Experimental Study On UbcH10 As A Marker For The Diagnosis And Therapy Of Colorectal Cancer

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 cancerAim: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 siRNAAim: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.