Cui et al. Journal of Experimental & Clinical Cancer Research (2020) 39:90 https://doi.org/10.1186/s13046-020-01591-1 RESEARCH Open Access ABCA8 is regulated by miR-374b-5p and inhibits proliferation and metastasis of hepatocellular carcinoma through the ERK/ ZEB1 pathway Yifeng Cui1,2†, Shuhang Liang1,2†, Shugeng Zhang1,2†, Congyi Zhang1,2†, Yunzheng Zhao1,2, Dehai Wu1,2, Jiabei Wang3, Ruipeng Song3, Jizhou Wang3, Dalong Yin3, Yao Liu3, Shangha Pan2, Xirui Liu1,2, Yan Wang4, Jihua Han1,2, Fanzheng Meng1,2, Bo Zhang1,2, Hongrui Guo1,2, Zhaoyang Lu1,2* and Lianxin Liu1,2,3* Abstract Background: ATP binding cassette subfamily A member 8 (ABCA8) belongs to the ATP binding cassette (ABC) transporter superfamily. ABCA8 is a transmembrane transporter responsible for the transport of organics, such as cholesterol, and drug efflux. Some members of the ABC subfamily, such as ABCA1, may inhibit cancer development. However, the mechanism of ABCA8 in the process of cancer activation is still ambiguous. Methods: The expression of ABCA8 in human hepatocellular carcinoma (HCC) tissues and cell lines was examined using qPCR, immunoblotting, and immunohistochemical staining. The effects of ABCA8 on the proliferation and metastasis of HCC were examined using in vitro and in vivo functional tests. A luciferase reporter assay was performed to explore the binding between microRNA-374b-5p (miR-374b-5p) and the ABCA8 3′-untranslated region (UTR). Results: ABCA8 was frequently down-regulated in HCC and this down-regulation was negatively correlated with prognosis. The overexpression of ABCA8 inhibited growth and metastasis in HCC, whereas the knockdown of ABCA8 exerted the antithetical effects both in vivo and in vitro. ABCA8 was down-regulated by miR-374b-5p; this down-regulation can induce epithelial transformation to mesenchyme via the ERK/ZEB1 signaling pathway and promote HCC progression. Conclusion: We exposed the prognostic value of ABCA8 in HCC, and illuminated a novel pathway in ABCA8- regulated inhibition of HCC tumorigenesis and metastasis. These findings may lead to a new targeted therapy for HCC through the regulation of ABCA8, and miR-374b-5p. Keywords: Hepatocellular carcinoma, ATP binding cassette subfamily a member 8, Epithelial to mesenchymal transition, Therapeutic target * Correspondence: [email protected]; [email protected] †Yifeng Cui, Shuhang Liang, Shugeng Zhang and Congyi Zhang contributed equally to this work. 1Department of Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Journal of Experimental & Clinical Cancer Research (2020) 39:90 Page 2 of 14 Background Table 1 Relationship between ABCA8 expression and Hepatocellular carcinoma (HCC) accounts for the ma- clinicopathologic features of HCC patients (n = 105) jority of primary liver cancer, ranks as the sixth most Features ABCA8 expression P Value common cancer, and was the fourth leading cause of Low(n = 47) High (n = 58) cancer death worldwide in 2018 [1]. The main patho- Age 0.8078 genic factors of HCC are viral hepatitis infection (HBV/ ≤ 60 24 31 HCV), ingestion of aflatoxin, diabetes, tobacco intake, >60 23 27 and heavy alcohol intake [2–4]. Surgical treatment is an effective treatment for liver cancer, but recurrence and Gender 0.9969 metastasis may still occur, limiting the overall survival of Male 30 37 HCC patients [5]. Therefore, further understanding of Female 17 21 the molecular mechanisms related to HCC can help us AFP (μg/L) 0.2034 determine effective therapies to combat the recurrence ≤ 20 11 8 and metastasis of HCC. >20 36 50 ATP binding cassette subfamily A member 8 (ABCA8) is a member of the ABC transporter superfamily, of HBV infection 0.6307 which, human beings have 48 transcriptionally active Yes 27 36 ABC transporter genes divided into 7 subfamilies, A-G No 20 22 [6]. ABCA1 and ABCA8 are homologous and belong to Tumor diameter (cm) 0.0121 the same subfamily. ABCA1 is believed to inhibit the ≤ 52140 proliferation and metastasis of many cancers [7–9]. > 5 26 18 However, the role of ABCA8 in tumorigenesis and the mechanism by which ABCA8 acts remain unclear, par- metastasis 0.0103 ticularly in HCC. Yes 28 20 In this study, we used clinical data and molecular bio- No 19 38 logical experiments to clarify the mechanism by which TNM stage 0.0106 ABCA8 impacts HCC. The results showed that ABCA8 I-II 12 29 was frequently down-regulated in HCC and that de- III-IV 35 29 creased ABCA8 was associated with poor prognosis, tumorigenesis, and metastasis. ABCA8 also proved to be down-regulated by miR-374b-5p, which in turn was up- lines were cultured in Dulbecco’s Modified Eagle regulated in HCC and resulted in the progression of Medium (Gibco, USA) supplemented with 10% fetal bo- HCC via the ABCA8/ERK/ZEB1 signal pathway. vine serum (Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were incubated in in- Methods cubators containing 5% CO2 at 37 °C. HCC specimens We collected matched HCC and adjacent non-tumor tis- Lentivirus sue from patients who underwent hepatectomy in the Lentiviral vectors for ABCA8 and miR-374b-5p gene up- First Affiliated Hospital of Harbin Medical University regulation (Lv-ABCA8, Lv-miR-374b-5p), down-regulation between August 2010 and September 2014. We invited (Lv-shABCA8, Lv-anti-miR-374b-5p), empty vectors (Lv- senior pathologists with senior professional titles to per- NC) and encoding human firefly luciferase were manufac- form pathological diagnosis on paraffin sections, and tured and obtained from GeneChem (Shanghai, China). only patients with pathological results of HCC were in- Details of the short hairpin RNA sequence against ABCA8 cluded in this study. All patients who participated in this are listed in Additional file 1:TableS1. study provided informed consent. This study was ap- proved by the Research Ethics Committee. Detailed clin- icopathological features of 105 HCC specimens involved Immunoblotting analysis in this study are shown in Table 1. Briefly, the protein samples extracted from cells or tis- sues were loaded, separated and then transferred onto HCC cells nitrocellulose membranes (Invitrogen, Carlsbad, USA). Human HCC cell lines, Huh7, HepG2, HCCLM3, and Subsequently, 5% bovine serum albumin was used to SK-Hep-1were obtained from the Chinese Academy of block the nitrocellulose membrane for 1 h. Finally, the Science (Shanghai, China). Normal liver cell line WRL- primary antibody and conjugated secondary antibody 68 was obtained from AcceGen (Fairfield, USA). All cell were added. Protein blots were detected using enhanced Cui et al. Journal of Experimental & Clinical Cancer Research (2020) 39:90 Page 3 of 14 chemiluminescence (Beyotime, Shanghai, China). Details Wound-healing assay of the antibodies are listed in Additional file 1: Table S2. Stably transfected cells were inoculated in 6-well plates and cultured until cell fusion occurred. A straight cut was made at the bottom of the plate with a 10-μL pip- Quantitative real-time polymerase chain reaction (qPCR) ette tip. The floating cells were washed away and the Total RNA was isolated from fresh frozen tissue and wound closure was photographed at 0 and 24 h. logarithmically growing cells using an RNA Miniprep Kit (Axygen, Jiangsu, China), and cDNA was synthesized Transwell migration and invasion assay using either a High Capacity RT Kit or TaqMan® Micro- Matrigel-coated (BD Biosciences, Franklin Lakes, NJ) or RNA RT kit (Applied Biosystems, Carlsbad, USA). qPCR non-Matrigel-coated Transwells were used to examine was performed using SYBR Green (Roche, Indianapolis, the invasion and migration ability of cells. Stably trans- USA) or TaqMan® qPCR Master Mix (Applied Biosys- fected cells were inoculated in the upper chamber of the tems, Carlsbad, USA) on a 7500 Fast PCR System. Then transwells and serum-free media was added. Normal the expression levels of mRNA and miRNA were nor- media was injected into the plate wells. After a 24 h in- malized to GAPDH and U6, respectively. Details of the cubation period, the cells in the upper layer filter were primers and probes for qPCR are listed in Additional file removed and the cells in the bottom layer were fixed, 1: Table S3. stained and counted. Immunohistochemical (IHC) staining Luciferase reporter assay After a series of processes including dewaxing, Cells were inoculated in 24-well plates, and the wild- rehydration, and antigen repair, the carcinoma and adja- type or mutated 3′-UTR sequence of ABCA8 were cent non-tumor sections were blocked with secondary cotransfected with pRL-TK Renilla. After incubation for antibody source serum. After blocking, the sections were 48 h, luciferase activities were measured by the dual lu- incubated with primary antibodies overnight.