Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:290 https://doi.org/10.1186/s13046-018-0951-8 RESEARCH Open Access Gain of UBE2D1 facilitates hepatocellular carcinoma progression and is associated with DNA damage caused by continuous IL-6 Chuanchuan Zhou1,2, Fengrui Bi1, Jihang Yuan1, Fu Yang1 and Shuhan Sun1* Abstract Background: Hepatocellular carcinoma (HCC) is the most common type of liver cancer with increasing incidence and poor prognosis. Ubiquitination regulators are reported to play crucial roles in HCC carcinogenesis. UBE2D1, one of family member of E2 ubiquitin conjugating enzyme, mediates the ubiquitination and degradation of tumor suppressor protein p53. However, the expression and functional roles of UBE2D1 in HCC was unknown. Methods: Immunohistochemistry (IHC), western blotting, and real-time PCR were used to detect the protein, transcription and genomic levels of UBE2D1 in HCC tissues with paired nontumor tissues, precancerous lesions and hepatitis liver tissues. Four HCC cell lines and two immortalized hepatic cell lines were used to evaluate the functional roles and underlying mechanisms of UBE2D1 in HCC initiation and progression in vitro and in vivo. The contributors to UBE2D1 genomic amplification were first evaluated by performing a correlation analysis between UBE2D1 genomic levels with clinical data of HCC patients, and then evaluated in HCC and hepatic cell lines. Results: Expression of UBE2D1 was significantly increased in HCC tissues and precancerous lesions and was associated with reduced survival of HCC patients. Upregulation of UBE2D1 promoted HCC growth in vitro and in vivo by decreasing the p53 in ubiquitination-dependent pathway. High expression of UBE2D1 was attributed to the recurrent genomic copy number gain, which was associated with high serum IL-6 level of HCC patients. Further experiments showed that continuous IL-6 activated the DNA damage response and genomic instability by repressing DNA damage checkpoint protein RAD51B. Moreover, continuous IL-6 could significantly facilitate the HCC growth especially with the genomic gain of UBE2D1. Conclusions: Our findings showed that UBE2D1 played a crucial role in HCC progression, and suggested a novel pattern of continuous IL-6 to promote cancers by inducing the genomic alterations of specific oncogenes. Keywords: UBE2D1, Hepatocellular carcinoma, Copy number variations, Continuous IL-6, RAD51B Background stage HCC patients can only benefit from the systemic Hepatocellular carcinoma (HCC) is one of the most com- treatment of sorafenib, which will lose efficacy due to in- mon and fatal human cancers with increasing incidence herent or acquired drug resistance [4]. Therefore, it is im- and mortality worldwide, especially in East Asia and South portant to unravel the molecular events underlying HCC Africa [1, 2]. Although several risk factors have been iden- initiation and to identify new therapeutic targets. tified to be responsible for the HCC initiation, such as Recent studies have identified a series of regulators to hepatitis B virus (HBV) and hepatitis C virus (HCV) infec- mediate the ubiquitination of tumor suppressor genes to tions, alcohol abuse and obesity, the diagnose of HCC at promote HCC growth and carcinogenesis [5, 6]. UBE2D1, an early stage is still difficult to realize [3]. And advanced a member of E2 ubiquitin conjugating enzyme, belongs to the UBE2D family. It has been reported that UBE2D1 me- diates the ubiquitination and degradation of tumor sup- * Correspondence: [email protected] – 1Department of Medical Genetics, Second Military Medical University, pressor protein p53 in vitro and in vivo [7 10], and Shanghai 200433, China UBE2D1 participates the ubiquitination of HSP90AB1, Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:290 Page 2 of 12 which contributes to the stability and transport to nucleus Western blot analysis of p53 [11, 12]. Recently, UBE2D1 was reported to con- Total cell and tissues protein extracts were separated by nect the overexpression of Aurora kinase A with Wnt and SDS-PAGE, transferred to nitrocellulose membrane and Ras-MAPK signaling pathways in colorectal cancer, sug- incubated with antibodies specific for UBE2D1(Abcam, gesting UBE2D1 may play an important role in the pro- Hong Kong, China), p53(Sigma-Aldrich, Saint Louis, gression from adenoma-to-carcinoma [13]. However, the MO), CHK1(Cell Signaling Technology, Boston, USA), functional roles of UBE2D1 in HCC were unclear. phosphorylated CHK1(Cell Signaling Technology), RPA(- Copy number variations(CNVs), as one of the import- Sigma-Aldrich), γ-H2Ax(Cell Signaling Technology), ant components of genomic variations in human, are STAT3 and phosphorylated STAT3(Cell Signaling Tech- closely associated with many genetic and developmental nology), RAD51B(Abcam), cleaved PARP, Caspase 9 and disorders [14, 15]. Differently expressed tumor suppres- Caspase 3 (Cell Signaling Technology) and β-actin (Sig- sor genes and oncogenes due to the CNVs were ma-Aldrich). IRdye800-conjugated goat anti-rabbit IgG regarded as the crucial cause of many types of cancer, and IRdye700-conjugated goat anti-mouse IgG were then including HCC [16, 17]. Genome instability and abnor- incubated with the blots. Antibody binding was detected mal DNA damage are the hallmarks of cancers [18, 19]. on Odyssey infrared scanner (Li-Cor Biosciences Inc.). And previous studies have implicated that DNA damage response and genomic instability would drive the copy Immunohistochemistry (IHC) number variants formation and common fragile sites Immunohistochemistry of UBE2D1 was performed on arising [20, 21]. Physical damage such as ionizing radi- 5 μm paraffin sections of tissue samples. UBE2D1 anti- ation, and many chemical agents can induced the DNA body (Abcam) was applied at 1:100 dilution. Vectastain damage response and genomic instability [22], while Elite ABC kits (Vector, Burlingame, CA, USA) and HBV integration events might increase the CNVs fre- ImmPACT DAB Substrate (Vector) were used to detect quency via inducing genomic instability [19]. Actually, UBE2D1 expression. The staining sections were visualized the factors driving DNA damage response and genomic in Olympus microscopy (IX71; Olympus, Japan).The score alteration in tumor microenvironment were unclear. was evaluated as four levels:0 (−), 1(+), 2(++), 3(+++). The In our study, we explored the roles of UBE2D1 in quantitative analyses of immunohistochemistry images HCC. UBE2D1 was significantly up-regulated in HCC were performed using Image Software Pro Plus 6.0. tissues, and high expression level of UBE2D1 could act as a poor prognosis factor for overall survival of HCC Lentivirus mediated the overexpression of target genes patients. UBE2D1 facilitated the HCC growth in vitro To obtain the overexpression of UBE2D1, STAT3 and and in vivo by decreasing the p53 protein level in an RAD51B, the indicated cells were transfected with re- ubiquitin-dependent manner. We also found that the combinant lentiviruses. Recombinant lentiviruses con- high expression of UBE2D1 was associated with recur- taining UBE2D1, STAT3 and RAD51B with negative rent genomic gain in HCC. Next, we confirmed the gain control were purchased from Obio Technology (Shang- of UBE2D1 was attributed to the high IL-6 level, and hai, China). For transfection of UBE2D1, STAT3 and continuous IL-6 could activate the DNA damage re- RAD51B into indicated cells, 1 × 106 transducing units sponse and genomic instability to drive genomic alter- were added to the medium of cultured cells, and super- ations. From our findings, we identified the IL-6/ natant was replaced with complete culture media after RAD51B/UBE2D1 axis, suggesting continuous IL-6 as a 24 h. The overexpression of genes was confirmed by factor to induce the genomic alterations of oncogenes in real-time PCR and Western blot with specific primers or HCC carcinogenesis. antibodies 96 h after infection. For construction of stably overexpressed UBE2D1, STAT3 and RAD51B, the cells Methods were transfected with 1 × 106 transducing units’ lenti- Cell culture and treatment virus, and the transfected cells were selected by using LO2, QSG -7701, SMMC -7721, LM3, Hep3B and Huh7 puromycin at the concentration of 0.5 μg/ml to 1.0 μg/ cells were achieved from the Cell Bank of the Chinese ml for indicated cells. The overexpression of target genes Academy of Sciences (Shanghai, China) and cultured in was confirmed by real-time PCR and Western blot. Dulbecco’s modified Eagle’s medium with 10% fetal bo- vine serum at 37 °C, 5% CO2. Where indicated, cells RNA interference were treated with IL-6 (PeproTech, Rocky Hill, NJ) for UBE2D1 and STAT3 interference was mediated by the indicated time. Without indications, cells were siRNA. RAD51B interference experiments were per- treated with IL-6 at 1 ng/ml for 48 h. IL-6-LT