Abstract
Hepatocellular carcinoma (HCC) is one of the most common types of liver cancer and is the second leading cause of cancer mortality with an estimated 745 500 deaths annually (Jemal et al., 2011). Although new therapeutic modalities including novel chemotherapeutic interventions and targeted therapy have been applied, the prognosis of HCC patients remains unsatisfactory due to the high incidence of intrahepatic and distal metastases (Siegel et al., 2018).
Keywords: miR-210, The Cancer Genome Atlas (TCGA), Hepatocellular carcinoma (HCC), Hypoxia, prognostic biomarker
Hepatocellular carcinoma (HCC) is one of the most common types of liver cancer and is the second leading cause of cancer mortality with an estimated 745 500 deaths annually (Jemal et al., 2011). Although new therapeutic modalities including novel chemotherapeutic interventions and targeted therapy have been applied, the prognosis of HCC patients remains unsatisfactory due to the high incidence of intrahepatic and distal metastases (Siegel et al., 2018).
Hypoxia or inadequate oxygen supply—a fundamental characteristic of the solid tumor microenvironment—activates adaptive transcriptional programs that promote cell survival, motility, cancer angiogenesis, invasion, metastasis, and dedifferentiation, and enhances glycolytic metabolism (Pugh and Ratcliffe, 2003; Semela and Dufour, 2004; Airley and Mobasheri, 2007; Wang et al., 2017). In HCC, hypoxia induces cell survival by activating growth factor signaling. However, the molecular mechanisms involved in the induction of HCC cell reprogramming, especially under hypoxic conditions, remain largely unknown (Heddleston et al., 2010; Brooks et al., 2016).
Various hypoxia-regulated microRNAs (miRNAs), also termed hypoxamiRs, have been recently identified (Bavelloni et al., 2017; Wan et al., 2017). The master hypoxamiR miR-210 is regulated by hypoxia-inducible factor 1α (HIF1α) in various tissues. Moreover, miR-210 has been reported to regulate responses and tolerate hypoxia-induced stress (Huang et al., 2010; Ivan and Huang, 2014). Despite abundant in vitro evidence on the relationship between miR-210 and hypoxia in HCC (Kai et al., 2016; Yang et al., 2016), in vivo evidence is lacking.
In the present study, we attempted to analyze the correlation between the expression of miR-210 and that of various hypoxia-related genes (HIF1Al, HIF3A, PTPN1, and BNIP3) as well as disease prognosis in patients with HCC by extracting a large amount of cases from The Cancer Genome Atlas (TCGA) database. All analyzed miRNA and mRNA data were extracted from TCGA database (https://portal.gdc. cancer.gov). Data including patients’ demographic information, primary tumor site, tumor morphology, tumor-node-metastasis (TNM) stage, first course of treatment, and follow-up for prognostic outcome were collected for the HCC tissue and the adjacent non-tumorous tissue samples. The data were processed according to the requirements of the data access policies and the National Institutes of Health (NIH) TCGA human subject protection.
All patients were pathologically diagnosed with HCC from 1995 to 2013 using the morphological code C22.0. Patients, who received pre-or post-operative treatments, died within 30 d post-surgery, or with unknown survival time, were excluded. As shown inTable S1, 424 cases conforming to the inclusion criteria were chosen for the present study. The surgical treatment for HCC in the study included segmental resection, lobectomy, and extended lobectomy.
The results were statistically analyzed using GraphPad Prism, Version 6 (GraphPad Software Inc., CA, USA). The inter-group comparison of the miR-210 expression level was performed using Student’s t-test. Survival analysis was computed using the Kaplan-Meier method and significance was assessed using the log-rank test. Multivariate analysis with the Cox regression model was used to examine the combined effects. Additionally, the linear regression model was used for discerning the relationship between miR-210 expression and the hypoxia-related genes (HIF1α, HIF3α, PTPN1, and BNIP3). For all statistical analyses, P-value of <0.05 was considered statistically significant.
During the 20-year study period, a total of 424 patients with HCC, including 141 males and 283 females, were identified. Of these, 169 were less than 60 years old, and 255 aged more than or equal to 60 years. The clinicopathological parameters of the patients are shown in Table S1. To explore the potential role of miR-210 as a hypoxamiR, miR-210 expression in the 424 patients with HCC was characterized. Intriguingly, miR-210 expression was robustly upregulated in the advanced T stages compared to that in the early T stages (T1 vs. T3, P=0.002; T1 vs. T4, P=0.045; T2 vs. T3, P=0.043; Fig. 1a). miR-210 expression also correlated with the pathological grade (G1 vs. G3, P=0.012) and the TNM stage (stage I vs. stages III+IV, P=0.001; stage II vs. stages III+IV, P=0.023; Figs. 1b and 1d). However, miR-210 expression was not associated with metastasis (Fig. 1c), gender, age at diagnosis, year of diagnosis, or fibrosis score (data not shown).
Fig. 1.
miR-210 expression in advanced hepatocellular carcinoma (HCC)
(a) miR-210 expression in the advanced T stages was higher than that in the early T stages. (b) miR-210 expression in G3 (poorly differentiated) was higher than that in G1 (well differentiated). (c) The difference in miR-210 expression between the non-metastatic HCC and metastatic HCC tissues was not statistically significant. (d) miR-210 expression in the advanced tumor-node-metastasis (TNM) stage was higher than that in the early TNM stage. * P<0.05, ** P<0.01, **** P<0.0001
Next, we investigated the correlation between miR-210 expression and disease prognosis in patients with HCC. Disease-free survival (DFS) was calculated along with overall survival (OS). Patients with upregulated miR-210 expression had shorter median DFS than those with low miR-210 expression (Fig. 2a, Table S2). The median DFS time in the patients with upregulated miR-210 expression was 1560.6 d and in those with low miR-210 expression was 1805.5 d (P=0.036; Table S2, Fig. 2a). However, the difference in the median OS time between the patients with low and high miR-210 expression was not significant (P=0.449; Table S2, Fig. 2b). Furthermore, previously reported clinicopathological characteristics, such as age at diagnosis (P=0.003), pathological grade (P=3.218×10−45), fibrosis score (P=0.002), T stage (P=0.001), M stage (P=0.042), and TNM stage (P=1.274×10−21), were associated with HCC prognosis (Table S2). However, in the multivariate Cox regression model, the difference in the median DFS time between the patients with low and high miR-210 expression was not significant (P=0.957; Table S2).
Fig. 2.
Correlation between miR-210 expression and the survival of patients with hepatocellular carcinoma (HCC)
(a) Kaplan-Meier curves of disease-free survival (DFS) in patients with HCC according to miR-210 expression. (b) Kaplan-Meier curves of overall survival (OS) in patients with HCC according to miR-210 expression
To further investigate the role of miR-210, the correlation between the expression of miR-210 and the hypoxia-related genes (HIF1α, HIF3α, PTPN1, and BNIP3) was investigated. A positive correlation between miR-210 and HIF1α expression (R=0.078, P=0.0068; Fig. 3a) and a negative correlation between miR-210 and HIF3α expression (R=−0.0102, P=0.0291; Fig. 3b) were observed. However, the associations between miR-210 and PTPN1 (P=0.3909; Fig. 3c), and between miR-210 and BNIP3 (P=0.7483; Fig. 3d) expression were not statistically significant.
Fig. 3.
Linear regression analysis demonstrating the correlation between the expression of miR-210 and the hypoxia-related genes
(a) miR-210 expression positively correlated with HIF1α expression. (b) miR-210 expression inversely correlated with HIF3α expression. (c, d) The associations between miR-210 and PTPN1 expression, and between miR-210 and BNIP3 expression were not significant
Similar to previous studies (Yang et al., 2016; Wang and Zheng, 2018), miR-210 along with other clinicopathological factors, including age, pathological grade, T, M, and TNM stages, was considered prognostic factors for DFS in this study. However, in the multivariate Cox regression model, miR-210 expression did not have a significant impact on DFS. Furthermore, univariate analysis demonstrated that the clinicopathological factors, but not miR-210 expression, affected OS. Based on this result, the multivariate Cox regression model was not applied for further analysis. Thus miR-210 expression is not an independent prognostic factor for DFS or OS.
In vitro evidence showed that miR-210 mediates hypoxia-induced HCC cell metastasis by promoting the migration and invasion of HCC cells (Ying et al., 2011). Several molecular mechanisms are involved in the induction of HCC metastasis, chemotherapeutic resistance, and radiotherapeutic resistance by miR-210 via the hypoxia pathway. miR-210 attenuates hypoxia-induced cell apoptosis by directly targeting HIF1α and inhibiting the HIF1α pathway in the hypoxic kidney lesions (Liu et al., 2017). It has also been reported that HIF1α suppression is regulated by a feedback loop consisting of HIF1α/miR-210/HIF3α (Kai et al., 2016). However, recent evidences have shown that HIF3α suppression by HIF1α-induced miR-210 downregulates HIF1α expression and constitutes a feed-forward hypoxic regulatory loop (Silakit et al., 2018). In the present study, a positive correlation between the expression of miR-210 and HIF1α, and a negative correlation between the expression of miR-210 and HIF3α were observed via linear regression analysis. Thus, the present study provides further evidence on the feed-forward hypoxic regulatory loop. The present evaluation of the correlation between the expression of miR-210 and BNIP3 and that between miR-210 and PTPN1 demonstrated no statistical significance. The findings of this study are contrary to those of previous studies. A possible reason may be the different tumor types included in this study. Further verification through in vitro studies in HCC cell lines is warranted.
In conclusion, upregulated miR-210 expression is significantly associated with the advanced TNM stage of HCC and poor DFS of HCC patients. However, miR-210 is not an independent prognostic factor for HCC patients. Moreover, miR-210 expression positively correlated with HIF1α expression and inversely correlated with HIF3α expression.
List of electronic supplementary materials
Clinicopathological parameters of the patients
Univariate and multivariate analyses of survival in 424 patients with HCC according to clinicopathologic factors and miR-210 expression
Footnotes
Project supported by the Education Department of Zhejiang Province (No. G201432123) and the Public Welfare Technology Application Research Plan Project of Zhejiang Science and Technology Department (No. LGC19H200005), China
Contributors: Yi DAI performed data analysis, and wrote and edited the manuscript. Ji-liang SHEN, Xue-yong ZHENG, and Tian-yu LIN collected the data. Hai-tao YU contributed to the study design, data analysis, and editing of the manuscript. All authors have read and approved the final manuscript and had full access to all the data in the study. All authors take responsibility for the integrity and security of the data.
Electronic supplementary materials: The online version of this article (https://doi.org/10.1631/jzus.B1900343) contains supplementary materials, which are available to authorized users
Compliance with ethics guidelines: Yi DAI, Ji-liang SHEN, Xue-yong ZHENG, Tian-yu LIN, and Hai-tao YU declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
References
- 1.Airley RE, Mobasheri A. Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics. Chemotherapy. 2007;53(4):233–256. doi: 10.1159/000104457. [DOI] [PubMed] [Google Scholar]
- 2.Bavelloni A, Ramazzotti G, Poli A, et al. MiRNA-210: a current overview. Anticancer Res. 2017;37(12):6511–6521. doi: 10.21873/anticanres.12107. [DOI] [PubMed] [Google Scholar]
- 3.Brooks DL, Schwab LP, Krutilina R, et al. ITGA6 is directly regulated by hypoxia-inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer models. Mol Cancer, 15:26. 2016 doi: 10.1186/s12943-016-0510-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Heddleston JM, Li Z, Lathia JD, et al. Hypoxia inducible factors in cancer stem cells. Br J Cancer. 2010;102(5):789–795. doi: 10.1038/sj.bjc.6605551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Huang X, Le QT, Giaccia AJ. MiR-210–micromanager of the hypoxia pathway. Trends Mol Med. 2010;16(5):230–237. doi: 10.1016/j.molmed.2010.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Ivan M, Huang X. MiR-210: fine-tuning the hypoxic response. In: Koumenis C Hammond E, Giaccia A , editors. Tumor Microenvironment and Cellular Stress: Signaling, Metabolism, Imaging, and Therapeutic Targets. Springer, New York; 2014. pp. 205–227. [DOI] [PubMed] [Google Scholar]
- 7.Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. doi: 10.3322/caac.20107. [DOI] [PubMed] [Google Scholar]
- 8.Kai AKL, Chan LK, Lo RCL, et al. Down-regulation of TIMP2 by HIF-1α/miR-210/HIF-3α regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma. Hepatology. 2016;64(2):473–487. doi: 10.1002/hep.28577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Liu LL, Li DH, He YL, et al. miR-210 protects renal cell against hypoxia-induced apoptosis by targeting HIF-1 alpha. Mol Med. 2017;23:258–271. doi: 10.2119/molmed.2017.00013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003;9(6):677–684. doi: 10.1038/nm0603-677. [DOI] [PubMed] [Google Scholar]
- 11.Semela D, Dufour JF. Angiogenesis and hepatocellular carcinoma. J Hepatol. 2004;41(5):864–880. doi: 10.1016/j.jhep.2004.09.006. [DOI] [PubMed] [Google Scholar]
- 12.Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7–30. doi: 10.3322/caac.21442. [DOI] [PubMed] [Google Scholar]
- 13.Silakit R, Kitirat Y, Thongchot S, et al. Potential role of HIF-1-responsive microRNA210/HIF3 axis on gemcitabine resistance in cholangiocarcinoma cells. PLoS ONE. 2018;13(6):e0199827. doi: 10.1371/journal.pone.0199827. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Wan LL, Zhang DQ, Zhang JN, et al. Anti-hepatocarcinoma activity of TT-1, an analog of melittin, combined with interferon-α via promoting the interaction of NKG2D and MICA. J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2017;18(6):522–531. doi: 10.1631/jzus.B1600369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Wang LY, Zheng SS. Advances in predicting the prognosis of hepatocellular carcinoma recipients after liver transplantation. J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2018;19(7):497–504. doi: 10.1631/jzus.B1700156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Wang ZD, Qu FY, Chen YY, et al. Involvement of microRNA-718, a new regulator of EGR3, in regulation of malignant phenotype of HCC cells. J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2017;18(1):27–36. doi: 10.1631/jzus.B1600205. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Yang Y, Zhang J, Xia T, et al. MicroRNA-210 promotes cancer angiogenesis by targeting fibroblast growth factor receptor-like 1 in hepatocellular carcinoma. Oncol Rep. 2016;36(5):2553–2562. doi: 10.3892/or.2016.5129. [DOI] [PubMed] [Google Scholar]
- 18.Ying Q, Liang LH, Guo WJ, et al. Hypoxia-inducible microRNA-210 augments the metastatic potential of tumor cells by targeting vacuole membrane protein 1 in hepatocellular carcinoma. Hepatology. 2011;54(6):2064–2075. doi: 10.1002/hep.24614. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Clinicopathological parameters of the patients
Univariate and multivariate analyses of survival in 424 patients with HCC according to clinicopathologic factors and miR-210 expression