MicroRNA-203 increase cell radiosensitivity via directly targeting Bmi

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MicroRNA-203 increase cell radiosensitivity via directly targeting Bmi-1 in hepatocellular carcinoma Yingjie Shao, Dachuan Zhang, Xiaodong Li, Jing Yang, Lujun Chen, Zhonghua Ning, Yun Xu, Guohua Deng, Min Tao, Yibei Zhu, and Jingting Jiang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00302 • Publication Date (Web): 15 Jun 2018 Downloaded from http://pubs.acs.org on June 16, 2018

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Molecular Pharmaceutics

MicroRNA-203 increase cell radiosensitivity via directly targeting Bmi-1 in hepatocellular carcinoma Running title: MiR-203 regulated HCC radiosensitivity by Bmi-1

Yingjie Shao1,2,3, Dachuan Zhang1,3,4, Xiaodong Li1,3,4, Jing Yang1,3,4, Lujun Chen1,3,4, Zhonghua Ning1,2,3, Yun Xu1,3,4, Guohua Deng1,3,4, Min Tao4, Yibei Zhu4, Jingting Jiang1,3,4,§

1

Department of Tumor Biological Treatment, The Third Affiliated Hospital of

Soochow University, Changzhou 213003, China 2

Department of Radiation Oncology, The Third Affiliated Hospital of Soochow

University, Changzhou 213003, China 3

Jiangsu Engineering Research Center for Tumor Immunotherapy, Changzhou

213003, China 4

§

Institute of Cell Therapy, Soochow University, Changzhou 213003, China

Corresponding author:

Jingting Jiang Department of Tumor Biological Treatment, the Third Affiliated Hospital of Soochow University, Changzhou 213003, China E-mail: [email protected] Tel: +86-519-68870978

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Abstract Background: B-cell-specific moloney leukemia virus insertion site 1 (Bmi-1) plays important roles in various cancers, but its regulation through microRNAs (miRNAs) and its functions in hepatocellular carcinoma (HCC) remains unclear. Methods: We evaluated the expression and prognostic significance of Bmi-1 in HCC by using tissue samples and The Cancer Genome Atlas (TCGA) datasets. The relationship between miRNAs and Bmi-1 was verified by bioinformatics prediction and immunofluorescence. Colony formation and apoptosis assays was used to reveal the effect of miR-203 on radiosensitivity. Results: The Bmi-1 mRNA and protein were up-regulated in HCC tissues. Cox regression multivariate analyses showed that Bmi-1 overexpression was an independent prognostic parameter for HCC patients. The expression level of Bmi-1 was negatively associated with miR-203 levels in HCC tissues. Dual-luciferase reporter assays showed miR-203 could target the 3’ un-translated region (3’-UTR) of Bmi-1 directly. Overexpression of miR-203 in HepG2 and Smmc-7721 cells increases their sensitivity to ionizing radiation in vitro and vivo. Moreover, the improved cell radiosensitivity induced by miR-203 could be rescued by restoration of Bmi-1 expression. Conclusions: Bmi-1 could improve the predictive accuracy for HCC patients’ survival. Moreover, miR-203 enhance cells radiosensitivity in vitro and vivo by targeting Bmi-1 in HCC. Keywords: Bmi-1; Hepatocellular carcinoma; Prognosis; miR-203; Radiosensitivity

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Introduction Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide, and there are approximately 750,000 new cases reported each year [1]. Over the past decade, with increased understanding of the epidemiological risk factors for HCC [2], as well as the development of imaging diagnostic, surgery and liver transplantation techniques, the survival rate has been greatly improved. . However, the prognosis of HCC is still very poor [3]. About 60% of cancer patients receive radiation therapy, However, HCC has been considered as a radiation-resistant tumor. Thus, it is necessary to explore the mechanisms of resistance to radiotherapy and develop new radiosensitizers in HCC. Recently, the B-cell-specific moloney leukemia virus insertion site 1 (Bmi-1) was found to be a new potential biomarker for HCC. It can regulates the transcription of p16Ink4a and p14ARF, two very important tumor suppressors [4, 5]. Bmi-1 is reported to be involved in the initiation, progression, and radioresistance of cancer (reviewed in [6]). Bmi-1 is overexpressed in HCC tissue compared to the corresponding non-tumor tissue [7-12]. However, the expression level and prognostic value of Bmi-1 in HCC is controversial. Some studies suggest that Bmi-1 expression is not related to any clinicopathological parameters, such as tumor size, differentiation degree, recurrence or distant metastasis [8-10], so that it can't predict the outcome of HCC patients [9]. While in other studies, Bmi-1 expression is closely associated with overall survival (OS) [11, 12]. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally by base-pairing with the 3’ un-translated regions (3’-UTR) of mRNAs [13]. Recently, miRNAs have been found to play an important role in the radiosensitivity of tumor cells [14]. However, the function and mechanism of miRNAs in HCC radiosensitivity

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are still not clear. In this study, we explored the expression of Bmi-1 in HCC and its prognostic roles. In addition, we also analyzed the relationship between Bmi-1 and miRNAs in HCC, as well as their effect on HCC radiosensitivity in HCC cells.

Experimental Section Patients We purchased the tissue microarray from Shanghai Outdo Biotech Co. Itd (Lot No. HLiv-HCC180Sur-02). Ninety HCC tissues that were surgically resected between August 2006 and November 2009 were enrolled in this study. Besides, ninety corresponding adjacent normal liver tissues were served as negative control.

Immunohistochemistry (IHC) IHC analysis of Bmi-1 was performed with the protocol in our previous study [15]. The tissue microarray was microwave-treated and boiled in citrate buffer. Each section was added with 1 drop 3% H2O2 and incubated for 10min at indoor temperature. Then, they were incubated with goat serum for 15min at indoor temperature and the serum was discarded. Then the tissue microarray was treated by primary mouse antibody anti-Bmi-1 (Merck Millipore, 1:300 dilution) overnight. Flushed with PBS, the tissue microarray was added with the secondary antibodies (Abcam, Cambridge, MA, USA) and incubated for 30-40min. Immunostaining was scored by two pathologists. The staining index was calculated by multiplying staining intensity and staining area. We defined high or low group by the staining index: 0-2, low expression; 3-9, high expression.

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The Cancer Genome Atlas (TCGA) datasets TCGA is a comprehensive and coordinated database to accelerate our understanding about the molecular changes of different tumor types by applying genome analysis technologies. We downloaded miRNA and mRNA expression data for HCC TCGA datasets from UCSC cancer genome browser (https://genome-cancer.ucsc.edu/). There were 367 cancer tissues that had both mRNA and miRNA expression data. Also, the datasets included 50 paired HCC and adjacent normal liver tissues that had both mRNA and miRNA expression data.

Cell culture and quantitative reverse transcription-polymerase chain reaction analysis The HCC cells Smmc-7721 and HepG2 used in this study were purchased from Shanghai Cell Bank of Chinese Academy of Sciences on 1 February 2015. The culture media used for the Smmc-7721 and HepG2 cells were RMPI1640 media including 10% fetal bovine serum. The Two cell lines were cultured at indoor temperature in the cell incubators with 5% CO2. The RNA from HCC cells was extracted using TRIzol reagent with instructions. 200ng total RNA was extracted for reverse transcriptase (Promega, Fitchburg, WI, USA) with the instructions. The relative levels of miRNA were normalized to U6 and the relative levels of mRNA were normalized to GAPDH.

Western blotting analysis Total proteins from cells were lysed with RIPA buffer. Quantification for protein concentration was conducted using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA), and 100µg proteins were isolated by SDS-PAGE, and then subjected to a polyvinylidene fluoride (PVDF) membrane. Then the membrane was immersed in

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TBST containing 5% defatted milk at indoor temperature for 1h and cultured overnight with primary antibodies at 4°C (anti-Bmi-1, 1:2000, Cell Signaling, CA, USA; anti-GAPDH, 1:2000, Santa Cruz Biotechnology, CA, USA). Flushed with TBST for 3 times and added with secondary antibodies (anti-mouse-HRP, 1:5000, Bio-Rad, Hercules, CA, USA), the membrane was incubated in a shaker at indoor temperature. GAPDH served as an internal control.

Cell transfection For the miR-203 over-expression experiments, HepG2 and Smmc-7721 cells engineered to stably express complete miR-203 gene sequence by transduction with a lentiviral vector that constitutively expresses precursor of miR-203 driven by a CMV (cytomegalovirus) promoter. The lentiviral vector system, purchased from Clontech. Bmi1 expression plasmid pcDNA3.1-Bmi1 was generated by cloning open reading frame (lacking the 3’-UTR) of Bmi1 into pcDNA3.1 vector and transfected for 24 h using Lipofectamine 2000 (2 ug/well in 6-well plate) with instructions.

Irradiation, colony formation and apoptosis assay The cells of the transfection exposed to radiation with 0-8 Gy as indicated at a dose rate of 5 Gy/min using a 6-MV x-ray generated by a linear accelerator. After 14 days, colonies containing >50 cells were counted under a light microscope. The surviving fraction (SF) was calculated as (number of colonies) / (number of cells plated) * (plating efficiency). For apoptosis assays, cells were stained with antibodies against PI and annexin V after irradiation 48 hours (5 Gy). The fluorescence intensity was detected by flow cytometer (FACS, BD biosciences) with manufacturer’s instructions.

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Dual-luciferase reporter assays The wild-type and mutant-type fluorescence plasmids psiCheck2-Bmi-1 3’UTR-Luc and psiCheck2-Bmi-1 3’UTR-MUT-Luc, respectively, were constructed. The Hepg2 cells were co-transfected with the miR-203, psiCheck2-Bmi-1 3’UTR-Luc and psiCheck2-Bmi-1 3’UTR-MUT-Luc vector. After 48h for transfection, the cells were flushed with PBS. 80µl 1×Passive Lysis Buffer was added into each well. After incubation for 15min at indoor temperature in a shaker, the cells were lysed. Then cell lysis buffer was collected at 4°C and centrifuged for 2min. The supernatant was collected and refrigerate at -80°C refrigerator. Cell lysis buffer was added to 96-well plate with 10µl per well. During the detection, 30 µl luciferase reagents II (Promega, WI, USA) were added. Then 30µl stop buffer were added to terminate the activities. Finally, luciferase activities were record.

Xenograft studies HepG2 cells (1×107 cells) stably expressing miR-NC (control) and miR-203 in serum-free DMEM were injected into either side of flank area of 6-week-old nude mice. Radiation treatment with a single 8-Gy dose was initiated 10 days after injection. Tumor size was measured and recorded five days for 35 days and tumor volumes were calculated using the formula (V=LW2/2, L: length, W: width). After 35 days, mice were sacrificed, and the tumors were collected for weighting and analysis of the miR-203 and Bmi-1 expression. Tumors were flash frozen in LN2, tissue was crushed and lysed and RNA extracted using the Qiagen miRNeasy extraction kit (Qiagen).

Statistical analysis Student’s t-test and one-way ANOVA were used to analyse the significant differences

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between the two groups and among the groups. Spearman’s correlation test was performed to analyze the correlation among variables. The nomogram was formulated using R software. The datas are presented as the means ± standard error. Statistical analysis was carried out by Graphpad Prism (version 6.01) software (La Jolla, CA, USA) and R software version 3.2.0 (http://www.r-project.org/) with Hmisc, rms, and survival ROC packages. P

MicroRNA-203 increase cell radiosensitivity via directly targeting Bmi

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