Study On The Autoantibodies To PcG Family And Its Related Target Genes In HCC

ObjectivesAt present, the incidence and mortality of hepatocellular cancer (HCC) rank second in the malignant tumor in our country, each year about 300,000 new cases increased, accounting for more than 50% of the world. The HCC develops very fast and is easy to metastasize. Currently it is one of the malignant tumors which are the most difficult in treatment with the poorest prognosis. However, with respect to breast cancer, lung cancer, prostate cancer and other types of malignant tumors, HCC is still lack of in-depth understanding of related tumor markers and molecular signaling network.In recent years, tumor biomarkers (TM) provide a new train of thought for the diagnosis and treatment for cancer by the wide application in malignant tumor early diagnosis, prognosis, treatment and curative effect evaluation and other aspects. For example, the gene mutation rate of human epidermal growth factor receptor (HER) is 25% in patients with breast cancer. In breast cancer and other cancer treatment and diagnosis, according to the HER2 classification diagnosis and treatment have achieved outstanding results. Herceptin is humanized monoclonal antibody to the HER2 receptor, approved by USA FDA for the treatment of breast cancer in 1998, has achieved encouraging curative effect. In addition, the in vivo and in vitro studies have confirmed the killing effect of Pertuzumab on HER2+ ovarian cancer, prostate cancer, lung cancer and colon cancer cells. But in addition to the above several kinds of cancer, especially for HCC and other types of malignant tumors, there is no or low activation occurs in HER. The key molecules further screening and identification of HCC will provide potential targets and strategies for clinical early diagnosis and treatment and individualized treatment, is the key to cure HCC and other malignant tumors.Recent studies show that aberrant histone methylation is widely involved in the occurrence and development of HCC. PcG proteins are a group of transcriptional repressors through chromatin modification to regulate target gene expression, which were originally identified in Drosophila, is an important factor in maintaining the stable expression of homeobox gene when Drosophila development. In terms of function, PcG family can be divided into two categories:PRC1 and PRC2. PRC2 family includes EZH2, SUZ12, EED and etc, mainly catalyze covalent modification of H3K27 histone methylation process; PRC1 family includes Bmil, CBX8, Ringl and etc, mainly mediate the extension of formed histone covalent modification of H3K27 methylation. Although studies showed that, PcG family is a potential HCC typing diagnosis and therapeutic target, because of the difficulty in detecting the nuclear proteins, which posed a challenge of them to be effective clinical diagnostic markers.In recent years, employing autoantibodies as markers for early diagnosis of cancer has been widely accepted, which has been commercial applied in Europe and North America. In this study, level of PcG family protein expression was detected by immunohistochemistry in cancer and paracancerous tissues, using optimized ELISA assay examined serum levels of autoantibodies to PcG family and its related target genes in HCC patients, and study its biological role in HCC development, seeking effective biomarkers for diagnosis and treatment of HCCMethodsIn his study, we selected PcG family and its related target genes as tumor associated antigens. Using bioinformatics database and software comprehensive analysis of protein structure and properties, to design polypeptide epitope antigen fragments for above molecules, employing the optimized ELISA method to detect the corresponding antibodies level in the serum of hepatic adenocarcinoma patients with above polypeptide epitope antigens, at the same time adopting immunohistochemistry test to determine the levels of PcG family protein expression in cancer and paracancerous tissues. Healthy volunteers were selected as control group,100 cases of healthy control mixed blood sample was used for quality control sample, t test Kaplan meier and ROC curve analysis was processed by SPSS20.0 software.Results:I. Expression of PcG family proteins in HCC:(1) Analysis of the expression of PcG family proteins in HCC:we detected 110 cases of HCC tissue, patients that level of Bmil expression in cancer tissue was higher than that of paracancerous tissues accounted for 46.15%(51/110), patients that no differences in the expression of Bmil between cancer and paracancerous tissues accounted for 53.85%(59/110). Patients that level of CBX8 expression in cancer tissue was higher than that of paracancerous tissues accounted for 48.18%(53/110), patients that no differences in the expression of CBX8 between cancer and paracancerous tissues accounted for 51.82%(57/110). Patients that level of EZH2 expression in cancer tissue was higher than that of paracancerous tissues accounted for 47.27%(52/110), patients that no differences in the expression of EZH2 between cancer and paracancerous tissues accounted for 52.73%(58/110).(2) In further study, according to the expression differences in, BMI1, CBX8 and EZH2 in immunohistochemistry test, we conducted statistical analysis about the correlation of survival curve in 110 cases of clinical HCC specimens. The results showed that the patients of which Bmil expression in cancer tissue was higher than that of paracancerous tissues, the tumor-free survival rate had no significantly difference compared with the patients that no differences in the Bmil expression between cancer tissue and paracancerous tissue (P=0.841 log-rank test); the patients of which CBX8 expression in cancer tissue was higher than that of paracancerous tissues, the tumor-free survival rate was significantly lower than the patients with no differences in the CBX8 expression between cancer tissue and paracancerous tissue(P=0.032 log-rank test); the patients of which EZH2 expression in cancer tissue was higher than that of paracancerous tissues, the tumor-free survival rate was either significantly lower than the patients with no differences in the EZH2 expression between cancer tissue and paracancerous tissue (P=0.009 log-rank test).2. PcG family genes and its key target genes(1) Designed fragments of polypeptide epitope antigen: P16:H-NSYGRLVVLHRAGARLDVRDAWGRLPVDLA-OH; C-myc:H-RVKLDSVRVLRQISNNRKCFELLPTPPLSPS-OH P53:H-LIRVEGNLRVEYPGTRVRAMAIYKQSQHMTE-OH; BMI1:H-AGELESDSGSDKANSPHPSADAANGSNEDRGED-OH CBX8:H-TCRAEAPRDRDRKGRKLDDTPSGAGKFPED-OH EZH2:H-DDGDDPEEREEKQKD YQPCDHPRQPCDSSCPED-OH(2) Expression of autoantibodiesThere was no significant difference in the levels of plasma autoantibody to EZH2 between the malignant tumor patients group and the healthy control group (P> 0.05); the levels of autoantibodies to BMI1 and CBX8 in malignant tumor group were significantly higher than that of healthy control group (P<0.05, P<0.05); in the detection of the key target genes, we found that the expression of autoantibodies to P16, C-myc and P53 in plasma of patients with HCC were significantly higher than those of healthy controls (P<0.05, P<0.05). Using ROC curve analysis we found that take the 90% of specificity, the sensitivity of each antigen in malignant tumor group is 12-30%.Conclusions:(1) In the HCC tissue, expression of PcG family BMI1, CBX8 and EZH2 proteins increased in different degree, suggesting that they may be potential markers for diagnosis and treatment of HCC.(2) The increased expression of PcG family BMI1, CBX8 and EZH2 proteins in HCC tissue correlated significantly with survival of HCC patients, indicating that the expression level of PcG family proteins might be closely related to the prognosis of patients.(3) In the peripheral blood of HCC patients, except EZH2, the autoantibody levels of other 5 genes were significantly increased, suggesting that the autoantibodies to PcG family and its key target genes may be novel diagnostic markers of HCC.