CTGF Promotes Osteosarcoma Angiogenesis by Regulating Mr-543

CAN13199_proof ■ 19 January 2017 ■ 1/10

Cancer Letters xxx (2017) 1e10

55 Contents lists available at ScienceDirect 56 57 Cancer Letters 58 59 60 journal homepage: www.elsevier.com/locate/canlet 61 62 63 Original Article 64 65 1 CTGF promotes osteosarcoma angiogenesis by regulating mR-543/ 66 2 67 3 Q7 angiopoietin 2 signaling 68 4 69 a b b b c 5 Q6 Li-Hong Wang , Hsiao-Chi Tsai , Yu-Che Cheng , Chih-Yang Lin , Yuan-Li Huang , 70 6 Chun-Hao Tsai d, e, Guo-Hong Xu a, Shih-Wei Wang f, Yi-Chin Fong g, h, 71 7 * Chih-Hsin Tang b, c, d, 72 8 73 9 a Department of Orthopedics, Dongyang People's Hospital, Wenzhou Medical University, Dongyang, China 74 10 b Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan c 75 11 Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan d School of Medicine, China Medical University, Taichung, Taiwan 76 12 e Department of Orthopedic Surgery, China Medical University Hospital, Taichung, Taiwan 77 13 f Department of Medicine, Mackay Medical College, New Taipei City, Taiwan 78 g 14 Department of Orthopaedic Surgery, China Medical University Beigang Hospital, Yun-Lin County, Taiwan 79 h Department of Sports Medicine, College of Health Care, China Medical University, Taichung, Taiwan 15 80 16 81 17 82 article info abstract 18 83 19 84 Article history: Osteosarcoma is the most common primary solid tumor of bone. It has a high metastatic potential and 20 85 Received 19 September 2016 occurs predominantly in adolescents and young adults. Angiopoietin 2 (Angpt2) is a key regulator in 21 86 Received in revised form tumor angiogenesis, facilitating tumor growth and metastasis. Connective tissue growth factor (CTGF, 22 23 December 2016 also known as CCN2), is a cysteine-rich protein that has been reported to promote metastasis of oste- 87 23 Accepted 11 January 2017 osarcoma. However, the effect of CTGF on Angpt2 regulation and angiogenesis in human osteosarcoma 88 24 remains largely unknown. We found that overexpression of CTGF in osteosarcoma cells increased Angpt2 89 Keywords: 25 production and induced angiogenesis, in vitro and in vivo. Our ﬁndings demonstrate that CTGF-enhanced 90 26 CTGF d Angiopoietin 2 Angpt2 expression and angiogenesis is mediated by the phospholipase C (PLC)/protein kinase C (PKC ) 91 27 Osteosarcoma signaling pathway. Moreover, endogenous microRNA-543 (miR-543) expression was negatively regulated 92 d ﬁ 28 Angiogenesis by CTGF via the PLC/PKC pathway. We also provide evidence showing clinical signi cance between 93 29 miR-543 CTGF, Angpt2, and miR-543 as well as tumor staging in human osteosarcoma tissue. CTGF may serve as a 94 30 therapeutic target in the process of osteosarcoma metastasis and angiogenesis. 95 © 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY- 31 96 NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 32 97 33 98 34 99 35 100 36 Introduction pro-angiogenic as well as anti-angiogenic factors [6]. Angiopoietin 101 37 2 (Angpt2) plays a key role in angiogenesis [7]. Angpt over- 102 38 Osteosarcoma is the most common primary malignant bone expression is associated with advanced stage hepatocellular and 103 39 tumor in children and adolescents [1]. The treatment of osteosar- gastric cancer [8,9]. However, the effect of Angpt2 in osteosarcoma 104 40 coma has undergone dramatic changes in the past 20 years. Up remains unknown. 105 41 until recently, a 5-year survival of 20% with surgical treatment Connective tissue growth factor (CTGF, also known as CCN2), is a 106 42 alone was considered acceptable. This suggests that 80% of the cysteine-rich protein that regulates cell-ECM interactions and dis- 107 43 patients were presenting with pulmonary metastasis [2]. Much plays multicellular functions including the regulation of cell 108 44 recent research has focused on the role of angiogenesis in osteo- adhesion, migration and apoptosis [2,10]. In cancer cells, CTGF has 109 45 sarcoma proliferation, migration, invasion and metastasis [3e5]. been reported to increase tumor metastasis in breast cancer and 110 46 Tumor angiogenesis has been recognized in the imbalance between chondrosarcoma [11,12], but inhibit cell migration in oral squamous 111 47 cell carcinoma and glioblastoma [13,14]. We previously reported 112 48 that CTGF promotes osteosarcoma migration and metastasis 113 49 through up-regulation of matrix metalloproteinase expression [15] 114 50 * Corresponding author. Graduate Institute of Basic Medical Science, China and enhances resistance to paclitaxel- and cisplatin-induced cell 115 51 Medical University, Taiwan. 116 E-mail address: [email protected] (C.-H. Tang). 52 117 53 http://dx.doi.org/10.1016/j.canlet.2017.01.013 118 54 0304-3835/© 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- 119 nc-nd/4.0/).

Please cite this article in press as: L.-H. Wang, et al., CTGF promotes osteosarcoma angiogenesis by regulating mR-543/angiopoietin 2 signaling, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.01.013 CAN13199_proof ■ 19 January 2017 ■ 2/10

2 L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10

1 apoptosis [16,17]. Nevertheless, the angiogenic effect of CTGF in HUVEC migration and tube formation assay 66 2 osteosarcoma remains unclear. HUVEC cells (1 104 cells/well) were seeded with MV2 complete medium onto 67 3 MicroRNAs (miRNAs) have emerged as crucial players regulating the upper chamber of Transwell system membrane inserts and incubated in the 68 4 the magnitude of gene expression in a variety of organisms [18]. bottom chamber with 50% MV2 complete medium and 50% osteosarcoma cell CM. 69 Migrated HUVECs were stained with 0.05% crystal violet after 24 h and quantiﬁed by 5 MiRNAs are short (z22 nucleotides) non-coding RNA molecules 70 counting the number of stained cells [27]. 6 that participate in many cellular processes and their dysregulation HUVECs were resuspended in a tube formation assay at a density of 5 104/ 71 7 is observed in different human pathologies, including cancer [19]. 100 mL in culture medium (50% MV2 medium and 50% osteosarcoma cell CM) and 72 8 Previous studies have shown that miRNAs inhibit tumor angio- added to the 48-well plates pre-coated with 150 mL matrigel. HUVEC tube formation 73 ﬁ 9 genesis and metastasis through dysregulation of the miRNA/ was photographed after 6 h and quanti ed by counting the tube branches [27]. 74

10 Angpt2 axis [20,21]. miR-543 has been indicated to be involved in Western blotting 75 11 various cancer progression, migration and metastasis [22e24]. 76 Cellular lysates were prepared as according to our previous study [31]. Proteins 12 77 However, the role of miR-543 in osteosarcoma has not yet been were resolved on SDS-polyacrylamide gel electrophoresis then transferred to pol- 13 clarified. yvinyldifluoride membranes. The blot membranes were blocked with 4% non-fat 78 14 We have previously documented that CTGF enhances cell milk for 1 h at room temperature, then incubated overnight with primary anti- 79 15 migration, invasion and drug resistance in human osteosarcoma bodies at 4 C. The blots were then incubated with anti-rabbit or anti-mouse HRP- 80 16 [15e17], which indicates that CTGF facilitates osteosarcoma conjugated secondary antibodies for 1 h at room temperature. Finally, the blots were 81 visualized by enhanced chemiluminescence, using a Fujifilm LAS-3000 chem- 17 metastasis. However, it remains unclear as to whether CTGF in- iluminescence detection system (Fujifilm; Tokyo, Japan). 82 18 creases Angpt2 expression to facilitate angiogenesis in human 83 19 osteosarcoma. In this present study, we examined the effect of Plasmid construction and transfection 84 20 CTGF in Angpt2 expression and angiogenesis, and evaluated the The CTGF cDNA was cloned in our laboratory [16]. Briefly, the full-length of CTGF 85 21 involvement of miRNA in human osteosarcoma cells. was amplified by reverse transcription (RT)-PCR, using Platinum Pfx DNA Polymer- 86 22 ase (Invitrogen, Groningen, Netherlands). The primers for amplified the CTGF were: 87 sense 50-CCAACCATGACCGCCGCCAG-30, and anti-sense 50-TCATGCCATGTCTCCGTA- 23 Materials and methods 0 88 CATCTTCCTG-3 . The PCR product was purified using the Viogene Gel/PCR DNA 24 Materials Isolation System (Viogene, CA, USA) and then cloned into the topoisomerase- 89 25 activated pcDNA3.1-TOPO vector (Invitrogen, Groningen, Netherlands). The vector 90 Rabbit monoclonal antibodies specificforCTGF,b-Actin,p-PLC,PLC,p-PKCd was transfected into cells using Lipofectamine™ 2000 according to the manufac- 26 and PKCd, as well as anti-mouse and anti-rabbit IgG-conjugated horseradish 91 turer's instructions. The neomycin was used as a selection marker. The single colony 27 peroxidase, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, 92 surviving cells were picked and expanded to make clonal cell populations. USA). Rabbit monoclonal antibodies specific for Angpt2 and control IgG were 28 The CTGF-shRNA plasmid was obtained from RNAi Core Facility (Academia 93 purchased from Abcam (Cambridge, MA, USA). The ELISA kit for Angpt2 was ® 29 Sinica, Taipei, Taiwan). The jetPEI -Polyplus-transfection reagent was used to 94 obtained from PerpoTech (Rocky Hill, NJ, USA). The recombinant human CTGF transfect CTGF-shRNA or control-shRNA into cells. After 48 h, the puromycin was 30 was obtained from R&D Systems (Minneapolis, MN, USA). The human osteosar- 95 added to the culture medium as a selection marker. The single colony surviving cells 31 coma tissue array was obtained from Biomax (Rockville, MD, USA). Matrigel was 96 were picked and expanded to make clonal cell populations. 32 purchased from BD Biosciences (Bedford, MA, USA). TRIzol reagent, Lipofect- 97 amine 2000, the MMLV RT kit, miR-543 mimic, and control miRNA were obtained 33 Quantitative real-time polymerase chain reaction (qPCR) 98 34 from Invitrogen (Carlsbad, CA, USA). Small interfering RNAs (siRNAs) against PLC, 99 PKCd, and control were obtained from Dharmacon Research (Lafayette, CO, USA). Total RNA was extracted from osteosarcoma cells by TRIzol reagent. Reverse 35 The TaqMan assay kit and TaqMan MicroRNA Reverse Transcription Kit were transcription of messenger RNA to complementary DNA was performed using MMLV 100 36 obtained from Thermo Fisher Scientific (Grand Island, NY, USA). U73122 and RT kit, followed by qPCR using the TaqMan assay kit. qPCR analysis of miR-543 101 37 other pharmacological inhibitors were purchased from Sigma-Aldrich (St. Louis, expression was performed on the StepOnePlus sequence detection system using 102 38 MO, USA). the TaqMan MicroRNA Reverse Transcription Kit and was normalized to U6 103 expression. The specific forward primer of miR-543 was as follows: 50-AAA- 39 0 0 104 Cell culture CATTCGCGGTGCACTTCTT-3 . Forward and reverse primers for U6 are 5 - 40 CTCGCTTCGGCAGCACATATACTA-30and 50-ACGAATTTGCGTGTCATCCTTGCG-30. 105 41 Human osteosarcoma cell lines MG-63 and 143B as well as human fetal osteo- 106 42 blastic cell line hFOB were purchased from the American Type Cell Culture Collection Plasmid construction and luciferase reporter assay 107 (Manassas, VA, USA). The MG-63 and 143B cells were maintained in Dulbecco's 0 43 fi Wild-type Angpt2-3 -UTR was constructed into the pmirGLO reporter vector 108 modi ed Eagle's medium (DMEM), which was supplemented with 20 mM HEPES, 0 44 m between the NheI and XhoI cutting sites. The sequence of wild-type Angpt2-3 -UTR 109 2 mM glutamine, penicillin (100 U/ml), streptomycin (100 g/ml) and 10% fetal 0 was amplified using PCR with primers (5 -GACAGTTTACAGACGCTGCTGTCA- 45 bovine serum (FBS) at 37 C with 5% CO2. 0 0 110 The hFOB was maintained in a 1∶1 mixture of phenol-free DMEM/Ham's F12 CAACCAAGAATGTT-3 and 5 -AACATTCTTGGTTGTGACAGCAGCGTCTGTAAACTGTC- 46 0 0 111 medium containing 10% FBS supplemented with geneticin (300 mg/ml) and antibi- 3 ). The mutation of Angpt2-3 -UTR was performed by the Quickchange site directed 47 112 otics at 33.5 C, the permissive temperature for the expression of the large T antigen. kit (Stratagene; La Jolla, CA, USA), according to the manufacturer's instructions. The 48 mutagenic oligonucleotide primers were 50-GACAGTTTACAGACGCTGCT GTCA- 113 All experiments with hFOB cells were carried out at the permissive temperature of 0 0 49 33.5 C. CAACCAAGACTGTT-3 and 5 -AACAGTCTTGGTTGTGACAGCAGCGTCTGTAAACTGTC- 114 30. To analyze 30-UTR luciferase activity, cells were transfected with wt-Angpt2-30- 50 Human umbilical vein endothelial cells (HUVEC) (ScienCell Research Labora- 0 115 fl UTR or mt-Angpt2-3 -UTR luciferase plasmids. After 24 h, cell lysates were harvested 51 tories, San Diego, CA, USA) were grown to con uence on 1% gelatin and main- 116 tained in MV2 complete medium consisting of MV2 basal medium and growth and detected using the luciferase assay system (Promega; Madison, WI, USA). 52 supplement (PromoCell, Heidelberg, Germany) supplied in 20% FBS (HyClone, 117 53 Logan, UT, USA). Chick chorioallantoic membrane (CAM) assay 118 54 Fertilized chicken eggs were incubated at 38 C in an 80% humidified atmo- 119 55 Q1 Patients and specimen preparation sphere. On day 7, CM from osteosarcoma cells was mixed with matrigel and 120 56 deposited in the center of the egg. CAM results were analyzed on the fourth day. 121 The study protocol was approved by the Institutional Review Board of China Chorioallantoid membranes were collected for microscopic and photographic 57 Medical University Hospital. All patients gave written consent before enrollment. documentation. At least 10 viable embryos were tested for each treatment. All an- 122 58 Tumor tissue specimens were collected from patients diagnosed with osteosarcoma imal experiments were done in accordance with a protocol approved by the China 123 59 who underwent surgical resection at China Medical University Hospital. Medical University (Taichung, Taiwan) Institutional Animal Care and Use 124 60 Committee. 125 Collection of conditioned medium and ELISA assay 61 126 In vivo matrigel plug assay 62 Osteosarcoma cells were stimulated with CTGF, or pretreated with pharmaco- 127 63 logical inhibitors for 30 min, or pre-transfected with siRNA or miR-543 mimic for Male BALB/c nude mice (4 weeks of age; purchased from the National Laboratory 128 24 h. Cells were then incubated with serum-free medium for 2 days. The medium Animal Center, Taipei, Taiwan) were subcutaneously injected with 0.2 mL matrigel 64 was collected as conditioned medium (CM) and examined for Angpt2 expression by containing 0.2 mL osteosarcoma cell CM. On day 10, matrigel plugs were excised and 129 65 Angpt2 ELISA kit, according to the manufacturer's procedure. used to measure the extent of blood vessel formation by hemoglobin assay. 130

L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10 3

1 In vivo tumor xenograft study on Angpt2 expression and angiogenesis remain largely unknown. 66 2 Four-week-old male BALB/c-nu mice were subcutaneously injected in the right Here, we found that the aggressive osteosarcoma cell line 143B 67 3 flank with 2 106 osteosarcoma cells resuspended in 200 mL of containing 50% [25] expresses higher levels of CTGF and Angpt2 than normal 68 a 4 serum-free DMEN/ -MEM and 50% matrigel. After 6 weeks, the tumor was removed osteoblasts (hFOB) and less invasive MG63 cells (Fig. 1A). The 69 and fixed in 10% formalin, and measured for tumor volume and weight. 5 CTGF-overexpression cell line MG63/CTGF was established, to 70 6 Statistics examine the link between CTGF expression and angiogenesis. 71 7 CTGF and Angpt2 expression were significantly higher in CTGF- 72 Data are expressed as the mean ± standard error. Between-group differences 8 were analyzed using the Student's t-test of variance. The difference was considered expressing cells (MG63/CTGF) than in control cells (MG63/vec- 73 9 significant if the p value was less than 0.05. tor) (Fig. 1B&C). We also observed dramatic increases in HUVEC 74 10 tube formation and migration ability in CM from MG63/CTGF 75 11 Results cells, which was abolished by Angpt2 monoclonal antibody (mAb) 76 12 (Fig. 1D&E). The VEGF-A is other important pro-angiogenesis 77 13 CTGF increases Angpt2 expression in osteosarcoma and promotes factor in osteosarcoma [26]. We found the VEGF-A mAb also 78 14 angiogenesis diminished CTGF-promoted HUVEC tube formation and migration 79 15 (Fig. 1D&E), implying VEGF-A also involved in CTGF-mediated 80 16 In a previous investigation, we found that CTGF promotes angiogenesis. In contrast, small hairpin (sh) RNA-mediated 81 17 human osteosarcoma cell migration [15], but the effects of CTGF knockdown of CTGF in 143B/shCTGF cells markedly reduced 82 18 83 19 84 20 85 21 86 22 87 23 88 24 89 25 90 26 91 27 92 28 93 29 94 30 95 31 96 32 97 33 98 34 99 35 100 36 101 37 102 38 103 39 104 40 105 41 106 42 107 43 108 44 109 45 110 46 111 47 112 48 113 49 114 50 115 51 116 52 117 53 118 54 119 55 120 56 121 57 122 58 123 59 124 60 125 61 Fig. 1. CTGF promotes Angpt2 expression in osteosarcoma and enhances angiogenesis.(AeC) CTGF and Angpt2 expression in indicated cells were examined by western blot and 126 62 ELISA. (D&E) Cell culture media were collected from osteosarcoma cells as CM and then applied to HUVECs for 24 h. The capillary-like structure formation and cell migration were 127 & 63 examined by tube formation and Transwell assays. (F) Chick embryos were incubated with osteosarcoma CM for 4 days, then photographed with a stereomicroscope. (G H) MG63 128 cells were stimulated with CTGF (1e10 ng/ml) for 24 h, and the Angpt2 expression was measured by qPCR, western blot and ELISA. (I) CM solution was added to HUVECs and tube 64 formation activity was examined. (J) CM was also added to the Transwell assay, to examine HUVEC migration activity. Results are expressed as mean ± S.E.M. *p < 0.05 compared 129 65 with MG63/vector group; $p < 0.05 compared with the MG63/CTGF group; #p < 0.05 compared with the 143B/shControl group. 130

4 L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10

1 CTGF and Angpt2 expression, as well as HUVEC tube formation PKCd can occur downstream of PLC [28,29]. We therefore 66 2 and migration ability, to a greater extent as compared with 143B/ examined whether PKCd was involved in CTGF-induced Angpt2 67 3 shControl cells (Fig. 1BeE). CTGF-mediated angiogenesis was expression. Pretreatment of cells with a pan-PKC inhibitor 68 4 further demonstrated by the in vivo CAM assay. We found that CM (GF109203X) or transfecting cells with a speciﬁcPKCd siRNA 69 5 from the MG63/CTGF group promoted angiogenesis, however, CM abolished CTGF-induced Angpt2 expression and HUVEC tube 70 6 from the 143B/shCTGF group decreased angiogenesis in the CAM formation as well as migration (Fig. 2BeD). Increasing phos- 71 7 model (Fig. 1F). In addition, exogenous recombinant Angpt2 phorylation of PLC and PKCd occurred in MG63/CTGF cells and 72 8 rescued CTGF shRNA-inhibited HUVEC tube formation and was inhibited in 143B/shCTGF cells (Fig. 2E). Our results suggest 73 9 migration as well as angiogenesis in CAM model (Fig. 1DeF). that CTGF induces Angpt2 expression and angiogenesis by acti- 74 10 Next, we applied recombinant CTGF protein to the low-CTGF- vating PLC/PKCd pathway in human osteosarcoma cells. Integrin 75 11 producing MG63 cells and observed the impact of exogenous avb3 receptor has been mediated in CTGF-induced cell migration 76 12 CTGF on Angpt2 expression and angiogenesis. We found that CTGF [11]. In a previous report, we found that osteosarcoma cells 77 13 increased mRNA expression, protein level and secretion level of expressed avandb3 integrin receptor [30]. Treatment of osteo- 78 14 Angpt2 in a concentration-dependent manner (Fig. 1G&H). sarcoma cells with avb3 integrin mAb abolished CTGF-induced 79 15 Notably, CM from CTGF-treated osteosarcoma cells enhanced Angpt2 expression (Fig. 2F). Therefore, avb3 integrin receptor is 80 16 HUVEC tube formation and migration in a concentration- involved in CTGF-promoted Angpt2 expression. 81 17 dependent manner (Fig. 1I&J). These data demonstrate that 82 18 CTGF promotes Angpt2 expression and angiogenesis in human CTGF promotes Angpt2 expression and angiogenesis by down- 83 19 osteosarcoma cells. regulating miR-543 expression 84 20 85 21 PLC and PKCd signaling pathways are involved in CTGF-induced Increasing evidence has reported the involvement of miRNAs in 86 22 Angpt2 expression and angiogenesis the angiogenic process and their potential therapeutic applications 87 23 for vascular diseases [19]. We therefore sought to determine which 88 24 PLC has been documented as playing a key role in tumor miRNAs are involved in CTGF-induced Angpt2 expression and 89 25 growth, metastasis, and angiogenesis [27]. To verify whether PLC angiogenesis. We used open-source software (miRWalk, TargetS- 90 26 activation is involved in CTGF-induced Angpt2 expression, we can, and the microRNA.org resource) to search for possible miRNAs 91 27 pretreated cells with a PLC inhibitor (U73122) or PLC siRNA in responsible for Angpt2, We ranked the top 9 miRNAs harbored in 92 28 CTGF-overexpressing cells. Both antagonists abolished CTGF- the binding sites of Angpt2 (Supplementary Fig. S1). miR-543 was 93 29 induced Angpt2 expression (Fig. 2A). Furthermore, they the most down-regulated miRNA in overexpressing-CTGF MG63 94 30 reduced CTGF-enhanced HUVEC tube formation and migration cells (Fig. 3A). Silencing CTGF enhanced miR-543 expression in 95 31 (Fig. 2C&D). In various types of cancers, the phosphorylation of 143B cells (Fig. 3A). Direct application of CTGF into MG63 cells 96 32 97 33 98 34 99 35 100 36 101 37 102 38 103 39 104 40 105 41 106 42 107 43 108 44 109 45 110 46 111 47 112 48 113 49 114 50 115 51 116 52 117 53 118 54 119 55 120 56 121 57 122 58 123 59 124 60 125 61 126 62 Fig. 2. PLC and PKCd signaling pathways are involved in CTGF-induced Angpt2 expression and angiogenesis.(A&B) MG63/CTGF cells were pretreated with indicated phar- 127 63 macological inhibitors or pretransfected with indicated siRNAs for 24 h. The Angpt2 mRNA level was measured by qPCR. CM was added to HUVECs and tube formation activity was 128 assessed (C). CM was also added to the Transwell assay and examined for HUVEC migration activity (D). PLC and PKCd expression in indicated cells were examined by western blot 64 (E). MG63 cells were pretreated with avb3 mAb followed by stimulation with CTGF, the Angpt2 expression was examined by qPCR. Results are expressed as mean ± S.E.M. *p < 0.05 129 65 compared with MG63/vector group; #p < 0.05 compared with the MG63/CTGF group. Q4 130

L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10 5

1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 84 20 85 21 86 22 87 23 88 24 89 25 90 26 91 27 92 28 93 29 94 30 95 31 96 32 97 33 98 34 99 35 Fig. 3. CTGF promotes Angpt2 expression and angiogenesis via downregulation of miR-543. (A) miR-543 expression in osteosarcoma cells was examined by qPCR. (B) MG63 100 36 cells were treated with CTGF (1e10 ng/ml) for 24 h, and miR-543 expression was detected by qPCR. (C&D) MG63/CTGF cells were pretransfected with miR-543 mimic for 24 h. The 101 37 Angpt2 mRNA and protein levels were measured by qPCR, western blot and ELISA. CM was added to HUVECs and tube formation activity was assessed (E). CM was also added to the 102 38 Transwell assay and HUVEC migration activity was examined (F). Chick embryos were incubated with osteosarcoma CM for 4 days, then photographed with a stereomicroscope (G). 103 Results are expressed as mean ± S.E.M. *p < 0.05 compared with MG63/vector group; #p < 0.05 compared with the MG63/CTGF group. 39 104 40 105 41 reduced miR-543 expression in a dose-dependent manner (Fig. 3B). 143B/shCTGF group abolished neovascularization in plugs, as 106 42 To explore miR-543 involvement in CTGF-induced Angpt2 expres- assessed by hemoglobin concentrations (Fig. 5A&B), and reduced 107 43 sion and angiogenesis, miR-543 mimic was used; transfection with microvessel formation, according to an analysis of CD31 expres- 108 44 miR-543 mimic diminished CTGF-induced Angpt2 expression sion (Fig. 5C). Moreover, vessel formation in plugs was increased 109 45 (Fig. 3C&D) and also diminished CTGF-promoted HUVEC tube for- to a greater extent by CM from the overexpressing-CTGF MG63 110 46 mation and migration as well as vessel formation in the CAM model cell line as compared with the MG63/vector group (Fig. 5AeC). In 111 47 (Fig. 3EeG). the tumor-induced angiogenesis model, we observed that 112 48 To determine whether miR-543 regulates Angpt2 expression by silencing CTGF reduced tumor growth in mice (Fig. 5DeF). In 113 0 49 binding to the Angpt2 3 UTR, we constructed luciferase reporter addition, silencing of CTGF expression reduced tumor-induced 114 50 vectors, using the pmiRGLO vector harboring the wild-type angiogenesis (Fig. 5D&G). Ex vivo analysis of excised murine tu- 115 0 51 (pmirGLO-WT; WT) or mutant (pmirGLO-MUT; Mut) 3 UTR of mors showed significantly decreased CD31 and Angpt2 expression 116 52 miR-543 (Fig. 4A), then transfected these vectors into MG63 cells. in the 143B/shCTGF group (Fig. 5H). Overall, these results suggest 117 53 As shown in Fig. 4B, CTGF promoted wild-type but not mutant that silencing CTGF inhibits angiogenesis and tumor growth 118 0 54 Angpt2 3 UTR luciferase activity. In addition, treatment with in vivo. 119 55 U73122 and GF109203X reversed CTGF-mediated miR-543 120 0 56 expression and wt-Angpt2-3 UTR luciferase activity (Fig. 4C&D), Clinical importance of CTGF, Angpt2, and miR-543 in osteosarcoma 121 57 suggesting that miR-543 inhibits the protein expression of Angpt2 patients 122 0 58 via integrating to the 3 UTR region of the human Angpt2 gene 123 59 through PLC/PKCd pathway. To characterize the role of CTGF in tumor angiogenesis in human 124 60 osteosarcoma, we analyzed the expression profile of CTGF and 125 61 Silencing CTGF expression suppresses tumor-induced angiogenesis Angpt2 in specimens obtained from patients with osteosarcoma. 126 62 in vivo Higher CTGF and Angpt2 expression was found in tumor specimens 127 63 than in normal bone (Fig. 6A) and was significantly correlated with 128 64 CTGF-mediated angiogenesis was further demonstrated by the tumor stage (Fig. 6B and C). Analysis of the IHC results revealed a 129 65 in vivo Matrigel plugs assay. Matrigel mixed with CM from the high positive relationship between CTGF and Angpt2 expression in 130

6 L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10

1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 84 20 85 21 86 22 87 23 88 24 89 25 90 26 91 27 92 28 93 29 94 30 95 0 31 Fig. 4. miR-543 directly represses Angpt2 expression through binding to the 3′UTR of the human Angpt2. (A) Schematic representation of the 3 UTR of the human Angpt2 96 containing a miR-543 binding site. (B) Cells were co-transfected with miR-543 mimic or miRNA control and wt-Angpt2-30UTR or mt-Angpt2-30UTR plasmid for 24 h, and the relative 32 97 luciferase activities were measured. (C&D) Cells were pretreated with indicated pharmacological inhibitors for 24 h, then miR-543 expression and Angpt2 promoter luciferase 33 activity were examined. Results are expressed as mean ± S.E.M. *p < 0.05 compared with MG63/vector group; #p < 0.05 compared with the MG63/CTGF group. 98 34 99 35 100 36 101 37 osteosarcoma cases (Fig. 6D). Similar results were observed with neck, as well as oral cancers [13,14,36]. In osteosarcoma, we have 102 38 CTGF and Angpt2 mRNA expression (Fig. 6E&F). Moreover, miR-543 previously indicated that CTGF enhances osteosarcoma progression 103 39 was downregulated (Fig. 6G) High Angpt2 expression was signifi- and metastasis [15]. Here, we have identified a further effect of 104 40 cantly associated with both clinical stage and metastasis (Table 1). CTGF in osteosarcoma angiogenesis; the upregulation of Angpt2 105 41 expression. Similar reports have implied that CTGF plays a role in 106 42 Discussion angiogenesis by increasing other angiogenic factors such as VEGF in 107 43 many cell types [37e39]. We also found that silencing CTGF 108 44 Osteosarcoma is malignant bone tumor with high morbidity expression reduced tumor growth and angiogenesis in vivo, which 109 45 that occurs predominantly in children and adolescents. Metastases suggests that inhibiting CTGF expression is a potential target for 110 46 in patients represent the most common cause of death [5,31]. osteosarcoma treatment. The CTGF mAb FG-3019 inhibits tumor 111 47 Angiogenesis in the osteosarcoma microenvironment facilitates growth and metastases in pancreatic cancer [40]. Future research is 112 48 tumor progression and metastasis [32,33]. It has been recognized needed to evaluate the effectiveness of FG-3019 in metastatic 113 49 that angiogenesis may be a potential therapeutic target for osteo- osteosarcoma. 114 50 sarcoma treatment [34]. Angpt2 is a key factor for tumor angio- Evidence indicates that PLC, a potential candidate signaling 115 51 genesis [35], however, the role of Angpt2 in osteosarcoma remains pathway, mediates cancer invasion, metastasis and angiogenesis 116 52 unclear. In this present study, we identified that Angpt2 expression [41]. Here, we report that both a PLC inhibitor and siRNA antago- 117 53 is associated with both clinical stage and metastasis in human os- nized CTGF-promoted production of Angpt2. These agonists also 118 54 teosarcoma. We found that the Angpt2 mAb blocked CTGF- diminished CTGF-increased tube formation and migration in 119 55 promoted tube formation and migration in HUVECs, which sug- HUVEC cells. Overexpression of CTGF promoted phosphorylation of 120 56 gests that Angpt2 is a key regulator for CTGF-mediated angiogen- PLC, suggesting that PLC activation plays a crucial role in CTGF- 121 57 esis. Our evidence shows that CTGF enhances the expression and induced Angpt2 production and angiogenesis. PKCd activation is 122 58 secretion of angiogenic factor Angpt2 by inhibiting miR-543 via an important downstream event of PLC signaling [42]. In the cur- 123 59 PLC/PKCd pathway in human osteosarcoma cells, and subsequently rent study, inhibition of PKCd by a pharmacological inhibitor or 124 60 enhances angiogenesis, indicating that CTGF and miR-543 may be genetic siRNA reduced Angpt2 production as well as HUVEC 125 61 novel molecular targets for restricting Angpt2-mediated angio- migration and tube formation. We also found that overexpression 126 62 genesis in the osteosarcoma microenvironment. of CTGF enhanced PKCd phosphorylation, while silencing of CTGF 127 63 The biphasic nature of CTGF has been fully discussed: CTGF diminished phosphorylation of PKCd. These findings show that the 128 64 enhances tumor migration or metastasis in breast and chon- PLC/PKCd pathway plays an essential role in CTGF-increased 129 65 drosarcoma [11,12], but reduces cancer metastasis in lung, head and Angpt2 expression and angiogenesis in osteosarcoma cells. 130

L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10 7

1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 84 20 85 21 86 22 87 23 88 24 89 25 90 26 91 27 92 28 93 29 94 30 95 31 96 32 97 33 98 34 99 35 100 36 101 37 102 38 103 39 104 40 105 41 106 42 107 43 108 44 109 45 110 46 111 47 112 48 113 49 114 50 115 51 Fig. 5. Silencing CTGF expression inhibits tumor-induced angiogenesis in vivo.(AeC) Mice were injected subcutaneously with matrigel mixed with osteosarcoma CM for 7 days, 116 fi e 52 after which time the plugs were excised, photographed, immunostained with CD31 antibody and quanti ed for hemoglobin content. (D H) At 28 days after mice were injected with 117 indicated cells, tumors were measured for size and weight, then embedded in paraffin. Sections were immunostained using CD31 and Angpt2 antibodies. Results are expressed as 53 mean ± S.E.M. *p < 0.05 compared with MG63/vector group; #p < 0.05 compared with the 143B/shControl group. 118 54 119 55 120 56 121 57 MiRNAs are short non-coding RNAs that function as negative Angpt2, and to rank the top 9 miRNAs harboring Angpt2 binding 122 58 regulators of gene expression [18], which is vital in homeostasis sites. miR-543 was downregulated most frequently in cells over- 123 59 and disease, including cancer development [19]. MiR-543 has been expressing CTGF. In osteosarcoma patients, miR-543 expression is 124 60 reported to be dysregulated in several human cancers [43]. How- negatively correlated with CTGF or Angpt2. Transfection of osteo- 125 61 ever, the function and mechanism of miR-543 in human osteosar- sarcoma cells with miR-543 mimic reduced CTGF-enhanced Angpt2 126 62 coma remains unclear. In the present study, we used open-source expression as well as tube formation and migration in HUVEC cells. 127 63 software (miRWalk, TargetScan, and the microrna.org resource) in Our results highlight the significance of miR-543 in CTGF-induced 128 64 an attempt to determine which miRNAs regulate the expression of Angpt2 expression and angiogenesis. We suggest that restoration 129 65 130

8 L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10

1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 84 20 85 21 86 22 87 23 88 24 89 25 90 26 91 27 92 28 93 29 94 30 95 31 96 32 97 33 98 34 99 35 100 36 101 37 102 38 103 39 104 40 105 41 106 42 107 43 108 44 109 45 110 46 111 47 112 48 113 49 114 50 115 51 116 52 117 53 118 54 119 55 120 56 121 57 122 58 123 59 124 Fig. 6. CTGF, Angpt2 and miR-543 expression have important clinical signiﬁcance in osteosarcoma patients. (A) Human normal bone or osteosarcoma tissues were IHC stained 60 125 with anti-CTGF or Angpt2 antibody. The quantitative data are shown in (B&C). (D) The correlation of CTGF, Angpt2 and tumor stage. (EeG) mRNA expression of CTGF, Angpt2 and 61 miR-543 in normal bone and osteosarcoma patients were examined by qPCR. Results are expressed as mean ± S.E.M. *p < 0.05 compared with normal bone group. 126 62 127 63 128 64 129 65 130

L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10 9

1 Table 1 [12] P.S. Chen, M.Y. Wang, S.N. Wu, J.L. Su, C.C. Hong, S.E. Chuang, et al., CTGF 66 2 Correlation of Angpt-2 expression with clinical status in osteosarcoma. enhances the motility of breast cancer cells via an integrin-alphavbeta3- 67 ERK1/2-dependent S100A4-upregulated pathway, J. Cell Sci. 120 (2007) 3 Characteristic Angpt2 low (n ¼ 15) Angpt2 high (n ¼ 27) p-value 2053e2065. 68 4 [13] J.Y. Chuang, W.Y. Yang, C.H. Lai, C.D. Lin, M.H. Tsai, C.H. Tang, CTGF inhibits cell 69 Age (year) motility and COX-2 expression in oral cancer cells, Int. Immunopharmacol. 11 5 <24 5 10 0.8103 70 (2011) 948e954. > 6 25 10 17 [14] L.F. Romao, F.A. Mendes, N.M. Feitosa, J.C. Faria, J.M. Coelho-Aguiar, J.M. de 71 7 Gender Souza, et al., Connective tissue growth factor (CTGF/CCN2) is negatively 72 8 Female 9 7 0.0293* regulated during neuron-glioblastoma interaction, Plos One 8 (2013) 73 Male 6 20 e55605. 9 Stage [15] H.C. Tsai, H.L. Su, C.Y. Huang, Y.C. Fong, C.J. Hsu, C.H. Tang, CTGF increases 74 10 I A 3 0 0.0219* matrix metalloproteinases expression and subsequently promotes tumor 75 11 II A 6 7 metastasis in human osteosarcoma through down-regulating miR-519d, 76 e 12 II B 6 14 Oncotarget 5 (2014) 3800 3812. 77 IVB 0 6 [16] H.C. Tsai, C.Y. Huang, H.L. Su, C.H. Tang, CCN2 enhances resistance to cisplatin- 13 78 Tumor status mediating cell apoptosis in human osteosarcoma, Plos One 9 (2014) e90159. 14 T1 7 9 0.3939 [17] H.C. Tsai, C.Y. Huang, H.L. Su, C.H. Tang, CTGF increases drug resistance to 79 paclitaxel by upregulating survivin expression in human osteosarcoma cells, 15 T2eT3 8 18 80 Biochim. Biophys. Acta 1843 (2014) 846e854. Distant metastasis 16 [18] N. Bushati, S.M. Cohen, microRNA functions, Annu. Rev. Cell Dev. Biol. 23 81 17 M0 15 21 0.0486* (2007) 175e205. 82 M1 0 6 [19] Y. Suarez, W.C. Sessa, MicroRNAs as novel regulators of angiogenesis, Circ. Res. 18 e 83 Q5 p < 0.05. 104 (2009) 442 454. 19 [20] W.J. Cao, J.D. Rosenblat, N.C. Roth, M.A. Kuliszewski, P.N. Matkar, D. Rudenko, 84 20 et al., Therapeutic angiogenesis by ultrasound-mediated MicroRNA-126-3p 85 21 delivery, Arterioscler. Thromb. Vasc. Biol. 35 (2015) 2401e2411. 86 of miR-543 may be a potential therapeutic strategy for osteosar- [21] T. He, F. Qi, L. Jia, S. Wang, N. Song, L. Guo, et al., MicroRNA-542-3p inhibits 22 87 coma angiogenesis. tumour angiogenesis by targeting angiopoietin-2, J. Pathol. 232 (2014) 23 499e508. 88 24 [22] J. Sun, J. Zhou, M. Dong, W. Sheng, Dysregulation of MicroRNA-543 expression 89 Acknowledgments in colorectal cancer promotes tumor migration and invasion, Mol. Carcinog. 25 90 (2016). Q3 26 91 This work was supported by grants from the Ministry of Science [23] C. Fan, Y. Lin, Y. Mao, Z. Huang, A.Y. Liu, H. Ma, et al., MicroRNA-543 sup- 27 presses colorectal cancer growth and metastasis by targeting KRAS, MTA1 and 92 and Technology of Taiwan (MOST103-2628-B-039-002-MY3; 105- e 28 HMGA2, Oncotarget 7 (2016) 21825 21839. 93 2320-B-039-015-MY3) and China Medical University (CMU 105- [24] J. Li, G. Dong, B. Wang, W. Gao, Q. Yang, miR-543 promotes gastric cancer cell 29 94 ASIA-20). proliferation by targeting SIRT1, Biochem. Biophys. Res. Commun. 469 (2016) 30 15e21. 95 31 [25] D.S. Geller, R. Gorlick, Osteosarcoma: a review of diagnosis, management, and 96 Q2 Conflict of interest treatment strategies, Clin. Adv. Hematol. Oncol. 8 (2010) 705e718. 32 [26] S.W. Wang, S.C. Liu, H.L. Sun, T.Y. Huang, C.H. Chan, C.Y. Yang, et al., CCL5/ 97 33 CCR5 axis induces vascular endothelial growth factor-mediated tumor 98 The authors declare that they have no conflicts of interest. 34 angiogenesis in human osteosarcoma microenvironment, Carcinogenesis 36 99 (2015) 104e114. 35 100 Appendix A. Supplementary data [27] L. Cocco, M.Y. Follo, L. Manzoli, P.G. Suh, Phosphoinositide-specific phospho- 36 lipase C in health and disease, J. Lipid Res. 56 (2015) 1853e1860. 101 37 [28] H.S. Yu, S.W. Wang, A.C. Chang, H.C. Tai, H.I. Yeh, Y.M. Lin, et al., Bradykinin 102 Supplementary data related to this article can be found at http:// promotes vascular endothelial growth factor expression and increases 38 103 dx.doi.org/10.1016/j.canlet.2017.01.013. angiogenesis in human prostate cancer cells, Biochem. Pharmacol. 87 (2014) 39 243e253. 104 40 [29] J.Y. Chuang, W.H. Yang, H.T. Chen, C.Y. Huang, T.W. Tan, Y.T. Lin, et al., CCL5/ 105 References CCR5 axis promotes the motility of human oral cancer cells, J. Cell Physiol. 220 41 (2009) 418e426. 106 42 [1] D.V. Rogozhin, I.V. Bulycheva, D.M. Konovalov, A.G. Talalaev, V.Y. Roshchin, [30] C.L. Wu, H.C. Tsai, Z.W. Chen, C.M. Wu, T.M. Li, Y.C. Fong, et al., Ras activation 107 43 A.P. Ektova, et al., Classical osteosarcoma in children and adolescent, Arkh mediates WISP-1-induced increases in cell motility and matrix metal- 108 44 Patol. 77 (2015) 68e74. loproteinase expression in human osteosarcoma, Cell. Signal. 25 (2013) 109 [2] P.C. Chen, H.C. Cheng, S.F. Yang, C.W. Lin, C.H. Tang, The CCN family proteins: 2812e2822. 45 modulators of bone development and novel targets in bone-associated tu- [31] H.F. Chen, K.J. Wu, Epigenetics, TET proteins, and hypoxia in epithelial- 110 46 mors, Biomed. Res. Int. 2014 (2014), 437096. mesenchymal transition and tumorigenesis, Biomedicine (Taipei) 6 (2016) 1. 111 47 [3] F. Giner, J.A. Lopez-Guerrero, I. Machado, Z. Garcia-Casado, A. Peydro-Olaya, [32] J. PosthumaDeBoer, M.A. Witlox, G.J. Kaspers, B.J. van Royen, Molecular al- 112 A. Llombart-Bosch, The early stages of tumor angiogenesis in human osteo- terations as target for therapy in metastatic osteosarcoma: a review of liter- 48 sarcoma: a nude mice xenotransplant model, Virchows Arch. 467 (2015) ature, Clin. Exp. Metastasis 28 (2011) 493e503. 113 49 193e201. [33] Y.Y. Liao, H.C. Tsai, P.Y. Chou, S.W. Wang, H.T. Chen, Y.M. Lin, et al., CCL3 114 50 [4] J.C. Chen, Y.C. Fong, C.H. Tang, Novel strategies for the treatment of chon- promotes angiogenesis by dysregulation of miR-374b/VEGF-A axis in human 115 drosarcomas: targeting integrins, Biomed. Res. Int. 2013 (2013), 396839. osteosarcoma cells, Oncotarget 7 (2016) 4310e4325. 51 [5] L.C. Chang, Y.L. Yu, Dietary components as epigenetic-regulating agents [34] F. Uehara, Y. Tome, S. Miwa, Y. Hiroshima, S. Yano, M. Yamamoto, et al., Os- 116 52 against cancer, Biomedicine (Taipei) 6 (2016) 2. teosarcoma cells enhance angiogenesis visualized by color-coded imaging in 117 53 [6] P. Carmeliet, R.K. Jain, Molecular mechanisms and clinical applications of the in vivo Gelfoam(R) assay, J. Cell Biochem. 115 (2014) 1490e1494. 118 e [35] M. Tampellini, C. Sonetto, G.V. Scagliotti, Novel anti-angiogenic therapeutic 54 angiogenesis, Nature 473 (2011) 298 307. 119 [7] N.M. Biel, D.W. Siemann, Targeting the Angiopoietin-2/Tie-2 axis in strategies in colorectal cancer, Expert Opin. Investig. Drugs 25 (2016) 55 conjunction with VEGF signal interference, Cancer Lett. (2014). 507e520. 120 56 [8] B. Veres, B. Radnai, F. Gallyas Jr., G. Varbiro, Z. Berente, E. Osz, et al., Regulation [36] M.H. Yang, B.R. Lin, C.H. Chang, S.T. Chen, S.K. Lin, M.Y. Kuo, et al., Connective 121 tissue growth factor modulates oral squamous cell carcinoma invasion by 57 of kinase cascades and transcription factors by a poly(ADP-ribose) polymer- 122 ase-1 inhibitor, 4-hydroxyquinazoline, in lipopolysaccharide-induced activating a miR-504/FOXP1 signalling, Oncogene 31 (2012) 2401e2411. 58 inflammation in mice, J. Pharmacol. Exp. Ther. 310 (2004) 247e255. [37] D.R. Brigstock, Regulation of angiogenesis and endothelial cell function by 123 59 [9] S.F. Hackett, H. Ozaki, R.W. Strauss, K. Wahlin, C. Suri, P. Maisonpierre, et al., connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61), Angio- 124 e 60 Angiopoietin 2 expression in the retina: upregulation during physiologic and genesis 5 (2002) 153 165. 125 pathologic neovascularization, J. Cell Physiol. 184 (2000) 275e284. [38] F. Yang, J.A. Tuxhorn, S.J. Ressler, S.J. McAlhany, T.D. Dang, D.R. Rowley, 61 [10] A. Dhar, A. Ray, The CCN family proteins in carcinogenesis, Exp. Oncol. 32 Stromal expression of connective tissue growth factor promotes angiogenesis 126 62 (2010) 2e9. and prostate cancer tumorigenesis, Cancer Res. 65 (2005) 8887e8895. 127 63 [11] T.W. Tan, C.H. Lai, C.Y. Huang, W.H. Yang, H.T. Chen, H.C. Hsu, et al., CTGF [39] S.C. Liu, S.M. Chuang, C.J. Hsu, C.H. Tsai, S.W. Wang, C.H. Tang, CTGF increases 128 enhances migration and MMP-13 up-regulation via alphavbeta3 integrin, FAK, vascular endothelial growth factor-dependent angiogenesis in human syno- 64 ERK, and NF-kappaB-dependent pathway in human chondrosarcoma cells, vial fibroblasts by increasing miR-210 expression, Cell Death Dis. 5 (2014) 129 65 J. Cell Biochem. 107 (2009) 345e356. e1485. 130

10 L.-H. Wang et al. / Cancer Letters xxx (2017) 1e10

1 [40] N. Dornhofer, S. Spong, K. Bennewith, A. Salim, S. Klaus, N. Kambham, et al., [42] J.S. Lee, E.J. Yang, I.S. Kim, The roles of MCP-1 and protein kinase C delta 7 ﬁ 2 Connective tissue growth factor-speci c monoclonal antibody therapy in- activation in human eosinophilic leukemia EoL-1 cells, Cytokine 48 (2009) 8 hibits pancreatic tumor growth and metastasis, Cancer Res. 66 (2006) 186e195. 3 5816e5827. [43] B. Teferedegne, J. Macauley, G. Foseh, E. Dragunsky, K. Chumakov, H. Murata, 9 4 [41] R. Lattanzio, M. Piantelli, M. Falasca, Role of phospholipase C in cell invasion et al., MicroRNAs as potential biomarkers for VERO cell tumorigenicity, Vac- 10 5 and metastasis, Adv. Biol. Regul. 53 (2013) 309e318. cine 32 (2014) 4799e4805. 11 6 12