The Expression And Function Of TPD52 In Primary Hepatocellular Carcinoma

Background and purposesPrimary hepatocarcinoma is the fifth most prevalent neoplasm and the third most frequent cause of cancer-related death,75% of which is hepatocellular carcinoma(HCC).The cancer statistic conducted in China in 2015 showed that the overall pick of liver cancer death cause has been the third place. Hence, liver cancer has become one of the most terrible health hazards to human. Currently, hepatectomy and local ablative therapies are the first-line therapy strategies for HCC. However, their therapeutic effect is usually unsatisfactory because of many factors, such as liver dysfunction, multifocality and patient condition. Therefore, this has given rise to various alternative treatments for HCC, including selective intra-arterial radiotherapy(SIRT), molecular targeting treatment(sorafenib) and so on.. As HCC is not sensitive to conventional chemotherapy or radiotherapy, adjuvant immunotherapy might benefit patients with HCC who received hepatectomy, as any residual lesions would probably be minimal. Unfortunately, HCC recurrence are likely to happen, especially intrahepatic recurrence.Thus,the long-term survival of patients is less than 30%. Hepatocarcinogenesis is a complex multifactorial procedure that involves genomic alterations, including cancer gene overexpression and related tumor-suppressor gene inactivation. Hence, further study of effective genes that closely associated with HCC and elucidating the role of these genes in HCC would help us understanding the mechanism of hpatocarcinogenesis,as well as increasing the levels of diagnosis, therapy and prognosis evaluation for HCC.The tumor protein D52(TPD52) gene was identified about 20 years ago, which are 180-200 amino acid residues and contain many sequential structures, including a coil-coil motif, PEST sequences, and N- and C-terminal-located proline, serine and glutamic acid in humans. Numerous studies have revealed that TPD52 is involved in regulating a variety of cellular functions, such as cell proliferation,survival,DNA repair, exocytosis, and vesicle trafficking. However, its roles in cancers are controversial. TPD52 is overexpressed in several cancers including breast cancer, multiple myeloma, burkitt lymphoma, melanoma, and ovarian cancer.,but it is also down-regulated in certain tumors, such as papillary renal cell cancer, clear cell renal cell cancer, leiomysarcoma, liposarcoma and lung cancer. However, the expression and prognostic role of TPD52 have not been reported yet in HCC.In this study, we investigated the expression of TPD52 in primary HCC via real-time quantitative RT-PCR, western blot and immunohistochemistry. Additionally, we explored the correlation of TPD52 with HCC tumor characteristics and clinical parameters, and analyzed its role in prognosis of the patients.. We further investigated the mechanism of TPD52 in hepatocarcinogenesis to provide a new, reliable theoretical basis for HCC diagnosis and therapy.MethodReal-time RT-PCR,western blotting and immunohistochemistry were conducted to detect TPD52 expression level in HCC tissues samples and cell lines, and its correlation with HCC tumor characteristics, clinical parameters and prognosis of patients was analyzed. The mechanism of TPD52 in p21-related pathway in HCC was also investigated through altering the expression levels of TPD52 in Bel7402 and HepG2 cells by transfection with a TPD52-overexpression vector or targeted siRNA.Statistical methods: MannWhitney U-test was used to compare TPD52 mRNA and protein expression between the tumor samples and the matched adjacent noncancerous liver tissue samples. The Pearson χ2 test and Fisher’s exact test were used to determine the correlation between TPD52 expression and the clinical variables or p21 expression.Survival analysis is used to compare the overall survival time and disease-free survival time between high TPD52 expression group and low TPD52 expression group. Finally, Univariate and multivariate regression analyses were performed with a Cox proportional-hazard model to investigate the effects of TPD52 expression and HCC clinical characteristics on survival. Results1、TPD52 mRNA transcription level(p<0.05) and protein expression level(p=0.039) were significantly lower in the tumor tissues than those in the matched adjacent non-tumor tissues. Likewise, TPD52 m RNA transcription level and protein expression level also decreased in HCC cell lines relative to the LO2 liver cell line.2、TPD52 is mainly lied on cytoplasm and membrane, showing strong expression in paracancer but low expression in carcinoma tissues. The correlation analysis suggested that TPD52 expression is significantly correlated with TNMstage. Overall survival(p=0.007) and disease-free survival(p=0.019) of patients with high TPD52 expression was significantly longer than those with low TPD52 expression. Multivariate analysis further indicated that TPD52 expression was an independent predictor for improved overall survival(p=0.019) and disease-free survival(p=0.019) of patients.3、In HCC samples, TPD52 expression showed a positive correlation with p21 expression(r=0.176, p=0.026). The combination of high TPD52 expression and high p21 expression significantly prolonged the overall survival(p=0.012) and disease-free survival(p=0.013) in HCC patients.4、The p53 and p21 expression were up-regulated when TPD52 was overexpressed in vitro, while MDM2、BCL2 and P-GSK-3βexpression were down – regulated. As predicted, TPD52 knockdown in vitro was associated with significant down-regulation of p53 and p21 expression and with evident up-regulation of MDM2、BCL2 and P-GSK-3βexpression. It is suggested that TPD52 may influence the occurrence and development of HCC directly or indirectly through MDM2-p53-p21 pathway. ConclusionTPD52 is a potential tumor suppressor which inhibits HCC occurrence and development. It can function as a favorable marker for HCC diagnosis, therapy and prognosis assessment, as well as a new target for HCC molecular target therapy..