MicroRNA-34a promotes MICB expression in hepatocytes

Carcinogenesis, 2018, Vol. 39, No. 12, 1477–1487 doi:10.1093/carcin/bgy128 Advance Access publication September 25, 2018 Original Article Original Article MicroRNA-34a promotes MICB expression in hepatocytes Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325015, China, 2Department of Gastroenterology, The 98th Hospital of PLA, Huzhou, Zhejiang 313000, China, 3Digestive Cancer Center, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325015, China, 4Institute of Genomic Medicine, Wenzhou Medical University, Wenzhou, Zhejiang 325015, China, 5Department of Gynaecology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325015, China, 6Department of Hepatobiliary Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400000, China, 7Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325015, China and 8Department of Dermato-Venereology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325015, China 1 *To whom correspondence should be addressed. Tel: +86 0577 88831271; Fax: +86 0577 88831359; Email: or † These authors contributed equally to this work. Abstract MicroRNA-34a (miR-34a) behaves as a tumor suppressor by decreasing the expression of oncogenes involved in multiple carcinogenic pathways. Intravenous delivery of miR-34a mimics has been investigated in clinical trials as a potential treatment for advanced cancers; however, the effect of miR-34a on cancer immune surveillance is controversial. In the current study, we found that miR-34a plays a dual role in the regulation of major histocompatibility complex class I-related sequence B (MICB) protein, a ligand of the NKG2D receptor. MiR-34a could both induce and reduce MICB expression by upregulating ataxia telangiectasia and Rad3-related (ATR) protein kinase and downregulating the transcription factor E2F1, respectively. The net effect of miR-34a on MICB expression depended on endogenous E2F1 levels. Overexpression of miR34a promoted MICB expression in hepatocytes and hepatocellular carcinoma (HCC) cells that have low E2F1 levels but not in HCC cells that have high E2F1 levels. In HCC patients, the expression of miR-34a and MICB showed positive correlation in paratumor liver tissues, which have low E2F1 levels, but not in HCC tissues, which have high E2F1 levels. We showed that miR-34a overexpression in non-transformed liver cells enhanced cytolysis and interferon-γ production by NK-92MI cells. Furthermore, higher miR-34a expression in tumor and paratumor tissues was associated with positive and negative outcomes, respectively, in HCC patients. Our findings suggest that miR-34a induces MICB expression in paratumor liver tissues, which may cause liver damage and serious cytokine release syndrome, thus disclosing potential side effects of systemic administration of miR-34a in anticancer therapy. Introduction MicroRNAs (miRNAs) are small non-coding RNAs with a length of 21–23 nucleotides that repress the expression of target mRNAs by binding to their 3′-untranslated regions (1), and it is known that deregulation of miRNA expression plays a pivotal role in Received: February 16, 2018; Revised: September 5, 2018; Accepted: September 21, 2018 © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: . 1477 Meng-Tao Zhou1,†, Chunming Zhao2,†, Xiao Chen1,3,†, Heng-Chao Zhang1, Guiling Li4, Hongyan Lou5, Wen-Jie Huang3, Lin-Jie Wei3, De-Wei Li6, Xiaoli Wu7, Zhe-Chao Zhang5, Hui Liu1, Rongying Ou5, Wen-Jun Yang1, Shanshan Hu1, Yunsheng Xu8 and Kai-Fu Tang1,3,*, 1478 | Carcinogenesis, 2018, Vol. 39, No. 12 Abbreviations cancer origination and progression (2). MicroRNA-34a (MiR-34a) is frequently lost or downregulated in tumor tissues and is recognized as a tumor suppressor targeting multiple oncogenes related to tumor cell proliferation, apoptosis and invasion in many types of cancer (3). Therefore, intravenous delivery of a liposomal miR-34a mimic has been investigated in clinical trials as a potential treatment for advanced cancers (4,5). However, these clinical trials were terminated because of immune-related adverse effects. In addition to inhibiting the expression of oncogenes, miR-34a promotes an antitumor immune response by regulating several immune molecules (5–9); it enhances T cell activation by targeting diacylglycerol kinase ζ and sensitizes programmed death-ligand 1 (PD-L1)-overexpressing tumor cells to T cell killing by targeting PD-L1 (5,6,9). Furthermore, miR-34a suppresses the recruitment of Fox-P3-expressing regulatory T (Treg) cells to the tumor microenvironment by targeting CCL22 (7). However, it has been reported that miR-34a may reduce the antitumor immune response by targeting the NKG2D ligand ULBP2 (8). NKG2D is a transmembrane protein expressed by natural killer (NK) cells, cytotoxic T lymphocytes and γδ T cells (10–12). In NK cells, the binding of NKG2D to its ligands triggers cytolysis and cytokine production, whereas in CD8+ T cells, it provides costimulatory signals (10–12). In humans, there are two families of NKG2D ligands: major histocompatibility complex class I-related molecules A and B (MICA and MICB, respectively) and human cytomegalovirus glycoprotein UL16-binding proteins (ULBP1– ULBP6) (13). NKG2D ligands are absent or expressed at low levels on the surface of normal cells but are induced by viral infection, oncogenic transformation and other cellular stresses (14). In particular, the expression of NKG2D ligands is increased in the early stage of tumorigenesis (15). Tumor cells ectopically expressing NKG2D ligands were rejected by NK cells and/or CD8+ T cells after grafting into syngeneic mice (16–18), whereas NKG2D knockout in mice led to increased incidence of spontaneous tumors (19). These findings indicate that NKG2D engagement provides an innate barrier against cancer development and progression. The expression of NKG2D ligands during tumorigenesis is regulated by several signaling pathways (14). The DNA damage response (DDR) is frequently activated in human tumors of different stages and precancerous lesions (20–26). Gasser et al. (27) demonstrated that DNA-damaging agents induced the transcription of NKG2D ligands by activating the ATM/ ATR-Chk1/2 DDR pathway. MiR-34a is a transcriptional target of Materials and methods Cell culture Liver cancer cell lines HepG2, Huh-7 and Hep3B and the NK cell line NK-92MI were originally purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cell lines L02 established from fetal liver tissues, QSG-7701 established from paratumor tissues of a female HCC patient and SMCC-7721 established from tumor tissues of a male HCC patient were obtained from the Cell Bank of th (...truncated)