Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

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Available online at www.sciencedirect.com

www.elsevier.com/locate/yexcr

Research Article

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro Wei Yanga,⁎, 1 , Ting Sunb, 1 , Jianping Caoa , Fenju Liua , Ye Tianc , Wei Zhua a

Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou, China Brain and Nerve Research Laboratory, The First Affiliated Hospital, Soochow University, Suzhou, China c Department of Radiotherapy and Oncology, The Second Affiliated Hospital, Soochow University, Suzhou, China b

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

Hypoxia is a common feature of solid tumors and an important contributor to tumor

Received 16 December 2011

radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia

Revised version received

in different types of tumor cells and normal cells. In the present study, to explore the feasibility of

31 January 2012

miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer

Accepted 16 February 2012

technique was employed to downregulate miR-210 expression in hypoxic human hepatoma

Available online 24 February 2012

SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia

Keywords:

in the G0/G1 phase in all cell lines. miR-210 downregulation significantly suppressed cell viability,

led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest miR-210

induced cell arrest in the G0/G1 phase, increased apoptotic rate and enhanced radiosensitivity in

AIFM3

hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated,

Hypoxia

3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA at-

Radiosensitivity

tenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells.

Hepatoma

Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. © 2012 Elsevier Inc. All rights reserved.

Introduction Owing to an imbalance in oxygen supply and demand, hypoxia is a common feature of pathological conditions such as tissue ischemia and inflammation, as well as solid tumors, including hepatocellular carcinoma (HCC). Hypoxia is an important contributor to tumor radioresistance [1,2]. Oxygen can chemically modify the radiation-induced DNA damage and produce adducts that are

difficult to repair by cells. Hypoxia can abolish oxygen effect and lead to decreased radiosensitivity [3]. In addition, hypoxia may also modulate tumor radiosensitivity through biological effects [4]. Signaling pathways stimulated by hypoxia are commonly activated in tumors. Hypoxia inducible factor-1 (HIF-1) is one of the key mediators of hypoxia signaling pathways [5]. HIF-1 is a heterodimeric transcription factor consisting of two subunits, HIF-1α and HIF-1β. HIF-1β is constitutively expressed while HIF-1α is rapidly degraded

⁎ Corresponding author at: Department of Radiobiology, School of Radiological Medicine and Protection, No.199 Renai Road, Soochow University, Suzhou, 215123, China. Tel.: +86 512 6588 0065. E-mail address: [email protected] (W. Yang). 1 Wei Yang and Ting Sun contributed equally to this work. 0014-4827/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2012.02.010

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in normoxic conditions and can be stabilized by hypoxia [6,7]. Therefore, hypoxia can induce HIF-1 expression, which modulates more than 100 genes involved in regulating important processes such as angiogenesis, metabolism, proliferation and apoptosis [8]. Recently, some microRNAs (miRs) have been found to be regulated by hypoxia [9,10]. miRs are a family of small (20 to 22 nucleotide in length) noncoding RNAs that regulate gene expression by sequence-selective targeting of mRNAs, inducing translational repression or mRNA degradation, depending on the degree of complementarities between miRs and the target sequences [11]. miRs have emerged as important regulators in developmental, physiological and pathological settings including cell growth, differentiation, apoptosis, metabolism and tumorigenesis [12]. Among these hypoxia-induced miRs, miR-210 is the most consistently and robustly induced miRNA under hypoxia in different types of tumor cells and normal cells [13,14]. Several miR-210 targets which influence cell proliferation, ATP metabolism, and angiogenesis have been identified such as E2F3, MYC antagonist (MNT), ephrin-A3 (EFNA3) and iron-sulfur cluster scaffold protein (ISCU) [15–20]. Based on the functions of these targets, we hypothesize that miR-210 might be a logical novel target to overcome hypoxia-induced radioresistance in cancer. In the present study, human hepatoma cells with stable integration of the anti-sense miR-210 were generated through lentiviral-mediated gene transfer, which followed by exposure to hypoxia. Cell proliferation, apoptosis and radiosensitivity were detected to explore the effect of miR-210 downregulation on biological function of hypoxic human hepatoma cells and its mechanism.

Materials and methods Plasmid construction MiR-210 anti-sense (5′-TCAGCCGCTGTCACACGCACAG-3′) or scramble (5′-TTCTCCGAACGTGTCACGTTTC-3′) oligos (GenePharma Co. Shanghai, China) and the target vector pGLV-H1-GFP (GenePharma Co. Shanghai, China) were cut with BamHI and EcoRI. After gel purification, the vector and insert were ligated overnight using T4 DNA Ligase, resulting in pGLV-anti-210-GFP and pGLV-scr-GFP, and sequenced.

Generation of stable cell lines Cells with stable integration of the anti-sense miR-210 or scramble sequence were generated through lentiviral-mediated gene transfer [21]. To generate the respective viruses, 293 T cells were transfected with the lentiviral vector, pGLV-anti-210-GFP or pGLV-scr-GFP, and the packaging plasmid PG-P1-VSVG, PG-P2REV and PG-P3-RRE according to standard protocols. The target human hepatocarcinoma cells SMMC-7721, HepG2 and HuH7 were infected with both of the viruses (encoding either antisense miR-210 or scramble sequence) and selected using puromycin. Clonal cell populations carrying anti-sense miR-210 or scramble sequence were obtained by limiting dilution of 100–300 cells in three 96-well plates. After 4 weeks, single clones were analyzed for positive GFP signals. The positive clones were expanded for additional testing.

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Cell line and cell culture The 293 T, p53 wild-type human hepatocarcinoma cell lines SMMC-7721 and HepG2 and p53 mutant human hepatocarcinoma cell line HuH7 were purchased from the Type Culture Collection of the Chinese Academy of Sciences and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, in a 37 °C incubator in a 5% CO2 humidified atmosphere. Cells treated with hypoxia were exposed to a steady flow of low-oxygen gas mixture (1% O2, 5% CO2, 94% N2) in a modular incubator chamber (MiniGalaxy, RSBiotech, Irvine, Scotland).

Real-time Reverse transcription-Polymerase Chain Reaction (RT-PCR) analysis of HIF-1 α mRNA, AIFM3 mRNA and miR-210 expression Total cellular RNA was isolated using Trizol reagent (Sangon Inc. Shanghai, China) according to the manufacturer's protocol. For HIF-1α and AIFM3 mRNA expression analysis total RNA was reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and analyzed using the SYBR Green PCR Master Mix (Applied Biosystems) on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) using standard conditions. Fold changes in HIF-1α and AIFM3 mRNA expression were quantified with the 2− ΔΔCT relative quantification method using β-actin as house keeping control. The primer sequences for HIF-1α were F-ATCGCGGGGACCGATT and R-CGACGTTCAGAACTTATCTTTTTCTT. The primer sequences for AIFM3 were F-GGGAGCCATCCACACTGGTC and R-TCTTGCCAAACATGGCGGTC. The primer sequences for β-actin were F-GATCATTGCTCCTCCTGAGC and R-TGTGGACTTGGGAGAGGACT. For miR-210 expression analysis total RNA was transcribed using TaqMan microRNA reverse transcription kit (Applied Biosystems) according to manufacturer's instructions. miR-210 expression was assessed by real-time PCR according to the TaqMan MicroRNA Assay protocol (Applied Biosystems). The 20 μL reactions were incubated in a 96-well optical plate at 95 °C for 3 min, followed by 40 cycles of 95 °C for 12 s, and 58 °C for 30 s. Fold changes in miR-210 expression between treatments and controls were determined by the 2− ΔΔCT method, normalizing the results to U6 RNA expression level.

Cell viability assay Cell viability was measured by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) assay (Sigma, St. Louis, MO, USA) after 24, 48 and 72 h normoxic or hypoxic culture. Cells were seeded into 96-well culture plates at 4.0 × 103 per well. After 24, 48 and 72 h normoxic or hypoxic culture, 200 μL MTT (5 mg/mL) was added to each well, and the cells were incubated at 37 °C for additional 4 h. After incubation, MTT-containing medium was removed and 150 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystals. Optical densities (OD) were determined on a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 490 nm. Cell viability was calculated as: OD value/cell number, and the cell viability of control cells after 24 h normoxic culture was taken as 100%.

Flow cytometric analysis of cell cycle and apoptosis Cells were harvested and fixed overnight with 70% ethanol at 4 °C, followed by resuspension in 500 μL of PBS. After addition of 10 μL

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RNase (10 mg/mL), cells were left for 30 min at 37 °C and stained with 10 μL propidium iodide (1 mg/mL). Cellular DNA content was determined on a flow cytometer Beckton Dickinson (BD) FACScan (BD Biosciences, San Jose, CA). Quantification of apoptotic cells was performed according to the Annexin-V-fluorescein isothiocyanate (FITC) manufacturer instructions (KeyGen Biotech. Nanjing, China). Analyses were performed by a flow cytometer (BD FACScan). FITCpositive and PI-negative cells were regarded as apoptotic cells.

Western blot analysis of protein expression Western blotting was performed using standard procedures as previously described [22]. The following primary antibodies were used: rabbit monoclonal anti-MYC antagonist (MNT), rabbit polyclonal anti-AIFM3 (Santa Cruz Inc. California, USA) and antiβ-actin (Sigma, St Louis, MO, USA).

Data analysis Clonogenic cell survival assay Cells were cultured in 35 mm tissue culture dishes. After 72 h hypoxic culture, cell dishes were sealed by sealing membrane and cells were irradiated with 0, 2, 4, 6, and 8 Gy X-ray irradiation (6 MV, the dose rate was 200 cGy/min) by a PRIMUS accelerator (SIEMENS Medical Solutions, Erlangen, Germany) at room temperature. After irradiation, cells were harvested using trypsinization, counted and a specific number of cells (100 for cells irradiated with 0 or 2 Gy, 200 for 4 Gy and 2000 for 6 or 8 Gy) was plated in petri dishes in triplicate for clonogenic assay. Then the cells were incubated for 11 days. Colonies were fixed by 37% formaldehyde solution and stained with crystal violet and colonies of more than 50 cells were counted. Plating efficiency (PE) was calculated as: PE = (colony number/plating cell number)× 100%, in triplicate. Furthermore, the cell survival fraction was counted out and the mean lethal dose (D0) was calculated by the linear quadratic model. Finally, the radiosensitization ratio was calculated (a ratio of D0).

Luciferase reporter assay

All data were calculated as Mean ± SEM. Comparisons between treatment groups and controls were made by t-test. A P-value less than 0.05 between groups was considered to be significant difference.

Results Time course of HIF-1α mRNA expression in human hepatoma cells exposed to 1% oxygen First we examined the hypoxia responsiveness of human hepatoma cells by assessing the HIF-1α gene expression upon hypoxic exposure. To study the time course of HIF-1α mRNA expression in SMMC-7721, HepG2 and HuH7 cells exposed to 1% oxygen, experiments were undertaken in cells exposed to 1% oxygen for 0, 6, 24, 48, and 72 h. As shown in Figs. 1A, C and E, hypoxia resulted in an increased HIF-1a expression reaching a plateau at 48 h exposure for all cell lines.

The 3′UTR of apoptosis-inducing factor, mitochondrionassociated, 3 (AIFM3) contains a putative miR-210-target site: 5′-ACCGTAAAATTAAAACGCACAA-3′ (297–318), and the 3′UTR of Programmed cell death protein 6 (AHRR) contains a putative miR-210-target site: 5′-TGCAGAGCTGT-GCATGCGCAG-3′ (110– 130). The 3′UTRs of AIFM3 and AHRR were isolated from cDNA of SMMC-7721 cells and were ligated into the pGL3 basic luciferase expression vector (pGL3-Luc) (Promega, Madison, WI, USA) at the 3′-end of the luciferase coding sequence, respectively. The pGL3-Luc vector containing 3′UTR of AIFM3 or AHRR and the internal control vector pRL-TK (Promega) were co-transfected into SMMC-7721 cells. Twenty four hours later, 20 nM of miR-210 oligos or scramble oligos (GenePharma Co. Shanghai, China) were transfected into SMMC-7721 cells. Luciferase activity was measured 48 h after vectors transfection using Dual-luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.

Time course of miR-210 expression in human hepatoma cells exposed to 1% oxygen

AIFM3-siRNA design and transfection

We investigated the effect of miR-210 downregulation on normoxic or hypoxic human hepatoma cell viability in vitro by MTT assay. Cell viability was detected after 24, 48 and 72 h culture. As shown in Figs. 2A, C and E, miR-210 downregulation showed no obvious change in viability of normoxic SMMC-7721, HepG2 and HuH7 cells (P > 0.05). Nevertheless, Figs. 2B, D and F showed that miR-210 downregulation decreased viability of hypoxic SMMC-7721, HepG2 and HuH7 cells in a time-dependent manner (P < 0.05 or P < 0.01) and the highest inhibitory rate was 31.13%, 37.38% and 31.52% at 72 h, respectively. All these results indicated that downregulation of miR-210 expression significantly suppressed hypoxic human hepatoma cell viability in vitro.

The cDNA sequence of AIFM3 was obtained from GenBank (NM_144704.1). The siRNA target design tools from Ambion were used to design AIFM3-siRNA. AIFM3-siRNA was designed and synthesized as follows (Sangon Inc. Shanghai, China): sense: 5′-ACCUCAUGAAGAUGUUUGAGAA-3′, antisense: 5′-UUCUCAA ACAUCUUCAUGAGGG-3′. Cells with stable integration of antisense miR-210 were plated 24 h prior to transfection. Cells were transfected in 6-well plates by use of Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). AIFM3-siRNA and negative control siRNA (ncsiRNA) were used at 100 nM final concentration.

Corresponding with the increased HIF-1α expression, induction of miR-210 by hypoxia was discernable at 6 h and showed a progressive increase in expression, becoming significant at 24 h and being maximal at 48 h time point in SMMC-7721, HepG2 and HuH7 cells and cells with stable integration of the scramble sequence. MiR210 expression in cells with stable integration of anti-sense miR210 was significantly decreased compared with that of control cells at each time point (P < 0.01; Figs. 1B, D and F). These results indicated that stable integration of anti-sense miR-210 significantly suppressed miR-210 expression in hypoxic human hepatoma cells.

Effect of miR-210 downregulation on normoxic or hypoxic SMMC-7721, HepG2 and HuH7 cell viability

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Fig. 1 – Real-time Reverse transcription-Polymerase Chain Reaction (RT-PCR) analysis of HIF-1α mRNA and miR-210 expression (fold change) in human hepatoma cells exposed to 1% oxygen. Error bars indicate the standard error of the mean of three individual experiments. (A) Time course of HIF-1α mRNA expression in SMMC-7721 cells. *P < 0.05, †P < 0.01 vs SMMC-7721 cells at 0 h. (B) Time course of miR-210 expression in SMMC-7721 cells. †P < 0.01 vs SMMC-7721 cells at each time point. (C) Time course of HIF-1α mRNA expression in HepG2 cells. †P < 0.01 vs HepG2 cells at 0 h. (D) Time course of miR-210 expression in HepG2 cells. † P < 0.01 vs HepG2 cells at each time point. (E) Time course of HIF-1α mRNA expression in HuH7 cells. *P < 0.05, †P < 0.01 vs HuH7 cells at 0 h. (F) Time course of miR-210 expression in HuH7 cells. †P < 0.01 vs HuH7 cells at each time point.

Effect of miR-210 downregulation on normoxic or hypoxic SMMC-7721, HepG2 and HuH7 cell cycle To investigate the mechanism underlying the miR-210 downregulation mediated cell proliferation suppression in hypoxia, we analyzed cell cycle of human hepatoma cells by flow cytometry after 72 h normoxic or hypoxic culture. Figs. 3A, C and E showed that miR-210 downregulation showed no obvious change in cell cycle of normoxic cells (P > 0.05). Nevertheless, As shown in Fig. 3B,

the percentage of G0/G1 phase cells in SMMC-7721 cells with stable integration of anti-sense miR-210 was increased (P < 0.05), and no significant change in S phase cells, while the percentage of G2/M phase cells was significantly reduced compared with those of SMMC-7721 cells (P < 0.05). As shown in Figs. 3D and F, the percentage of G0/G1 phase cells in HepG2 and HuH7 cells with stable integration of anti-sense miR-210 was increased (P < 0.01), while the percentage of G2/M and S phase cells was significantly reduced compared with those of HepG2 and HuH7 cells (P < 0.05). These

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Fig. 2 – Effect of miR-210 downregulation on normoxic or hypoxic human hepatoma cell viability. Cell viability was detected after 24, 48 and 72 h culture by MTT assay. Error bars indicate the standard error of the mean of five individual experiments. (A) Cell viability of normoxic SMMC-7721 cells. (B) Cell viability of hypoxic SMMC-7721 cells. *P < 0.05, †P < 0.01 vs SMMC-7721 cells at each time point. (C) Cell viability of normoxic HepG2 cells. (D) Cell viability of hypoxic HepG2 cells. *P < 0.05, †P < 0.01 vs HepG2 cells at each time point. (E) Cell viability of normoxic HuH7 cells. (F) Cell viability of hypoxic HuH7 cells. †P < 0.01 vs HuH7 cells at each time point. results indicated that hypoxic human hepatoma cells with miR210 downregulation arrested in the G0/G1 phase of cell cycle.

Effect of miR-210 downregulation on apoptosis of normoxic or hypoxic SMMC-7721, HepG2 and HuH7 cells Apoptosis of human hepatoma cells was quantitatively measured using flow cytometry after 24, 48 and 72 h normoxic or hypoxic culture. As shown in Figs. 4A, C and E, miR-210 downregulation showed no obvious change in apoptotic rate of normoxic cells (P > 0.05). Nevertheless, as shown in Figs. 4B, D and F, the average apoptotic rate of cells with stable integration of anti-sense miR-210

was significantly increased compared with that of control cells after 48 and 72 h hypoxic culture (P < 0.01). No obvious change of apoptotic rate was observed in control cells or cells with stable integration of the scramble sequence after 24, 48 and 72 h hypoxic culture. These results indicated that miR-210 downregulation induced apoptosis in hypoxic human hepatoma cells.

Effect of miR-210 downregulation on radiosensitivity of normoxic or hypoxic SMMC-7721, HepG2 and HuH7 cells After exposure to 0, 2, 4, 6, and 8 Gy X-ray irradiation, cell survival fractions were examined using a clonogenic assay. The PE of

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Fig. 3 – Effect of miR-210 downregulation on normoxic or hypoxic human hepatoma cell cycle. Cell cycle was analyzed by flow cytometry after 72 h culture. Error bars indicate the standard error of the mean of six individual experiments. (A) Cell cycle of normoxic SMMC-7721 cells. (B) Cell cycle of hypoxic SMMC-7721 cells. *P < 0.05 vs SMMC-7721 cells. (C) Cell cycle of normoxic HepG2 cells. (D) Cell cycle of hypoxic HepG2 cells. *P < 0.05, †P < 0.01 vs HepG2 cells. (E) Cell cycle of normoxic HuH7 cells. (F) Cell cycle of hypoxic HuH7 cells. *P < 0.05, †P < 0.01 vs HuH7 cells.

normoxic SMMC-7721, HepG2 and HuH7 cells was 67.8%, 78.9% and 75.6%, respectively. The survival curves of cells, as shown in Figs. 5A, B and C, were obtained from data fitting according to the linear quadratic model. No obvious change of survival fraction was observed in normoxic cells and normoxic cells with stable integration of the scramble sequence or anti-sense miR-210. It is clear that hypoxic SMMC-7721 cells with miR-210 downregulation (D0 = 2.80 Gy) were more sensitive to X-ray irradiation than hypoxic SMMC-7721 cells (D0 = 3.38 Gy) and the radiosensitization ratio was 1.21. The hypoxic HepG2 cells with miR-210 downregulation (D0 = 4.42 Gy) were more sensitive to X-ray irradiation

than hypoxic HepG2 cells (D0 = 5.26 Gy) and the radiosensitization ratio was 1.18. The hypoxic HuH7 cells with miR-210 downregulation (D0 = 4.76 Gy) were more sensitive to X-ray irradiation than hypoxic HuH7 cells (D0 = 6.13 Gy) and the radiosensitization ratio was 1.29. Thus, we concluded that the downregulation of miR-210 expression could radiosensitize hypoxic human hepatoma cells. The oxygen enhancement ratio (OER) was calculated as the ratio of D0 (hypoxia) to D0 (normoxia). The OER value of SMMC-7721, SMMC/Lv-scr and SMMC/Lv-anti210 cells was 1.93, 2.06 and 1.61, respectively. The OER value of HepG2, HepG2/Lv-scr and HepG2/Lv-anti-210 cells was 2.31,

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Fig. 4 – Effect of miR-210 downregulation on apoptosis of normoxic or hypoxic human hepatoma cells. Apoptosis of human hepatoma cells was quantitatively measured using flow cytometry after 24, 48 and 72 h normoxic or hypoxic culture. Error bars indicate the standard error of the mean of six individual experiments. (A) Apoptosis of normoxic SMMC-7721 cells. (B) Apoptosis of hypoxic SMMC-7721 cells. †P < 0.01 vs SMMC-7721 cells at each time point. (C) Apoptosis of normoxic HepG2 cells. (D) Apoptosis of hypoxic HepG2 cells. †P < 0.01 vs HepG2 cells at each time point. (E) Apoptosis of normoxic HuH7 cells. (F) Apoptosis of hypoxic HuH7 cells. †P < 0.01 vs HepG2 cells at each time point.

2.51 and 1.95, respectively. The OER value of HuH7, HuH7/Lv-scr and HuH7/Lv-anti-210 cells was 2.84, 2.35 and 1.90, respectively.

miR-210 directly targets AIFM3 3′UTR In order to investigate the mechanism underlying the miR-210 downregulation mediated apoptosis and radiosensitization in hypoxic human hepatoma cells, we examined whether AIFM3 or AHRR gene, which is associated with apoptosis and predicted as a miR-210 target gene by Microcosm, is a direct target of miR-

210. The 3′UTR of AIFM3 or AHRR was cloned into a pGL3-Luc vector to perform reporter assay. As shown in Fig. 6C, when miR-210 oligos were transfected into SMMC-7721 cells with the reporter construct pGL3-Luc-AIFM3, luciferase activity was repressed more than 50% compared with transfection of scramble oligos. Meanwhile, luciferase activity in cells transfected with reporter constructs containing the 3′UTR of AHRR was not repressed by the transfection of miR-210 oligos. These results suggest that AIFM3 is a direct and robust target gene of miR-210 in human hepatoma cells.

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Surviving fraction

A

Normoxic HepG2 Normoxic HepG2/Lv-scr Normoxic HepG2/Lv-anti-210 Hypoxic HepG2 Hypoxic HepG2/Lv-scr Hypoxic HepG2/Lv-anti-210

.1

.01

Normoxic HuH7 Normoxic HuH7/Lv-scr Normoxic HuH7/Lv-anti-210 Hypoxic HuH7 Hypoxic HuH7/Lv-scr Hypoxic HuH7/Lv-anti-210

Fig. 5 – Effect of miR-210 downregulation on radiosensitivity of hypoxic and normoxic human hepatoma cells. After exposure to 0, 2, 4, 6, and 8 Gy X-ray irradiation, cell survival fractions were examined using a clonogenic assay. Error bars indicate the standard error of the mean of three individual experiments. Curves represent data fitting according to the linear quadratic model. (A) Survival curves of SMMC-7721 cells. (B) Survival curves of HepG2 cells. (C) Survival curves of HuH7 cells.

A 3’-agucggcgacAGUGUGCGUGUc-5’

miR-210

Effect of AIFM3-siRNA on radiation induced apoptosis in miR-210 downregulated hypoxic SMMC-7721, HepG2 and HuH7 cells

| | ||||||| 5’-accgtaaaatTAAAACGCACAa-3’

B 3’-agucggCGACAGUGUGCGUGUC-5’

AIFM3 3’UTR (297-318)

miR-210

||||| :||:||:||| 5’-tgcagaGCTGT-GCATGCGCAG-3’

Relative luciferase activity (%)

C

AHRR 3’UTR (110-130)

scr miR-210

120 100 80 60

†

40 20 0

pGL3-Luc

pGL3-Luc-AIFM3 pGL3-Luc-AHRR

Fig. 6 – miR-210 directly targets AIFM3 3′UTR. The pGL3-Luc vector containing 3′UTR of AIFM3 or AHRR and the internal control vector pRL-TK were co-transfected into SMMC-7721 cells. Twenty four hours later, 20 nM of miR-210 oligos or scramble oligos were transfected into SMMC-7721 cells. Luciferase activity was measured 48 h after vectors transfection using Dual-luciferase Reporter Assay System. Error bars indicate the standard error of the mean of three individual experiments. (A) A putative miR-210-target site in the 3′UTR of AIFM3. (B) A putative miR-210-target site in the 3′UTR of AHRR. (C) Relative luciferase activity in SMMC-7721 cells transfected with pGL3-Luc vector containing 3′UTR of AIFM3 or AHRR. †P < 0.01 vs cells transfected with scramble oligos.

Human hepatoma cells were irradiated with 4 Gy X-ray irradiation after 24 h hypoxic culture. Twenty four hours later, apoptosis of cells was quantitatively measured using flow cytometry. As shown in Fig. 7A, the average apoptotic rate of cells with stable integration of anti-sense miR-210 was significantly increased compared with that of cells with stable integration of the scramble sequence after irradiated with 4 Gy X-ray irradiation (P < 0.01). AIFM3-siRNA and negative control siRNA (ncsiRNA) were transfected into cells with stable integration of anti-sense miR-210. Twenty four hours later, cells were cultured in hypoxia for 24 h. AIFM3 expression was assessed by real-time PCR. As shown in Fig. 7B, AIFM3 expression in cells transfected with AIFM3-siRNA was significantly decreased compared with that of cells transfected with ncsiRNA (P<0.01). Then, miR-210 downregulated hypoxic cells transfected with AIFM3-siRNA or ncsiRNA were irradiated with 4 Gy X-ray irradiation. Twenty four hours later, apoptosis of cells was quantitatively measured using flow cytometry. As shown in Fig. 7C, the average apoptotic rate of cells transfected with AIFM3-siRNA was significantly decreased compared with that of cells transfected with ncsiRNA after irradiation (P <0.01). These results indicated that AIFM3 downregulation attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells.

Effect of miR-210 downregulation on protein expression of miR-210 targeted AIFM3 and MNT genes in hypoxic SMMC-7721, HepG2 and HuH7 cells We analyzed protein expression of AIFM3 and MNT genes in human hepatoma cells after 72 h hypoxic culture by Western blot. Experiments were repeated three times. The relative levels of miR-210 targeted protein expression were normalized against protein levels of an internal control gene, β-actin, performed in the same run. As

952

EX PE R IM EN TA L C E LL RE S EA RC H 31 8 ( 20 1 2) 9 4 4– 9 5 4

shown in Figs. 8A and B, the level of AIFM3 and MNT protein expression in cells with miR-210 downregulation was significantly increased compared with that of cells with stable integration of the scramble sequence (P < 0.01). These results indicated that miR-210 downregulation partially rescued protein expression of miR-210 targeted genes in hypoxic human hepatoma cells.

Discussion The primary liver cancers, in which 85–90% are HCC, is the third most common cause of death worldwide. HCC remains one of

A

40

4 Gy Lv-scr+4 Gy

Apoptotic rate (%)

Lv-anti-210+4 Gy

30

20

10

0

B

SMMC

HepG2

1.6

Control

1.4

AIFM3-siRNA

HuH7

Relative AIFM3 expression

NCsiRNA

1.2 1.0 0.8 0.6 0.4 0.2 0.0

SMMC/Lv-anti-210

C

HepG2/Lv-anti-210

HuH7/Lv-anti-210

Control+4 Gy

32

NCsiRNA+4 Gy AIFM3-siRNA+4 Gy

28

Apoptotic rate (%)

the most common cancer and a leading cause of cancer death in Asia, and its incidence is also rising in Western countries [23,24]. Management of HCC continues to be challenging because of high recurrence rate after surgical resection and resistance to chemotherapy and radiotherapy [25]. Radiotherapy is becoming more and more important due to the development of advanced conformal techniques especially for unresectable HCC. In order to improve its therapeutic ratio, there has been much interest in augmenting the effect of radiation on tumors by combining it with molecularly targeted therapeutics [22]. Hypoxia is a well-characterized environmental factor that halts cell cycle and cell proliferation. Since energy availability from oxidative phosphorylation is reduced in hypoxia, cell cycle arrest is likely to prevent excessive cell death under reduced oxygen [26]. The hypoxic regulation of miR-210 was first identified by miRs microarray in 2007 [27]. The stem–loop of miR-210 is located in an intron of a noncoding RNA, which is transcribed from AK123483 on chromosome 11p15.5. HIF1α regulates miR-210 by directly binding to a hypoxia responsive element (HRE) on the proximal miR-210 promoter [13]. miR-210 could override hypoxiainduced cell cycle arrest by downregulating MNT [16]. MNT is a member of the Myc/Max/Mad network with a basic–helix–loop– helix–zipper domain (bHLHzip). MNT represses Myc target genes by binding the E box DNA sequence (CANNTG) after forming heterodimers with Max [28,29]. To explore the possibility of miR-210 as an effective therapeutic target, we employed lentiviral-mediated anti-sense miR-210 gene transfer technique to downregulate miR-210 expression and analyze phenotypic changes in hypoxic SMMC-7721, HepG2 and HuH7 cells. In the present study, we found that hypoxia led to an increased HIF-1a and miR-210 expression and cell arrest in the G0/G1 phase in all cell lines. Moreover, miR-210 downregulation significantly suppressed cell viability, partially rescued MNT protein expression and induced cell arrest in the G0/G1 phase in hypoxic human hepatoma cells. It was demonstrated that miR-210 influenced survival/death in hypoxia by targeting genes that are not directly involved in

24 20 16 12 8 4 0

SMMC/Lv-anti-210

HepG2/Lv-anti-210

HuH7/Lv-anti-210

Fig. 7 – Effect of AIFM3-siRNA on radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Error bars indicate the standard error of the mean of three individual experiments. Human hepatoma cells were irradiated with 4 Gy X-ray irradiation after 24 h hypoxic culture. Twenty four hours later, apoptosis of cells was quantitatively measured using flow cytometry. (A) Apoptosis rate of hypoxic human hepatoma cells irradiated with 4 Gy X-ray irradiation. †P < 0.01 vs Lv-scr + 4 Gy group. AIFM3-siRNA and negative control siRNA (ncsiRNA) were transfected into cells with stable integration of anti-sense miR-210. Twenty four hours later, cells were cultured in hypoxia for 24 h. AIFM3 expression was assessed by real-time PCR. (B) Relative AIFM3 expression in miR-210 downregulated hypoxic human hepatoma cells after transfection with AIFM3-siRNA. †P<0.01 vs ncsiRNA group. miR-210 downregulated hypoxic cells transfected with AIFM3-siRNA or ncsiRNA were irradiated with 4 Gy X-ray irradiation. Twenty four hours later, apoptosis of cells was quantitatively measured using flow cytometry. (C) Apoptosis rate of miR-210 downregulated hypoxic human hepatoma cells transfected with AIFM3-siRNA after irradiation with 4 Gy X-ray. †P<0.01 vs ncsiRNA+4 Gy group.

EX PE R IM E NTA L CE L L RE S E ARC H 31 8 (20 1 2 ) 9 4 4 –9 5 4

A

Lv-scr

Lv-anti-210

Lv-scr

Lv-anti-210

Lv-scr Lv-anti-210

AIFM3 MNT Beta actin

B

Relative protein expression

SMMC-7721

HepG2

HuH7

7 AIFM3 MNT

6 5

† †

4

†

†

† †

3 2 1 0

Lv-scr Lv-anti-210 Lv-scr Lv-anti-210 Lv-scr Lv-anti-210

SMMC-7721

HepG2

HuH7

Fig. 8 – Detection of protein expression of miR-210 targeted AIFM3 and MNT genes in hypoxic SMMC-7721, HepG2 and HuH7 cells after 72 h hypoxic culture by Western blot. The relative levels of miR-210 targeted protein expression were normalized against protein levels of an internal control gene, β-actin, performed in the same run. (A) Western blot of miR-210 targeted AIFM3 and MNT protein. (B) Relative levels of AIFM3 and MNT protein expression. Error bars indicate the standard error of the mean of three individual experiments. †P < 0.01 vs cells with stable integration of the scramble sequence.

apoptosis, such as neuronal pentraxin 1, which has been shown to mediate apoptosis in ischemic neurons [18]. miR-210 decreases the activity of Caspase 3/7 and exerts anti-apoptotic effect in hypoxia [30]. In the present study, we also found that miR-210 downregulation led to increased apoptotic rate and enhanced radiation induced apoptosis in hypoxic human hepatoma cells. Furthermore, we examined the changes in radiosensitivity of the miR-210 downregulated hypoxic human hepatoma cells by clonogenic survival assay. Results indicated that miR-210 downregulation could significantly enhance radiosensitivity, which was probably associated with its effects on cell proliferation and radiation induced apoptosis as described previously. The OER values were lower than expected, which might result from the long time exposure to hypoxia. It was reported that reduced OER was also observed in some cell lines, especially p53 mutant cell lines under chronic hypoxia (>24 h), and energy depletion might be the cause of a decreased ability to repair radiation damage and the observed increase in radiosensitivity [31,32]. The lowest OER values were found in cells with stable integration of anti-sense miR-210, which might result from miR-210 downregulation decreasing D0 in hypoxia, but having no significant effect on D0 in normoxia. In order to investigate the mechanism underlying the miR-210 downregulation mediated apoptosis and radiosensitization in hypoxic human hepatoma cells, we identified miR-210 targeted gene associated with apoptosis by luciferase reporter assay. The results suggest that AIFM3, a gene homologous to the apoptosis-inducing factor (AIF), which is also named AIFlike (AIFL), is a direct target of miR-210 in human hepatoma cells. Human AIFM3 has 598 amino acids, with a characteristic Rieske domain and a pyridine nucleotide-disulfide oxidoreductase domain

953

(Pyr_redox). AIFM3 shares 35% homology with AIF, mainly in the Pyr_redox domain. AIFM3 is predominantly localized to the mitochondria, which increases cytochrome c release and induces apoptosis in a caspase-dependent manner [33,34]. Our results showed that AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells, which suggest miR-210 downregulation mediate enhanced radiation induced apoptosis in hypoxic human hepatoma cells through AIFM3 gene at least in part. In conclusion, our findings suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy would be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. On the contrary, Quero et al. demonstrated that miR-210 could be a marker of chronic hypoxia, but no enhanced effect was observed when miR-210 inhibitor was combined with irradiation in prostate cancer [32]. The contradictory findings maybe result from a different cell type and different hypoxic condition and duration, which need to be further studied.

Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 81071958 and 30600160), Priority Academic Program Development of Jiangsu Higher Education Institutions and Jiangsu Province's Key Medical Department in 2011.

REFERENCES

[1] B.J. Moeller, M.R. Dreher, Z.N. Rabbani, T. Schroeder, Y. Cao, C.Y. Li, M.W. Dewhirst, Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity, Cancer Cell 8 (2005) 99–110. [2] J.Y. Liu, J. Zhang, X.W. Wang, Y. Li, Y.B. Chen, K.C. Li, J. Zhang, L.B. Yao, G.Z. Guo, HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells, Exp. Cell Res. 316 (2010) 1985–1993. [3] B.J. Moeller, M.W. Dewhirst, HIF-1 and tumour radiosensitivity, Br. J. Cancer 95 (2006) 1–5. [4] B.J. Moeller, R.A. Richardson, M.W. Dewhirst, Hypoxia and radiotherapy: opportunities for improved outcomes in cancer treatment, Cancer Metastasis Rev. 26 (2007) 241–248. [5] K.L. Lan, K.H. Lan, M.L. Sheu, M.Y. Chen, Y.S. Shih, F.C. Hsu, H.M. Wang, R.S. Liu, S.H. Yen, Honokiol inhibits hypoxia-inducible factor-1 pathway, Int. J. Radiat. Biol. 87 (2011) 579–590. [6] K.J. Williams, B.A. Telfer, D. Xenaki, M.R. Sheridan, I. Desbaillets, H.J. Peters, D. Honess, A.L. Harris, G.U. Dachs, A. van der Kogel, I.J. Stratford, Enhanced response to radiotherapy in tumours deficient in the function of hypoxia-inducible factor-1, Radiother. Oncol. 75 (2005) 89–98. [7] W. Yang, T. Sun, J.P. Cao, S.J. Fan, Hypoxia-inducible factor-1α downregulation by small interfering RNA inhibits proliferation, induces apoptosis, and enhances radiosensitivity in chemical hypoxic human hepatoma SMMC-7721 cells, Cancer Biother. Radiol. 6 (2011) 565–571.

954

EX PE R IM EN TA L C E LL RE S EA RC H 31 8 ( 20 1 2) 9 4 4– 9 5 4

[8] L. Marignol, M. Coffey, M. Lawler, D. Hollywood, Hypoxia in prostate cancer: a powerful shield against tumour destruction? Cancer Treat. Rev. 34 (2008) 313–327. [9] M.E. Crosby, C.M. Devlin, P.M. Glazer, G.A. Calin, M. Ivan, Emerging roles of microRNAs in the molecular responses to hypoxia, Curr. Pharm. Design 15 (2009) 3861–3866. [10] X. Huang, Q.T. Le, A.J. Giaccia, MiR-210—micromanager of the hypoxia pathway, Trends Mol. Med. 16 (2010) 230–237. [11] N. Bushati, S.M. Cohen, MicroRNA functions, Annu. Rev. Cell Dev. Biol. 23 (2007) 175–205. [12] A. Ventura, T. Jacks, MicroRNAs and cancer: short RNAs go a long way, Cell 136 (2009) 586–591. [13] X. Huang, L. Ding, K.L. Bennewith, R.T. Tong, S.M. Welford, K.K. Ang, M. Story, Q.T. Le, A.J. Giaccia, Hypoxia-inducible miR-210 regulates normoxic gene expression involved in tumor initiation, Mol. Cell 35 (2009) 856–867. [14] R. Kulshreshtha, M. Ferracin, M. Negrini, G.A. Calin, R.V. Davuluri, M. Ivan, Regulation of microRNA expression: the hypoxic component, Cell Cycle 6 (2007) 1426–1431. [15] A. Giannakakis, R. Sandaltaopoulos, J. Greshock, S. Liang, J. Huang, K. Hasegawa, C. Li, A. O'Brien-Jenkins, D. Katsaros, B.L. Weber, C. Simon, G. Coukos, L. Zhang, miR-210 links hypoxia with cell cycle regulation and is deleted in human epithelial ovarian cancer, Cancer Biol. Ther. 7 (2008) 255–264. [16] Z. Zhang, H. Sun, H.Y. Dai, R.M. Walsh, M. Imakura, J. Schelter, J. Burchard, X. Dai, A.N. Chang, R.L. Diaz, J.R. Marszalek, S.R. Bartz, M. Carleton, M.A. Cleary, P.S. Linsley, C. Grandori, MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT, Cell Cycle 8 (2009) 2756–2768. [17] S.Y. Chan, Y.Y. Zhang, C. Hemann, C.E. Mahoney, J.L. Zweier, J. Loscalzo, MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2, Cell Metab. 10 (2009) 273–284. [18] K. Pulkkinen, T. Malm, M. Turunen, J. Koistinaho, S. Ylä-Herttuala, Hypoxia induces microRNA miR-210 in vitro and in vivo EphrinA3 and neuronal pentraxin 1 are potentially regulated by miR-210, FEBS Lett. 582 (2008) 2397–2401. [19] P. Fasanaro, Y. D'Alessandra, V. Di Stefano, R. Melchionna, S. Romani, G. Pompilio, M.C. Capogrossi, F. Martelli, MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand ephrin-A3, J. Biol. Chem. 283 (2008) 15878–15883. [20] D.C. Lee, R. Romero, J.S. Kim, A.L. Tarca, D. Montenegro, B.L. Pineles, E. Kim, J. Lee, S.Y. Kim, S. Draghici, P. Mittal, J.P. Kusanovic, T. Chaiworapongsa, S.S. Hassan, C.J. Kim, miR-210 targets iron-sulfur cluster scaffold homologue in human trophoblast cell lines: siderosis of interstitial trophoblasts as a novel pathology of preterm preeclampsia and small-for-gestational-age pregnancies, Am. J. Pathol. 179 (2011) 590–602.

[21] F. Wolf, W.R. Li, F. Li, C.Y. Li, Novel luciferase-based reporter system to monitor activation of ErbB2/Her2/neu pathway noninvasively during radiotherapy, Int. J. Radiat. Oncol. 79 (2011) 233–238. [22] W. Yang, T. Sun, J.P. Cao, F.J. Liu, Survivin downregulation by siRNA/ cationic liposome complex radiosensitizes human hepatoma cells in vitro and in vivo, Int. J. Radiat. Bio. 86 (2010) 445–457. [23] G.D. Kirk, E. Bah, R. Montesano, Molecular epidemiology of human liver cancer: insights into etiology, pathogenesis and prevention from The Gambia, West Africa, Carcinogenesis 27 (2006) 2070–2082. [24] C.D. Mann, C.P. Neal, G. Garcea, M.M. Manson, A.R. Dennison, D.P. Berry, Prognostic molecular markers in hepatocellular carcinoma: a systematic review, Eur. J. Cancer 43 (2007) 979–992. [25] T.F. Jakobs, R.T. Hoffmann, K. Tatsch, C. Trumm, M.F. Reiser, Therapy response of liver tumors after selective internal radiation therapy, Radiologe 48 (2008) 839–849. [26] O. Amellem, E.O. Pettersen, Cell inactivation and cell cycle inhibition as induced by extreme hypoxia: the possible role of cell cycle arrest as a protection against hypoxia-induced lethal damage, Cell Prolif. 24 (1991) 127–141. [27] R. Kulshreshtha, M. Ferracin, S.E. Wojcik, R. Garzon, H. Alder, F.J. Agosto-Perez, R. Davuluri, C.G. Liu, C.M. Croce, M. Negrini, G.A. Calin, M. Ivan, A microRNA signature of hypoxia, Mol. Cell. Biol. 27 (2007) 1859–1867. [28] P.J. Hurlin, C. Queva, R.N. Eisenman, Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites, Genes Dev. 11 (1997) 44–58. [29] P.J. Hurlin, J. Huang, The MAX-interacting transcription factor network, Semin. Cancer Biol. 16 (2006) 265–274. [30] H.W. Kim, H.K. Haider, S. Jiang, M. Ashraf, Ischemic preconditioning augments survival of stem cells via miR-210 expression by targeting caspase-8-associated protein 2, J. Biol. Chem. 284 (2009) 33161–33168. [31] F. Zölzer, C. Streffer, Increased radiosensitivity with chronic hypoxia in four human tumor cell lines, Int. J. Radiat. Oncol. Biol. Phys. 54 (2002) 910–920. [32] L. Quero, L. Dubois, N.G. Lieuwes, C. Hennequin, P. Lambin, miR-210 as a marker of chronic hypoxia, but not a therapeutic target in prostate cancer, Radiother. Oncol. 101 (2011) 203–208. [33] Q. Xie, T. Lin, Y. Zhang, J. Zheng, J.A. Bonanno, Molecular cloning and characterization of a human AIF-like gene with ability to induce apoptosis, J. Biol. Chem. 280 (2005) 19673–19681. [34] R.K. Mutharasan, V. Nagpal, Y. Ichikawa, H. Ardehali, microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53-dependent pathways and exerts cytoprotective effects, Am. J. Physiol. Heart Circ. Physiol. 301 (2011) H1519–H1530.