Integrated analysis of hypoxia-induced miR-210 signature as a potential prognostic biomarker of hepatocellular carcinoma: a study based on The Cancer Genome Atlas#

Integrated analysis of hypoxia-induced miR-210 signature as a potential prognostic biomarker of hepatocellular carcinoma: a study based on The Cancer Genome Atlas # # 一项基于肿瘤基因数据库关于综合分析缺氧导致的肝细胞癌的蛋白预测标志物miR-210变化的研究 1 first-author 2 2 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:3411053@zju.edu.cn †E-mail:3411053@zju.edu.cn 3411053@zju.edu.cn Zhejiang Provincial Key Laboratory of Laparoscopic Technology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China Zhejiang Provincial Key Laboratory of Laparoscopic Technology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China 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 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 2018 ). 目的 2 研究肝癌细胞缺氧微环境导致miR-210表达变化与肿瘤进展、预后等相关性. 创新点 2 首次阐明了miR-210表达与肝癌预后及缺氧相关基因的关系. 方法 2 选取肿瘤基因数据库(TCGA)中424位肝癌患者的miR-210表达水平、临床病理参数及缺氧相关基因( HIF1α 、 HIF3α 、 PTPN1 和 BNIP3 )表达量,研究miR-210与肝癌预后及缺氧基因之间的相关性. 结论 2 miR-210的表达与肝细胞癌进展分期呈正相关,它的高表达预示更低的无瘤生存率.因此,推测miR-210可能与肿瘤细胞缺氧相关性死亡有关. miR-210 The Cancer Genome Atlas (TCGA) Hepatocellular carcinoma (HCC) Hypoxia prognostic biomarker miR-210 肿瘤基因数据库(TCGA) 肝细胞癌(HCC) 缺氧 预后意义 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 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 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 2003 ; Semela and Dufour, 2004 2004 ; Airley and Mobasheri, 2007 2007 ; Wang et al., 2017 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 2010 ; Brooks et al., 2016 2016 ). Various hypoxia-regulated microRNAs (miRNAs), also termed hypoxamiRs, have been recently identified (Bavelloni et al., 2017 2017 ; Wan et al., 2017 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 2010 ; Ivan and Huang, 2014 2014 ). Despite abundant in vitro evidence on the relationship between miR-210 and hypoxia in HCC (Kai et al., 2016 2016 ; Yang et al., 2016 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 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 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 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 1b and 1d ). However, miR-210 expression was not associated with metastasis (Fig. 1c 1c ), gender, age at diagnosis, year of diagnosis, or fibrosis score (data not shown). (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 2a , Table S2 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 S2 , Fig. 2a 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 S2 , Fig. 2b 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 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 S2 ). (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 3a ) and a negative correlation between miR-210 and HIF3α expression ( R =−0.0102, P =0.0291; Fig. 3b 3b ) were observed. However, the associations between miR-210 and PTPN1 ( P =0.3909; Fig. 3c 3c ), and between miR-210 and BNIP3 ( P =0.7483; Fig. 3d 3d ) expression were not statistically significant. (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 2016 ; Wang and Zheng, 2018 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 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 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 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 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. References RE Airley , A Mobasheri . Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics . Chemotherapy , 2007 . 53 ( 4 ): 233 - 256 . 17595539 DOI: 10.1159/000104457 http://doi.org/10.1159/000104457 . Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics A Bavelloni , G Ramazzotti , A Poli , 等 . MiRNA-210: a current overview . Anticancer Res , 2017 . 37 ( 12 ): 6511 - 6521 . 29187425 . MiRNA-210: a current overview DL Brooks , LP Schwab , R Krutilina , 等 . 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 http://doi.org/10.1186/s12943-016-0510-x . ITGA6 is directly regulated by hypoxia-inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer models JM Heddleston , Z Li , JD Lathia , 等 . Hypoxia inducible factors in cancer stem cells . Br J Cancer , 2010 . 102 ( 5 ): 789 - 795 . 20104230 DOI: 10.1038/sj.bjc.6605551 http://doi.org/10.1038/sj.bjc.6605551 . Hypoxia inducible factors in cancer stem cells X Huang , QT Le , AJ Giaccia . MiR-210–micromanager of the hypoxia pathway . Trends Mol Med , 2010 . 16 ( 5 ): 230 - 237 . 20434954 DOI: 10.1016/j.molmed.2010.03.004 http://doi.org/10.1016/j.molmed.2010.03.004 . MiR-210–micromanager of the hypoxia pathway M Ivan , X Huang . Hammond E Koumenis C , Giaccia A . MiR-210: fine-tuning the hypoxic response . Tumor Microenvironment and Cellular Stress: Signaling, Metabolism, Imaging, and Therapeutic Targets , 2014 . Springer, New York , 205 - 227 . DOI: 10.1007/978-1-4614-5915-6_10 http://doi.org/10.1007/978-1-4614-5915-6_10 . MiR-210: fine-tuning the hypoxic response A Jemal , F Bray , MM Center , 等 . Global cancer statistics . CA Cancer J Clin , 2011 . 61 ( 2 ): 69 - 90 . 21296855 DOI: 10.3322/caac.20107 http://doi.org/10.3322/caac.20107 . Global cancer statistics AKL Kai , LK Chan , RCL Lo , 等 . 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 . 27018975 DOI: 10.1002/hep.28577 http://doi.org/10.1002/hep.28577 . Down-regulation of TIMP2 by HIF-1α/miR-210/HIF-3α regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma LL Liu , DH Li , YL He , 等 . miR-210 protects renal cell against hypoxia-induced apoptosis by targeting HIF-1 alpha . Mol Med , 2017 . 23 258 - 271 . 29387863 DOI: 10.2119/molmed.2017.00013 http://doi.org/10.2119/molmed.2017.00013 . miR-210 protects renal cell against hypoxia-induced apoptosis by targeting HIF-1 alpha CW Pugh , PJ Ratcliffe . Regulation of angiogenesis by hypoxia: role of the HIF system . Nat Med , 2003 . 9 ( 6 ): 677 - 684 . 12778166 DOI: 10.1038/nm0603-677 http://doi.org/10.1038/nm0603-677 . Regulation of angiogenesis by hypoxia: role of the HIF system D Semela , JF Dufour . Angiogenesis and hepatocellular carcinoma . J Hepatol , 2004 . 41 ( 5 ): 864 - 880 . 15519663 DOI: 10.1016/j.jhep.2004.09.006 http://doi.org/10.1016/j.jhep.2004.09.006 . Angiogenesis and hepatocellular carcinoma RL Siegel , KD Miller , A Jemal . Cancer statistics, 2018 . CA Cancer J Clin , 2018 . 68 ( 1 ): 7 - 30 . 29313949 DOI: 10.3322/caac.21442 http://doi.org/10.3322/caac.21442 . Cancer statistics, 2018 R Silakit , Y Kitirat , S Thongchot , 等 . Potential role of HIF-1-responsive microRNA210/HIF3 axis on gemcitabine resistance in cholangiocarcinoma cells . PLoS ONE , 2018 . 13 ( 6 ): e0199827 29953500 DOI: 10.1371/journal.pone.0199827 http://doi.org/10.1371/journal.pone.0199827 . Potential role of HIF-1-responsive microRNA210/HIF3 axis on gemcitabine resistance in cholangiocarcinoma cells LL Wan , DQ Zhang , JN Zhang , 等 . 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 http://doi.org/10.1631/jzus.B1600369 . Anti-hepatocarcinoma activity of TT-1, an analog of melittin, combined with interferon-α via promoting the interaction of NKG2D and MICA LY Wang , SS Zheng . 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 http://doi.org/10.1631/jzus.B1700156 . Advances in predicting the prognosis of hepatocellular carcinoma recipients after liver transplantation ZD Wang , FY Qu , YY Chen , 等 . 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 http://doi.org/10.1631/jzus.B1600205 . Involvement of microRNA-718, a new regulator of EGR3, in regulation of malignant phenotype of HCC cells Y Yang , J Zhang , T Xia , 等 . MicroRNA-210 promotes cancer angiogenesis by targeting fibroblast growth factor receptor-like 1 in hepatocellular carcinoma . Oncol Rep , 2016 . 36 ( 5 ): 2553 - 2562 . 27666683 DOI: 10.3892/or.2016.5129 http://doi.org/10.3892/or.2016.5129 . MicroRNA-210 promotes cancer angiogenesis by targeting fibroblast growth factor receptor-like 1 in hepatocellular carcinoma Q Ying , LH Liang , WJ Guo , 等 . 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 . 22144109 DOI: 10.1002/hep.24614 http://doi.org/10.1002/hep.24614 . Hypoxia-inducible microRNA-210 augments the metastatic potential of tumor cells by targeting vacuole membrane protein 1 in hepatocellular carcinoma 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. 10.1631/jzus.B1900343 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 2019-07-03 2019-09-12 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 20