Fat 10 Protein As A Potential Serological Marker In Diagnosis Of Hepatocellular Carcinoma And Other Cancers

Background and ObjectivesHepatocellular carcinoma(HCC) and colon cancer were the most common malignancy worldwide. Their mortality rates were respectively ranked third and fifth in the Asia Pacific region. The morbidity and mortality of colon cancer were significantly increased in recent years. The onset of HCC was usually not so noticeable due to the lack of typical symptoms at early stage. Most of the patients with overt clinical symptoms had already entered the middle or late stages. At present, surgical resection was the best therapeutic method for HCC. Several treatment regimens were available for intermediate stage HCC such as transarterial chemoembolisation(TACE), percutaneous ethanol injection(PEI) and radiofrequency ablation. Despite all these effects, early diagnosis of HCC is an important way to improve the prognosis. The routine screening and surveillance for HCC mainly included serum AFP and liver ultrasonography(US). US had some limitation for the nodules less than 10 mm. AFP had been widely used based on serological diagnosis due to its advantages on non-invasive. Recently, it was noticed that the sensitivity and specificity of AFP in the early stage HCC and small HCC were not satisfactory.The American Society for Study Liver Diseases(AASLD) guidelines published in2010 had no longer used the AFP as a screening indicator. However, liver biopsycould not be accepted by most suspected HCC patients due to its invasive examination. Therefore, the research for novel biomarkers has an important clinical significance to improve the early diagnosis of HCC.FAT10 gene was first discovered in the HLA-F locus of the major histocompatibility complex class I. FAT10 protein contained 165 amino acids and was named double ubiquitin(UBD) initially because it had highly homologous N and C terminal structural domain with ubiquitin. FAT10 protein belongs to UBLs and promotes protein degradation by proteasome system. Liu proved that overexpression of FAT10 gene involved in cell cycle management through non-covalent bind with human spindle checkpoint sites MAD2, which was closely related to the stability of chromosomes. Meanwhile, FAT10 gene has been shown participate in mediating the function of tumor necrosis factor-α(TNF-α) by inducing exception management of cell cycle and chromosomal instability. The mechanism of chromosomal instability may lead to tumorigenesis.Previous studies indicated that FAT10 gene showed up-regulated expression in HCC tissues. However, few intensive researches on whether expression level of FAT10 m RNA and protein in plasma of HCC or colon cancer could be used as a non-invasive diagnosis index were conducted. Therefore, the aim of the paper is to determine the expression level of FAT10 m RNA and protein in plasma, and assess the diagnostic value by analyzing the sensitivity and specificity of FAT10 in patients with HCC or colon cancer.Materials and Methods1. MaterialsPatients and blood samples: Blood samples from 70 HCC patients were collected from the Department of Hepatobiliary Surgery and Gastrointestinal Surgery at Henan-Provincial People’s Hospital, Zhengzhou University, Zhengzhou, China,from June, 2014 to April, 2015. Meantime, we collected blood samples from 60 liver cirrhosis, 64 colon cancer, and 64 healthy volunteers as the control group. The patients of HCC included 51 male and 19 female with an average age of 54.03 ± 8.93.The Child-Pugh system was used to classify the clinical function of the liver. In addition, all HCC patients were subjected to Tumor-node-metastasis(TNM)classification according to the Union of International Cancer Control. Patients with colon cancer patients covered 38 males and 26 females, with an average age of 53.55± 6.56, were divided by Duke’s stage. HCC and colon caner samples without radiotherapy or chemotherapy before collection should be diagnosed based on multiple methods such as ultrasonography, CT, MRI, then subsequently confirmed by pathological. Small HCC was defined as a single cancer nodule with maximum diameter less 3 cm, or two cancer nodules less than 3 cm in total diameter. Health controls were selected from donors with normal liver, and without history of liver disease or other malignancies. The study was approved by the Research Institute of Ethics Committee and all of the participants signed informed consent.2. Methods2.1 Real-time PCR: Total RNA were respectively extracted from plasma of patients with HCC, colon cancer,cirrhosis and healthy volunteers. A260/A280 absorbance ratio was detected by UV spectrophotometer. If the A260 / A280 ratio was 1.8-2.0, this indicated good purity of the extracted RNA. It could be synthesized by reverse transcription c DNA. Sequently, we have detected positive rate of FAT10 m RNA in different groups accordance with the RT-PCR kit purchased from manufacturers(TAKARA,Japan). Melting curve and amplification curve were appeared after the end of the PCR reaction. CT value between 18-35 is defined as a positive expression, the CT value was obtained to calculate the relative expression of the products.2.2 ELISA: A part of plasma was selected randomly for further ELISA experiments. In this study, 70 HCC, 40 liver cirrhosis, 32 colon cancer and 32 healthy volunteers were selected. The operation of the kit should be completed strictly according to the manufacture’s recommendation. 96-well microtiter plate reader(Bio-Rad Laboratories, Hercules, MA, USA) was used to measure the optical density(OD) at 450 nm. The concentrations of FAT10 protein and AFP were calculated according to the standard curve, which were obtained with a four-parameter logistic based on the standard value.3. Statistic methodsLevels of FAT10 were indicated as mean±standard deviation value.Kruskal-Wallis test were used to compare FAT10 and AFP in HCC group with the controls respectively, and independent-sample t test or Mann-Whitney U test were used to compare their difference in two groups. The relationship between expression of FAT10 and AFP in plasma and clinical characteristic of HCC was analyzed through Mann-Whitney U test or Kruskal-Wallis test. Receiver operating characteristics(ROC) curves and area under the curves(AUCs) were used to analyze the diagnostic value of FAT10 protein and AFP. Statistical software(version SPSS22.0) was applied to analyze the different expression levels of FAT10 m RNA and protein. Graph Pad Prism version 6.0(Graph Pad Software, La Jolla, CA, USA) was used to make figures. P values lower than 0.05(two sides) was considered to be statistical significant.Results1. Real-time PCR : The positive rates of FAT10 m RNA in serum of HCC,cirrhosis and healthy controls was 72.85%, 28.3% and 12.5%, respectively,significantly differences were found in these three groups(P<0.05). We found that FAT 10 m RNA in plasma of HCC were significantly higher than the two groups( P<0.01). While there was no obvious difference between cirrhosis and healthy volunteer(P>0.05). The quantitative data of FAT10 m RNA in HCC group, liver cirrhosis group and healthy volunteer were 6.83 士 3.37, 2.14 士 0.70, 1.08 士0.46,respectively. There were significantly differences in three groups. However,no significantly differences were found between cirrhosis and healthy volunteer.The expression levels of FAT10 m RNA in HCC were related to the TNM stage and lymph node metastasis(P<0.05). Meanwhile, higher positive rates were revealed when compared the colon cancer with healthy controls(59.4%). The quantitative data of FAT10 m RNA in colon group were 4.04 士 3.33,The up-regulated of FAT10 m RNA in CC was closely related to Duke’s stage and distance metastasis. FAT10 m RNA had a sensitivity of 66.6% in AFP-negative HCC. Furthermore, the FAT10 m RNA had sensitivity of 68.8% and specificity of 79.8% in distinguished small HCC from non-HCC individuals(cirrhosis and healthy controls).2. ELISA: The concentrations of FAT10 protein in HCC, cirrhosis, healthy volunteers were respectively 293.855±127.61 pg/ml, 169.30±58.659 pg/ml,142.87±70.08 pg/ml. We noticed that FAT 10 protein in plasma of HCC were significantly higher than cirrhosis or health group(χ2 = 53.34, P <0.01). While there was no obvious difference between cirrhosis and health group, regardless of that the mean concentration of FAT10 protein in plasma was a little higher in cirrhosis controls(P=0.086). The levels of FAT10 protein in HCC were related to the TNM stage(P=0.034, <0.05) and distant metastasis(P = 0.032, <0.05).Levels of FAT10 in AFP-negative HCC and small HCC were respectively271.57±128.68 pg/ml and 299.71±110.02 pg/ml. Significantly increased FAT10 proteins were observed in AFP-negative HCC compared with healthy volunteers.Significantly differences were also found when compared small HCC with healthy volunteers. The average concentration of FAT10 protein in patients with colon cancer was 192.806±61.69pg/ml, which was significantly different from healthy volunteers(P<0.05). Levels of FAT10 protein in colon cancer were correlated to Duck’s stage and distant metastasis(P<0.05).3. ROC analysis: The concentration of FAT 10 protein in plasma was analyzed by ROC and the accuracy of this diagnostic method was described based on sensitivity and specificity. 20ng/ml was selected as the cutoff value for AFP according to international recommendations. The sensitivity and specificity of AFP protein in HCC were 78.6% and 81.9% respectively with distinguish difference from non-HCC controls. ROC curves showed the optimum cutoff value for FAT10 in HCC was 194.14pg/ml. When comparing HCC with healthy groups, the ROC curve showed that AUC value of FAT10 protein was(0.874)(95%CI: 0.800-0.949,sensitivity 78.6%, specificity of 87.5%). Furthermore, the ROC curve revealed a lager AUC value for FAT10(0.826; 95%CI: 0.749-0.903, sensitivity 78.6%,specificity of 77.5%) when distinguishing HCC from cirrhosis groups.The ROC for FAT10 protein respectively showed AUC of 0.865( 95%CI :0.754-0.975) when distinguished small HCC from non-HCC. In small HCC, theFAT10 protein has sensitivity of 75.0% and specificity of 77.5% in contrast to cirrhosis, sensitivity of 75% and specificity of 84.4% by contrast healthy volunteers.The sensitivity of AFP in small HCC could increase from 37.5% to 81.3% when combined FAT10 protein and AFP. Meanwhile, ROC analysis demonstrated that AUC value at 0.836(95CI:0.724-0.947) when compared AFP-negative HCC with non-HCC. Further study, the ROC curve of FAT10 protein shows a good diagnosis values for AFP-negative HCC when compared to cirrhosis(AUC 0.797, 95% CI:0.660-0.934) and healthy volunteers(AUC 0.884, 95%CI: 0.785-0.982), respectively.Conclusions1. The expression levels of FAT10 m RNA and protein in HCC were both significantly higher than the controls. In our study, the expression of FAT10 m RNA in HCC was closely related to stage of TNM and lymph node metastasis.Meanwhile, we found plasma of FAT10 protein was associated with stage of TNM and distance metastasis in HCC. The expression levels of FAT10 m RNA and protein in colon cancer were higher than that in healthy volunteers. Higher levels of FAT10 m RNA and protein in colon cancer were correlated with tumor metastasis and Duke’s stage. These suggest that FAT10 maybe participate in the development and progression of HCC and colon cancer.2. ROC curve showed that FAT10 protein has a good diagnostic value in HCC.FAT10 may be a new serological marker in HCC and colon cancer. Furthermore, it could be a supplement for AFP in diagnosis of AFP negative HCC and early stage of HCC. Combined FAT10 and AFP protein can improve the diagnostic accuracy of liver cancer.