European Review for Medical and Pharmacological Sciences 2016; 20: 3011-3016 HIFs-MiR-33a-Twsit1 axis can regulate invasiveness of hepatocellular cancer cells X.-F. GUO1, A.-Y. WANG2, J. LIU3 1Department of Infectious Liver Diseases, Zaozhuang Municipal Hospital, Zaozhuang, Shandong, China 2Endoscopy Room, Shouguang People’s Hospital, Weifang, Shandong, China 3Department of Oncology, Taixing People’s Hospital, Taixing, Jiangsu, China Xiangfei Guo and Aiyan Wang are co-first authors Abstract. – OBJECTIVE: In this study, we in- including hepatocellular carcinoma (HCC)1. The vestigated whether miR-33a downregulation in chronic hypoxia exerts strong selective pressure HCC is a result of hypoxia-inducible factors to tumor cells and changes cell behaviors such (HIFs) overexpression. Then, we further studied as extracellular matrix remodeling, increased mi- the regulative effects of miR-33a on Twist1 and 2,3 their regulation in HCC cell invasiveness. gratory and metastatic potential . Hypoxia-in- MATERIALS AND METHODS: Human hepato- ducible factors (HIFs) are among the most im- cellular cancer (HCC) cell lines (HepG2 and BEL- portant transcriptional regulators regulating the 7402) were transfected with miR-33a mimics, HIFs cellular response to hypoxia4. In human, three siRNA or Twist1 siRNA. MiR-33a level was mea- isoforms of HIFα, including HIF-1a, HIF-2a, and sured using QRT-PCR. The binding between miR- HIF-3a are involved in hypoxic responses5. 33a and Twist1 3’UTR was verified using Western MiRNA are conserved small noncoding RNA, blot analysis and dual luciferase assay. E-cadher- in and N-cadherin expression levels were detect- which negatively regulate gene expression via ba- ed by western blot analysis. Tumor cell invasion se-pairing with a complementary sequence within was assessed using transwell assay. mRNAs6,7. Previous studies found that hypoxia RESULTS: MiR-33a downregulation in HCC could induce changes in miRNA expression and cells is hypoxia-induced and is a result of HIFs subsequent tumor cell behaviors via HIFs media- upregulation. HIF-1α and HIF-2α suppression ted mechanisms. For example, miR-210 can be partly rescued miR-33a expression under hy- 8 poxia. Both HepG2 and BEL-7402 cells with miR- induced via a HIF-1 or HIF-2 dependent manner 33a overexpression had significantly decreased and miR-210 augments the metastatic potential E-cadherin expression and increased N-cadher- of HCC cells by targeting vacuole membrane in level. Transwell analysis confirmed that miR- protein9 and tissue inhibitor of metalloproteina- 33a overexpression significantly suppressed ses 2 (TIMP2)10. Hypoxia-suppressed miR-199a the tumor cell invasion capability. Twist1 is a di- can inhibit glycolysis in HCC cells by targeting rect target of miR-33a in HCC. HepG2 cells with hexokinase-2 (Hk2) and pyruvate kinase-M2 Twist1 knockdown had significantly increased 11 E-cadherin, decreased N-cadherin and sup- (Pkm2) . The MiR-30c expression is inhibited by pressed invasion capability. hypoxia in a HIFs-dependent manner and promo- CONCLUSIONS: MiR-33a downregulation in tes epithelial-mesenchymal transition (EMT) in HCC cells is hypoxia-induced and is a result of human renal cell carcinoma12. HIFs upregulation. MiR-33a can modulate EMT MiR-33a is a tumor suppressive miRNA in and invasion of hepatocellular cancer cells at HCC. One recent study found that miR-33a is least partly via downregulating Twist1. usually downregulated in HCC cells13. Functio- Key Words: nally, miR-33a can directly target β-catenin and HIFs, miR-33a, Twsit1, Invasion, Hepatocellular cancer. downregulate its expression, thereby weakening the β-catenin signaling pathway and inhibiting cell growth13. However, how it is downregulated Introduction in HCC and whether other downstream targets are involved in its tumor suppressive effects are Hypoxic mass within the neoplastic mass is not clear. In this study, we investigated whether one of the key features of solid malignant tumors, miR-33a downregulation in HCC is a result of Corresponding Author: Jun Liu, MD; e-mail: [email protected] 3011 X.-F. Guo, A.-Y. Wang, J. Liu HIFs overexpression. In addition, by performing denatured and then 25 μg proteins were loaded in bioinformatics analysis, we found that Twist1, each lane for separation using SDS-PAGE with which is an EMT-promoting transcription fac- 10% acrylamide gels. After that, the proteins were tor14,15 is a possible target of miR-33a. Therefore, transferred to nitrocellulose membrane. The pri- we further studied the regulative effects of miR- mary antibodies used include anti-HIF-1α (1:1000, 33a on Twist1 and their regulation in HCC cell ab82832, Abcam, Cambridge, UK), anti-HIF-2α invasiveness. (1:1000, ab73895, Abcam), anti-E-cadherin (1:1000, #3195, Cell Signaling, Danvers, MA, USA), an- Materials and Methods ti-N-cadherin (1:1000, #13116, Cell Signaling, Dan- vers, MA, USA) and anti-Twist1 (1: 2000, ab175430) Cell Culture and Treatment and anti-β-actin (ab3280, Abcam). The signals were HCC cell line HepG2 and BEL-7402 were visualized by using the ECL Western blotting sub- obtained from the Cell Bank of the Chinese Aca- strate (Promega, Madison, WI, USA) and the gray demy of Sciences (Shanghai, China) and were scale of the protein bands were quantified by using cultured in Eagle’s minimum essential medium the Image-J software. supplemented with 10% fetal-bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. All Transwell Analysis of Cell Invasion 5 cells were cultured in 37°C with 5% CO2 in humi- In brief, 24 hours after transfection, 1 × 10 dified atmosphere. For hypoxic treatment,oxygen cells were placed into chambers coated with 150 supply was set to 1%. HepG2 and BEL-7402 were μg of Matrigel. The chambers were then inserted cultured under hypoxic condition for 12 or 24 into the wells of a 24-well plate and incubated hours and then subjected to Western blot analysis for 48 hours in Roswell Park Memorial Institu- of HIF-1α and HIF-2α expression and qRT-PCR te-1640 (RPMI-1640) medium with 20% fetal analysis of miR-33a expression. bovine serum. Then, The invading cells on the MiR-33a mimics, HIF-1α and HIF-2α siRNA bottom side were fixed with 4% polyoxymethy- (si-HIFs), Twist1 siRNA and the corresponding lene and stained with 0.1% crystal violet. Cell negative controls were purchased from Ribobio counting was performed at 100× magnification (Guangzhou, China). HepG2 and BEL-7402 cells under a microscope. were transfected with 100 nM miR-33a, 100 nM Twist1 siRNA, or 50 nM HIF-1α siRNA and 50 nM Dual Luciferase Assay HIF-2α siRNA in combination using Lipofectamine Based on bioinformatics data, two short sequen- 2000 (Invitrogen, Carlsbad, CA, USA) according to ces of oligonucleotides containing the predicted the manufacturer’s protocol. HepG2 and BEL-7402 targeting sites between miR-33a and 3’UTR of cells transfected with si-HIFs were treated under Twist1 and the corresponding mutant sequences hypoxic condition for 24 hours and then subjected were chemically synthesized. The sequences were, to qRT-PCR analysis of miR-33a expression. then, cloned into the downstream of the luciferase gene of pmirGLO Dual-Luciferase miRNA Tar- QRT-PCR Analysis get Expression Vector (Promega, Madison, WI, Total RNA from cells samples was extracted USA). The recombinant vectors were named as using TRIzol reagent (Invitrogen, Carlsbad, CA, pGLO-Twist1-WT and pGLO-Twist1-MT respecti- USA). Then, MiRNAs specific cDNA was syn- vely. HepG2 cells were co-transfected with 200 thesized using stem-loop primers and the Taq- ng luciferase reporter vector and 100 nM miR-33a Man MicroRNA Reverse Transcription Kit (Ap- mimics or the negative controls. Luciferase activity plied Biosystems, Foster City, CA, USA). After was examined 24 hours after the transfection using the reverse transcription, mature miR-33a expres- the Dual-Luciferase Assay kit (Promega) according sion level was quantified using qRT-PCR analysis to manufacturer’s instruction. with TaqMan MicroRNA Assay Kit (Applied Biosystems). The relative expression of miR-33a Statistical Analysis was calculated using the 2-ΔΔCt method. The statistical analysis was performed using Graphpad Prism 5.1. Data were presented in the Western Blot Analysis form of means ± standard deviation based on at Conventional Western blot analysis was perfor- least three repeats. The comparison was perfor- med. The cancer cells were collected and lyzed for med using the unpaired t-test. p-value of <0.05 protein extraction. Then, the protein samples were was considered as statistically significant. 3012 HIFs-MiR-33a-Twsit1 axis can regulate invasiveness of hepatocellular cancer cells Figure 1. Hypoxia-induced significant downregulation of miR-33a in HCC cells. A-B. Western blot analysis of HIF-1α and HIF-2α levels (A) and qRT-PCR analysis of miR-33a levels (B) in HepG2 and BEL-7402 cells after 0, 12 and 24 hours hypoxic culture. C-D. Western blot analysis of HIF-1α and HIF-2α levels (C) and qRT-PCR analysis of miR-33a levels (B) in HepG2 and BEL-7402 cells with or without transfection of HIF-1α siRNA (50 nM) and HIF-2α siRNA (50 nM) in combination 24 hours after hypoxic culture. N: normoxia; H: hypoxia; *p<0.05; **p<0.01. Results MiR-33a Can Modulate EMT and Inva- sion of Hepatocellular Cancer Cells Hypoxia-Induced Significant One previous study13 reported that miR-33a has Downregulation of miR-33a in HCC Cells inhibitive effects on carcinogenesis of HCC via de- Previous studies16,17 reported that HIF-1α and creasing β-catenin expression and thereby weake- HIF-2α expression is associated with poor pro- ning the Wnt/β-Catenin signaling pathway. In this gnosis of hepatocellular carcinoma. In this in- work, we further investigated the role of miR-33a vestigation, we firstly examined the level of in HCC cells. HepG2 and BEL-7402 cells were fir- HIF-1α and HIF-2α expression in HCC cell lines stly transfected for miR-33a overexpression (Figure under hypoxic conditions.