Abstract
Background /Aim: Reticulon 3 (RTN3), resides predominantly in the endoplasmic reticulum and has opposite regulatory effects on Hepatitis C virus (HCV) replication through interacting with NS5A and NS4B proteins. This study aimed to unravel the actual effect of RTN3 on HCV replication. Materials and Methods: A total of 115 HCV-related hepatocellular carcinoma patients receiving hepatectomy was enrolled in this study. The hepatic HCV RNA and RTN3 protein levels in the non-cancerous liver tissues were examined for clinical analysis. Results: Of the 115 patients, 16 (11.5%) were occult HBV infection (positive for tissue HBV DNA. Univariate followed by multivariate analysis revealed that intrahepatic RTN3 levels were independently associated with higher HCV viral load (p=0.018) and HCV genotype 1 (p=0.017). Multivariate analysis revealed that HCV genotype 1, tumor size, albumin and aspartate transaminase associated with a shorter recurrence-free survival (p<0.05). Conclusion: Higher intrahepatic RTN3 levels independently correlated with higher intrahepatic HCV RNA levels and genotype 1 HCV.
Chronic hepatitis C virus (HCV) infection is a major global health problem that can lead to severe consequences such as liver cirrhosis and hepatocellular carcinoma (HCC), and an overall increase in the liver-related morbidity and mortality (1-3). HCV, a member of the Flaviviridae family, has a genome that contains a positive-sense, single-stranded RNA of approximately 9.6 kb in length encoding a polyprotein of ~3,000 amino acid (4, 5). The structural (components of the mature virus) and non-structural (NS) proteins (functional elements to help viral replication) are produced through a proteolytical process (6-8). The seven NS proteins (P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) are involved in different stages of viral replication (9).
Similar to other positive-strand RNA viruses, the sites of HCV RNA replication is believed to be concentrated on ER-associated membranous compartments, although the molecular details remain to be elucidated (6). Of the NS proteins, NS4B is a 27-kDa ER membrane-associated protein, which comprises of four transmembrane (TM) domains at its central portion, two consecutive amphipathic α -helical domains (AH1 and AH2) at the N-terminal region, and two helices (H1 and H2) at the C-terminal portion (9-11). NS4B self-oligomerizes via the AH2 domain, facilitating the formation of the “membranous web”, which is believed to be critical for efficient HCV viral replication (12). On the other hand, NS5A is an RNA binding hydrophilic phosphoprotein, that exists in a dimeric form without trans-membrane helices (13). It consists of three domains with distinct functions (14). NS5A also modulates the polymerase activity of NS5B, an RNA-dependent RNA polymerase (15). Consequently, NS4B and NS5A have emerged as attractive and promising drug targets for antiviral therapy.
The reticulon (RTN) family proteins reside predominantly in ER with more than 200 members. They are 200-1,200 amino acids (aa) in length (16), sharing a reticulon homology domain of 150-201 aa in carboxyl-terminal regions. Based on the sequence homology, it was classified into four families: reticulon 1, 2, 3 and 4/Nogo. In general, RTN family proteins mainly exist in nervous tissues, whereas RTN3 and RTN4B are expressed ubiquitously (9, 17). RTN primarily plays a role in promoting membrane curvature development, nuclear pore complex formation, vesicle maturation, DNA binding autophagy, and inflammatory-related functions (18, 19). Notably, an interaction between RTN3 and NS4B proteins has been confirmed from screening a liver cDNA library using a yeast-two hybrid system (20). It has been reported that RTN3 acts as a restriction factor for HCV replication due to its ability to compete for and bind to the AH2 domain of NS4B (9). However, another study discovered that RTN3 promotes HCV propagation by interfering with the NS5A protein (14).
RTN3 has been associated with both inhibition and promotion of HCV life cycle. This controversial role of RTN3 in the HCV life cycle and its actual function in HCV-infected patients remains elusive. In this study, we clarified this important issue regarding use of clinic liver tissues from patients with HCV-related HCC.
Patients and Methods
Ethics. The research was ethically conducted in accordance with the World Medical Association Declaration of Helsinki. All patients enrolled, signed an informed consent before submitting their surgical samples to the tissue bank of Chang Gung Medical Center for research use. This study was conducted under the approval of Institutional Review Board, Chang Gung Medical Center, Taiwan. All patients were identified by numbers and no real names were used during the study.
Patients. A total of 115 patients diagnosed as HCC with chronic HCV infection and receiving surgical resection were enrolled during July 2002 to August 2007 in Chang Gung Memorial Hospital. All patients were negative for serum Hepatitis B virus (HBV) surface antigen (HBsAg). The diagnosis of HCC was confirmed by liver biopsy, aspiration cytology and/or high alpha-fetoprotein (AFP) levels (>400 ng/ml) plus two dynamic image studies (dynamic computer tomography and angiography). Patients with anti-HCV seropositive tissues and detectable HCV RNA for more than 24 weeks were identified as infected with chronic HCV. The occult HBV infection was defined as the persistence of HBV genomes in the blood or liver of individuals with negative serum HBsAg testing (21). Clinicopathological parameters were recorded, including gender, age, HBsAg, alcohol usage, clinical cirrhosis, Edmondson's histology grade, microvascular invasion, macrovascular invasion, presence of tumor capsule, number of tumors, largest tumor size (in diameter) and AFP. The albumin, aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, creatinine, and prothrombin time were determined with biochemistry and hemogram analysis.
Serum anti-HCV antibody was assayed by a third-generation enzyme immunoassay kit (AxSYM HCV Version 3.0; Abbott Laboratories). Serum HBsAg was detected by radioimmunoassay (Ausria-II, HBsAg-RIA; Abbott Laboratories).
HCV RNA quantification and genotype determination of non-cancerous liver tissues. Hepatic HCV-RNA levels were quantified following a previous study (22). Briefly, RNA was extracted from liver tissues (para-neoplastic, non-cancerous parts) using TRIzol reagents (Invitrogen, Carlsbad, CA). HCV RNA detected by COBAS TagMan HCV test (Roche Diagnostics, Tokyo, Japan) according to the manufacturer's protocol. The final levels of hepatic HCV RNA were calculated as IU per gram with a lower detected limit of 1,500 IU/g. HCV genotype was determined using the InoLipa method (COBAS AmpliPrep/COBAS TaqMan HCV Test, Roche Diagnostics, Basel, Switzerland).
Hepatic HBV DNA detection. Liver tissues were homogenized in DNA extraction buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl and 100 μg/mL proteinase K) and incubated at 56°C for 3 h. After addition of RNase A and incubated at room temperature for 1 h, the homogenates were extracted once with phenol/chloroform followed by chloroform extraction. The DNA was precipitated by adding NaOAc, glycogen, and alcohol. The DNA pellet was washed with 70% alcohol, dried, and dissolved in pure water. The quantitation of HBV DNA was examined by COBAS TagMan HBV test (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's protocol. The lowest detection limit of this assay was 60 IU/g (23).
Hepatic RTN3 protein extraction and Western blot analysis. Total protein was extracted from liver tissues using RIPA lysis buffer. Proteins were separated in a 10% SDS-PAGE gel and transferred to a PVDF membrane (PerkinElmer, Boston, MA). Antibodies against RTN3 used in Western blot analysis were raised against full-length human RTN3 (9). The protein-antibody complex was detected by the chemiluminescent substrate (Cell Signaling Technology, Danvers, MA). The intensities of bands were semi-quantified by Image J software. GAPDH was usedas a loading control, and was detected using anti-GAPDH antibody (6C5; Novus Biologicals, Littleton, CO).
Statistical analysis. Dichotomous data were reported as numbers and percentages and compared using chi-square test or Fisher's exact tests, where appropriate. Parametric data were reported as means±SD for continuous variables with normal distribution and as median (range) for continuous variables with a non-normal distribution. For comparisons between groups, the two-sample Student's t-test was used for continuous variables with normal distribution, and the Mann-Whitney U-test was used for continuous variables with a non-normal distribution. The parametric data were dichotomized into two groups, with the mean values used as cut-offs. To evaluate the relationship between the intrahepatic RTN3 levels and clinicopathological parameters as well as virological parameters, univariate and multivariate logistic regression analysis were performed. Recurrence-free survival was calculated from the date of treatment to the date of HCC recurrence or last follow-up. Overall survival was calculated from the date of treatment to the date the patient deceased or had the last follow-up. Univariate and multivariate Cox proportional hazard models were used to estimate recurrence-free survival and overall survival for clinicopathological parameters, virological parameters, and intrahepatic RTN3 levels. Those variables that were statistically significant in the univariate analysis at the level of p<0.05 were included in the multivariate analysis. All tests were two-tailed, and a p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using the Statistical package for the social sciences (SPSS) for Windows Version 18.0 (SPSS, Chicago, IL).
Results
Basic characteristics of patients. A total of 115 HCV-related HCC patients receiving hepatectomy were enrolled in the study. Their baseline demographic and clinical features are summarized in Table I. The mean age was 62.8 (range=41-78) ±11.0 years and 78 (67.8%) patients were male. The mean AST and ALT levels were mildly higher than the upper limit of normal (51 U/L and 59 U/L, respectively). Sixteen (11.5%) patients had occult HBV infection with detectable intrahepatic HBV DNA in the non-cancerous liver tissue. The pathological parameters of most tissues revealed, liver cirrhosis, HCV genotype 1, Edmondson's histologic grade >2, tumor capsule, and tumor numbers ≥1.
Higher RTN3 expression is independently correlated with higher HCV viral loads and genotype 1. To clarify the role of RTN3 in HCV replication under physiological conditions, total protein extracted from liver tissues was examined for RTN3 expression by western blot (Figure 1). After normalization with actin, the RTN3 expression in HepG2 cells was defined as 1 to estimate the relative fold difference of RTN3 expression levels in liver tissues. The RTN3 levels were dichotomized into low- and high-expressing groups by using mean values as a cut-off. The RNA extraction from the same specimens was used for HCV RNA analysis. The correlation between the expression level of intrahepatic RTN3 and the clinical and virological factors was analyzed. As shown in Table II, the positive correlation between expression levels of RTN3 and HCV-RNA was observed. The patients with higher intrahepatic RTN3 levels had higher HCV-RNA levels (2493.3 IU/g vs. 517.5 IU/g; p=0.005). The high expression of intrahepatic RTN3 was also found to be significantly correlated with genotype 1 (42/55 (76.4%) versus 25/60 (41.7%); p<0.001). As for other clinical parameters including age, sex, occult HBV infection, liver cirrhosis, alcoholism, Edmondson's histological grade, microvascular invasion, macrovascular invasion, presence of tumor capsule, tumor numbers, largest tumor size, AFP, albumin, AST, ALT, bilirubin, creatinine, and prothrombin time, no statistically significant correlation was found with intrahepatic RTN3 expression levels.
Multivariate logistic regression analysis showed that higher HCV viral load (OR=1.053; 95%CI=1.009-1.099; p=0.018) and HCV genotype 1 (OR=2.846; 95%CI=1.202-6.734; p=0.017) independently correlated with high RTN3 (Table III).
Clinicopathological and virological factors associated with postoperative survival in HCV-related HCC. The cox proportional hazard model was used to examine the relationship between the recurrence-free survival and clinicopathological factors, virological factors, and intrahepatic RTN3 levels. As shown in Table IV, HCV genotype 1, combined occult HBV infection, tumor size >4.4 cm, albumin ≤3.9 g/dl, and ALT >51 U/l were associated with a shorter recurrence-free survival using Univariate analysis. After adjusting other confounders, multivariate analysis revealed that HCV genotype 1 (p=0.001), tumor size >4.4 cm (p=0.018), albumin ≤3.9 g/dL (p=0.028), and ALT >51 U/L (p=0.001) were significantly associated with a shorter recurrence-free survival. Similarly, the association between overall survival and clinicopathological factors, virological factors, and intrahepatic RTN3 levels was also analysed using the Cox proportional hazard model (Table IV). Only an AFP level greater than 38 ng/ml associated with a shorter overall survival in Univariate analysis.
Discussion
Owing to the discovery of HCV in 1989, the technique of HCV RNA detection can now be used to accurately identify patients that are infected with HCV. It has been found that most HCV-infected patients (80-85%) fail to clear the virus following acute infection, and thus subsequently progress to chronic stages. Chronic HCV infection may lead to various complications including cirrhosis, portal hypertension, hepatic decompensation, and the development of HCC. Up to 350,000 patients die per year due to HCV infection (24). Therefore, how to eradicate chronic HCV infection remains presently a critical issue. As such, identification of viral and host factors involved in HCV replication is a key prerequisite for the discovery of new antiviral drug targets in the future. RTN3 plays a crucial role in HCV replication (9, 14, 20), however, the conflicting regulatory effects of RTN3 in HCV replication were found from studying different HCV viral proteins. Two HCV non-structural proteins (NS4B and NS5A) which play important roles in HCV replication physically interact with RTN3. Studies have shown that, RTN3 can compete for and bind to the AH2 domain of NS4B restricting HCV replication by abolishing AH2-mediated NS4B self-interaction (9). On the contrary, RTN3 promotes HCV propagation by interfering NS5A (14). To date, no studies have clarified the interaction between these two, NS4B and NS5A, non-structural proteins and RTN3 at the same time and its effect on HCV replication. In this study we tried to shed light on this puzzling issue through direct assessment of intrahepatic RTN3 levels and investigation of its correlation with various clinicopathological and virological factors.
According to our clinical analysis, the higher intrahepatic RTN3 levels were significantly associated with higher intrahepatic HCV viral loads in liver tissue. This finding is consistent with the study which enhanced HCV propagation through the effects of NS5A-RTN3 interaction, suggesting that increased viral propagation may overcome the interference of viral replication (NS4B-RTN3 interaction effect) in order to maintain a higher HCV RNA level.
Apart from proving the clinical correlation between RTN3 and HCV replication, we discovered that higher intrahepatic RTN3 levels also associated with HCV genotype 1 (42/67 vs. 13/48, p<0.001). In addition, HCV genotype 1 was shown to be an independent factor for predicting high intrahepatic RTN3 levels (p=0.017). To our knowledge, this finding has never been reported before. The molecular mechanism of this novel finding remains unclear. Nevertheless, since HCV viral load and genotype 1 are important predictors of response to antiviral treatment (24), we speculate the intrahepatic RTN3 levels could potentially predict outcome of antiviral therapies.
Taiwan is an area hyperendemic for HBV infection. The majority of patients are infected by the age of 3 through perinatal transmission (25, 26). However, HBV-infected persons rarely progress into a chronic status if the infection occurs after the age of 3 (21). Therefore, the cases with occult HBV infection included in this study were probably chronic HBV carriers superinfected by HCV, resulting in loss of HBV surface antigen (27). In this view, HBV served as an initiator and HCV served as a promoter of hepatocarcinogenesis. In the present study, the presence of occult HBV infection (p=0.031) was associated with unfavorable recurrence-free survival in univariate Cox analysis. However, the expression levels of intrahepatic HCV RNA was unable to predict the postoperative survival of patients. These findings are in accordance with a previous study (22). Other survival predictors included tumor size, albumin, ALT, and AFP, suggesting the prognostic role of tumor burden, liver functional reserve, and continuous hepatic inflammation (28). In our analysis, the RTN3 levels did not play a role in predicting postoperative outcome.
The major limitation of the current study is that the number of liver tissues obtained from HCV-associated HCCs remained relatively small because most HCC patients (>60%) in Taiwan were HBsAg-positive.
Conclusion
This study demonstrated that higher intrahepatic RTN3 levels were independently associated with higher intrahepatic HCV viral loads and genotype 1 in HCV-related HCC.
Acknowledgements
This study is supported by grants from Chang Gung Medical Research Council, Taiwan (CLRPG3C0012 and CMRPG290152). The Authors would like to thank Dr. Horng JT from Chang Gung University for providing anti-RTN3 antibodies.
Footnotes
This article is freely accessible online.
Conflicts of Interest
All Authors declare no conflict of interest.
- Received April 13, 2017.
- Revision received April 25, 2017.
- Accepted April 26, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved