| 1. Background and objectives:The liver is one of most important organs in the internal environment to maintain the stability and health of the body. Fatty liver, hepatitis, cirrhosis and primary liver cancer are the important reasons for the four most common serious liver diseases, but also endanger human health. In fact, there are nearly one million people died of HCC leaded by hepatitis virus infection and cirrhosis. Particularly, as the most common primary malignant liver primary hepatocellular carcinoma (Hepatocellular carcinoma HCC) because of the higher incidence found difficult, less treatment reasons has led to the death of a variety of human diseases in the forefront. Currently, primary hepatocellular carcinoma ranked fifth worldwide cancer death rate, while in some African and Asian countries, primary hepatocellular carcinoma in tumor death has been ranked.Consume caused by human hepatocellular carcinoma, financial, material and medical resources more obvious in our country, mainly because almost half of the new cases of the world. For example, in2000years the total number of cases worldwide emerging624000people, it is a bit shocking which have accounted for about55%of this figure occurred in our country. According to Ministry of Health statistics, from the1990s hepatocellular carcinoma (PHC) has evolved into the second cancer killer just behind lung cancer in the city, and gastric cancer in the countryside. This corresponds with the reality is:the HCC cases each year has reached564,000cases, while there are549,000cases of deaths as much. Since There is no effective method for early diagnosis, much of the cases found in the middle and late stage, combined with the absence of reasonable and effective solution to cure, most of the HCC had poor surgical results, the effect of radiotherapy and chemotherapy were not evident, resulted5-year survival rate of patients with HCC is very low. The recurrence rate and mortality rate is very high. There have been reports that5years after radical resection of HCC recurrence rate was51.6%, of which a small hepatocellular carcinoma recurrence rate as high as43.6%. There have been reports that5years after radical resection of HCC recurrence rate was51.6%, of which a small hepatocellular carcinoma recurrence rate as high as43.6%. Although surgical resection besides, there are some new treatments into clinical (such as radiofrequency ablation, microwave therapy, cryoablation, the intratumoral injection of ethanol,hepatic artery chemoembolization local treatment intervention, etc.) but these methods have little effect for improving the overall quality of life of HCC.Early detection and early treatment, therefore, early diagnosis of liver cancer had already become the focus of attention, actually effective treatments still lacks, early detection of liver cancer remains a huge challenge. Fast growing in medical science today, although investment in the field of diagnosis has been increasing, but a few markers were effective. In fact, many of early stage of HCC may has occurred long-standing and metastasis, the patient has a tumor in the middle and late, various phenomena indicate that current testing technology is still not timely detection and effective detection of early-stage of HCC. So, looking for tumor markers remains an important direction and it is also an important method for the early detection of the cancer.Hepatocellular carcinoma is a common highly malignant tumor, the regional distribution of certain characteristics of its incidence exist where hepatitis virus is endemic in Asia and Africa, the incidence is relatively high. In the census of liver cancer, liver cancer susceptible individuals routine screening can significantly prolong the survival of many patients, but for many patients with advanced hepatocellular carcinoma, the effect is still not optimistic. For the part of the patients with advanced hepatocellular carcinoma, although only a small tumor, surgery is completely removed, but to improve the quality of long-term survival after surgery is still unknown.For the part of the patients with advanced hepatocellular carcinoma, although only a small tumor, surgery is completely removed, but much of the cancer can not removed by the surgery, to improve the quality of long-term survival after surgery is very important. Many intrahepatic or extrahepatic metastasis and recurrence is poor prognosis of patients with liver cancer the main reason. In fact, the current estimates of survival are often inadequate or excessive, often caused by improper postoperative treatment, for patients these reasons undoubtedly were negative factors.Many studies have confirmed that inflammation, immunity, cancer three closely related. Now that the inflammation in tumor incidence, progression, malignant transformation, invasion and metastasis have played an important role. We also understand very clearly between the inflammatory process and the immune response and has a close relationship between the two, there has been some scholars have proposed cancer1/5is associated with chronic infection. Inflammation and cancer is a contradiction, Dvorak HF, such as tumor called a "non-healing wounds" Schrader J, infered proinflammatory made of the tumor microenvironment for tissue remodeling, angiogenesis, invasion and metastasis occurs in one hand. to promote the inhibition of CTLs by the inhibition of macrophage production, aggregation Treg cells, thereby preventing immunization system to attack tumor cells in the other hand. As a cancer on the body damage, occurred in the course of its development, inflammatory response, especially for cancer, because it is destructive relatively strong, sparking the patient’s self-defense mechanisms will be relatively obvious. Given the close links between inflammation and cancer, and for participating in the process of inflammation-related factors in tumorigenesis has become an important research direction.It was known that there has been very significantly progress in the development of anti-cancer agents, including tyrosine kinase inhibitors, angiogenesis inhibitors, and agents that interact with the cell cycle and cell death (apoptosis). HTATIP2/TIP30was an evolutionarily conserved gene that is expressed ubiquitously in human tissues and some tumor tissues. The protein displayed a serinethreonine kinase activity that could phosphorylate the carboxyl terminal domain of RNA polymerase II in a TAT-dependent manner. So, it was necessary to evaluate its potential value. It was evidenced that HTATIP2/TIP30and EGFR had a close link in signaling pathways in liver. As reported, Endothelial Growth Factor Receptor (EGFR) was also central to the promotion of cell growth and had a role in the development of HCC. With the role of VEGF as a major antigenic factor in HCC, preventing EGFR activity was an attractive method for anti-HCC. Zhang, C, et al found that HTATIP2/TIP30banding with Endophilin B and ACSL4, form a complex in the cytoplasm, then participate in the transport of EGFR. In HTATIP2/TIP30gene knockout studies, after HTATIP2/TIP30knocked out, EGF-induced EGFR degradation were inhibited. The reason was that HTATIP2/TIP30, CSL4, Endophilin B1(EndoB1) formed a complex protein, which promoted EGFR from early endosomes into the liposomal sorting, and then could accelerate the degradation of EGFR in liver cells in mice and mammary cells. HTATIP2/TIP30gene knockout cells could not make the access of early endosomes for EGFR to the liposomal degradation, resulted in the activation of EGFR and its downstream signaling and ultimately promoted cell proliferation.The immunosuppressive microenvironment by the inflammatory state revealed the relationship between cytokine changes and tumor recurrence and metastasis TNF-a and IL-1β in primary liver cancer patients were higher than normal, the other side of these two factors have increased cancer, in addition to the blood vessels, such as generating MIF gene are associated with liver cancer liver metastasis TNF-a as a has anti-tumor cytokine, Intracellular NF-kβ and STAT3signaling pathway, leading to tumor proliferation, anti-apoptosis, angiogenesis, invasion and metastasis related genes to the development of liver cancer. Apart Th17cytokines secreted IL-17(IL-17A), but also includes IL-17F, and IL-21, IL-22, IL-6, TNF-a and other cytokines, Thl7cells are considered an important group important mediated inflammation can activate dendritic cells and T cells in lymphoid helper cells, release of cytokines to maintain the state of chronic inflammation and carcinogenesis environmentIL-17is an important mediator of inflammation, it can be induced by other inflammatory cytokines such as IL-6, the expression of TNF-a, and chemokines such as MCP-1, MIP-2, etc., to a local inflammatory cell mediated infiltration and tissue damage. Interleukin27(interleukine27, IL-27) was discovered in2002and named a new cytokine, IL-12, and it is with another newly discovered cytokine IL-23together constitute the IL-12family of molecules with complex biological functions in the anti-infection immunity, anti-tumor immunity and autoimmunity plays an important role. Based on existing research on HTATIP2/TIP30and inflammatory mediators and cancer and liver cancer in a variety of relevant reports, down HTATIP2/TIP30gene may be associated with the incidence of primary liver cancer, and therefore, the protein is detected through blood serum, determine the expression levels of normal liver cancer occurrence and development of research-related aspects are very necessary and critical. In addition, combined with the existing AFP clinical value, relevance and evaluating HTATIP2/TIP30, AFP in HCC clinical data also appears to make sense, This is more conducive to systematically analyze and test the performance of the two tests, we detected through the use of Luminex xMAP technology, high efficiency, high sensitivity,52cases of hepatocellular carcinoma in patients with hepatocellular carcinoma in patients with serum IL-1β,IL-6, IL-10, IL-17, IL-27, TNF-α levels were detected, and further explore these inflammatory factors may predict liver cancer metastasis. To be better utilized, and this is where the purpose of the present study.2Methods:2.1PatientsA total of84subjects, which included52patients diagnosed on the basis of National Comprehensive Cancer Network (NCCN) guidelines and32healthy individuals were recruited. All the subjects were from Hunan Provincial People’s Hospital between September2013and December2013. Before therapy, CT scan, ultrasound, biochemical, and serological parameters were performed to determine the presence of tumors and asides. According to our routine clinical approach,2ml of blood were taken from each patient. This study was approved by the Local Human Ethics Committee of the Ministry of Health. The clinical characteristics of the study population are shown in Table1. Table (1-1) Clinical datas in each group.2.2Blood samplingBlood samples were collected from these patients and healthy individuals and put them in biochemical tubes and then immediately taken to the biochemistry laboratory, where they were centrifuged at4,000rpm for10minutes at4℃.The serum were kept at-80℃until the biochemical measurements for HTATIP2/TIP30and other evaluation were conducted.2.3Biochemical analysesHTATIP2/TIP30concentration was determined using a commercially available ELISA kit (Cusabio Biotech. catalog number:CSB-E14917H ELISA kit). For this kit, the minimum detectable dose of human HTATIP2\TIP30was0.16ng/ml. The sensitivity of this assay, or lower limit of detection (LLD), was defined as the lowest protein concentration that could be differentiated from zero. Intra-assay precision (precision within an assay) was CV%<8%, and inter-assay precision (precision between assays) was CV%<10%.2.4Roche electrochemiluminescence assay HBeAg statusAll subjects enrolled HBeAg determined by electrochemiluminescence detection of HBeAg SOP sample handling processes in accordance with Roche chemiluminescence, aircraft, Elecsys software analysis determined that the specimens HBeAg positive and negative.2.5Quantitative PCR serum HBV DNA levelsAll enrolled subjects by HBV fluorescence quantitative detection kit instructions to operate the experimental subjects serum PCR reaction mixture and mix well with a common standard quantitative HBV PCR amplification, to be amplified by ABI7100after the data analysis2.6Luminex xMAP serum cytokines in patients with liver cancerLuminex xMAP technology (multi-anlyte profiling beads) eBioscience the ProcartaPlex kit standards cytokines (including IL-Iβ, IL-10, IL-6, IL-17, TNF-a, IL-27) cells in a total of6standard and sample dilution factor. Data analyzed by Luminex200results analysis software that captures images by detecting the sample, and then through image analysis, the standard curve, calculated from the data sample to be tested.2.7Statistical analysesSPSS Statistics17computer program package for statistical calculations. The results are expressed as mean±standard deviation (SD). ANOVA and multiple comparison analysis of the healthy control group and two groups between HCC and each stage of HCC.(Operating Characteristic curve) ROC analysis of various indicators of HCC group and the control group, metastatic and nonmetastatic, early diagnosis of HCC and control group. Spearman clinical detection of cytokines and biochemical indices and indicators of relevance. Multivariate logistic regression analysis and the establishment of joint diagnostic model indicators, Hosrner-Lemeshow goodness of fit of the logistic regression test, P values are accurate to three decimal places, with P<0.05as statistically significant. Medcalc draw ROC curve, Graphpade Prism5draw a histogram and associated line graph.3Results:3.1Serum level of HTATIP2/TIP30The HTATIP2/TIP30level was significantly lower in the HCC group (mean,4.505ng/ml; range,0.191-11.07) compared with the healthy group (mean,9.499ng/ml; range,5.759-13.278, unpaired t-test,*P<0.001, P<0.05) For the subgroups, the serum level of HTATIP2/TIP30was significantly lower in the (mean,3.667ng/ml; range,0.191-8.965) than in the SD group (mean,6.089ng/ml; range,2.452-11.07),(unpaired t-test,**P=0.001, P<0.05) for the diagnosis of the subgroups, the PD group or SD group was detected by CT scan and evaluated by response evaluation criteria in solid tumors RECIST,NCI2000.3.2ROC curves of HTATIP2/TIP30and AFPAlthough the level of HTATIP2/TIP30was elevated in majority of the HCC patients, there was considerable overlap between hepatocellular carcinoma patients and the healthy controls. Therefore, ROC analysis was used to detect the optimal cut-off points of HTATIP2/TIP30and AFP for discrimination HCC patients and the healthy individuals The Area under the curves (AUCs) of HTATIP2/TIP30and AFP were0.928(0.926±0.027) and0.888(0.888±0.043) respectively.(P<0.001, P <0.05) ROC analysis was also used to detect the optimal cut-off points of HTATIP2/TIP30and AFP for discrimination for the subgroup. The Area under the curves (AUCs) of HTATIP2/TIP30(0.769±0.066, P=0.001, P<0.05) was greater than that of AFP (versus0.668±0.086, P=0.043, P<0.05).3.3HTATIP2/TIP30and AFP for diagnosisIf combined the HTATIP2/TIP30and AFP to diagnosis of HCC and healthy individuals, with the optical cut-points (7.271ng/ml for HTATIP2/TIP30,11.930ng/ml for AFP), the sensitivity, specificity and Youden index was showed in If combined the HTATIP2/TIP30and AFP to diagnosis of HCC progressed or not, with optical cut-points (6.240ng/ml for HTATIP2/TIP30, and31.190ng/ml for AFP), the sensitivity, specificity and Youden index was showed in.3.4Relationships between clinical characteristics and HTATIP2/TIP30 We analyzed relationships between HTATIP2/TIP30level, tumor characteristics, serological parameters and Child-Pugh score. HTATIP2/TIP30level showed a statistically significantly negative correlation with HBV DNA load (r=-0.364, P=0.002) and Child-Pugh score (r=-0.419, P=0.008), although not associated with tumor size and AFP.3.5Logistic regression model to predict HCCBy HTATIP2/TIP30, HBvDNA level, AFP concentration of three factors do backward stepwise logistic regression analysis, the probability choose to enter the variable standard of0.10, excluding the probability standard variable is0.15, derived regression equation P=1/[1+e-(4.449-0.717×1+0.042×2)]:where×1representatives HTATIP2/TIP30, x2behalf of AFP, Odd Ratio (OR) values were:0.488and1.043;95%CI (95%confidence interval) were (0.331-0.791) and (0.998-1.089). When AUC Logistic regression analysis model predictors PRE ROC curve was0.963, more than HTATIP2/TIP30and the individual indicators AFP, its largest Youden index corresponding sensitivity of90.6%and a specificity of90.4%compared with the two indicators alone diagnosis significantly increased.3.6Detection serum of multiple inflammatory cytokines in HCCUsing Luminex xMAP method for detecting HCC metastasis and without metastasis in serum samples content (IL-1β, IL-6, IL-10, IL-17A, TNFa, IL-27) and the results showed no metastasis group the cytokines levels were (IL-1β,3.93±2.07; IL-6,6.61±4.63; IL-10,7.77±5.49; IL-17A,255.49±94.33; IL-27,2.67±2.53), the transfer group content of each group of cytokines (IL-1β,4.11±1.95; IL-6,6.30±5.39; IL-10,8.70±4.77; IL-17A,5.56±2.85IL-27,353.19±177.55), which IL-17A, IL-27cytokine groups was significantly higher in the transfer group, the difference was statistically significant. P values were (0.015, P<0.001)3.7Correlation analysis HTATIP2/TIP30each serum levels of inflammatory cytokinesPearson correlation analysis HTATIP2/TIP30, IL-1β,IL-6, IL-10, IL-17, IL-27, TNF-a and other cytokines correlation was, IL-17and TIP30negative correlation (r=-0.601, P<0.001),3.8ROC analysis of IL-17A and IL-27predict liver cancer metastasisROC analysis of IL-17and IL-27both indicators of liver metastasis and without metastasis, respectively, the area under the curve (0.818, P=0.044) and (0.664, P <0.001), both as a predictor of liver cancer metastasis statistically significant. To IL-27is238.95pg/mL threshold is, the sensitivity was:95.8%, specificity was:64.3%maximum Youden index. IL-17select critical value2.005pg/ml, the largest Youden index, the sensitivity was83.3%and specificity of35.7%. IL-27and IL-17compared to a better prediction of liver cancer metastasis.3.9Logistic regression model to predict HCC metastasisLogistic regression analysis HTATIP2/TIP30and IL-27, the dependent variable select "0" for the metastasis,"1" for the transfer. Choose to enter the variable probability standard is0.10, excluding the probability standard variable is0.15, derived regression equation:P=1/[1+e-(-1.264-0.642×1+0.013x2)],×1representatives HTATIP2/TIP30, x2behalf of IL-27, P is a logistic model to predict the probability ranges between0-1, e is the natural logarithm (e=2.718), Odd Ratio (OR) values were:0.526and1.013;95%CI (95%confidence interval), respectively (0.346-0.799) and (1.003-1.026). The regression equation prompt independent factors HTATIP2/TIP30and IL-27are HCC metastasis, the risk of high HTATIP2/TIP30serum levels in patients with metastasis was0.526times its low level of patient transfer risks. IL27-risk as a risk factor, high serum levels of IL-27in patients with metastasis was1.013times its low level of patient transfer risk. PRE regression model to predict both the area under the curve of HCC metastasis (AUC) was0.903±0.043, P<0.001, sensitivity87.5%, specificity82.1%, significantly higher than the two indicators predict when alone.Novelty1. This study is the first time to detect the level of HTATIP2/TIP30in the serum of HCC patients, and found it is a powerful indicator in dignose and predict metastasis.2. For the first time by liquid chip found IL-17and IL-27associated with hepatocellular carcinoma metastasis, two indicators may be used to predict HCC metastasis. |
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