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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 3  |  Page : 753-761

mRNA expression of interleukin-17 in viral hepatitis B and hepatocellular carcinoma patients


1 Medical Biochemistry Department, Faculty of Medicine, National Liver Institute, Menoufia University, Menoufia, Egypt
2 Hepatology Department, National Liver Institute, Menoufia University, Menoufia, Egypt

Date of Submission03-Apr-2016
Date of Acceptance06-Jun-2016
Date of Web Publication31-Dec-2018

Correspondence Address:
Sherin S El-Naidany
Medical Biochemistry Department, Faculty of Medicine, Menoufia University, Shebin Elkom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.248729

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  Abstract 


Objective
We aimed to study the mRNA expression of interleukin-17 (IL-17) in patients with chronic viral hepatitis B (HBV) and evaluate its role in the development of liver cirrhosis and hepatocellular carcinoma (HCC).
Background
IL-17 is a cytokine produced by a newly defined subset of helper T cells. The IL-17 family of cytokines has been reported to be involved in many immune processes, most notably in inducing and mediating proinflammatory responses. Recent studies have found that both natural and acquired immunity play important roles in regulating liver fibrosis, tissue repair, and recovery, which are vital for the maintenance of tissue homeostasis. Inflammation is a pathological hallmark feature of chronic liver diseases. Sustained inflammation then promotes liver fibrosis and as an end-stage liver cirrhosis or HCC.
Participants and methods
This study was carried out on 50 participants classified into the following groups: group I included 15 patients with chronic HBV; group II included 15 patients with HBV-related cirrhosis; group III included 10 patients with HCC with HBV infection; and group IV included 10 age-matched and sex-matched healthy individuals as controls. Liver function tests, HBV and hepatitis C virus serology, AFP alpha-fetoprotein and HBV DNA analysis by PCR were performed for all individuals. IL-17 level was estimated by enzyme-linked immunosorbent assay, and mRNA expression of IL-17 was determined by RT-PCR in peripheral blood.
Results
The results showed that both serum IL-17 level and mRNA expression of IL-17 increased in HCC patients compared with the cirrhosis and control groups. In patients with posthepatitis B cirrhosis, there was a significant rise in serum IL-17 and mRNA expression of IL-17 when compared with the control group. There was significant positive correlation between IL-17 and mRNA expression of IL-17 and aminotransferases and total and direct bilirubin in all groups. There was a negative correlation between serum IL-17 and mRNA expression of IL-17 and serum albumin in all groups.
Conclusion
Serum IL-17 and mRNA expression of IL-17 may play a role in disease progression and development of HCC in chronic HBV patients; they might be candidates for novel biological markers of HCC.

Keywords: chronic viral hepatitis B, cirrhosis, hepatocellular carcinoma, interleukin-17


How to cite this article:
El Sebaii HM, Abd El-Hamid AK, Abou Elnour ES, El-Azab GI, El-Helbawy NG, El-Naidany SS. mRNA expression of interleukin-17 in viral hepatitis B and hepatocellular carcinoma patients. Menoufia Med J 2018;31:753-61

How to cite this URL:
El Sebaii HM, Abd El-Hamid AK, Abou Elnour ES, El-Azab GI, El-Helbawy NG, El-Naidany SS. mRNA expression of interleukin-17 in viral hepatitis B and hepatocellular carcinoma patients. Menoufia Med J [serial online] 2018 [cited 2019 Mar 25];31:753-61. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/753/248729




  Introduction Top


Chronic hepatitis B virus (HBV) infection is one of the most common viral infections worldwide. More than 90% of HBV infections in adults are self-limited within 6 months[1].

There are more than 350 million people around the world suffering from persistent HBV infection. Of these, 10–20% will develop liver cirrhosis and in 1–5% the disease will progress to hepatocellular carcinomas (HCCs). The study of the pathogenesis of chronic hepatitis B and the early prevention of disease progression are important to reduce the incidence of liver cirrhosis and HCC[2].

Exposure to blood and body fluids is a major risk factor for the development of HBV infection; the risk of acquisition of HBV infection after a single exposure to HBV-infected blood or body fluid ranges from 6 to 30%[3].

Interleukin-17 (IL-17) is a cytokine produced by a newly defined subset of helper T cells. The IL-17 family of cytokines has been reported to be involved in many immune processes, most notably in inducing and mediating proinflammatory responses[4].

There are six IL-17 family ligands (IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F) and five receptors (IL-17RA, IL-17RB/IL-25R, IL-17RC, IL-17RD/SEF, and IL-17RE)[5].

Human IL-17-producing CD4+ T cells (Th17) are characterized by the secretion of IL-17. The cytokine transforming growth factor-β, in the presence of IL-6, promotes the differentiation of native T lymphocytes into Th17 cells, which promote autoimmunity and inflammation. As the liver is known to be an important source of transforming growth factor-β and IL-6, Th17 differentiation may be favored in the liver[6].

The objectives of this study were to evaluate the mRNA expression of IL-17 in patients with chronic HBV and to evaluate its role in the development of liver cirrhosis and HCC.


  Participants and Methods Top


This study was carried out in the Medical Biochemistry Department, Faculty of Medicine, Menoufia University, and Internal Medicine Department of the National Liver Institute, Menoufia University, from May 2014 to June 2015. We included 50 participants; there were 10 females and 40 males with ages ranging from 20 to 57 years. This study was approved by the ethics committee of the Faculty of Medicine, Menoufia University.

The participants were classified into the following groups:

Group I included 15 patients with chronic hepatitis B without cirrhosis; group II included 15 patients with chronic hepatitis B-related cirrhosis; group III included 10 patients with HCC along with hepatitis B infection; and group IV included 10 healthy age-matched and sex-matched controls with no history of liver disease.

Chronic hepatitis B was defined as positivity for hepatitis B surface antigen for 6 months or longer

The diagnosis of cirrhosis was based on clinical, laboratory, and imaging findings. The diagnosis of HCC was made by presence of HCC radiological hallmarks in imaging technique [computed tomography (CT) and/or dynamic MRI]±histopathological examination if needed[7].

Individuals with hepatitis C, history of autoimmune diseases, hematological malignancy, and history of tumors in any organ other than the liver were excluded from the study.

Methods

All the participants underwent complete history analysis, general and local clinical examination, radiological investigations including abdominal ultrasonography and CT to detect changes in the liver, and ultrasound-based transient elastography (fibroscan). The following laboratory investigations were carried out: (a) complete blood count (CBC); (b) liver function tests to detect albumin, total protein, total bilirubin, direct bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and prothombin time; (c) α-fetoprotein analysis; (d) detection of hepatitis B surface antigen and hepatitis B (e) antigen, HBeAb, and HBV DNA by PCR; (e) hepatitis C virus Ab detection; (f) serum IL-17 level by enzyme-linked immunosorbent assay (ELISA) technique; and (g) mRNA expression of IL-17 by RT-PCR in peripheral blood.

Sample collection

Ten milliliters of venous blood was collected from each participant and divided as follows: 5 ml of venous blood was transferred to a vacutainer plain test tube. The samples were allowed to clot, and serum was separated after centrifugation at 3000 rpm for 10 min. This was followed by liver function tests and analyses of liver enzymes, AFP level, and hepatitis viral markers. All serum samples were stored at −70°C until IL-17 analysis by ELISA kits. Three milliliters of venous blood was transferred to a vacutainer plastic tube containing EDTA for CBC, and 2 ml of venous blood was transferred to a vacutainer plastic tube containing EDTA for the detection of IL-17 mRNA expression by RT-PCR.

Assay methods

For CBC, an automated homogram was performed for all cases. Qualitative immunoenzymatic determination of hepatitis B surface antigen in serum was applied[8]. Hepatitis C virus antibody was determined by eletrochemiluminescence immunoassay ‘ECLA’ using Cobas immunoassay analyzer (Roche Diagnostics, Ryan Dempsey, Indianapolis, Japan)[9].

Liver function tests

Serum ALT was determined by a kinetic UV-optimized method IFCC, and serum AST was determined by a kinetic UV optimized method IFCC[10]. For serum bilirubin test (total and direct), total bilirubin reagent was used to measure total bilirubin concentration by the timed end point Diazo method[11]. Prothrombin time determination was performed on a STA-Stago compact CT autoanalyzer (Holliston, MA, USA)[12].

Determination of human IL-17 in serum by ELISA (Chongqing Biospes Co. Ltd., Chongqing, China) was based on sandwich ELISA technology[13]. Real-time PCR for IL-17 mRNA expression was performed: total RNA was prepared from peripheral blood mononuclear cells (PBMCs) using QIAamp RNA blood mini kit (2013; Qiagen, Ermantown Road Germantown, MD, USA). Complementary DNA was synthesized using Thermo Scientific (Thermo Fisher Scientific Inc., Lithuania, V. A. Graiciuno g. 8, LT-02241 Vilnius). Real-time PCRs were performed using Quanti Tect SYBR Green PCR kit with readymade quantiTect primer assay (Qiagen). For measurement of IL-17A mRNA levels, the following primers were used: the forward and reverse primers of human for IL-17 were 5-AGA GAT ATC CCT CTG TG ATC-3 and 5-TACCCC AAA GTTATC TCA GG-3; and the forward and reverse primers for human β-actin were 5-CATGTACGTTGCTATCCAGGC-3 and 5-CTCCTTAATGTCACGCACGAT-3. PCR was performed under the following conditions in 45 cycles: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s.

Statistical analysis

The data collected were tabulated and analyzed by SPSS (Statistical Package for the Social Science software) statistical package version 20 (SPSS Inc., Chicago, Illinois, USA) on an IBM-compatible computer; values less than 0.05 were considered as significant.


  Results Top


Group I included 12 males and three females, and their ages ranged from 20 to 49 years. Group II included 11 males and four females, and their ages ranged from 35 to 54 years. Group III included 10 patients with HCC – nine males and one female – and their ages ranged from 27 to 57 years. Group IV was the control group, including eight males and two females, and their ages ranged from 22 to 55 years.

There was a significant decrease in hemoglobin, platelet count, and white blood cell count in HCC patients in comparison with the other groups. There was a highly significant increase in international normalized ratio (INR) in the HCC patient group and in the cirrhotic group in comparison with the other two groups. There was a highly significant statistical difference in AFP between the studied groups – AFP was markedly increased in HCC patients compared with the cirrhosis and control groups [Table 1].
Table 1: Comparison among the studied groups with regard to CBC parameters, INR, and AFP

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There was a significant increase in the levels of AST, ALT, ALP, and GGT in HCC patients in comparison with the other groups. There was a highly significant decrease in the levels of total protein and serum albumin in HCC patients in comparison with the other groups. In addition, there was a significant increase in the levels of total bilirubin and direct bilirubin in HCC patients in comparison with the other groups [Table 2].
Table 2: Comparison among the studied groups with regard to liver function tests

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There were highly significant statistical differences in serum levels of IL-17 and IL-17 mRNA expression (RQ) between the studied groups. There was a significant increase in serum levels of IL-17 and IL-17 mRNA expression (RQ) in the HCC group when compared with either the Chronic HBV group or the control group [Table 3].
Table 3: Comparison among the studied groups with regard to serum IL-17 and IL-17 mRNA expressions (RQ)

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There was a significant positive correlation between serum IL-17 and aminotransferases (ALT and AST) in patients with chronic HBV infection. There was also a significant positive correlation between serum IL-17 and aminotransferases (ALT and AST) and total and direct bilirubin in cirrhotic patients. In addition, there was a significant negative correlation between IL-17 and total protein and serum albumin. Similarly, a significant positive correlation was found between serum IL-17 and aminotransferases, AFP, and total and direct bilirubin in HCC patients. In addition, there was a significant negative correlation between serum IL-17 and total protein and serum albumin [Table 4].
Table 4: Correlation between serum IL-17 and other assessed laboratory parameters among the studied patient groups

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There was a significant positive correlation between IL-17 mRNA expression (RQ) and aminotransferases (ALT and AST) in patients with chronic HBV infection. A significant positive correlation was also found between IL-17 mRNA expression (RQ) and aminotransferases (ALT and AST) and ALP in cirrhotic patients. A significant positive correlation was documented between IL-17 mRNA expression (RQ) and ALT, AST, GGT, and AFP in HCC patients. In addition, there was a significant negative correlation between IL-17 and serum albumin and total protein in all groups [Table 5].
Table 5: Correlation between RQ and other assessed laboratory parameters among the studied patient groups

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Regarding chronic viral hepatitis B, at a cutoff point greater than 32.85 pg/ml, the sensitivity of serum IL-17 was 80%, the specificity was 29%, and the accuracy was 44%. The positive predictive value was 32%, and the negative predictive value was 77%. Regarding liver cirrhosis, at a cutoff point greater than 50.55 pg/ml, the sensitivity of serum IL-17 was 87%, the specificity was 75%, and the accuracy was 78%. The positive predictive value was 59%, and the negative predictive value was 93%. In the HCC group, at a cutoff point greater than 53.75 pg/ml, the sensitivity of serum IL-17 was 80%, the specificity was 70%, and the accuracy was 72%. The positive predictive value was 40%, and the negative predictive value was 93% [Table 6].
Table 6: Validity of serum IL-17 among the studied cases of chronic viral hepatitis B, liver cirrhosis, and HCC

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Regarding chronic viral hepatitis B, at a cutoff point greater than 3, the sensitivity of RQ was 86%, the specificity was 30%, and the accuracy was 46%. The positive predictive value was 34%, and the negative predictive value was 83%. Regarding liver cirrhosis, at a cutoff point greater than 5.55, the sensitivity of RQ was 93%, the specificity was 80%, and the accuracy was 84%. The positive predictive value was 67%, and the negative predictive value was 97%. In the HCC group, at a cutoff point greater than 5.15 pg/ml, the sensitivity of RQ was 90%, the specificity was 63%, and the accuracy was (68%). The positive predictive value was 38%, and the negative predictive value was 96% [Table 7].
Table 7: Validity of RQ among the studied cases of chronic viral hepatitis B, liver cirrhosis, and HCC

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In the chronic viral hepatitis B group, for the combination of serum IL-17 and RQ, the sensitivity was 80%, the specificity was 30%, and the accuracy was 45%. It had a positive predictive value of 33% and a negative predictive value of 90%. In liver cirrhosis group, for the combination of serum IL-17 and RQ, the sensitivity was 87%, the specificity was 80%, and the accuracy was 82%. It had a positive predictive value of 65% and a higher negative predictive value of 93%. In the HCC group, for the combination serum IL-17 and RQ, the sensitivity was 89%, the specificity was 70%, and the accuracy was 74%. The positive predictive value was 43%, and the negative predictive value was 97% [Table 8].
Table 8: Validity of combined IL-17 and RQ among the studied cases of chronic viral hepatitis B, liver cirrhosis, and HCC

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Multivariate regression analysis showed that both mRNA expression of IL-17 (RQ) and serum IL-17 were independent risk factors for developing cirrhosis and HCC among HBV patients [Table 9].
Table 9: Multivariate regression analysis for risk factors in cirrhotic patients and HCC patients

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  Discussion Top


IL-17 produced by Th17 cells serves as a chemoattractant for neutrophils, recruiting them to sites of infection and inflammation. Th17 cells also produce a number of other cytokines such as IL-22 and IL-21 that have been shown to synergize with IL-17 and enhance the expression of antimicrobial peptides in mucosal tissues[14].

HBV infection remains a global public health problem. More than 350 million people worldwide are suffering from persistent HBV infection. Chronic HBV infection is a serious clinical problem due to the potentially adverse sequelae of hepatic decompensation and the development of cirrhosis or HCC[15].

In addition to its potent proinflammatory capacity, IL-17 exerts its effects through the recruitment of monocytes and neutrophils, facilitating T-cell infiltration and activation and amplification of immune response by inducing the production of IL-6. In addition, IL-17 synergizes with other cytokines, particularly IL-1β and tumor necrosis factor-α[16].

This study aimed to evaluate the mRNA expression of IL-17 in patients with chronic HBV and evaluate its role in the development of liver cirrhosis and HCC.

The incidence of HCC started to increase over 45 years[17]. Similarly, the greatest proportional increase in cases of HCC has been observed between 45 and 60 years of age[18]. A study stated that liver cancer incidence rates increase after 20 years of age and peak at about age 50[19]. Another study revealed that the most predominant age group is between 40 and 59 years[20].

These results are in agreement with this study.

In this study, HCC was more predominant in males than in females; this result is in accordance with the most recent world health report (WHO) that indicated a total of 714 600 new cases of HCC worldwide, with 71% among men[21],[22], and have also reported that the male sex is an important risk factor for HCC[23].

In the current study, platelet count and hemoglobin were significantly lower in cirrhotic patients compared with the control group.

Another study reported that there are various theories about thrombocytopenia in chronic liver diseases. These theories include decreased thrombopoietin levels and splenic sequestration of platelets due to portal hypertension[24]. The associated anemia likely results from increased portal pressure generated by the resulting cirrhosis, which leads to a relative hypersplenism[25].

In the current study, there was a significant increase in PC% (INR) in patients groups (HCC and cirrhotic) compared with the control group as a result of a decreased rate of synthesis by the injured and exhausted liver cells.

The decrease in prothrombin concentration and the increase in INR had happened as a result of the decreased production of tissue factor, factor VII, and vitamin–K-dependent factors, which are synthesized by hepatocytes, and coagulation factors in the common pathway (prothrombin, factors V and X, and fi brinogen)[24].

The present study also revealed a significantly higher level of AFP in HCC patients compared with HBV patients and controls.

A significant elevation in serum AFP was found in the HCC group compared with cirrhosis patients and the control group[26],[27]. The increased production of AFP in HCC patients is most probably due to certain genes that are reactivated as a result of malignant transformation of cells. It was demonstrated that intracellular AFP may function as a signal molecule by binding key proteins involved in growth or apoptosis signal pathways[28].

ALT, AST, GGT, and ALP and total bilirubin in the liver were significantly higher in HCC patients and hepatitis B patients compared with the control group. A highly significant decrease in total protein and Alb and a significant increase in PC% (INR) in the patient groups (HCC and cirrhotic) compared with the control group were a result of decreased rate of synthesis by the injured and exhausted liver cells.

Aminotransferases are the most frequently utilized indicators of hepatocellular necrosis and are expected to be elevated in liver cell injury whatever the cause. The conventional tests for hepatic function do not distinguish HCC from cirrhosis, and therefore they contribute little to the diagnosis of the tumor with the exception of serum ALP, GGT, and total bilirubin, which were significantly elevated in the HCC group when compared with the HBV group due to the more prominent cholestatic effect of the tumor[29].

It was found that GGT levels were highly significant in the HCC group than in the liver cirrhosis group. This was in accordance with the results found by Carr[30].

In this study, serum albumin levels were significantly lower in HCC and cirrhotic patients compared with the control group.

Another study reported a significant deterioration in liver function in cirrhosis and HCC patients compared with the control group[31].

In the current study, a significant increase in the levels of total bilirubin and direct bilirubin in HCC patients in comparison with the other groups was found.

There was a significant elevation of bilirubin in HCC patients, and elevated bilirubin levels were associated with higher AFP levels, worse prognosis, and multifocality. This could reflect an infiltrative destruction of liver parenchyma by diffusely growing HCC[32].

This study showed a significant statistical difference in serum levels of IL-17 of the studied groups, which increased gradually from the chronic group to the cirrhotic and HCC groups, which were all higher compared with the control group.

Serum IL-17A levels in HCC patients were higher than those in cirrhosis or chronic hepatitis patients, with no significant difference between cirrhosis and chronic hepatitis patients. IL-17 level was significantly associated with HCC risk in a multivariate analysis. IL-17 has been shown to promote carcinogenesis and HCC metastasis and mediate immune tolerance in tumors[33].

Serum IL-17 protein levels were found to be significantly higher in HBV-infected patients when compared with normal controls. The highest IL-17 levels in the serum and liver were observed in cirrhotic patients and HCC patients, suggesting that IL-17 might contribute to the pathogenesis and/or progression of liver fibrosis[34].

IL-17 cytokines mainly produced by Th17 cells could dramatically promote the activation and proliferation of hepatic stellate cells, the cornerstone of liver fibrosis. Activated hepatic stellate cells play a critical role in collagen and extracellular matrix production[35].

Interestingly, in this study, there was a significant positive correlation between serum IL-17 and inflammatory markers of the liver – namely, AST, ALT, and serum bilirubin levels – and there was a significant negative correlation between IL-17 and serum albumin and total protein.

Blood Th17 and plasma IL-17A levels positively correlated with ALT and total bilirubin levels in these chronic HBV-infected participants. However, there were positive correlations between both Th17 frequency and plasma IL-17A levels and serum ALT levels[36].

Significant correlations between the serum concentration of IL-17 and some indicators of liver function – for example, ALT, prothrombin time, and serum albumin – were found, suggesting that IL-17 is, to a certain extent, associated with the extent of liver damage[37].

Correlation analysis identified that serum IL-17 levels were negatively correlated with albumin, and serum IL-17 levels were significantly positively correlated with total bilirubin, ALT, and the Child Pugh classification[38].

In the present study, there was a significant correlation between serum IL-17 and HBV DNA and AFP.

A previous study showed that serum IL-17 levels were positively correlated with serum AFP; this may act as a noninvasive marker for HCC screening and recurrence monitoring[37].

This study also showed a highly significant statistical difference with respect to IL-17 mRNA expression (RQ) in the studied groups. There was a significant increase in IL-17 mRNA expression (RQ) in the HCC group when compared with each of the CHB and control groups.

IL-17 mRNA expression in PBMCs of the patient groups was enhanced (especially in patients of the hepatic cirrhosis group, where mRNA expressions of IL-17 were significantly higher than that in the other groups). Meanwhile, RNA expressions of IL-17 in hepatic cancer were found to be higher than that in the CHB and severe hepatitis groups. PBMC IL-17A mRNA levels were higher in patients with HCC than in patients with CHB and liver failure[34].

Expression levels of IL-17 mRNA in patients with liver cirrhosis were significantly higher than that in the CHB group. PBMC IL-17A mRNA levels were found to be significantly higher in HBV-infected patients when compared with normal controls. IL-17 expression in the liver tissues of patients was positively correlated with inflammation grade and fibrosis stage, suggesting that IL-17 takes part in chronic HBV infection[38].

The association between Th17 frequency and worse clinical outcome of HCC is attributed to IL-17 – the main cytokine produced by Th17 that can promote tumor growth both by angiogenesis and recruitment of neutrophils. IL-17 acts as an angiogenic factor that stimulates the migration and cord formation of vascular endothelial cells in vitro and elicits vessel formation in vivo[39].

The present study showed a significant positive correlation between IL-17 mRNA expression (RQ) and aminotransferases ALT and AST and negative correlation between it and serum albumin and total protein.

Serum IL-17 protein levels and IL-17 expression in the liver tissue were negatively correlated with albumin levels, and the ratio of albumin to globulin suggested that IL-17 is, to a certain extent, positively correlated with liver damage. ALT can be easily affected by drugs that decrease the enzyme, which may explain why the hepatic histological inflammation grades in some of the patients were not positively correlated with the ALT[34].

IL-17 expression was negatively correlated with albumin, but positively correlated with total bilirubin and ALT. It was also correlated with liver inflammation, necrosis, and synthetic function, suggesting an important function in the occurrence and development of liver fibrosis[38].


  Conclusion Top


Our study confirmed that serum IL-17 and mRNA expression of IL-17 have an association with fibrosis progression and HCC development in patients with chronic HBV infection, and hence may contribute to disease evolution and consequences in such patients, and serum IL-17 and mRNA expression of IL-17 may be potential candidates for biological markers of HCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]



 

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