| Background and objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, ranking fifth in clinical morbidity of all the malignant tumors worldwide. At present, there is a clear ascendant trend in the incidence of HCC and about 625000 new cases each year according to the latest data. In recent years, the morality of HCC remains very high though more and more patients can be diagnosed and undergone operations. There are about 600000 deaths due to HCC worldwide, taking the third place just after stomach cancer and esophageal cancer. The incidence of HCC in our country was higher than any other countries in the world and the numbers of cases occupy 55% among all the HCC patients worldwide. The lack of effective treatments make liver cancer become the second cancer killer in our country, threatening people’s health and life.The incidence and Development of HCC is a multi-step, gradual process, accompanied by the expression regulation disorders of many oncogenes and tumor suppressor genes. Many studies have shown the process is related to these disorders, for examples, the inactivation of tumor suppressor genes such as PTEN, or the upregulation or mutation of oncogenes such as GPC3 and TGF-β1. However, these genes, value for early diagnosis and prognostic judgment as molecular markers is very limited. Therefore, studying the molecular mechanism of developing liver cancer, looking for liver cancer diagnosis and treatment of new molecular markers is still an important part of the study of liver cancer. So, it is a very significant work in the study of HCC to explore its molecular mechanisms and search for novel diagnostic markers and treatment targets.Ubiquitin-conjugating enzyme E2T (UBE2T) is a component of ubiquitin-conjugating enzyme (E2) family, with a characteristic conserved domain-ubiquitin binding domain (UBC), with a C-terminal expanding sequence, which is about 16-18kDa, belongs to the secondary category of ubiquitin-conjugating. UBE2T is an important component of Fanconi anemia pathway (FA), DNA damage can activate the pathway, in which the ubiquitin-activating enzyme passes ubiquitins to the ubiquitin-conjugating enzyme (UBE2T) ubiquitin ligase UBE2T, then UBE2T passes ubiquitins to FA core complex (including FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FANCM), which can stimulate FANCD2 monoubiquitination as an ubiquitin ligase, then the monoubiquited FANCD2 targets the cell nucleus to repair the damaged DNA.More and more researches show that ubiquitin-conjugating enzymes (E2), as an important member in the ubiquitin-proteasome system, have a close relationship with cell proliferation, apoptosis and oncogenesis. In the early study, Takata et al. found UBE2T was upregulated in the specimens of bladder cancer, lung cancer, and prostate cancer. Then Yuichi et al. found UBE2T expression can adjust the level of single ubiquitin FANCD2, thereby regulating FA pathway, blocking cell DNA damage repair pathways, cause the occurrence of tumor. In recent years, studies have shown that UBE2T as a tumor related genes in a wide variety of tumor development plays an important role in regulating, Hao et al. found increased expression in lung cancer tissue and express UBE2T lung cancer cells colony-forming ability significantly higher, indicating UBE2T expression level is closely related to the initiation of lung cancer. Study of Ueki et al. suggested that suppressing UBE2T expression or inhibiting its activity can restrain the degradation of BRCA1, inhibiting the proliferation of breast cancer cells. Ramaekers et al. found that in the condition of hypoxi, UBE2T expression can increase the chemotherapy sensitivity of cancer cells. These results suggest that UBE2T involves in the development of oncogenesis as an oncogene.First of all, we used tissue microarray and immunohistochemistry to explore the expression level of UBE2T in the digestive system malignant tumor tissues. Then we focused on the the level of gene and protein in hepatocellular carcinoma to analyze how UBE2T have effect on the prognosis of HCC patients, making clear its role in the initiation and development in HCC, also illustrating its influence on the invasion and metastasis, finally laying the theoretical basis for HCC new prognostic molecular markers.Methods1. The expression level of UBE2T in digestive system tumors.We applied tissue microarray of digestive system purchased from xi’an Alenabio biological technology limited company, a total of 96 points, including 37 cases of malignant tumors (7 cases each for esophageal cancer, gastric cancer and colon cancer,6 cases of liver cancer,4 cases of colorectal cancer,5 cases of small bowel cancer,1 case of cholangiocarcinoma) and 11 cases of normal tissues (2 cases each for esophagus, stomach, liver, colon and rectum tissue, and 1 case of intestinal tissue).15 cases of female,33 cases of male, aging from 22 to 79 years old, at a mean of (50.87±13.534) years old, all the tumors were classified according to the WHO standard classification, pathologically confirmed. We used immunohistochemistry to detect the expression of UBE2T in digestive system tumors. Two-sample nonparametric tests to analyze the immunohistochemistry scores, comparing the difference between the expression level of UBE2T in tumor tissues and normal tissues.2. The expression of UBE2T in HCC.We use immunohistochemistry to detect the expression of UBE2T in the paraffin sections of primary HCC, then grading its expression level. We apply Kaplan-Meier method to analyse the influence of UBE2T on prognosis of HCC, Spearman rank correlation method to analyse the relationship between the UBE2T expression and clinical pathological parameters, and COX proportional hazards regression model to do univarible and multivariable factor analysis on the relationship between the clinical pathological parameters and the prognosis of primary HCC to make clear UBE2T whether can be an independent risk factor for the prognosis of primary HCC; Real time-PCR method was used for the detection the mRNA level of UBE2T of 30 cases of HCC tissues and adjacent tissues to verify its difference between them; Western blot was used for the protein level.3. The biological effect and the mechanism during invasion and metastasis of UBE2T in HCC.We used Real time-PCR to detect UBE2T mRNA expression level in HCC cell lines, Western blot for the protein level. We used siRNA to interfere UBE2T in the cell lines that have the higher expression. We used Transwell to detect the changes in HCC cells invasion and metastasis ability. We transfected pEGFP2-UBE2T to the cell lines that expressed a low level of UBE2T, used fluorescence microscopy to observe the transfection efficiency, then used G418 to pick out positive clones, also Transwell to detect the changes in HCC cells invasion and metastasis ability after the overexpression of UBE2T.After the overexpression or interference of UBE2T, we used Western blot to detect EMT related protein expression in HCC.Results1. The expression of UBE2T in digestive system tumors.Results from tissue microarray using immunochemistry method showed that the expression of UBE2T was pervasively upregulated in many kinds of digestive system neoplasms. In 37 tumor tissue samples from patients with different tumors, the positive expression rate of UBE2T was nearly 100%, which the medium-strong positive rate was up to 83.8%, while in 11 normal tissue samples instead of the apparently low positive expression rat, the expression was mainly negative or weak positive (10/11,90.9%). There were significant difference about the expression of UBE2 between tumor and normal tissue samples (P< 0.001).2. Analysis of UBE2T expression in liver cancer.qRT-PCR was used to detect the UBE2T expression level of mRNA in HCC tissues and non-tumor tissues in 30 patients. The paired t-test was used to find out the difference of UBE2T mRNA expression level between HCC tissues and non-tumor tissues. In addition, their protein expressions of UBE2T were detected by Western blot in 26 cases. There is statistically significant that UBE2T expression in HCC tissue is higher than non-tumor (P<0.001).The UBE2T expression in 133 cases of liver cancer paraffin specimen was tested by immunohistochemical method. And the result showed that UBE2T expression was higher in HCC-tissue compare to non-tumor tissues. Spearman rank correlation was used to analyze the relationship between the expression of UBE2T and HCC clinicopathological parameters. And we found that the expression was significantly relate to the number of the tumors (P=0.001), BCLC staging (P=0.000), portal vein tumor thrombus (P=0.002), distant metastasis (P=0.033). On contrast, there was no significant relationship between the expression and age, gender, pathological grade, tumor size, recurrence.We performed the Kaplan-Meier to analyze UBE2T and primary liver cancer prognosis. By using COX proportional hazards regression model single factor and multi-factor to analyze the relationship between various clinicopathological parameters and primary liver cancer prognosis, it was found that UBE2T and recurrence were both the risk factors of HCC prognosis and ALB was the protect factor.Two risk groups of liver cancer:high and low were distinguished according to the above three independent prognostic factors. The low risk group included high expression UBE2T, no recurrence, HB≥40 g/L or UBE2T low expression, recurrence, ALB≥40 g/L or UBE2T low expression, no recurrence, ALB≤40 g/L. And the rest was the high risk group. The result showed that there was a significant relationship between the low risk group and prognosis by using the Kaplan-Meier.3. The biological role of UBE2T in HCC cells and the mechanism of invasion and metastasis of HCC.We found that the ability of invasion and metastasis of HCC was decreased by transwell after interference of UBE2T, and increased after overexpression. We also found that the epithelial marker E-cadherin was increased significantly and the mesenchymal marker Vimentin was decreased significantly after interference of UBE2T by western blot method, and we got adverse result after overexpression of UBE2T.Conclusions1. UBE2T’s expression is generally higher in the malignant gastrointestinal tumor tissues and it can act as a valuable molecular diagnostic marker.2. UBE2T was upregulated in HCC, and was closely related to the prognosis, and it can be used as an independent prognostic risk factor of HCC.3. UBE2T can promote the invasion and metastasis of HCC through the induction of EMT. |
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