Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Expression quantitative trait loci for PAX8 contributes to the prognosis of hepatocellular carcinoma

  • Shijie Ma ,

    Contributed equally to this work with: Shijie Ma, Jianshui Yang, Ci Song

    Affiliations Department of Gastroenterology, Huai'an First People's Hospital, Nanjing Medical University, Nanjing, China, Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, Nanjing Medical University, Nanjing,China

  • Jianshui Yang ,

    Contributed equally to this work with: Shijie Ma, Jianshui Yang, Ci Song

    Affiliations Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, Nanjing Medical University, Nanjing,China

  • Ci Song ,

    Contributed equally to this work with: Shijie Ma, Jianshui Yang, Ci Song

    Affiliations Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, Nanjing Medical University, Nanjing,China

  • Zijun Ge,

    Affiliations Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, Nanjing Medical University, Nanjing,China

  • Jing Zhou,

    Affiliations Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, Nanjing Medical University, Nanjing,China

  • Guoxin Zhang ,

    zhibin_hu@njmu.edu.cn (ZH); guoxinz@njmu.edu.cn (GZ)

    Affiliation Departmentof Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China

  • Zhibin Hu

    zhibin_hu@njmu.edu.cn (ZH); guoxinz@njmu.edu.cn (GZ)

    Affiliations Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, Nanjing Medical University, Nanjing,China

Expression quantitative trait loci for PAX8 contributes to the prognosis of hepatocellular carcinoma

  • Shijie Ma, 
  • Jianshui Yang, 
  • Ci Song, 
  • Zijun Ge, 
  • Jing Zhou, 
  • Guoxin Zhang, 
  • Zhibin Hu
PLOS
x

Abstract

Paired-box family member PAX8 encodes a transcription factor that has a role in cell differentiation and cell growth and may participate in the prognosis of hepatocellular carcinoma (HCC). By bioinformatics analysis, we identified several single nucleotide polymorphisms (SNPs) within a newly identified long non-coding RNA (lncRNA) AC016683.6 as expression quantitative trait loci (eQTLs) for PAX8. Hence, we hypothesized that PAX8eQTLs in lncRNA AC016683.6 may influence the HCC prognosis. We then performed a case-only study to assess the association between the two SNPs as well as the prognosis of HCC in 331 HBV-positive HCC patients without surgical treatment. Cox proportional hazard models were used for survival analysis with adjustments for the age, gender, smoking status, drinking status, Barcelona-Clinic Liver Cancer (BCLC) stage, and chemotherapy or TACE (transcatheter hepatic arterial chemoembolization) status. We found that the G allele of rs1110839 and the T allele of rs4848320 in PAX8was significantly associated with a better prognosis compared with the T allele of rs1110839 and the C allele of rs4848320 (adjusted HR = 0.74, 95% CI = 0.61–0.91, P = 0.004 for rs1110839 and adjusted HR = 0.71, 95% CI = 0.54–0.94, P = 0.015 for rs4848320 in the additive model). Furthermore, the combined effect of the variant genotypes for these two SNPs was more prominent in patients with the BCLC-C stage orpatients with chemotherapy or TACE. Although the exact biological function remains to be explored, our findings suggest a possible association of PAX8eQTLs in lncRNA AC016683.6 with the HCC prognosis inthe Chinese population. Further large and functional studies are needed to confirm our findings.

Introduction

Liver cancer is the fifth most common cancer worldwide and the third greatest cause of cancer-related death worldwide [1]. Hepatocellular carcinoma (HCC) is the most common type of liver cancer [2]. Although surgical resection, liver transplantation, radiotherapy and some other therapies are potentially effective treatments for HCC, studies have shown that HCC has an increasing incidence and a poor 5-year survival rate of approximately 7% despite treatment [35]. The Barcelona Clinic Liver Cancer (BCLC) staging classification has been widely endorsed to predict the prognosis of HCC patients; however, remarkably different survival outcomes among HCC patients at the same stage suggest that the existence of other important factors might affect the prognosis. This current status has inspired researchers to focus on identifying molecular biomarkers to guide individualized treatment and improve the prognosis of cancer patients.

Paired-box gene 8 (PAX8), a member of the PAX gene family, encodes a transcription factor that has a role in cell differentiation and cell growth, and silencing of PAX8 in cell culture results in cell death [6]. PAX8 is known to activate the transcription of BCl2, which is an anti-apoptotic gene that is also involved in p53suppression, suggesting it plays a role in tumor initiation and progression [7]. A recent study also showed that overexpression of PAX8 protein by endometrial cancer is associated with poor disease outcomes[6]. Accumulating data have revealed that PAX8is expressed in a high percentage of kidney, thyroid, ovarian, and lung carcinomas [811]. However,the potential role of PAX8 in HCC prognosis has been rarely explored.

Long non-coding RNAs (lncRNAs) are defined as non-protein-coding transcripts,which are usuallylonger than 200 nucleotides.Based on the recent studiesonlncRNAs, it is reasonable to believe thatlncRNAsare important for regulating gene expression in the nucleus, exerting their biological functions. Recent studieshave provideda comprehensive generalization on the functions of lncRNAsthatmay modulate transcription or post-transcription via targeting the splicing, stability, or translation of mRNAs [12]. LncRNAAC016683.6is located in the intron region of PAX8, which belongs to chromosome 2q13. Using bioinformatics analyses, we identified two single nucleotide polymorphisms (SNPs) (rs1110839 and rs4848320) in AC016683.6that may be the expression quantitative trait loci (eQTLs) for PAX8 (http://www.regulomedb.org) [13]. Therefore, it is likely that the two SNPs could influence the interaction between AC016683.6 and PAX8, regulating the expression of PAX8. According to the potential role of PAX8, we hypothesized that PAX8eQTLsmayinfluence the development and progression of HCC. To validate our hypothesis, we examined the associations between the two SNPs of PAX8and the prognosis of 331patients with intermediate or advanced HCC in China.

Materials and methods

Study subjects

This study was approved by the institutional review board of Nanjing Medical University. All participants provided written informed consent to participate in this study and the ethics committees approved of this consent procedure.Theenrollment of participants was previously described[14,15]. In consideration for constructing a relatively homogenous population, our current study was restrained to HCC patients who did not undergo surgery in the intermediate stage (B) or advanced stage (C) according to the Barcelona Clinic Liver Cancer (BCLC) staging system [16,17]. Briefly, 414 intermediate or advanced HCC patients were consecutively recruited from Nantong Tumor Hospital and the First Affiliated Hospital of Nanjing Medical University, Jiangsu, China.All participants were newly diagnosed and histopathologically confirmed HCC cases. Then, we prospectively conducted a follow-up study every 3 months from the time of enrollment by personal or family contacts until death or final follow-up. As a result, 331 intermediate or advanced HCC patients who had completed follow-ups and whose clinical information was available were enrolled in our study with the response rate of 80.0%. The maximum follow-up time (MFT) for the 331 patients was 60.7 months (final follow-up in January 2013), and the median survival time (MST) was 14.5 months.

Serological testing

After performingthe enzyme-linked immunosorbent assay(Kehua Bio-engineering Co., Ltd., Shanghai, China) according tothe manufacturer’s protocol[14], we detected HBsAg, anti-HBs, anti-HBc and anti-HCV in the serum collected from eachpatient respectively.

Genotyping

Followingthe traditional method[18], genomic DNA was extracted from a leukocyte pellet by a series of treatments. Then, all SNPs were genotyped using the Sequenom Mass ARRAY iPLEX platform (SequenomInc). To reduce the false positive and error rates, three blank (water) controls were detected in each384-well plate during the sample testingevery time, and more than 10% samples were randomly repeated for quality control, checking whether the latter results coincided with the former. The success rates of genotyping for the two SNPs were all above 98%.

Statistical analysis

The survival time was calculated from the date of HCC diagnosis to the date of patient death or the last follow-up. The associations between the median survival time(MST) and demographic characteristics, clinical features and genotypes were estimated using the Kaplan–Meier method and log-rank test. Univariate and multivariable Cox proportional hazard regression analyses were conducted to estimate the crude or adjusted hazard ratio (HR) and their 95% confidence intervals (CI) with adjustment for the age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE (transcatheter hepatic arterial chemoembolization) status. The Cox stepwise regression model was also performed to determine the predictive factors of HCC prognosis with a significance level of 0.050 for entering and 0.051 for removing the respective explanatory variables. The heterogeneity between subgroups was assessed with the chi-square-based Q-test, and the heterogeneity was considered significant for P<0.10.Analysiswas performed using Stata software (version 11.0;Stataconference, Chicago). All tests were two-sided, and the criterion of statistical significance was set at P<0.05.

Results

Patients’ characteristics and clinical features

The demographic characteristics and clinical information of the 331 HCC patients in stage B or C included in this study were previously described[15]. In brief, 258 of the 331 patients died from HCC, and 2 died from other causes during a period of up to 60.7 months of follow-up. For the disease-specific survival analysis, the latter were considered censored data in the analyses. Chemotherapy or TACE and the drinking status were significantly associated with the patient survival time (log-rank P ≤0.001 and 0.006 for the drinking status and chemotherapy or TACE status, respectively). Compared to those who received neither chemotherapy nor TACE therapy (MST = 3.4 months), patients with chemotherapy or TACE therapy (MST = 16.8 months) had a significantly decreased risk of death (61%, HR = 0.39; 95% CI = 0.29–0.51).

Effects of PAX8 polymorphisms on HCC survival

The associations of the two SNPs with HCC survival were examined in an additive model by the Kaplan–Meier method. As shown in Table 1, patients carrying rs1110839 GT/GG genotypes and rs4848320 CT/TT genotypes had a longer survival time(MST:14.3 months for rs1110839 GT/GG and 15.4 months for rs4848320 CT/TT) than those carrying the rs1110839 TT and rs4848320 CC genotypes (MST:13.4months for rs1110839 TT and 13.0 months for rs4848320 CC). Furthermore, multivariable Cox regression analysis showed that rs1110839 and rs4848320 could be considered significant prognostic markers for HCC (Table 1). After adjusting for the age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE status, variant genotypes of rs1110839 and rs4848320 were significantly associated with a favorable HCC prognosis (adjusted HR = 0.74, 95% CI = 0.61–0.91, P = 0.004 for rs1110839 andadjusted HR = 0.71, 95% CI = 0.54–0.94, P = 0.015 for rs4848320).

Stratified analysis and interaction effects

We then examined the combined effect of these two variants on the HCC survival and observed a significant locus-dose effect between favorable genotypes and the risk of death (P for trend <0.001). Compared to patients without favorable genotypes (MST = 12.6 months), those patients with 1 or 2–4 favorable genotypes had a significantly longer MST (13.3 and 14.9 months, respectively). After adjusting for the age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE status, patients with 1 or 2–4 favorable genotypes had 0.22- and 0.43-fold decreased risks of HCC-specific deaths, respectively (Table 2).

thumbnail
Table 2. Combined effect of two SNPs genotypes associated with HCC patients’ survival.

https://doi.org/10.1371/journal.pone.0173700.t002

Theassociations between PAX8SNPs and HCC survival were further investigated by stratified analysis of the age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE status. As shown in Table 3, we found that the protective effect of combined variant genotypes was more prominent in B stage patients and patients without chemotherapy and TACE (adjusted HR = 0.73, 95% CI = 0.62–0.86; adjusted HR = 0.66, 95% CI = 0.49–0.87, respectively) than in C stage patients or patients with chemotherapy or TACE therapy (adjusted HR = 1.30, 95% CI = 0.77–2.21 and adjusted HR = 0.96, 95% CI = 0.80–1.15; P = 0.040 and 0.026 for the heterogeneity test, respectively). Therefore, the gene-BCLC stage and gene-chemotherapy or TACE status interaction analysis were performed, and statistically significant multiplicative interactionswere observed, as shown in Tables 4 and 5 (P for multiplicative interaction = 0.029 and <0.001, respectively).

thumbnail
Table 3. Stratified analyses of combined effect of two SNPs genotypes associated with HCC patients’ survival.

https://doi.org/10.1371/journal.pone.0173700.t003

thumbnail
Table 4. Interactive effect of combined effect of two SNPs genotypes and bclc stage associated with HCC patients’ survival.

https://doi.org/10.1371/journal.pone.0173700.t004

thumbnail
Table 5. Interactive effect of combined effect of two SNPs genotypes and Chemotherapy/TACE associated with HCC patients’ survival.

https://doi.org/10.1371/journal.pone.0173700.t005

Stepwise Cox regression analyses

We finally performed stepwise Cox proportional hazard analysis to evaluate the effects of the age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE status and PAX8eQTLs on HCC survival. Four variables (Chemotherapy or TACE status, age, drinking status and PAX8eQTLs) were selectedfor use in the final regression model. Furthermore, when the gender, drinking status, chemotherapy or TACE status, and PAX8eQTLswere included in the final model, the PAX8eQTLs remained an independent protective factor for HCC survival (HR = 0.78, 95% CI = 0.67–0.91, P = 0.001)(Table 6).

thumbnail
Table 6. Multivariate Cox regression analysis on HCC patients’ survival.

https://doi.org/10.1371/journal.pone.0173700.t006

Discussion

In the present case-cohort study, we demonstrated that PAX8eQTLs (rs1110839 and rs4848320) may be an independent candidate biomarker for predicting HCC survival in Chinese with a prospective study design. Furthermore, the combined effect of these two SNPs was more prominent in patients with BCLC-B stage who did not receivechemotherapy or TACE.

Since the ENCODE Project and RNA-seq analysis have identified thousands of new lncRNAs, the genetic variants and biological function of lncRNAs are becoming a focus in studies of complex diseases. We previously reported that ZNRD1(Zinc ribbon domain containing 1)eQTLsincreased the risk of HCCLncRNA AC016683.6, located in the intron region of PAX8, may influence the expression of PAX8. A PAX family member, PAX8, is implicated in the development of the kidney, thyroid gland, Mullerian and Wolffian ducts, and others [19,20]. Tumors derived from these organs, as mentioned above, also typically express PAX8[21]. One study showed that PAX8 activates the transcription of the NCAM gene through binding sequences resembling paired domain binding sites in the NCAM promoter [22]. NCAMis expressed on the surface of immune cells [23], and it has been shown to mediate adhesion between immune cells, which is particularly important in inflammation [24]. Previous studies have shown that abnormal cell growth and proliferation are often associated with high expression levels of PAX genes. In fact, overexpression of PAX proteins does not appear to be an initiating or transforming molecular event in tumor pathogenesis, but it facilitates malignant development through the effects of PAX genes on apoptosis resistance, tumor cell proliferation and migration, and repression of terminal differentiation [25]. Our results showed that the variant genotypes (rs1110839 GT/GG and rs4848320 CT/TT genotypes) of the two PAX8eQTLs in lncRNAAC016683.6promotedbetter prognosis in HCC patients with intermediate or advanced disease.

There are several limitations ofthe study that need to be addressed by further research. First, further validation needs to be conducted. Large-scale studies are required to validate the associations between the two eQTLs in AC016683.6 and the HCC prognosis. Second, the biological function of the two eQTLsin AC016683.6 was not further investigated in this study. Additionally, our previous study found that ZNRD1eQTLs rs3757328 in ZNRD1-AS1 (ZNRD1 antisense RNA1) was associated with an increased risk for HCC, and further eQTL analysis indicated the significant association between the genotypes of rs3757328 and the expression of ZNRD1 and ZNRD1-AS1. In vitro experiments have also demonstrated that ZNRD1 knockdown inhibits the expression of HBV mRNA and promotes the proliferation of HepG2.2.15 cells. Given the findings from our previous study[26], we hypothesized that lncRNAAC016683.6 might regulate the expression of a related protein (PAX8) based on its variation, influencing the prognosis of hepatic tumors.

To the best of ourknowledge, this is the first study investigating the association between PAX8eQTLs in the lncRNAAC016683.6andHCC prognosis. This study showed that the variant rs1110839 GT/GG and rs4848320 CT/TT genotypes in lncRNA AC016683.6 influenced the prognosis of hepatic tumor patients. These results suggested that AC016683.6 rs1110839 and rs4848320 might serve as susceptibility markers for HCC survival. Further studies incorporating diverse populations and functional assays are warranted to validate and extend our findings.

Supporting information

Author Contributions

  1. Conceptualization: ZH.
  2. Data curation: CS.
  3. Formal analysis: SM.
  4. Funding acquisition: ZH.
  5. Investigation: CS.
  6. Methodology: CS.
  7. Project administration: GZ.
  8. Resources: ZH.
  9. Software: SM.
  10. Supervision: ZH GZ.
  11. Validation: SM CS JY ZG JZ.
  12. Writing – original draft: SM.
  13. Writing – review & editing: ZH GZ.

References

  1. 1. Llovet JM, Burroughs A, Bruix J (2003) Hepatocellular carcinoma. Lancet 362: 1907–1917. pmid:14667750
  2. 2. Lau WY, Lai EC (2008) Hepatocellular carcinoma: current management and recent advances. Hepatobiliary Pancreat Dis Int 7: 237–257. pmid:18522878
  3. 3. Cheung TT, Ng KK, Chok KS, Chan SC, Poon RT, et al. (2010) Combined resection and radiofrequency ablation for multifocal hepatocellular carcinoma: prognosis and outcomes. World J Gastroenterol 16: 3056–3062. pmid:20572310
  4. 4. Carr BI (2004) Hepatocellular carcinoma: current management and future trends. Gastroenterology 127: S218–224. pmid:15508087
  5. 5. Merion RM (2010) Current status and future of liver transplantation. Semin Liver Dis 30: 411–421. pmid:20960380
  6. 6. Mhawech-Fauceglia P, Wang D, Samrao D, Godoy H, Pejovic T, et al. (2012) Pair-Box (PAX8) protein-positive expression is associated with poor disease outcome in women with endometrial cancer. Br J Cancer 107: 370–374. pmid:22644304
  7. 7. Filippone MG, Di Palma T, Lucci V, Zannini M (2014) Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells. BMC Mol Biol 15: 21. pmid:25270402
  8. 8. Nonaka D, Chiriboga L, Soslow RA (2008) Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas. Am J Surg Pathol 32: 1566–1571. pmid:18724243
  9. 9. Tong GX, Weeden EM, Hamele-Bena D, Huan Y, Unger P, et al. (2008) Expression of PAX8 in nephrogenic adenoma and clear cell adenocarcinoma of the lower urinary tract: evidence of related histogenesis? Am J Surg Pathol 32: 1380–1387. pmid:18670350
  10. 10. Tong GX, Yu WM, Beaubier NT, Weeden EM, Hamele-Bena D, et al. (2009) Expression of PAX8 in normal and neoplastic renal tissues: an immunohistochemical study. Mod Pathol 22: 1218–1227. pmid:19525927
  11. 11. Becker N, Chernock RD, Nussenbaum B, Lewis JS Jr. (2014) Prognostic significance of beta-human chorionic gonadotropin and PAX8 expression in anaplastic thyroid carcinoma. Thyroid 24: 319–326. pmid:23806007
  12. 12. Atianand MK, Fitzgerald KA (2014) Long non-coding RNAs and control of gene expression in the immune system. Trends Mol Med 20: 623–631. pmid:25262537
  13. 13. Boyle AP, Hong EL, Hariharan M, Cheng Y, Schaub MA, et al. (2012) Annotation of functional variation in personal genomes using RegulomeDB. Genome Res 22: 1790–1797. pmid:22955989
  14. 14. Hu L, Zhai X, Liu J, Chu M, Pan S, et al. (2012) Genetic variants in human leukocyte antigen/DP-DQ influence both hepatitis B virus clearance and hepatocellular carcinoma development. Hepatology 55: 1426–1431. pmid:22105689
  15. 15. Xie K, Liu J, Zhu L, Liu Y, Pan Y, et al. (2013) A potentially functional polymorphism in the promoter region of let-7 family is associated with survival of hepatocellular carcinoma. Cancer Epidemiol 37: 998–1002. pmid:24103425
  16. 16. Gomez-Rodriguez R, Romero-Gutierrez M, Artaza-Varasa T, Gonzalez-Frutos C, Ciampi-Dopazo JJ, et al. (2012) The value of the Barcelona Clinic Liver Cancer and alpha-fetoprotein in the prognosis of hepatocellular carcinoma. Rev Esp Enferm Dig 104: 298–304. pmid:22738699
  17. 17. Forner A, Llovet JM, Bruix J (2012) Hepatocellular carcinoma. Lancet 379: 1245–1255. pmid:22353262
  18. 18. Yin J, Wen J, Hang D, Han J, Jiang J, et al. (2015) Expression Quantitative Trait Loci for CARD8 Contributes to Risk of Two Infection-Related Cancers—Hepatocellular Carcinoma and Cervical Cancer. PLoS One 10: e0132352. pmid:26147888
  19. 19. Plachov D, Chowdhury K, Walther C, Simon D, Guenet JL, et al. (1990) Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland. Development 110: 643–651. pmid:1723950
  20. 20. Chi N, Epstein JA (2002) Getting your Pax straight: Pax proteins in development and disease. Trends Genet 18: 41–47. pmid:11750700
  21. 21. Ozcan A, Shen SS, Hamilton C, Anjana K, Coffey D, et al. (2011) PAX 8 expression in non-neoplastic tissues, primary tumors, and metastatic tumors: a comprehensive immunohistochemical study. Mod Pathol 24: 751–764. pmid:21317881
  22. 22. Holst BD, Goomer RS, Wood IC, Edelman GM, Jones FS (1994) Binding and activation of the promoter for the neural cell adhesion molecule by Pax-8. J Biol Chem 269: 22245–22252. pmid:8071351
  23. 23. Kowitz A, Kadmon G, Eckert M, Schirrmacher V, Schachner M, et al. (1992) Expression and function of the neural cell adhesion molecule L1 in mouse leukocytes. Eur J Immunol 22: 1199–1205. pmid:1577062
  24. 24. Kos FJ, Chin CS (2002) Costimulation of T cell receptor-triggered IL-2 production by Jurkat T cells via fibroblast growth factor receptor 1 upon its engagement by CD56. Immunol Cell Biol 80: 364–369. pmid:12121226
  25. 25. Muratovska A, Zhou C, He S, Goodyer P, Eccles MR (2003) Paired-Box genes are frequently expressed in cancer and often required for cancer cell survival. Oncogene 22: 7989–7997. pmid:12970747
  26. 26. Wen J, Liu Y, Liu J, Liu L, Song C, et al. (2015) Expression quantitative trait loci in long non-coding RNA ZNRD1-AS1 influence both HBV infection and hepatocellular carcinoma development. Mol Carcinog 54: 1275–1282. pmid:25110835