Tumor Biol. (2014) 35:295–301 DOI 10.1007/s13277-013-1039-x

RESEARCH ARTICLE

Association between an insertion/deletion polymorphism within 3′UTR of SGSM3 and risk of hepatocellular carcinoma Chaoqun Wang & Hua Zhao & Xiankun Zhao & Jiao Wan & Dayong Wang & Wanli Bi & Xinghong Jiang & Yuzhen Gao

Received: 24 June 2013 / Accepted: 18 July 2013 / Published online: 6 August 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy, and multiple host genetic factors are believed to contribute to HCC development. The small G protein signaling modulator 3 (SGSM3) has been shown to be associated with small G protein coupled receptor signal transduction pathway, suggesting a potential role in HCC susceptibility. We carried out a case–control study in a Chinese population (502 cases and 513 controls) to determine whether the 4-bp insertion/deletion polymorphism (rs56228771) in 3′ untranslated region of SGSM3 could affect HCC susceptibility. Logistic regression analysis showed that compared with the del/del genotype, the ins/del genotype of rs56228771 was associated with a significantly decreased risk of HCC [adjusted odd ratio=0.55, 95 % confidence interval (CI)=0.42–0.73, P=1.93×10−5]. The combined ins/del+ins/ins genotypes

Chaoqun Wang, Hua Zhao, and Xiankun Zhao contributed equally to this work. C. Wang : X. Zhao : J. Wan : Y. Gao (*) Department of Forensic Medicine, Medical College of Soochow University, Suzhou 215123, Jiangsu, People’s Republic of China e-mail: [email protected] C. Wang : X. Jiang (*) Department of Physiology and Neurobiology, Key Laboratory of Pain Research AND Therapy, Medical College of Soochow University, Suzhou 215123, Jiangsu, People’s Republic of China e-mail: [email protected] H. Zhao Department of General Surgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu, People’s Republic of China D. Wang : W. Bi Nuhigh Biotechnology, Suzhou 215123, Jiangsu, People’s Republic of China

contributed to a 45 % decreased HCC risk (95 % CI=0.42– 0.73, P=1.03×10−5). This protective trend was more prominent in the HBsAg-negative subgroup. Furthermore, in vivo experiments showed that mRNA levels of SGSM3 from HCC tumor tissues and adjacent non-HCC tissues were correlated with rs56228771 genotypes. Tissue samples with ins/ins genotype have the highest level of SGSM3, which are 2.85–3.00fold and 1.46–1.57-fold higher than that with ins/del and del/del genotype, respectively. Similar results were also observed with four common hepatoma cell lines in vitro. In addition, compared with HCC tissues, significantly higher SGSM3 expression was observed in adjacent non-HCC tissues (fold change=2.48), implying its tumor suppressor roles in HCC. Bioinformatics prediction showed that the insertion allele disrupted a binding site for microRNA (miRNA)-151-5p, which would upregulate SGSM3. Taken together, we provided initial evidence that rs56228771 may contribute to hepatocarcinogenesis, possibly by affecting SGSM3 expression through a miRNA-mediated regulation. The replication of our studies in other populations and functional analysis will further strengthen the underlining mechanism. Keywords Hepatocellular carcinoma . SGSM3 . rs56228771 . microRNA-151-5p Abbreviations HCC Hepatocellular carcinoma SGSM3 Small G protein signaling modulator 3 Indel Insertion/deletion 3′UTR 3′ Untranslated region miR MicroRNA PAGE Polyacrylamide gel electrophoresis OR Odds ratios CI Confidence interval GAP GTPase-activating protein ANOVA Analysis of variance

296

HBV HCV HDV HIV

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Hepatitis B virus Hepatitis C virus Hepatitis D virus Human immunodeficiency virus

using a case–control study. Additional experimental and in silico studies were used to assess the possible functional significance of this polymorphism.

Materials and methods Introduction

Study populations

Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy which ranks among the ten most common cancers worldwide [1, 2]. Its incidence and prevalence is globally heterogeneous with the highest rates in Southeast Asia and Sub-Saharan Africa [3]. HCC is caused by multiple risk factors. Most patients with HCC have an underlying liver disease caused by either chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection or non-viral etiologic risk factors such as alcohol, fatty liver disease, dietary aflatoxin exposure, smoking, and diabetes mellitus [4]. In addition to these factors, multiple host genetic factors are believed to contribute to HCC development [5, 6]. Despite great progress in diagnosis and management of HCC, the molecular pathogenesis of HCC remains poorly understood [7]. In most cases, HCC is diagnosed at a late stage. Thus, the prognosis of HCC patients is generally poor. Early HCC detection in susceptible individuals becomes one of critical strategies to get better prognosis. Therefore, it is of particular interest in identifying HCC susceptibility genes, which will definitely benefit the prediction of HCC risk, and the exploration of approaches to prevent HCC carcinogenesis. The small G protein signaling modulator 3 (SGSM3, also called Merlin-associated protein) belongs to a novel protein family consisting of three members and appears to be associated with small G protein coupled receptor signal transduction pathway [8]. As a GAP, SGSM3 could regulate small G proteins and control their cellular functions. It has been revealed that human SGSM3 protein could coprecipitated with Rap and Rab, indicating its functional regulating roles for the Ras-mediated signaling pathway [8]. Recent studies have linked Rab dysfunction to various human diseases including cancer [9]. Several members of the Rab family have been shown to be aberrantly expressed in various cancer tissues [10]. Furthermore, SGSM3 has been shown to be interacted with Merlin, a key component of the Hippo signaling pathway [11]. It has been proposed that Hippo signaling plays a vital role in hepatocarcinogenesis [12]. Therefore, as an upstream regulator of Hippo signaling pathway, SGSM3 might play a potential role in hepatocarcinogenesis. In light of this information, we hypothesized that the genetic factors governing SGSM3 expression may contribute to individual susceptibility to HCC. To test this hypothesis, we evaluated the association between a 4-bp insertion/deletion (Indel) polymorphism (rs56228771) within 3′ untranslated region (3′UTR) of SGSM3 and HCC risk in a Chinese population

The case–control study was performed on genomic DNA extracted from peripheral blood of newly diagnosed incident HCC cases together with healthy controls. All of the subjects were genetically unrelated and of ethnic Han Chinese descent. The case series is comprised of 502 HCC patients diagnosed, hospitalized, and treated in the affiliated hospitals of Soochow University from 2008 to 2011. All of the patients had not received any radiotherapy or chemotherapy. The diagnosis of the cases, the inclusion and exclusion criteria for the cases and controls, and the definition of smokers and drinkers were described in detail previously [13–15]. Briefly, the diagnosis of these patients was confirmed by a pathological examination combined with positive imaging (magnetic resonance imaging and/or computerized tomography). Tumor stages were assigned according to a modified American Joint Committee on Cancer and international union against cancer (UICC) standard. Since HBV infection is the predominant cause of HCC in China, and significant clinical-pathologic differences exist among HCC patients with different hepatitis infection, those participants who were positive for antibodies against HCV, HDV, or HIV were excluded. A total of 513 healthy controls with matched age (5-year interval) and gender to each set of HCC individuals were cancer-free individuals randomly selected from a communitybased screening program that was conducted in the same regions during the same period as recruitment of HCC patients. Additional 48 tumor tissues and adjacent non-HCC tissues from patients with a diagnosis of HCC were collected from Department of General Surgery, the First Affiliated Hospital of Soochow University from 2011 to 2012. All cases had histological confirmation of their tumor diagnosis and none of these patients had received preoperative chemotherapy or radiotherapy. After surgical resection, the fresh tissues were immediately stored at −80 °C until the DNA/RNA extraction was processed. All participants were voluntary and had completed the informed consent in written before participating this study. The design of the study was approved by the Ethical Committee of Soochow University. DNA extraction and genotyping Genomic DNA of peripheral blood samples and tissues were isolated using genomic DNA purification kit (Qiagen). DNA fragments containing rs56228771 were amplified with a pair of genotyping primers (forward primer: 5′- GGTCCTTAGG

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GATGCTCT-3′, reverse primer: 5′- CAGAAGACAGGTAG GTGCTT-3′). The PCR products were analyzed by 7 % nondenaturing PAGE and visualized by silver staining [16]. The genotypes were determined by the numbers and positions of the band on the gels. The 4-bp deletion allele yielded a 180-bp band whereas the insertion allele yielded a 184-bp band. Genotyping was performed by individuals blinded to all clinical and laboratory data concerning to the participants. Approximately, 10 % samples were randomly selected and further tested in duplicate by different researchers, and the reproducibility was 100 %. To validate the genotyping method, we also analyzed 20 randomly selected DNA samples by direct sequencing; the results for these two methods were 100 % concordant.

Real-time RT-PCR analysis The Hep-G2, HuH-7, SK-Hep-1, and BEL-7704 hepatoma cell lines were obtained directly from Shanghai Cell Bank of Table 1 Clinical characteristics of HCC cases and controls Characteristics

Case

Control

P value

Chinese Academy of Sciences. Cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin at 37 °C in a humidified 5 % CO2 incubator before DNA/RNA extraction. Total RNA was isolated from tissue specimens and hepatoma cell lines using RNA isolation kit (Qiagen). cDNA was generated using random primers and Superscript II reverse transcriptase (Invitrogen). A SYBR® Green real-time PCR was performed using Roche LightCycler® 480 to quantify relative SGSM3 expression in these samples. Glyceraldehyde 3phosphate dehydrogenase (GAPDH) was chosen as the internal control. Primer sequences used for SGSM3 and GAPDH were as follows: SGSM3-F: 5′-CTGTCCTACCGCGAGATTGTG3′, SGSM3-R: 5′-CCGATGCTACCCATGCTGG-3′, GAPDHF: 5′-CTCTCTGCTCCTCCTGTTCGAC-3′, GAPDH-R: 5′TGAGCGATGTGGCTCGGCT-3′. The 25 μl total volume final reaction mixture consisted of 1 μM of each primer, 12.5 μl of Master Mix (Applied Biosystems), and 50–100 ng of cDNA. The negative control experiments were performed with distilled H2O as template. The expression level of target gene was normalized with GAPDH using a 2−ΔΔCT method [17]. In addition, the melting curve analysis was performed for the PCR products to verify primer specificity.

n=502 Frequencies n=513 Frequencies (%) (%) Age (mean±S.D.) 51.2±8.3 Gender Male 318 63.3 Female 184 39.0 Smoking status Smokers 185 36.9 Non-smokers 317 63.1 Drinking status Drinker 219 43.6 Nondrinker 283 56.4 Family history of HCC Yes No Tumor stages Ia+Ib IIa+IIb IIIa+IIIb Serum AFP <20 μg/L 21–400 μg/L ≥400 μg/L HBsAg, N (%) Positive Negative

57 445

11.4 88.6

343 104 55

68.3 20.7 11.0

106 155 241

21.1 30.9 48.0

50.8±8.9

0.89a

316 197

61.6 38.4

0.57b

168 345

32.8 67.2

0.17b

208 305

40.5 59.5

0.32 b

377

75.1

56

10.9

125

24.9

457

89.1

a

Two-sided two-sample t test between cases and controls

b

χ2 test for differences between cases and controls

b

0.00

Fig. 1 Example genotyping and sequencing output for rs56228771 polymorphism. The upper panel (a) displays an example of the genotyping assay results. Lanes 1, 2, 4, 5, 6, and 10, del/del genotype; lane 3, 7, and 8, ins/del genotype; lane 9, ins/ins genotype. The middle (b) and lower (c) panels show the sequence of deletion and insertion allele, respectively. The underlined base pairs indicate the inserted “GTCT” sequences

298 Table 2 Associations between rs56228771 and HCC susceptibility

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Genetic model

Genotype

Cases

(%)

Control

(%)

OR (95 % CI)a

Codominant model

del/del ins/del ins/ins del/del ins/del+ins/ins

368 123 11 368 134

73.3 24.5 2.2 73.3 26.7

308 188 17 308 205

60.0 36.7 3.3 60.0 40.0

1.00 (Reference) 0.55 (0.42–0.73) 0.54 (0.23–1.24) 1.00 (Reference) 0.55 (0.42–0.73)

del allele ins allele

859 145

85.6 14.4

804 222

78.4 21.6

1.00 (Reference) 0.61 (0.48–0.77)

Dominant model a

Adjusted for sex, age, smoking status, drinking status, and HBV infection

Additive model

Bioinformatics prediction of microRNA binding The human SGSM3 3′UTR containing rs56228771 was identified according to the UCSC genome browser (http://genome. ucsc.edu). The mature human microRNA (miRNA) sequences were obtained from the microRNA database, miRBase (http://microrna.sanger.ac.uk). The hybridization of putative microRNA and the SGSM3 3′UTR harboring either the 4-bp insertion or deletion allele were predicted by miRanda with default parameters [18].

P 1.93×10−5 0.12 1.03×10−5 2.54×10−5

among the stratum variable-related odd ratios (ORs). The normalized SGSM3 expression of HCC tissues and adjacent non-HCC tissues was analyzed using paired t test. Nonparametric Mann–Whitney U test and ANOVA test were used for evaluating the SGSM3 mRNA levels in tissue and hepatoma cell line samples, respectively. All tests were two-sided and were performed with the Statistic Analysis System software (version 8.0, SAS Institute). A P value of <0.05 was considered statistically significant.

Results Statistical analysis The genotype distribution was analyzed for Hardy–Weinberg equilibrium using chi-square test. Unconditional logistic regression was used to analyze the association between rs56228771 and HCC risk, adjusted for sex, age, smoking status, drinking status, and HBV infection. Data were further stratified by common risk factors to evaluate the homogeneity Table 3 Stratification analysis of rs56228771 genotypes associated with HCC susceptibility

Variables

Total Gender Male Female Age (median) <45 years ≥45 years Smoking status Non-smokers Smokers Drinking status Non-drinkers

a

Adjusted for sex, age, smoking status, drinking status, and HBV infection

Drinkers HBsAg Positive Negative

Association analysis between rs56228771 genotypes and risk of HCC The distribution of selected characteristics of the 502 HCC patients and 513 controls included in the current study were summarized in Table 1. There were no significant differences between cases and controls in terms of sex, age, smoking, and

Genotypes (cases/controls)

OR (95 % C.I.)a

Heterogeneity P

del/del

ins/del+ins/ins

368/308

134/205

0.55 (0.42–0.73)

223/182 145/126

95/134 39/71

0.58 (0.41–0.81) 0.47 (0.29–0.77)

0.50

180/151 188/157

65/107 69/98

0.52 (0.35–0.77) 0.59 (0.40–0.87)

0.57

257/228 111/80

60/117 74/88

0.46 (0.32–0.67) 0.59 (0.38–0.92)

0.31

0.10

215/179

68/126

0.45 (0.31–0.65)

153/129

66/79

0.70 (0.46–1.07)

263/40 105/268

114/16 20/189

1.01 (0.53–1.94) 0.26 (0.15–0.44)

0.0007

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Fig. 2 SGSM3 expression in HCC tumor tissues vs. non-tumor tissues and its correlations between rs56228771 indel polymorphism. a Relative SGSM3 expression in paired HCC tumor tissues vs. adjacent non-tumor tissues (N=48). **P<0.001 compared with HCC tumor tissues; b Relative SGSM3 expression among three genotypic groups of rs56228771 in vivo

(ins/ins, N=2, ins/del, N=12, del/del, N=34). *P<0.01, **P<0.001 compared with del/del genotypic group within the same group (HCC tissue or non-HCC tissue); c Relative SGSM3 expression in vitro. **P<0.01 compared with three hepatoma cell lines with del/del genotype (fold change=1 for Hep-G2 cell). Data represented as mean±SEM

drinking status. Compared to controls, cases had a significantly higher rate of HBsAg positive (75.1 vs. 10.9 %, P<0.0001), in accordance with the fact that HBV infection indeed was a major risk factor for HCC. Exampled output from genotyping assays and sequencing results of rs56228771 polymorphism were shown in Fig. 1. Genotype distributions had no deviation from Hardy–Weinberg equilibrium in both case and control groups (P>0.05). The genotype distributions of rs56228771 between the cases and controls are shown in Table 2. Multivariate logistic regression analysis showed that the ins/ del genotype was associated with a significantly decreased risk of HCC (adjusted OR = 0.55, 95% CI = 0.42–0.73, P=1.93×10−5), compared with the del/del genotype. Under dominant model, similarly, the combined ins/del+ins/ins genotypes contributed to a 45 % decreased HCC risk (95 % CI=0.42–0.73, P=1.03×10−5). Each additional copy of the 4-bp insertion allele was associated with a 39 % decreased HCC risk (OR=0.61, 95% CI =0.48–0.77, P=2.54×10−5). Stratified analyses based on common risk factors are summarized in Table 3. The decreased risk of combined ins/del+ins/ ins genotypes was more significant in HBsAg-negative subgroup (heterogeneity P=0.0007). No other significant heterogeneity between the subgroups was detected concerning sex, age, and drinking status.

Effects of rs56228771 polymorphism on SGSM3 mRNA expression

Fig. 3 In silico analysis of miRNA-binding. rs56228771 is located within the seed region (2 to 8 bp from 5′ end of miRs) of SGSM3-3′UTR and has-miR-151-5p hybrid. The underlined base pairs indicate the inserted “GTCT” sequences

To further investigate the correlation between rs56228771 genotypes and SGSM3 expression, SGSM3 expression were examined by real-time RT-PCR of SGSM3 mRNA in 48 pairs of HCC tissue and adjacent non-HCC tissue samples as well as four common hepatoma cell lines with different genotypes. As shown in Fig. 2a, compared with HCC tissues, significantly higher SGSM3 expression (normalized against GAPDH) was observed in the adjacent non-HCC tissues (fold change=2.48, P<0.001). Furthermore, the results revealed that SGSM3 mRNA levels were significantly higher in subjects with ins/ del or ins/ins genotype than in those with del/del genotype in both HCC and adjacent non-HCC tissue samples (Fig. 2b). As for hepatoma cell lines, similar trend was observed. The SGSM3 mRNA level of HuH-7 cells (ins/ins genotype) was significantly higher than that of other three cell lines with del/ del genotype (Fig. 2c, P<0.001). Together, these data demonstrated that the insertion allele of rs56228771 was significantly correlated with higher SGSM3 expression in vitro and in vivo.

Bioinformatics prediction of miRNA-binding Since genetic variations within 3′UTR of target genes could potentially affect miRNA–mRNA interactions, through which modulating expression levels of target genes, in silico prediction was performed to investigate whether rs56228771 could interrupt miRNA-binding. As shown in Fig. 3, rs56228771 was located within the seed region (2 to 8 bp from 5′ end of miRs) of SGSM3-3′UTR and has-miR-151-5p hybrid. Computational modeling suggests that the thermodynamics of binding between has-miR-151-5p and the SGSM3 3′UTR harboring either the 4-bp insertion or deletion allele is distinct,

300

and the presence of the 4-bp insertion allele would disrupt the binding of has-miR-151-5p and SGSM3 3′UTR.

Discussion In this case–control study of 502 HCC patients and 513 cancerfree controls, we investigated the associations between the indel polymorphism (rs56228771) within 3′UTR of SGSM3 gene and risk of HCC in a Chinese population. Our findings suggested, for the first time, that the presence of 4-bp insertion allele of rs56228771 conferred lower risk for HCC when compared with those noncarriers, particularly of those HBsAg-negative subjects. Additional functional analyses and in silico prediction showed that this potentially functional polymorphism may play a role in the development of HCC in a miRNA-dependent manner. The current study provided genetic evidence at the population level that SGSM3 might be involved in the development of HCC and further highlighted the importance of SGSM3 in HCC tumorigenesis. It is worthy of note that the protective roles of ins/del and ins/ins genotypes is more prominent in HBVAg-negative subgroup. It has been demonstrated that the HBV X protein activates Ras and rapidly induces a cytoplasmic signaling cascade linking Ras, Raf, and mitogen-activated protein kinase, leading to transcriptional transactivation [19]. In comparison to HBV-negative cell line (HepG2), activation and upregulation of Ras has been observed in Hep3B cells with integrated HBV [20]. Furthermore, studies have demonstrated that the level of the central transcription factor TATA-binding protein is increased in cells expressing the HBV X protein through the activation of the Ras signaling pathway [21]. In view of the regulating roles of SGSM3 in the Ras-mediated signaling pathway, our results imply that SGSM3 may be involved in regulating HBV infection or HBV-related HCC. The miRNAs are small noncoding RNAs that extensively regulate gene expression [22]. In most cases, they inhibit target mRNA and translation levels by basepairing to the mRNA of 3′UTR [23]. The binding of miRNAs to mRNA is critical for regulating the mRNA level and protein expression. Therefore, polymorphisms present at or near miRNA-binding sites of functional genes can affect gene expression by interfering with a miRNA function. The biological relevance of these polymorphisms in predicted miRNA-binding sites has begun to be examined in numerous case–control studies [24]. Based on bioinformatics analyses, rs56228771 lies within a predicted binding site (seed region) for human miR-1515p (Fig. 3). Thus, it is possible that miR-151-5p would bind tightly to SGSM3 mRNA transcripts containing the 4-bp deletion allele, repressing SGSM3 expression. On the contrary, the binding with mRNA transcripts containing the 4-bp insertion allele would be disrupted, resulting

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in higher SGSM3 expression. In consistent with this prediction, our functional studies have revealed that both HCC tissue samples and hepatoma cell lines with ins/ins genotype confer higher SGSM3 mRNA expression. Recent studies have shown that the genomic regions of miR-151 were amplified on half of HCC tumor tissues, and the expression of miR-151 is significantly correlated with DNA copy number, indicating the potential roles of miR-151 in HCC tumorgenesis [25]. In addition, it has been demonstrated that miR-151-5p significantly increases HCC cell migration and invasion by directly targeting RhoGDIA, a putative metastasis suppressor in HCC [26]. Since many individual miRNAs could regulate multiple mRNAs simultaneously, there is such possibility that both SGSM3 and RhoGDIA are the targets of miR-151-5p. Our results have demonstrated a differential expression of SGSM3 in HCC tissue and adjacent non-HCC tissue samples, indicating its tumor suppressor roles in HCC. Moreover, in silico prediction has also suggested that SGSM3 is very likely the target of miR-151-5p, a validated miRNA associated with HCC. Therefore, how rs56228771 can influence SGSM3 expression deserves to be fully elucidated both at genetic and functional levels. Taken together, we have provided initial evidence that rs56228771 may contribute to hepatocarcinogenesis, possibly by affecting SGSM3 expression through a miRNA-mediated regulation. It may be used as genetic biomarkers for HCC susceptibility in Chinese population. The replication of our studies in other populations and further functional analysis will strengthen the underling mechanism. Acknowledgments This study is supported by grants from National Science Foundation of China (nos. 81171893, 81201574, and 31271258). We gratefully acknowledge the participation of patients with HCC and cancer-free individuals.

Conflicts of interest None

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