcH10的表达能影响细胞周期分布,这也是对UbcH10表达影响细胞增殖的进一步证实。最后,我们采用裸鼠皮下移植瘤模型研究了shRNA沉默UbcH10基因对体内大肠癌生长的抑制作用。研究结果为大肠癌的基因诊断及基因治疗的研究提供了理论依据和技术对策。
第一部分大肠癌组织中UbcH10的表达及临床病理资料分析
实验目的：检测泛素结合酶UbcH10在大肠癌组织中的表达,分析UbcH10与大肠癌临床病理学特征的相关性,探讨UbcH10作为大肠癌诊断标志物的可行性。
实验方法：
1.收集手术切除的原发性结直肠癌患者肿瘤组织标本及相应癌旁正常组织(距离肿瘤组织边缘＞5cm)冻存于-80℃冰箱。
2.采用TRIzol一步法抽提组织总RNA,RT-PCR获得UbcH10 cDNA,实时荧光定量PCR检测大肠癌患者肿瘤组织与相应癌旁正常组织中UbcH10 mRNA的丰度。
3.S-P(链霉卵蛋白-辣根过氧化物酶)免疫组化染色分析大肠癌组织中UbcH10
4.统计分析UbcH10表达与大肠癌临床病理学特征的相关性。
实验结果：
1.所有肿瘤组织样本中UbcH10 mRNA丰度均明显高于癌旁正常组织样本的UbcH10 mRNA丰度(p＜0.01)。
2.与癌旁正常组织相比,大肠癌组织中UbcH10蛋白表达明显增强(p＜0.01)。
3.大肠癌组织中UbcH10的表达与肿瘤的分化程度呈负相关,与淋巴结转移呈正相关(p＜0.05)。
结论：UbcH10基因在大肠癌患者肿瘤组织中过度表达,并与肿瘤的分化程度及淋巴结转移相关,提示UbcH10基因表达可作为大肠癌诊断的潜在肿瘤标记物及大肠癌基因治疗的靶点。
第二部分UbcH10及其siRNA真核表达载体的构建
实验目的：构建UbcH10基因及其siRNA真核表达载体。
实验方法：
1.应用RT-PCR方法扩增出UbcH10 mRNA 559bp的基因片断,插入pMD-18T载体中,经EcoRⅠ、HindⅢ酶切回收,插入真核表达载体pcDNA3.1形成重组质粒pcDNA3.1-UbcH10。
2.参考文献报道设计UbcH10基因的siRNA片段,表达载体pGPU6/GFP/Neo/siRN A-UbcH10的合成与构建由上海吉玛制药技术有限公司完成。
3.表达载体pcDNA3.1-UbcH10、pGPU6/GFP/Neo/siRNA-UbcH 10及其对照转染大肠癌细胞后,Western blot分析证实UbcH10及其siRNA真核表达载体的有效性。
实验结果：
1.测序证实RT-PCR方法从大肠癌细胞扩增出559bp的UbcH10片断序列正确。
2.对转染细胞行Western blot分析表明：与对照组相比,pcDNA3.1-UbcH10组UbcH10蛋白表达明显增强：pGPU6/GFP/Neo/UbcH10-siRNA组UbcH10蛋白表达下调。
结论：成功构建了UbcH10及其siRNA真核表达载体,为进一步研究UbcH10基因表达作为大肠癌诊治靶标的可行性提供了必要的工具。
第三部分UbcH10基因表达对大肠癌细胞增殖活性及侵袭能力影响的体外研究
实验目的：研究pcDNA3.1-UbcH10和pGPU6/GFP/Neo/UbcH10-siRNA调控的UbcH10表达改变对大肠癌细胞增殖、侵袭能力以及细胞周期分布的影响。
实验方法：
1.利用脂质体转染法将pGPU6/GFP/Neo/UbcH10-siRNA, pcDNA3.1-UbcH10及其相应空载体分别转染大肠癌细胞HT-29及LoVo,采用FACS和荧光显微镜检测转染细胞中GFP表达,以测定大肠癌HT-29和LoVo细胞的基因转染效率。
2.采用细胞增殖活性分析试剂盒Cell Kounting Kit-8 (CCK-8)检测转染pcDNA3.1-UbcH 10、pGPU6/GFP/Neo/UbcH10-siRNA及其相应对照载体的大肠癌细胞的增殖能力。
3.采用流式细胞术检测pcDNA3.1-UbcH10和pGPU6/GFP/Neo/UbcH 10-siRNA对HT-29和LoVo细胞周期分布的影响。
4.采用Matrigel侵袭实验分析UbcH10对大肠癌细胞侵袭活性的影响。
实验结果：
1.基因转染效率实验结果显示：转染48小时后GFP表达阳性细胞超过50%,72小时GFP表达阳性细胞接近80%。
2.与对照组相比,pcDNA3.1-UbcH10转染组大肠癌细胞增殖和侵袭能力明显增强；而pGPU6/GFP/Neo/UbcH10-siRNA转染组大肠癌细胞增殖和侵袭能力明显减弱。
3.流式细胞术结果显示：pcDNA3.1-UbcH10转染组大肠癌细胞周期分布在G2／M期明显减少,而pGPU6/GFP/Neo/UbcH 10-siRNA转染组大肠癌细胞在G2／M期明显增多。
结论：UbcH10表达能改变大肠癌细胞周期分布,UbcH10过表达大肠癌细胞增殖和侵袭能力增强,而UbcH10基因沉默的大肠癌细胞增殖和侵袭能力受抑制,提示UbcH10基因作为大肠癌治疗的靶标是可行的,为大肠癌的基因治疗研究提供了新的思路和技术对策。
第四部分UbcH10基因沉默对大肠癌抑制作用的体内研究
实验目的：建立裸鼠皮下大肠癌移植瘤模型,通过RNA干扰沉默移植瘤内UbcH10基因的表达,并观察其对裸鼠移植大肠癌的抑制作用。
实验方法：
1.利用脂质体转染法分别将pGPU6/GFP/Neo/UbcH 10-siRNA(pUbcH 10-RNAi)及其对照载体pGPU6/GFP/Neo/siRNA-NC(pRNAi-NC)分别转染大肠癌细胞并通过G418筛选建立沉默细胞系HT-29/UbcH10-RNAi、HT-29/RNAi-NC以及LoVo/UbcH10-RNAi和LoVo/RNAi-NC。
2.裸鼠皮下注射大肠癌细胞HT-29/UbcH10-RNAi、HT-29/PBS和HT-29/RNAi-NC以及LoVo/UbcH10-RNAi、LoVo/PBS和LoVo/RNAi-NC建立移植瘤模型,观察肿瘤的发生和生长状况,记录肿瘤的出现时间并绘制肿瘤生长曲线。
3.肿瘤细胞接种36天后断颈处死裸鼠,手术剥离肿瘤拍照并称取瘤体重量。
实验结果：
1. Western blot分析证实沉默细胞系HT-29/UbcH10-RNAi和LoVo/UbcH10-RNAi中UbcH10的表达得到有效抑制。
2.裸鼠皮下大肠癌移植瘤模型显示HT-29/UbcH10-RNAi及LoVo/UbcH10-RNAi组裸鼠的肿瘤生长明显低于对照组,致死裸鼠时治疗组肿瘤的体积不足对照组的一半,肿瘤重量仅是对照组肿瘤的40%左右。
结论：pUbcH10-RNAi沉默UbcH10基因能有效抑制裸鼠移植瘤的增殖,提示UbcH10基因可能是大肠癌基因治疗的潜在靶标。
UbcH10, also named cyclin-selective ubiquitin carrier protein E2-C, belongs to E2 (ubiquitin conjugating enzyme, Ubc) family. In cell cycle progression, UbcH10 along with the anaphase promoting complex/cyclosome (APC/C) affect the activation of the spindle checkpoint and regulate the cell cycle. In ubiquitin-dependent proteolysis, the E1 (ubiquitin activating enzyme) protein activates ubiquitin and then transfers it to the E2 (ubiquitin conjugating enzyme) protein. The ubiquitin forms an adduct to the E2 protein via a thiol ester linkage between the active site cysteine of E2 and the carboxyl terminus of ubiquitin. The E2 then donates the ubiquitin to the target protein, either directly or in conjunction with the E3 (ubiquitin ligase) activity. Ultimately, a polyubiquitin-target protein conjugate is formed that then is recognized and hydrolyzed by the proteasome. Ubiquitin-proteasome system (UP-S) plays a pivotal role in protein homeostasis and is critical in regulating normal and cancer-related cellular processes. In the study about the potential contribution of E2 protein to the tumorigenic response mediated by ubiquitination-dependent proteolysis, Yoshiaki Okamoto et al. report that UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme. With the first in class proteasome inhibitory agent Bortezomib (Velcade) obtaining regulatory approval for the treatment of multiple myeloma, the hierarchical nature of the UP-S provides a rich source of molecular targets for specific intervention and has therefore arisen as a promising approach to innovative anticancer therapies.
Colorectal cancer (CRC), including rectal carcinoma and colon cancer, is one of the most common malignancies. It is also the second leading cause of cancer mortality around the world. Recently, the data come from CSCO show that the morbidity has been 2 parts per million in total population of whole country and the fourth incidence of tumor, becoming higher and younger. In 2008, the morbidity of CRC was the second of all the cancer in Shanghai. At present, first-line therapy is radical surgery with adjuvant chemotherapy. But overall 5 year survival rate for this disease is around 40%, it is not satisfactory in patient outcome and survival rate. Therefore, it is important to find a novel non-surgical intervention for treating colorectal cancer.
In the present study, firstly, we examined the expression levels of UbcH 10 in human malignant colorectal carcinoma tissues and their adjacent normal tissues in 45 patients with malignant colorectal carcinoma and analyzed the correlations of UbcH 10 expression to the clinicalpathologic characteristics of the colorectal cancer. The results showed that the expression of UbcH10 in colorectal carcinoma tissues was significantly higher than that in noncancerous tissues (p<0.01), and the UbcH 10 overexpression was related to the degree of tumor differentiation and lymph node metastasis of colorectal cancer patients (p<0.05). Secondly, we constructed the UbcH10 expression plasmid pcDNA3.1/UbcH10 and UbcH 10 RNA interference vector pUbcH10-RNAi, then evaluated the effect of UbcH10 on the properties of tumor cell growth and invasiveness by cell proliferation and Matrigel invasion assays. Lastly, we examined the antitumor effects of pUbcH10-RNAi in a nude mouse xenografts model. All the studies were conducted to better understand the association of UbcH 10 with colorectal cancer carcinogenesis, and to provide a possible prognostic marker in these neoplasias. At the same time, because of the antitumor activity of RNA interference-mediated silencing of UbcH10 gene on colorectal cancer, it may be therapeutic potential for the treatment of colorectal cancer.
Part 1 Association of clinicopathological features with UbcH10 expression in colorectal cancer
Aim:To detect the expression of UbcH10 in the tissues of CRC patients, analyze the association of UbcH10 gene expression with the clinicopathological features and investigate the feasibility of UbcH10 as a diagnostic and prognostic marker in CRC.
Methods:
1. Tumor tissues and their adjacent normal tissues (The distance from normal tissue to tumor was beyond 5 centimeter) used in this study were obtained from surgical specimens and verified by histological examination, then stored at-80℃refrigeratory.
2. Total RNA was isolated using the Trizol reagent (Invitrogen, USA) according to the manufacturer's recommendations. UbcH10 cDNA was obtained by RT-PCR. The expression levels of UbcH10 in human malignant colorectal carcinoma tissues and their adjacent normal tissues were examined using real time quantitative RT-PCR.
3. The protein level of UbcH10 expression in human colorectal carcinoma tissues was examined by immunohistochemical analysis (streptavidin-perosidase method, viz. S-P method).
4. The correlations of UbcH10 expression to the clinicalpathologic characteristics of the colorectal cancer were analyzed.
Results:
1. The mRNA levels of UbcH10 in all of human malignant colorectal carcinoma tissues were markedly higher than their adjacent normal tissues (p<0.01)
2. Compared with noncancerous tissues, the expression of UbcH10 in colorectal carcinoma tissues was significantly increased (p<0.01)
3. UbcH10 overexpression was related to the degree of tumor differentiation and lymph node metastasis of colorectal cancer patients (p<0.05). Conclusion:Overexpression of UbcH10 gene plays a critical role in the carcinogenesis and tumor progression of colorectal cancer. It may be a new marker in diagnosis and prognosis of colorectal cancer, and the inhibition of UbcH10 may be a therapeutic potential for the treatment of colorectal cancer.
Part2. Construction of eukaryotic recombinant expression vector of UbcH10 and its siRNA
Aim:To construct eukaryotic expression plasmid pcDNA3.1-UbcH10 and pGPU6/GFP/Neo/siRNA-UbcH10.
Methods:
1. The UbcH10 cDNA was obtained by RT-PCR method with total RNA extracted from the tissue and cloned into the pMD-18T. The recombinant expression plasmid pcDNA3.1-UbcH10 was constructed by EcoRI and HindⅢrestriction site.
2. The siRNA-UbcH10 segment was designed according to the reference. Expression plasmid pGPU6/GFP/Neo/siRNA-UbcH10 was constructed by GenePharma Company.
3. Both of the expression plasmids were transfected into human colon cancer cell line HT-29 and LoVo using liposome and detected by Western Blot.
Results:
1. Verified by sequencing, the length and sequence of the cloned UbcH 10 segment was correct.
2. Compared with the control cells, the UbcH10 expression in the group of pcDNA3.1-UbcH10 increased obviously but in the group of pGPU6/GFP/Neo/siRNA-UbcH10 reduced obviously.
Conclusion:The eukaryotic expression plasmids pcDNA3.1-UbcH10 and pGPU6/GFP/Neo/siRNA-UbcH10 were successfully constructed and expressed effectively in the colorectal cells. It lays a foundation on the feasibility study of UbcH10 as a marker in CRC diagnosis and therapy.
Part3. Effects of UbcH10 expression on colorectal cancer cell proliferation and invasion in vitro.
Aim:To study the effects of UbcH10 expression on colorectal cancer cell proliferation, invasion and cell cycle distribution.
Methods:
1. The pcDNA3.1-UbcH10, pUbcH10-RNAi and control plasmids were transfected into colorectal cells using LipofectaminTM 2000, respectively. The successfully transfected cells could express enhanced green fluorescent protein (GFP) which were verified by FACS and fluorescent microscopy.
2. Cell Counting Kit-8 was performed to assess properties of tumor cell growth in vitro.
3. Effects of UbcH10 expression on the cell cycle of colorectal cancer cells were analyzed by Flow cytometry.
4. Matrigel invasion assays were performed in HT-29 cells transfected with UbcH10 expression plasmid pcDNA3.1-UbcH10, UbcH10 RNA interference vector pUbcH10-RNAi as well as their control vectors.
Results:
1. The transfection was in high efficiency. Cells expressing enhanced green fluorescent protein (GFP) were more than 50%at 48h after transfection and near 80%after 72h.
2. Compared with control group cells, the overexpression of UbcH10 promoted cell proliferation and tumor invasiveness, but the downregulation of UbcH10 expression significantly reduced the growth rate and the invasiveness activity of tumor cell line.
3. Downregulation of UbcH10 markedly arrested colorectal cancer cells in the G2-M phase. Consistently, the distributions of pcDNA3.1-UbcH10 group cells at G2-M phases of the cell cycle were decreased significantly.
Conclusion:Colorectal cancer cell cycle was influenced by UbcH10 expression. The overexpression of UbcH 10 promoted cell proliferation and tumor invasiveness, but the silencing of UbcH10 gene significantly reduced the growth rate and the invasiveness activity of tumor cell line.Specific intervention to UbcH10 laid the foundation for gene therapy in colorectal cancer.
Part4. Inhibition effects of UbcH10 silencing on colorectal cancer in vivo.
Aim:To study the effects of UbcH10 silencing on colorectal cancer in vivo by a nude mouse xenografts model.
Methods:
1. The cells were transfected with pGPU6/GFP/Neo/UbcH10-siRNA (pUbcH10-RNAi) and pGPU6/GFP/Neo/siRNA-NC (pRNAi-NC), and selected with G418 (200μg/ml) starting 48h after transfection. The successfully transfected cells were verified by fluorescent microscopy.
2. The cell suspensions including HT-29/UbcH10-RNAi, HT-29/PBS, HT-29/RNAi-NC and LoVo/UbcH10-RNAi, LoVo/PBS, LoVo/RNAi-NC were injected subcutaneously into BALB/C nude mice. Tumor growth was monitored every 4 days starting from the 8th day after injection.
3. On the 36th day after inoculation, all mice were sacrificed, and the tumor masses were weighed.
Results:
1. Western blot verified UbcH10 expression was inhibited in HT-29/UbcH 10-RNAi and LoVo/UbcH10-RNAi cells.
2. HT-29/UbcH10-RNAi and LoVo/UbcH10-RNAi cells xenografts demonstrated growth suppression compared to control colorectal cancer cell xenografts. The size and weight of tumors from pUbcH10-RNAi groups were significantly decreased. The average weight of tumors in HT-29/UbcH10-RNAi and LoVo/UbcH10-RNAi groups was only-40%of that in control groups, demonstrating an in vivo growth-inhibitory effect.
Conclusion:Our study suggests that RNA interference-mediated silencing of UbcH10 gene has antitumor activity on colorectal cancer and has therapeutic potential for the treatment of colorectal cancer.
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