INTERNATIONAL JOURNAL OF MOleCular meDICine 42: 489-496, 2018 lncRNA PVT1 promotes the angiogenesis of vascular endothelial cell by targeting miR‑26b to activate CTGF/ANGPT2 JIFU ZHENG1,2*, LILI HU1,3*, JING CHENG2, JING XU2, ZHIQIANG ZHONG2, YUAN YANG2 and ZHENG YUAN2 Departments of 1Cardiovascular Medicine, 2Hematology and 3Nephrology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China Received September 12, 2017; Accepted March 22, 2018 DOI: 10.3892/ijmm.2018.3595 Abstract. Angiogenesis is essential for various biological migration and in vitro vascular tube formation of HUVECs processes, including tumor blood supply delivery, cancer cell via binding 3'‑UTR regions and downregulating CTGF and growth, invasion and metastasis. Plasmacytoma variant trans- ANGPT2 expression levels. The current findings may improve location 1 (PVT1) long noncoding RNA (lncRNA) has been the understanding of the underlying mechanism of PVT1 previously reported to affect angiogenesis of glioma micro- contributing to angiogenesis of vascular endothelial cells vascular endothelial cells by regulating microRNA (miR)-186 and offer rationale for targeting PVT1 to treat angiogenesis expression level. However, the specific underlying molecular dysfunction-associated diseases, including cancer metastasis. mechanism of PVT1 regulation of angiogenesis in vascular endothelial cells remains to be elucidated. The present study Introduction investigated the role of PVT1 in cell proliferation, migration and vascular tube formation of human umbilical vein endothe- A previous study suggested that various biological processes lial cells (HUVECs) using MTT assay, Transwell migration are determined by the regulatory potential of the noncoding assay and in vitro vascular tube formation assay, respectively. portions of the genome. It has been previously estimated that In order to determine the effect of miR-26b on cell prolif- ~1.5% of the genome is responsible for protein coding, whereas eration, migration and vascular tube formation of HUVECs, a number of noncoding regulatory elements are transcribed miR-26 mimic or miR-26b inhibitor were transfected into into noncoding RNA (ncRNA) (1). Long noncoding (lncRNAs) HUVECs. Reverse transcription-quantitative polymerase (>200 nucleotides) are a novel form of ncRNAs, which have chain reaction and western blotting were conducted to quantify been identified to exert their gene transcription regulatory effect the mRNA and protein expression levels of target genes. The through the epigenetic regulatory mechanism. LncRNA plas- present study confirmed that miR‑26b bound 3'‑untranslated macytoma variant translocation 1 (PVT1) has been reported to region (3'‑UTR) and subsequently influenced gene expression be associated with cell proliferation, invasion and metastasis, level using dual luciferase reporter assay. The current study apoptosis and tumor prognosis (2-7). Although, some previous observed that PVT1 affected cell proliferation, migration and studies have revealed that lncRNAs such as MEG3 maternally in vitro vascular tube formation of HUVECs. In addition, expressed 3, HOX transcript antisense RNA had an important it was determined that PVT1 was able to bind and degrade role in angiogenesis by directly regulating vascular endothe- miR-26b to promote connective tissue growth factor (CTGF) lial growth factor (VEGF) and angiopoietin 2 (ANGPT2); and angiopoietin 2 (ANGPT2) expression. miR-26b was also however, whether PVT1 had a similar function remains to be identified to have a suppressive role in cell proliferation, elucidated (8-10). A recent study by Ma et al (11) observed that PVT1 regulated growth, migration and angiogenesis of glioma microvascular endothelial cells by targeting microRNA (miR)-186; therefore, it should be considered if other pathways or downstream targets mediate PVT1 regulation of angiogen- Correspondence to: Dr Lili Hu, Department of Cardiovascular esis in vascular endothelial cells. Medicine and Nephrology, The Second Affiliated Hospital of MicroRNA is a class of ~22 nucleotide noncoding RNAs. Nanchang University, 1 Minde Road, Nanchang, Jiangxi 330006, They may regulate gene expression through recognizing P. R. Ch i na E‑mail: [email protected] and binding to the 3'‑untranslated region (3'‑UTR) of target gene mRNAs, leading to mRNA degradation or translational *Contributed equally suppression (12). It has been estimated that approximately one out of three human genes are regulated by miRNAs (13,14). Key words: angiogenesis, vascular endothelial cell, microRNA-26b, Previous studies have revealed that miRNAs have a critical connective tissue growth factor, angiopoietin 2 role in a variety of cellular processes in healthy and ailing individuals, including pro-angiogenic therapeutics to reconstruct vasculature for patients with ischemic heart and 490 ZHENG et al: lncRNA PVT1 PROMOTES ANGIOGENESIS BY TARGETING THE miR-26b/CTGF AXIS peripheral vascular diseases (15-18). A previous study reported 24-well plates at 4x104 cells/well the day before transfection that miR-26a regulates pathological and physiological angio- and transfected with the CTGF wild type or mutant 3'‑UTR genesis by targeting its downstream bone morphogenetic reporter vector (Shanghai GenePharma Co., Ltd.), PVT1 protein/SMAD family member 1 signaling (19). Another or PVT1 mutant using Lipofectamine® 2000 (Invitrogen; study demonstrated that lncRNA PVT1 is a miR-26b sponge Thermo Fisher Scientific, Inc.). The cells were harvested and and promoted melanoma progression, which indicated that lysed 48 h after transfection and the luciferase activity was lncRNA PVT1 may regulate angiogenesis via interaction with assayed using the Dual-Luciferase Reporter system (Promega miR-26b (20). Corporation, Madison, WI, USA). The β-lactamase gene of Connective tissue growth factor (CTGF) is a member of the pGL3 luciferase vector was used for the normalization of the CCN family which consists of cysteine-rich proteins. It the luminescence levels. Three independent experiments were has been previously demonstrated that CTGF is associated performed. with fibrosis, tissue remodeling and tumorigenesis (21). It is of note that CTGF has a promoter role in the regulation of vessel Transwell migration assay. The cells were transfected with growth during development, wound healing and vascular miR-26b mimic using Lipofectamine® 2000, miR-26b inhib- disease, suggesting CTGF is an angiogenetic inducer (22). itor (both from Shanghai GenePharma Co., Ltd.), PVT1 or In previous studies, CTGF has been revealed to be a direct shPVT1. After 24 h, the cells were starved in medium without downstream target of miR-26b, indicating that miR-26b may serum for another 12 h and then digested with trypsin and influence angiogenesis via downregulation of CTGF gene then 3x104 cells were seeded in the top chamber of 24-well expression (20). Transwell culture inserts (Promega Corporation). The medium The present study revealed that PVT1 directly interacts with supplemented with 20% FBS used as chemoattractant was miR-26b to reduce the expression level, which subsequently added to bottom chamber. After 24 h incubation, the cells were promoted CTGF and ANGPT2 expression levels, which fixed for 10 min with 4% paraformalin and cells which had not contributed to cell proliferation, migration and angiogenesis migrated were removed. The cells on the lower side of the filter of vascular endothelial cells. were stained with 0.005% crystal violet for 30 min in 25˚C and then the number of cells was counted and photographed with Materials and methods an inverted microscope at magnification of x100 and three fields of view. Cell line. Human umbilical vein endothelial cells (HUVECs) and 293T cells were purchased from America Type Culture Cell proliferation assay. The proliferation of HUVECs from Collection (Manassas, VA, USA). HUVECs were cultured in various groups were examined via MTT assay. The cells vascular cell basal medium (ScienCell Research Laboratories, were counted and plated into 96-well plates at a density of Inc., San Diego, CA, USA) supplemented with 10 ng/ml VEGF 2x103 cells/well for 24 h, then 0.1 mg/ml MTT was added to at 37˚C, 5% CO2. 293T cells were cultured in Dulbecco's cells at 37˚C for 3 h and lysed in DMSO at room temperature modified Eagle's medium (DMEM) (HyClone; GE Healthcare, for 30 min. Finally, absorbance was quantified at 490 nm Logan, UT, USA) supplemented with 10% fetal bovine using a microplate reader (Omega Bio-Tek, Inc., Norcross, GA, serum (FBS) (BI) and 1% penicillin (100 U/ml)/streptomycin USA). (100 U/ml) (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). In vitro vascular tube formation assay. To examine the ability of endothelial tube formation in vitro, the present study used Plasmid transfection and lentivirus package. PVT1 full-length 15‑well µ‑slides (ibidi GmbH, Martinsried, Germany) coated cDNA was cloned into a pEX2 plasmid and the short hairpin with 10 µl Matrigel as previously described (23). To determine RNA (shRNA) targeting PVT1 was cloned into a pLKO.1-TRC the effect of PVT1 and miR-26b on tube formation, HUVECs vector (all from Shanghai GenePharma Co., Ltd., Shanghai, were pretreated with CoCl2 and transfected with PVT1 and China), where the transcription was under the control of the miR-26b mimics or siRNAs against PVT1 or miR-26b. The U6 promoter. shPVT1 target sequence was 5'‑CAG CCA TCA cells were grown in Medium DMEM (ScienCell Research TGA TGG TAC T‑3'. In order to generate