PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of oncotargetLink to Publisher's site
 
Oncotarget. 2016 September 13; 7(37): 60303–60309.
Published online 2016 August 11. doi:  10.18632/oncotarget.11206
PMCID: PMC5312385

MiR-155 and its functional variant rs767649 contribute to the susceptibility and survival of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) ranks the fourth common cancer and the third common cause of cancer mortality among Chinese population. The development of hepatocellular carcinoma (HCC) were confirmed to be involved in complex interactions between environmental and genetic factors. MicroRNAs (miRNAs) have been found to play an important role in tumorigenesis and metastasis. Emerging evidence suggested that upregulation of miR-155, one of the best characterized miRNAs, could serve as a promising marker for the diagnosis and prognosis of many cancers, except for HCC. In current we tested the hypothesis that functional variant rs767649 located in the flanking region of miR-155 gene contributes to the development and survival of HCC. We identified that functional variant rs767649 in miR-155 regulation region was associated with risk and survival of HCC. The minor allele of rs767649 was significantly associated with an increased risk of HCC (OR=1.23, 95% CI=1.11-1.36, P = 7.97×10-5). The genotype TT of rs767649 was significantly associated with a 1.94 fold poor survival of HCC (HR=1.94, 95% CI=1.01-3.79), while 1.15 fold for genotype AT (HR=1.15, 95% CI=1.06-1.25). Results showed that miR-155 was highly overexpressed in HCC tissues, compared with the adjacent normal tissues (P<0.001). The allele T contributes to higher expression of miR-155 in both the HCC tissues and the adjacent non-tumor tissues (P< 0.01). Our findings suggested that miR-155 and its functional variant rs767649 might contribute to the increased risk and poor prognosis of HCC, highlighting the importance of miR-155 in the prevention and prognosis of HCC.

Keywords: miRNA, microRNA-155, susceptibility

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. According to a report from the National Office for Cancer Prevention and Control in China, HCC is the fourth common cancer and the third common cause of cancer mortality among Chinese population [1]. It is estimated that there will be 0.466 million new HCC cases and 0.422 new deaths occurring in 2015 [1]. Although chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) has a major role in the development of the disease, the exact molecular mechanism of HCC still remains uncertain.

MicroRNAs (miRNAs), a class of 21–25 nucleotide (nt) single-stranded non-coding RNAs and post-transcriptionally regulate gene expression by base pairing to their target sequences, have been found to play an important role in tumorigenesis and metastasis [24]. MiRNAs were found to be involved in many important functions in development, cell differentiation, and regulation of cell cycle and apoptosis, and expression was abnormally regulated in cancer by a variety of mechanisms including amplification, deletion, mutation, and epigenetic silencing [58]. Identifying clinical biomarkers and molecular targets for HCC, like microRNAs, may contribute to improving the rate of early diagnosis and survival of patients with this lethal disease [9, 10]. This would allow the clinicians to detect an early stage of HCC, monitor tumor dynamics, and predict sensitivity to treatment and prognosis [5, 11]. Among them, MiR-155 was one of the best characterized miRNAs [9, 12]. Its expression level is significantly changed and is associated with susceptibility and prognosis of many cancers, including breast cancer, lung cancer, esophageal cancer, and papillary thyroid cancer [1218]. Very recently, Xie et al [17] identified that a functional variant rs767649 in miR-155 regulation region contributed to lung cancer risk and survival. However, the intensive roles and mechanism of miR-155 in susceptibility and prognosis of HCC are poorly understood.

In current study, we hypothesize that functional variant rs767649 located in the flanking region of miR-155 gene contributes to the development and survival of HCC. Thus, we conducted this study which aims to identify an useful biomarker for screening HCC, monitoring and predicting poor prognosis.

RESULTS

Characteristics of the study population

Totally we recruited 1500 HCC patients and 1500 matched healthy controls in this study. The distribution of the studied population was presented in Table Table1.1. Similar age, sex ratio, consumption of alcohol and smoking in two groups of the samples in two stages (All P value > 0.05), while HBV infection significantly contributed to susceptibility of HCC (P<0.01).

Table 1
Comparison of HCC patients and controls by selective characteristics

Functional variant rs767649 contributes to the development of HCC

First, we genotyped the functional variant rs767649 of miR-155, which has been identified to be associated with risk and survival of lung cancer previously. The genotype distributions of rs767649 between HCC cases and cancer-free controls in stage 1 are shown in Table Table2.2. Logistic regression analysis revealed that the minor allele of rs767649 was significantly associated with an increased risk of HCC [additive model: adjusted Odds ratio (OR) = 1.25, 95% confidence interval (CI) = 1.04-1.49, P = 0.016] after adjusting for age, gender, HBV infection, smoking and drinking. Compared with the subjects with genotype AA, those with AT were more likely to infect HCC (OR=1.34, 95% CI=1.02-1.76), as well as those with TT (OR=1.48, 95% CI=1.03-2.15).

Table 2
Functional variant rs767649 and HCC risk in stage 1

Then, the results were replicated in an independent population (stage 2). As shown in Table Table3,3, the association was also significant in stage 2. When merged together, T allele was significantly associated with HCC risk, compared with A allele (OR=1.23, 95% CI=1.11-1.36, P = 7.97×10-5). We also evaluated the association between rs767649 and HCC risk, stratified by HBV infection status. The results showed that HBV infection status didn’t changed the association materially (Table (Table44).

Table 3
Functional variant rs767649 and HCC risk in stage 2 and the total effect
Table 4
Stratified analyses of association between rs767649 and HCC risk

Functional variant rs767649 contributes to the survival of HCC

We also evaluated the relationship between rs767649 and HCC survival. In the follow-up period of 9 years, 1422 patients were kept and 1125 of them died. As shown in Table Table5,5, we found rs767649 was significantly associated with poor HCC survival. The results of Cox regression analyses showed that the genotype TT of rs767649 was significantly associated with a 1.15 fold poor survival of HCC (HR=1.15, 95% CI=1.06-1.25), while 1.11 fold for genotype AT (HR=1.11, 95% CI=1.04-1.19).

Table 5
Functional variant rs767649 and HCC risk survival

Variant rs767649, expression level of miR-155 and HCC tissues

To further explore the function of miR-155 in the development of HCC, we also tested the hypothesis that that expression level of miR-155 was associated with susceptibility of HCC in 200 randomly selected HCC patients. As shown in Figure Figure1A,1A, the relative expression levels of miR-155 in the HCC tissues were significantly higher than those in adjacent non-tumor tissues (P< 0.001), which suggested that miR-155 may function as an oncogenic gene in the initiation of HCC. The allele T contributes to higher expression of miR-155 in both the HCC tissues and the adjacent non-tumor tissues (P< 0.01, Figure Figure1B),1B), which is consistent with our previous hypothesis that T allele of rs767649 contributes to the higher risk of HCC.

Figure 1
Comparison of miR-155 expression levels between HCC tissues and adjacent normal tissues

DISCUSSION

In this study, we investigated the association between miR-155 and its functional variant rs767649 and susceptibility and survival in the Chinese population using a two-stage case-control design. Consistent with our hypotheses, our findings suggested that miR-155 and its functional variant rs767649 which was located in the regulation region were associated with the increased risk and poor prognosis of HCC. To the best of our knowledge, this should be the first genetic association study to investigate miR-155 and its functional variant rs767649 on risk and survival of HCC.

HCC is one of the most aggressive tumors in human medicine. Previous studies revealed the necessity of genetic predisposing factors in the carcinogenesis of HCC [1926]. Recently, microRNAs (miRNAs) has been revealed a pivotal role in cancer with increasing evidence showing that they may drive and potentiate oncogenesis [2730]. Studies also found upregulation of miR-146b, miR-221, and miR-222, is observed in HCC and also in differentiated thyroid cancer, which indicated that these miRNAs’ overexpression was essential in maintaining tumorigenesis [31]. In current study, we found miR-155 was highly overexpressed in HCC tissues, compared with the adjacent normal tissues (P<0.001). These results were consistent with those in other related studies of cancers, including breast cancer, lung cancer, liver cancer, and so on [12, 17, 29, 32]. Consistent with Xie et al [17], we identified that functional variant rs767649 in miR-155 regulation region was also associated with risk and survival of HCC. MiR-155 was located at chr21:25573994-25574044, according to the UCSC genome browser, while SNP rs767649 was located at the intronic region of miR-155. According to HaploReg v4.1 [33, 34], a tool for exploring annotations of the noncoding genome at variants on haplotype blocks, such as candidate regulatory SNPs at disease-associated loci, we found that could change motifs, including Irf, Mrg1, Hoxa9, and PRDM1, which have been potential role in carcinogenesis.

Several limitations need to be addressed in our study. First, although we adopt the two-stage, case-control study design, which aims to make the results more reliable, the potential selection bias was Inevitable, and we still have limited statistical power to evaluated the gene-environment interaction relationship among the carcinogenesis of HCC. Second, due to the limited information about disease-free survival, we were unable to evaluate the role of rs767649 in disease-free survival of HCC. Strength this study should be the first and largest genetic association study to investigate candidate genes on risk and survival of HCC, the combination study of gene expression and functional variant which could in-depth study the essential role miR-155 in the tumorigenesis and development of HCC. Although further larger-scale studies are warranted to examine gene-gene and gene-environment interactions, this study had the acceptable statistical power to distinguish relatively small genotype association for rs767649.

In conclusion, this study provides strong evidence that miR-155 and its functional variant rs767649 may contribute to HCC susceptibility and prognosis, indicating biological significance of miR-155 in HCC carcinogenesis. However, larger, well designed studies with different populations and, in vitro and in vivo functional evaluations are warranted to confirm these findings.

MATERIALS AND METHODS

Patients and study samples

The study population has been described previously [35]. In brief, totally included in this study was samples from 1500 Chinese patients with sporadic HCC. The age and gender matched control group contained 1500 healthy Chinese population without any history of cancers, who were recruited from subjects who had annual physical examinations. Using a formatted questionnaire, which was used to collect demographic information, including age, sex, HBV infection status, smoking status, alcohol use, and family history of all cancer, face-to-face interviews were conducted among cases and healthy controls. Both approval from the appropriate institutional review board and written informed consents from patients who were included in this study were obtained. All patients were followed-up by personal or family contacts from the time of enrollment until death or the last scheduled of follow-up (July, 2014).

DNA and RNA extraction, quantitative real-time PCR assay

Genomic DNA was extracted from 5 ml blood samples of all patients and controls using the GoldMag Whole Blood Genomic DNA Extraction kit in accordance with the manufacturer's instructions. Total RNA from the ovarian tissues were extracted with the Qiagen miRNeasy Mini kit (Qiagen), then was reversely transcribed to complementary DNA by using the TaqMan miRNA RT Kit (Applied Biosystems). MiRNA expression levels were tested using the TaqMan PCR kit, using SYBR PCR Master Mix reagent kits (TaKaRa). The results of miRNA expression were normalized using the threshold cycle (Ct) of β-actin.

Genotyping

Genotyping was performed using SEQUENOM's MassARRAY® iPLEX assay (SEQUENOM Inc., San Diego, CA). The MassARRAY Typer 4.0 software was used for proper data acquisition and analysis. Genotypes were called after cluster analysis using the default setting of Gaussian mixture model. Inspection of the clusters was done to ensure a clear cluster separation with good signal to noise cutoff. A manual review was done to further clarify uncertain genotype calls. We selected 5% of the random samples for genotyping repeatedly, yielding a 100% concordance.

Statistical analysis

SAS 9.3 (SAS Institute Inc., Cary, NC, USA) for Windows was used for statistical analysis. All p-values were two-tailed, and p<0.05 was considered statistically significant. Differences in the frequencies of the variants between the groups were analyzed using the two-tailed Fisher's exact test, the Student's t-test, or chi-square test. Odds ratios with 95% confidence intervals (CIs) were calculated for the risk estimate with logistic regression model. The joint effect of variables for survival of HCC was examined using the Cox Proportional Hazard regression model.

Acknowledgments

The work was sponsored by Zheng Shu Medical Elite Scholarship Fund, the Major Social Development Program of Major Science and Technology Project of Zhejiang (Grant No. 2013C03010), and the National Natural Science Foundation of China (81573657).

Footnotes

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

REFERENCES

1. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin. 2016
2. Katz B, Trope CG, Reich R, Davidson B. MicroRNAs in Ovarian Cancer. Hum Pathol. 2015;46:1245–1256. [PubMed]
3. Galdiero F, Romano A, Pasquinelli R, Pignata S, Greggi S, Vuttariello E, Bello AM, Calise C, Scaffa C, Pisano C, Losito NS, Fusco A, Califano D, Chiappetta G. Detection of high mobility group A2 specific mRNA in the plasma of patients affected by epithelial ovarian cancer. Oncotarget. 2015;6:19328–19335. doi: 10.18632/oncotarget.2896. [PMC free article] [PubMed] [Cross Ref]
4. Zhang Y, Zhao FJ, Chen LL, Wang LQ, Nephew KP, Wu YL, Zhang S. MiR-373 targeting of the Rab22a oncogene suppresses tumor invasion and metastasis in ovarian cancer. Oncotarget. 2014;5:12291–12303. doi: 10.18632/oncotarget.2577. [PMC free article] [PubMed] [Cross Ref]
5. Garzon R, Calin GA, Croce CM. MicroRNAs in Cancer. Annu Rev Med. 2009;60:167–179. [PubMed]
6. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–233. [PMC free article] [PubMed]
7. Ruvkun G. Clarifications on miRNA and cancer. Science. 2006;311:36–37. [PubMed]
8. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. [PubMed]
9. Thomsen KG, Terp MG, Lund RR, Sokilde R, Elias D, Bak M, Litman T, Beck HC, Lyng MB, Ditzel HJ. miR-155, identified as anti-metastatic by global miRNA profiling of a metastasis model, inhibits cancer cell extravasation and colonization in vivo and causes significant signaling alterations. Oncotarget. 2015;6:29224–29239. doi: 10.18632/oncotarget.4942. [PMC free article] [PubMed] [Cross Ref]
10. Schetter AJ, Harris CC. Plasma microRNAs: a potential biomarker for colorectal cancer? Gut. 2009;58:1318–1319. [PubMed]
11. Paranjape T, Slack FJ, Weidhaas JB. MicroRNAs: tools for cancer diagnostics. Gut. 2009;58:1546–1554. [PMC free article] [PubMed]
12. Mattiske S, Suetani RJ, Neilsen PM, Callen DF. The oncogenic role of miR-155 in breast cancer. Cancer Epidemiol Biomarkers Prev. 2012;21:1236–1243. [PubMed]
13. Lee YS, Lim YS, Lee JC, Wang SG, Park HY, Kim SY, Lee BJ. Differential expression levels of plasma-derived miR-146b and miR-155 in papillary thyroid cancer. Oral oncology. 2015;51:77–83. [PubMed]
14. Liu R, Liao J, Yang M, Shi Y, Peng Y, Wang Y, Pan E, Guo W, Pu Y, Yin L. Circulating miR-155 expression in plasma: a potential biomarker for early diagnosis of esophageal cancer in humans. J Toxicol Environ Health A. 2012;75:1154–1162. [PubMed]
15. Wang J, Wu J. Role of miR-155 in breast cancer. Front Biosci (Landmark Ed) 2012;17:2350–2355. [PubMed]
16. Donnem T, Eklo K, Berg T, Sorbye SW, Lonvik K, Al-Saad S, Al-Shibli K, Andersen S, Stenvold H, Bremnes RM, Busund LT. Prognostic impact of MiR-155 in non-small cell lung cancer evaluated by in situ hybridization. Journal of translational medicine. 2011;9:6. [PMC free article] [PubMed]
17. Xie K, Ma H, Liang C, Wang C, Qin N, Shen W, Gu Y, Yan C, Zhang K, Dai N, Zhu M, Wu S, Wang H, Dai J, Jin G, Shen H, et al. A functional variant in miR-155 regulation region contributes to lung cancer risk and survival. Oncotarget. 2015;6:42781–42792. doi: 10.18632/oncotarget.5840. [PMC free article] [PubMed] [Cross Ref]
18. Yu DD, Lv MM, Chen WX, Zhong SL, Zhang XH, Chen L, Ma TF, Tang JH, Zhao JH. Role of miR-155 in drug resistance of breast cancer. Tumour Biol. 2015;36:1395–1401. [PubMed]
19. Chung IH, Chen CY, Lin YH, Chi HC, Huang YH, Tai PJ, Liao CJ, Tsai CY, Lin SL, Wu MH, Lin KH. Thyroid hormone-mediated regulation of lipocalin 2 through the Met/FAK pathway in liver cancer. Oncotarget. 2015;6:15050–15064. doi: 10.18632/oncotarget.3670. [PMC free article] [PubMed] [Cross Ref]
20. Fan C, Zheng W, Fu X, Li X, Wong YS, Chen T. Strategy to enhance the therapeutic effect of doxorubicin in human hepatocellular carcinoma by selenocystine, a synergistic agent that regulates the ROS-mediated signaling. Oncotarget. 2014;5:2853–2863. doi: 10.18632/oncotarget.1854. [PMC free article] [PubMed] [Cross Ref]
21. Kim SW, Yang HG, Kang MC, Lee S, Namkoong H, Lee SW, Sung YC. KIAA1114, a full-length protein encoded by the trophinin gene, is a novel surface marker for isolating tumor-initiating cells of multiple hepatocellular carcinoma subtypes. Oncotarget. 2014;5:1226–1240. doi: 10.18632/oncotarget.1677. [PMC free article] [PubMed] [Cross Ref]
22. Li C, Wang J, Zhang H, Zhu M, Chen F, Hu Y, Liu H, Zhu H. Interferon-stimulated gene 15 (ISG15) is a trigger for tumorigenesis and metastasis of hepatocellular carcinoma. Oncotarget. 2014;5:8429–8441. doi: 10.18632/oncotarget.2316. [PMC free article] [PubMed] [Cross Ref]
23. Ma L, Wang X, Jia T, Wei W, Chua MS, So S. Tankyrase inhibitors attenuate WNT/beta-catenin signaling and inhibit growth of hepatocellular carcinoma cells. Oncotarget. 2015;6:25390–25401. doi: 10.18632/oncotarget.4455. [PMC free article] [PubMed] [Cross Ref]
24. Wang F, Ying HQ, He BS, Pan YQ, Deng QW, Sun HL, Chen J, Liu X, Wang SK. Upregulated lncRNA-UCA1 contributes to progression of hepatocellular carcinoma through inhibition of miR-216b and activation of FGFR1/ERK signaling pathway. Oncotarget. 2015;6:7899–7917. doi: 10.18632/oncotarget.3219. [PMC free article] [PubMed] [Cross Ref]
25. Wang J, Yin D, Xie C, Zheng T, Liang Y, Hong X, Lu Z, Song X, Song R, Yang H, Sun B, Bhatta N, Meng X, Pan S, Jiang H, Liu L. The iron chelator Dp44mT inhibits hepatocellular carcinoma metastasis via N-Myc downstream-regulated gene 2 (NDRG2)/gp130/STAT3 pathway. Oncotarget. 2014;5:8478–8491. doi: 10.18632/oncotarget.2328. [PMC free article] [PubMed] [Cross Ref]
26. Zhou DS, Wang HB, Zhou ZG, Zhang YJ, Zhong Q, Xu L, Huang YH, Yeung SC, Chen MS, Zeng MS. TACC3 promotes stemness and is a potential therapeutic target in hepatocellular carcinoma. Oncotarget. 2015;6:24163–24177. doi: 10.18632/oncotarget.4643. [PMC free article] [PubMed] [Cross Ref]
27. Carvalheira G, Nozima BH, Cerutti JM. microRNA-106b-mediated down-regulation of C1orf24 expression induces apoptosis and suppresses invasion of thyroid cancer. Oncotarget. 2015;6:28357–28370. doi: 10.18632/oncotarget.4947. [PMC free article] [PubMed] [Cross Ref]
28. Cui R, Kim T, Fassan M, Meng W, Sun HL, Jeon YJ, Vicentini C, Tili E, Peng Y, Scarpa A, Liang G, Zhang YK, Chakravarti A, Croce CM. MicroRNA-224 is implicated in lung cancer pathogenesis through targeting caspase-3 and caspase-7. Oncotarget. 2015;6:21802–21815. doi: 10.18632/oncotarget.5224. [PMC free article] [PubMed] [Cross Ref]
29. Czubak K, Lewandowska MA, Klonowska K, Roszkowski K, Kowalewski J, Figlerowicz M, Kozlowski P. High copy number variation of cancer-related microRNA genes and frequent amplification of DICER1 and DROSHA in lung cancer. Oncotarget. 2015;6:23399–23416. doi: 10.18632/oncotarget.4351. [PMC free article] [PubMed] [Cross Ref]
30. Drayton RM, Peter S, Myers K, Miah S, Dudziec E, Bryant HE, Catto JW. MicroRNA-99a and 100 mediated upregulation of FOXA1 in bladder cancer. Oncotarget. 2014;5:6375–6386. doi: 10.18632/oncotarget.2221. [PMC free article] [PubMed] [Cross Ref]
31. Fuziwara CS, Kimura ET. MicroRNA Deregulation in Anaplastic Thyroid Cancer Biology. International journal of endocrinology. 2014;2014:743450. [PMC free article] [PubMed]
32. Tili E, Croce CM, Michaille JJ. miR-155: on the crosstalk between inflammation and cancer. International reviews of immunology. 2009;28:264–284. [PubMed]
33. Ward LD, Kellis M. HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease. Nucleic Acids Res. 2016;44:D877–881. [PMC free article] [PubMed]
34. Ward LD, Kellis M. HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants. Nucleic Acids Res. 2012;40:D930–934. [PMC free article] [PubMed]
35. Ji J, Xu M, Zhao Z, Tu J, Gao J, Lu C, Song J, Chen W, Chen M, Fan X, Cheng X, Lan X, Li J. SMAD7 loci contribute to risk of hepatocellular carcinoma and clinicopathologic development among Chinese Han population. Oncotarget. 2016;7:22186–22192. doi: 10.18632/oncotarget.8065. [PMC free article] [PubMed] [Cross Ref]

Articles from Oncotarget are provided here courtesy of Impact Journals, LLC