Introduction

HCC is one of the most common malignancies with the fourth highest incidence rate in the world. More than a half million people have been diagnosed with HCC in the world every year [1]. HCC is the second leading cause of cancer deaths in China, and nearly half of all new cases of liver cancer (50.5%) and related deaths (51.4%) worldwide, are estimated to occur in China [2, 3]. Many causes for HCC have been proposed, and it’s clear that a hereditary predisposition to HCC development exists [4–7]. And genetics of HCC pathogenesis is complex and largely unknown [8].

RTN4 gene, mapped to chromosome 2p12–14, plays an important role in the apoptosis and inhibition of tumor, vascular remodeling, inhibition of axonal regeneration [9]. A tremendous amount of researchers have been focusing on two insertion/deletion polymorphisms sites [CAA (rs34917480), (TATC (rs71682890)] in the 3′- UTR of RTN4, and confirmed the associations with diseases such as CSCC [10], UL [11] and NSCLC [12], schizophrenia [13], dilated cardiomyopathy (DCM) [14], congenital heart disease [15]. However, the association of RTN4 CAA/TATC polymorphisms with HCC remained unclear. Therefore, a hospital based case-control study was conducted to explore whether these two genetic variations are associated with HCC risk in Chinese Han people.

Material and Methods

Study Population

Our study conform the ethics committee guidelines and all the participants provided written informed consent. Peripheral blood was taken from each participant and stored at −20 °C with EDTA anticoagulating agent until use. The case-control study enrolled 768 subjects including 284 unrelated HCC patients (243 men and 41 women) from the West China Hospital of Sichuan University between 2008 and October 2010. Clinical characteristics were abstracted from the participants medical records regarding the individual’s gender, age, family history of HCC, and the state of hepatitis B surface antigen (HBs Ag) which indicates whether to be infected by HBV or not. 484 healthy individuals (394 men and 90 women) from a routine health survey as controls. The diagnosis of HCC was confirmed by tissue pathology examination. The control group inclusive criteria were no evidence of any personal or family history of cancer or other serious diseases, especially any disease in liver, such as infected by hepatitis virus, alcoholic hepatitis, or cirrhosis were excluded from the study. Patients with other cancers or family history of cancer were also excluded. The demographics of the patients and controls enrolled in this study were presented in Table 1.

Table 1 Clinical characteristics of the HCC patients and controls
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Genotyping

Genomic DNA of each individual was extracted from 200 μl EDTA-anticoagulated peripheral blood samples according to the instruction manual by DNA isolation kit from Bioteke (Peking, China).

The PCR assay was used to genotype the CAA/TATC polymorphisms of RTN4. The sequence of primers and condition for amplification was according to the previously published study [11]. PCR products were analyzed directly by vertical nondenaturing polyacrylamide gel electrophoresis and visualized by silver staining. The results were confirmed by two persons each time. To confirm the accuracy of the method used, different genotypes of PCR products were analyzed by direct sequencing, and the results were 100% agreed.

Statistical Analysis

All data analyses were carried out using SPSS for Windows software package version 13.0 (SPSS Inc., Chicago, IL). Demographic and clinical data of both groups were compared by the chi-square test. Allele and genotype frequencies of RTN4 CAA/TATC polymorphisms were obtained by using Modified-Powerstates standard edition software. Hardy–Weinberg equilibrium was evaluated by the χ2 test. Odds ratio (OR) and 95% confidence intervals (CI) were used to evaluate the effects of any difference between genotypes or alleles. Statistical significance was assumed at P < 0.05 level.

Results

The genotypes of the RTN4 CAA/TATC polymorphisms were successfully gained in all 768 subjects. The genotype and allele frequency distributions of the two polymorphisms in the control group met the requirements of the Hardy-Weinberg equilibrium (Table 2). As shown in Table 2, the ID/DD genotypes of CAA were significantly associated with an increased risk of HCC compared with the II genotype (CAA ID vs. II: OR = 1.50, 95% CI: 1.10–2.04, p = 0.01; DD vs. II: OR = 2.00, 95%CI: 1.15–3.46, p = 0.01). At the same time, the frequency of D allele of CAA was significantly associated with an increased risk of HCC compared with the I allele (CAA D vs. I: OR = 1.39, 95% CI: 1.12–1.73, p = 0.003). Meanwhile, compared with the DD genotype, the ID genotypes of TATC was significantly increased risk of HCC (ID vs. DD: OR = 1.70, 95% CI: 1.23–2.33, p = 0.01).

Table 2 Genotype and allele frequencies of RNT4 CAA/TATA polymorphisms between the HCC patients and controls
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Discussion

HCC is clinically silent for most of its course, and the majority of patients present with advanced disease that has little chance of effective treatment. Despite the recent 20 years in the pathogenesis of the HCC involved in epigenetic changes have been greatly developed, genetic pathogenesis of HCC is complex and still largely unknown yet [8]. The present study sheds light on the potential association between HCC and RTN4 CAA/TATC polymorphisms. Our results identified that the RTN4 CAA polymorphisms (ID/DD genotypes and D allele) and the TATC polymorphisms (ID genotype) were significantly associated with an increased risk of HCC.

RTN4 gene contains eight introns and nine exons, and located on chromosome 2p12–14 [16]. Derived from differential splicing and varied promoter usage, the RTN4 produces 3 major isoforms, named neurite growth inhibitor (Nogo)-A, Nogo-B and Nogo-C [16, 17]. Nogo-A, mainly express in the central nervous system, which has been identified as an inhibitor of axonal regeneration. Nogo-B is found in most tissues, while Nogo-C is highly express in skeletal muscles [17, 18]. Recent investigations have indicated that the Nogo protein induces apoptosis in various tissue or cell lines, such as HCC tissue [19], Human HCC cell line(SMMC-7721) [19] and hepatic stellate cell [20], human glioma cell line [21], other cancer cell lines (SaOS-2, HT-1080, MeWo, CGL4) [22], and cardiomyocytes [23]. Suggesting that RTN4 may act a role in suppressing tumor development [24]. The 3′-UTRs of eukaryotic mRNA, which is among noncodingregions, have been shown to be involved in regulating mRNA stability, cellular and subcellular localization, and translation efficiency [25–27]. Former research revealed mutations in 3′-UTR was associated with neuroblastoma, myotonic dystrophy and a-thalassemia [28]. The CAA/TATC polymorphisms are located at 4068–4071 and 4548–4554 in 3′-UTR of RNT4 mRNA, respectively [27]. Novak G.et.al reported that RNT4 CAA/TATC polymorphisms induce abnormal regulation of RTN4 expression [29]. Recently, numbers of case-control studies were conducted to investigate the association between CAA/TATC polymorphisms and cancer risk [11, 12, 30]. De-Yi L et al. conducted the case-control study including 411 NSCLC patients and 471 unrelated healthy controls. They found the D allele and ID/DD genotypes of RNT4 CAA polymorphisms distributions were significantly different between cases and controls. Therefore, they conclude that the RNT4 CAA polymorphisms contribute to NSCLC risk in Chinese population [12]. This result was consistent with K Zhang et al.’s study, which including 286 UL patients and 450 control subjects, and they declared the DD genotypes carriers had significantly increased association of UL risk when compared with other genotypes [11]. Shi S et al. determined the genotypes of the RNT4 CAA/TATC polymorphisms in 336 CSCC patients and 450 unrelated control subjects, but they didn’t find any difference allele frequencies between patients and control subjects. While stratified analysis results revealed both CAA/TATC polymorphisms were associated with high clinical stage, and the CAA polymorphisms was also associated with positive parametrial invasion [30]. In the present study, significantly increased HCC risk was found to be associated with CAA polymorphisms (ID/DD genotypes and D allele) and TATC polymorphisms (ID genotype). The data indicated that RTN4 CAA/TATC polymorphisms may be involved in the development of HCC.

There were a few limitations in this study. The relatively small sample size may cause instability to the result. And the information of environmental exposure was not detailed. Further studies with a larger size of samples and the genetic and environmental interaction analysis could help to confirm the exact significance of the association between these polymorphisms and the susceptibility of HCC.

In conclusion, the present study demonstrated that CAA/TATC polymorphisms in RTN4 were linked to increased HCC risk. Suggesting the RTN4 CAA/TATC polymorphisms may participate in the development and progression of HCC. Nevertheless, larger sample size and genetic and environmental interaction studies will be needed to clarify the findings in the future.