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1 IUBMB Life, 63(7): , July 2011 Research Communication mir-200a Modulate HUVECs Viability and Migration Yi-Xuan Li*, Da-Quan Liu*, Chen Zheng*, Shu-Qi Zheng, Min Liu, Xin Li and Hua Tang Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China Summary The posttranscriptional regulation of mirnas is important for organism development. To investigate the role of mirnas in angiogenesis, we performed a loss-of-function screening assay in human umbilical vein endothelial cells (HUVECs) and found that knockdown of 7 mirnas (mir-95a, mir-126, mir-129, mir-137, mir-139, mir-200a, and mir-335) significantly suppressed cell viability. As mir-200a was highly expressed in HUVECs, blocking endogenous mir-200a using 2 0 -OMe antisense oligonucleotide (ASOs) resulted in a decrease of cell viability and migration. Bioinformatics analysis indicates the 3 0 untranslated region (UTR) of thrombospondin-1 (THBS1) has a putative binding site for mir-200a. MiR-200a can directly bind to THBS1 3 0 UTR and negatively regulate THBS1 expression. The identification of endothelial cells (ECs) related mirna and its target gene may gain new insight into the mechanism of angiogenesis. Ó 2011 IUBMB IUBMB Life, 63(7): , 2011 Keywords mir-200a; angiogenesis; HUVECs; thrombospondin-1; cell viability; cell migration. Abbreviations ASO, antisense oligonucleotide; ECs, endothelial cells; EGFP, enhanced green fluorescence protein; HUVECs, human umbilical vein endothelial cells; mirna, microrna; THBS1, thrombospondin-1; UTR, untranslated region; VEGF, vascular endothelial growth factor. INTRODUCTION Angiogenesis is a process of growth of new blood vessels, which is a very complex process involving a number of molecular and cellular regulators and keeping a finely tuned balance between stimulatory and inhibitory signals (1). Imbalance of Additional Supporting Information may be found in the online version of this article. *These authors contributed equally to this work. Received 9 February 2011; accepted 2 April 2011 Address correspondence to: Hua Tang, Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, No. 22 Qi-Xiang-Tai Road, Tianjin , China. Tel: Fax: htang2002@yahoo.com this physiological process is involved in varieties of diseases such as cancer, diabetic retinopathy, thrombosis, and inflammatory disorders. MicroRNAs (mirnas) are highly conserved small RNA molecules (22 nucleotides) which mostly regulate gene expression and participate in almost every cellular process (2). Recently, some labs have demonstrated that mirnas play an important role in regulating the different aspects of angiogenic process (3). Evidence for the importance of micrornas in the regulation of angiogenesis comes from observations that Dicer is required for embryonic angiogenesis and that knocking it out resulted in severely compromised embryos and yolk sacs (4). Silencing Dicer in adult endothelial cells (ECs) results in the downregulation of lef-7f and mir-27b which target antiangiogenic genes (5). Being consistent with this founding, changes in microrna expression profiles in response to angiogenesis have been reported (5 7). This observation suggests that microrna expression is generally necessary for angiogenesis. But the roles of these mirnas in angiogenesis and endothelial function are totally different. MiR-126, mir-210, and mir-296 are considered as proangiomirs (8 10); while mir-221/222, mir-15, and mir-16 are believed to be anti-angiomirs (4, 6, 11, 12). Endothelial mirnas regulate the angiogenic response to multiple growth factors by targeting angiogenic factors, receptors and signaling molecules. In addition to their specific role in ECs phenotype and angiogenesis, dysregulation of some mirnas contribute to tumor angiogenesis, such as mir-221/222, mir- 296, mir-210, and the mir cluster (4, 6, 9 11, 13). At present, some endothelial mirnas had been identified, but the role of mirnas in angiogenesis is still little known. Here, we report that blocking mir-200a suppresses HUVECs viability and migration by targeting an angiogenesis inhibitor, thrombospondin-1 (THBS1). MATERIALS AND METHODS Cell Culture and Transfection Human umbilical vein endothelial cells (HUVECs) were cultured as previously described (14). For all experiments, ISSN print/issn online DOI: /iub.486

2 554 LI ET AL. HUVECs between passages 2 and 5 were used. Human embryonic kidney (HEK), HeLa and HepG2 cells were propagated and maintained in MEM alpha medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and antibiotics. Transfection was performed with Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA) following the manufacturer s protocol. 150 nm mirnas ASO (IDT, Coralville, IA) or control ASO was transfected into HUVECs respectively for screening. Vectors Construction To construct the mir-200a expression plasmid pcdna3/primir-200a, a 577-bp DNA fragment carrying pri-mir-200a was amplified by PCR from genomic DNA using specific primers (mir-200a-s, 5 0 -CGC GGA TCC ACA GCC ATC TTC CCT CCT G-3 0 ; mir-200a-as, 5 0 -CCG GAA TTC GGA GCT GAC ACA GGC CCT C-3 0 ). This fragment was then cloned into modified pcdna3.1b at BamH I and EcoR I sites. The EGFP (enhanced green fluorescence protein) reporter vectors were constructed as previously described (15). The 3 0 UTR of THBS1 containing the mir-200a binding site was cloned into pcdna3/egfp. Similarly, the fragment of the THBS1 3 0 UTR mutant, which contained a triple point mutation in mir-200a binding site, was also cloned into pcdna3/egfp. MTT Assay To determine relative cell viability, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed at 48 h posttransfection in 96-well plates. The absorbance at 570 nm was detected using lquant Universal Microplate Spectrophotometer (BioTek, Winooski, VT). Migration Assay For transwell assays, HUVECs were transfected with 100 nm mir-200a ASO or control ASO. 48 h later, cells which were suspended in serum-free medium were plated onto gelatin-coated 8.0 lm pore size polycarbonate membrane in 24- well plates (Corning, NY). The lower chamber contained MEM-a medium with 20% FBS and 20 ng/ml VEGF. After 6 or 18 h, the migrated cells were fixed with crystal violet stain and photographed for counting. For scratch assay, until cells reach 100% confluence and form a monolayer, a 200 ll pipette tip was used to create a scratch on cell monolayer. Wash the plate once with 13 PBS and replace with the 2% FBS medium. Migration was quantified by measuring the distance of the scratch. Fluorescent Report Assay Fluorescent Report Assay were performed as described by Zhang et al. (15). Quantitative Reverse Transcriptase Real-Time PCR Quantitation of mirnas was carried out using SYBR Greenbased real-time RT-PCR as previously described (15). To detect the relative level of THBS1 transcription, quantitative RT real-time RT-PCR was performed. SYBR Premix Ex Taq TM kit (TaKaRa, Madison, WI) was used following the instructions, and the real-time PCR was performed on iq5 Real- Time PCR system (Bio-rad, Hercules, CA). The real-time PCR results were analyzed and expressed as relative expression of CT (Threshold Cycle) value using 2 -DDCT method (16). Western Blot Analysis Total proteins from transfected HUVECs cells were extracted 72 h posttransfection using RIPA buffer, and protein expression was analyzed by western blot. GAPDH served as a loading control. The following antibodies were used: rabbit anti-thbs1, rabbit anti-gapdh, and goat anti-rabbit (Tianjin Saier Biotech, China). Bands were quantified with Labworks 4.0 software. Statistics and Data Analysis Statistical significance was determined using the Student s t test. In all figures, values are expressed as mean 6 standard deviation (SD), and statistical significance (P \ 0.05) is indicated by a single asterisk. The data generated in vitro are representative of at least three separate experiments conducted in triplicate. RESULTS Blocking mir-200a Suppressed Cell Viability of HUVECs To investigate the role of mirnas in angiogenesis, we used HUVECs as a model for in vitro study. A loss-of-function screening assay was first performed by transfecting cells with a library of mirna 2 0 -OMe antisense oligomers (ASOs) as previously described (15). At 48 h posttransfection, cell viability was assessed by MTT assay. Comparing with control oligomer, 7 mirnas (mir-95a, mir-126, mir-129, mir-137, mir-139, mir-200a, and mir-335) ASOs significantly suppressed HUVECs cell viability (Fig. 1A). Among these mirnas, mir- 200a ASO could efficiently block endogenous mir-200a level (Supporting Information Fig. 1) and caused a nearly 80% reduction of cell viability (Fig. 1A). To explore the role of mir-200a in HUVECs activity, we detected the endogenous expression level of mir-200a in HUVECs, as well as other cell types, such as HEK, HeLa and HepG2 cells. Although mir-200a is differently expressed in these cells (Supporting Information Fig. 2A), blocking mir-200a could only affect HUVECs viability (Supporting Information Fig. 2B), indicating mir-200a is specifically essential for HUVECs activity. In addition, we also compared the expression level of mir-200a with other mirnas in HUVECs using real-time PCR. As mir-27a was reported relatively down-regulated and mir-21 was highly expressed in HUVECs (5, 7), the threshold cycle (CT) of each mirna was normalized to mir-27a and expressed as relative expression of CT value using 2 -DCT formula. Comparing with mir-27a, mir- 200a is highly expressed in HUVECs, even higher than the level of mir-21 (Fig. 1B). So we further studied the specific

3 mir-200a MODULATE HUVECs VIABILITY AND MIGRATION 555 Figure 1. MiR-200a is essential for HUVECs activity. (A) HUVECs were transfected with a library of 150 nm mirnas ASO. At 48 h posttransfection, cell viability was measured by MTT assay. *P \ (B) Quantification of mir-200a, mir-21, and mir- 27a was carried out using QRT real-time PCR. The left is real-time PCR curves, and the right is relative mirnas expression level. The threshold cycle (CT) of each mirna was normalized to mir-27a and expressed as relative CT value using 2 -DCT formula. effect of mir-200a on HUVECs phenotypes using a loss-offunction assay. To assess whether mir-200a ASO suppressed HUVECs activity in a dose-dependent manner, different amounts (50, 100, and 150 nm) of mir-200a ASO or control ASO were transfected into HUVECs respectively. Due to mir-200a is highly expressed in endothelial cells, MTT assay showed 150 nm mir-200a ASO could significantly induce a decrease of cell viability, but low level of mir-200a ASO is not sufficient to suppress cell activity (Fig. 2A). Knockdown of mir-200a Suppressed HUVECs Migration In Vitro To explore the role of mir-200a in cell migration, an important endothelial phenotype, HUVECs were transfected with either 100 nm mir-200a ASO or control ASO. At 48 h posttransfection, HUVECs were allowed to migrate through gelatin coated filters in response to 20% FBS and 20 ng/ml VEGF for 6 or 18 h. Cells migration activity was significantly inhibited after knockdown of mir-200a (Figs. 2B and 2C). Because the low concentration of oligonucleotides had no effect on cell viability, the reduction of migrated mir-200a-aso-transfected cells indicated mir-200a also participate in HUVECs migration. We found the same results in wound-healing assay. The average recovery percentage of the wounded portion was measured and calculated. Knockdown of mir-200a resulted in a decrease of recovery rate (Fig. 2D). MiR-200a-ASO-transfected cells moved at a much lower velocity (1.5 lm/h) than that of the control cells (4.5 lm/h) (Fig. 2D). All these findings indicated that blocking of mir-200a suppressed HUVECs migration.

4 556 LI ET AL. Figure 2. The effect of mir-200a on HUVECs viability and migration. (A) Different amounts of mir-200a ASO or control ASO were transfected into HUVECs. After 48 h, MTT assay was performed. *P \ (B, C) HUVECs were transfected with 100 nm mirnas ASO or control ASO and were allowed to migrate through filters for 6 and 18 h. Migrated cells were fixed with crystal violet Stain and photographed for counting (magnification, 3400). *P \ (D) For scratch assay, black lines indicate width of wound. The wound areas were calculated and the results shown are mean (6SD) from three independent experiments. *P \ THBS1 gene 3 0 UTR Carries a Putative mir-200a Binding Site and is Negatively Regulated by mir-200a We hypothesized that mir-200a blocking might inhibit the angiogenesis phenotype of HUVECs by regulating anti-angiogenic genes. Using two algorithm programs (PicTar and TargetScan Release 5.1), thrombospondin-1 (THBS1) was found to have a putative mir-200a binding site within its 3 0 UTR which are highly conserved in some species (Fig. 3A). We constructed an EGFP reporter vector which carry the THBS1 3 0 UTR downstream of EGFP stop codon. The EGFP intensity in reportervector-transfected cells is lower than the control group indicating endogenous mirna could negatively regulate EGFP expression by targeting its 3 0 UTR (Fig. 3B). Then HUVECs were transfected with the reporter vector along with mir-200a ASO

5 mir-200a MODULATE HUVECs VIABILITY AND MIGRATION 557 Figure 3. The direct effect of mir-200a on THBS1 3 0 UTR. (A) The complementary sequences and evolutionary conservation of the mir-200a binding site in THBS1 3 0 UTR were shown. The arrows indicate the mutated nucleotides. (B) HUVECs were transfected with either pcdna3/egfp or pcdna3/egfp-thbs1-3 0 UTR. pdsred2-n1 (Clontech) expressing RFP was spiked in for normalization. *P \ (C, D) HUVECs were transfected with either EGFP reporter vector or mutant vector, along with mirna ASO or expression vector. The fluorescence value in the control group was set to 1. *P \ or expression vector. The results showed that mir-200a blocking could enhance EGFP expression, and ectopic expression of mir-200a could reduce the intensity of EGFP fluorescence (Fig. 3C). However, the EGFP with the mutated 3 0 UTR was not affected by mir-200a (Fig. 3D). These results suggested that mir-200a could directly bind to the 3 0 UTR of THBS1 mrna and specifically suppress target gene expression. mir-200a Regulates THBS1 mrna and Protein Expression in HUVECs To assess whether mir-200a negatively regulates endogenous THBS1 expression, HUVECs were transfected with either ASO or overexpression vectors. The THBS1 mrna and protein level was measured by real-time PCR and western blot. In contrast to the controls, knockdown of mir-200a in HUVECs resulted in a significant increase of THBS1 mrna and protein levels (Figs. 4A and 4C), whereas overexpression of mir-200a reduced THBS1 mrna and protein level (Figs. 4B and 4D). These results indicated that mir-200a could reduce THBS1 expression through both translation inhibition and RNA degradation. DISCUSSION Increasing evidences suggest that mirnas may be key modulators of physiological or pathological angiogenesis (3). In our study, using a loss-of-function screening assay, 7 mirnas were found to be related with HUVECs viability. Among these mirnas, mir-126 had been reported to be an endothelial cell specific microrna and a positive regulator of angiogenesis in response to angiogenic growth factors (8). Here we investigated the role of mir-200a in the angiogenic properties of HUVECs. Knockdown of mir-200a could significantly suppress cell viability and migration, but ectopic overexpression of mir-200a had no significant effect on cell phenotype (data

6 558 LI ET AL. angiogenesis. Recently, mir-200 family has received much attention for potentially regulating tumor progression in bladder cancer, gastric carcinoma and meningiomas (20 22). Whether mir-200a-mediated angiogenesis is involved in malignant phenotype is still unknown. In conclusion, our results indicated that mir-200a targets THBS1 and regulate in vitro ECs behaviors, including cell viability and migration. The identification of the proangiomirs mir-200a and its target gene, anti-angiogenic protein THBS1, in HUVECs may help us to understand the molecular mechanism of angiogenesis. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No: ) and the Natural Science Foundation of Tianjin (08JCZDJC23300). Figure 4. MiR-200a negatively regulates THBS1 expression. (A, B) The expression of THBS1 mrna was measured by QRT real-time PCR. b-actin mrna was used for endogenous normalization. (C, D) The THBS1 protein level was detected by western blot. GAPDH was used for endogenous normalization. *P \ not shown). It may be due to very high level of endogenous mir-200a in HUVEC cells, which may be sufficient to maintain cell behaviors. Although mir-200a is demonstrated to be a proangiomirs that is required for the maintenance of ECs behaviors, next we identified thrombospondin-1 (THBS1) as its target gene that may participate in this process. THBS1, the first identified endogenous angiogenesis inhibitor, has been shown to play roles in angiogenesis, inflammation, tumorigenesis and several chronic diseases. Recent studies indicated THBS1 expression is regulated by mirna-mediated RNAi pathway. Dicer and Drosha silencing significantly increased the expression of THBS1, as well as several Dicer-dependent endothelial micrornas were found downregulating THBS1 expression (5, 7). In transformed cells, mir cluster was also negatively regulate THBS1 expression and involved in Myc-induced neovascularization (17). THBS1 is targeted not only by cellular mirnas, but also by KSHV (Kaposi sarcoma-associated herpesvirus)-encoded mirnas (18). As THBS1 stimulates apoptosis of the endothelial cells and possesses both anti-proliferative and anti-angiogenic activity (19), highly expressed mir-200a negatively regulates THBS1 expression contributing to physiological REFERENCES 1. Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J., Holash, J. (2000) Vascular-specific growth factors and blood vessel formation. Nature 407, Eulalio, A., Huntzinger, E., and Izaurralde, E. (2008) Getting to the root of mirna-mediated gene silencing. Cell 132, Suarez, Y., and Sessa, W. C. (2009) MicroRNAs as novel regulators of angiogenesis. Circ. Res. 104, Suarez, Y., Fernandez-Hernando, C., Pober, J. S., and Sessa, W. C. (2007) Dicer dependent micrornas regulate gene expression and functions in human endothelial cells. Circ. Res. 100, Kuehbacher, A., Urbich, C., Zeiher, A. M., and Dimmeler, S. (2007) Role of Dicer and Drosha for endothelial microrna expression and angiogenesis. Circ. Res. 101, Poliseno, L., Tuccoli, A., Mariani, L., Evangelista, M., Citti, L., et al. (2006) MicroRNAs modulate the angiogenic properties of HUVECs. Blood 108, Suárez, Y., Fernández-Hernando, C., Yu, J., Gerber, S. A., Harrison, K. D., et al. (2008) Dicer-dependent endothelial micrornas are necessary for postnatal angiogenesis. Proc. Natl. Acad. Sci. USA 105, Fish, J. E., Santoro, M. M., Morton, S. U., Yu, S., Yeh, R. F., et al. (2008) mir-126 regulates angiogenic signaling and vascular integrity. Dev. Cell 15, Fasanaro, P., D Alessandra, Y., Di Stefano, V., Melchionna, R., Romani, S., et al. (2008) MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J. Biol. Chem. 283, Würdinger, T., Tannous, B. A., Saydam, O., Skog, J., Grau, S., et al. (2008) mir-296 regulates growth factor receptor overexpression in angiogenic endothelial cells. Cancer Cell 14, le Sage, C., Nagel, R., Egan, D. A., Schrier, M., Mesman, E., et al. (2007) Regulation of the p27(kip1) tumor suppressor by mir-221 and mir-222 promotes cancer cell proliferation. EMBO. J. 26, Cimmino, A., Calin, G. A., Fabbri, M., Iorio, M. V., Ferracin, M., et al. (2005) mir-15 and mir-16 induce apoptosis by targeting BCL2. Proc. Natl. Acad. Sci. USA 102, Bonauer, A., Carmona, G., Iwasaki, M., Mione, M., Koyanagi, M., et al. (2009) MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science 324, Liu, Y., Wang, W., Wang, J., Wang, Y., Yuan, Z., et al. (2010) Blood compatibility evaluation of poly(d,l-lactide-co-beta-malic acid)

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