GENE-40385; No. of pages: 5; 4C: Gene xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

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Yinghua Chen a, Carla A. Evans a, Xiaofeng Zhou b, Xianghong Luan c, Thomas G.H. Diekwisch c,⁎, Phimon Atsawasuwan a,⁎⁎

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Article history: Received 27 August 2014 Received in revised form 26 February 2015 Accepted 24 March 2015 Available online xxxx

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Keywords: MicroRNA Periodontal ligament Extracellular matrix Cyclic stretch Compression

University of Illinois at Chicago, College of Dentistry, Department of Orthodontics, United States University of Illinois at Chicago, College of Dentistry, Department of Periodontics and Center of Molecular Biology of Oral Diseases, United States University of Illinois at Chicago, College of Dentistry, Department of Oral Biology, United States

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MicroRNAs (miRs) play an important role in the development and remodeling of tissues through the regulation of large cohorts of extracellular matrix (ECM) genes. The purpose of the present study was to determine the response of miR-29 family expression to loading forces and their effects on ECM gene expression in periodontal ligament cells, the key effector cell population during orthodontic tooth movement. In a comparison between miRs from human periodontal ligament cells (PDLCs) and alveolar bone cells (ABCs) from healthy human subjects, the ABC cohort of miRs was substantially greater than the corresponding PDLC cohort. Cyclic mechanical stretch forces at 12% deformation at 0.1 Hz for 24 h decreased expression of miR-29 family member miRs about 0.5 fold while 2 g/cm2 compression force for 24 h increased miR-29 family member expression in PDLCs 1.8–4 folds. Cyclic stretch up-regulated major ECM genes in PDLCs, such as COL1A1, COL3A1 and COL5A1, while the compression force resulted in a down-regulation of these ECM genes. Direct interactions of miR-29 and Col1a1, Col3a1 and Col5a1 were confirmed using a dual luciferase reporter gene assay. In addition, transient transfection of a miR-29b mimic in mouse PDLCs down-regulated Col1a1, Col3a1 and Col5a1 while the transfection of miR-29b inhibitor up-regulated these genes compared to control transfection indicating that these target ECM genes directly responded to the altered level of miR-29b. These results provided a possible explanation for the effects of the miR-29 family on loaded PDLCS and their roles in extracellular matrix gene expression. © 2015 Published by Elsevier B.V.

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Cyclic stretch and compression forces alter microRNA-29 expression of human periodontal ligament cells

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1. Introduction

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MicroRNAs (miRs) are a family of highly conserved small noncoding RNA molecules involved in posttranscriptional gene regulation. MiRs have been known to negatively regulate the expression of their target mRNAs (Cech and Steitz, 2014). In the extracellular matrix (ECM), miRs have substantial roles in the synthesis of matrix as well as its maintenance and remodeling (Rutnam et al., 2013). miRs play a significant role in the development and remodeling of tissues through the regulation of large cohorts of ECM genes (Mouw et al., 2014).

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Abbreviations: miRNAs, microRNAs; miRs, microRNAs; PDLCs, periodontal ligament cells; ABCs, alveolar bone cells; mPDLCs, mouse periodontal ligament cells; ECM, extracellular matrix; IL-1, interleukin-1 beta; TGF-β, transforming growth factor-beta; IGF1, insulinlike growth factor-1; VEGF, vascular endothelial growth factor; PGE2, prostaglandin E2; POSTN, periostin; ASPN, asporin. ⁎ Correspondence to: T.G.H. Diekwisch, Department of Oral Biology, UIC College of Dentistry, 801 South Paulina Street, MC 690, Chicago, IL 60612, United States. ⁎⁎ Correspondence to: P. Atsawasuwan, University of Illinois College of Dentistry, Department of Orthodontics, 801 South Paulina Street, MC 841, Chicago, IL 60612, United States. E-mail addresses: [email protected] (T.G.H. Diekwisch), [email protected] (P. Atsawasuwan).

Through their control of ECM gene expression, miRs regulate many of the cellular processes that are affected by ECM, including cell cycle regulation, attachment, matrix secretion, survival and death (Ameres and Zamore, 2013). The ECM of the periodontium is an ideal model to study the role of miRs in matrix remodeling and control of gene expression. The periodontium is characterized by extreme changes in the extracellular load environment, subjecting cells such as periodontal ligament cells, alveolar bone cells, and cementoblasts/cementocytes to varying degrees of mechanical stress (Meikle, 2006). These cells respond to stress and strain from masticatory or orthodontic forces by expressing and secreting biological mediators such as interleukin1-beta (IL-1β), transforming growth factor-beta (TGF-β), insulin like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE2) (Iwasaki et al., 2006; Kaku et al., 2008; Riddle and Donahue, 2009), RANKL and osteoprotegerin and extracellular matrix (ECM) proteins such as collagens, elastin, proteoglycans and their modifying enzymes and proteases (Meikle, 2006). During orthodontic loading, it is known that increased expression of classic anabolic matrix genes such as collagen I, III, and V on the stretch side and decreased expression of these matrix molecules on the compression side are essential for remodeling of alveolar bone

http://dx.doi.org/10.1016/j.gene.2015.03.055 0378-1119/© 2015 Published by Elsevier B.V.

Please cite this article as: Chen, Y., et al., Cyclic stretch and compression forces alter microRNA-29 expression of human periodontal ligament cells, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.055

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2.1. Sample recruitments and cell isolation

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Human periodontal ligament cells (PDLCs), alveolar bone cells (ABCs) and gingival fibroblasts (GFs) were isolated using an explant culture technique (Dangaria et al., 2011a), from 4 healthy patients aged 18–25 years old undergoing third molar removal. This study is in compliance with the Institutional Review Board at the University of Illinois at Chicago. Only the 3rd–5th passages of PDLCs, ABCs and GFs were used in the experiments.

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2.2. Cell culture

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2 × 105 cells of PDLCs, ABCs, GFs and mouse periodontal ligament cells (mPDLCs) (Dangaria et al., 2011b) were maintained in low glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 25 ng/ml Amphotericin B in a 5% CO2 atmosphere at 37 °C. The medium was changed twice a week. To study cell differentiation, the human cells were subjected to mineralizing media containing 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate and 10−8 M dexamethasone and cultured for 28 days. Upon terminating the culture, cells were used for alkaline phosphatase activity test and alizarin red staining, or protein and RNA preparation.

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2.3. Western blot analysis

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After 7 days of culture, cell-matrices of PDLCs, ABCs and GF cultures were scraped and lysed in a lysis buffer as in a previous report (Atsawasuwan et al., 2013). Aliquots of cell lysate were subjected to Western blotting (primary antibody dilutions listed in Appendix Table 1).

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2.4. Alkaline phosphatase and in vitro mineralization assay

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After 5 days of culture in the mineralizing media, a set of PDLCs, ABCs and GF cells were washed and stained with alkaline phosphatase substrate (Roche Diagnostic, Indianapolis, IN) to verify early mineralization activity. After 28 days of culture in the mineralizing media, another set of the cultured cells was fixed with cold methanol, stained with 10% alizarin red solution, and mineralized nodules were identified as red spots on the culture dish.

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2.5. Mechanical force application

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Three independent isolated PDLCs were plated onto six-well, flexible-bottomed uniflex-plates at a density of 4 × 105 cells/well.

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Total RNA of 24 h cultures from PDLCs and ABCs was isolated using miRNAeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Each sample was labeled with Hy3. A mixture comprising equal volumes of each sample was labeled with Hy5 as a reference. Using a miRCURY LNA microRNA Array 7th generation (Exiqon A/S, Vedbaek, Denmark) containing 3100 probes, the miR expression profiles were compared and analyzed comprehensively. Normalization of the data was performed using Lowess (Locally Weighted Scatterplot Smoothing) normalization (Yang YH et al., 2002). Genes showing significant differences of 0.5-fold were extracted after filtering. To confirm the microarray data, quantitative real-time reverse transcription-polymerase reaction (qRT-PCR) was performed as described below for selected miRs.

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2.7. Quantitative real-time RT-PCR procedure

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For mRNA PCR assays, total RNA was extracted from PDLCs at 24 h after being subjected to the forces using miRNAeasy kit (Qiagen). To quantify mRNA expression levels, qRT-PCR was performed using sequence specific Sybergreen primers (Appendix Table 2) and ABI Prism 7000 detection system (Applied Biosystems, Carlsbad, CA). Relative expression levels were calculated using the 2− ΔΔCt method (Livak and Schmittgen, 2001). The expression of specific mRNA of unloaded PDLCs was used as baseline (=1) and β-actin was used as an endogenous control, and all PCR products were sequenced at the UIC DNA facility for sequence verification. For miR PCR assay, the total RNA from each sample underwent quantitative RT-real-time PCR (qRT-PCR) performed with Taqman miR PCR assays (Life Technologies, USA) according to the manufacturer's instructions. The miR PCR assay kits and numbers are listed in Appendix Table 3. qRT-PCR reactions were performed in ABI 7500 PCR system (Life Technologies, USA). Each reaction was analyzed in triplicate. Negative control reactions without RT reaction and template were also performed. To confirm its validity qRT-PCR was performed with primer of cel-miR-39-3p, which should not be present in human samples. Relative expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001). The expression of specific miR of unloaded PDLCs was used as baseline (=1) and U6snRNA was used as an endogenous control.

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2.8. Transient transfection

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The mPDLCs cells were seeded in 6-well plates in DMEM supplemented with 10% fetal bovine serum then transfected using Lipofectamine 2000 (Life Technologies) the following day, according to the manufacturer's instructions. MiR-29b mimic, inhibitor and negative control (Thermoscientific, Waltham, MA, USA) were added at the concentration of 62.5 nM per well. The cells were cultured for 72 h then total RNA was extracted and subjected to qRT-PCR using sequence specific Sybergreen primers (Appendix Table 2).

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After overnight incubation, the cells were confluent and subjected to cyclic stretch of 12% deformation at 6 cycles/min for 24 h using a Flexercell Strain Unit (FX 4000, Flexcell International). The parameter used in the study was based on numerical data derived from a finite element model (Natali et al., 2004). For compressive force loading, the PDLCs were seeded in 12-well culture plates and subjected to 2 g/cm2 of constant force from sterile glass beads for 24 h. This loading approach was modified from a previous report (Nishijima et al., 2006). The illustrations of mechanical force application to the cells are shown in Fig. 1A–C.

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resulting in tooth movement (Meikle, 2006; Xu et al., 2014). Several miRs have been reported in periodontal ligament cells (PDLCs) (Hung et al., 2010; Li et al., 2012; Liu et al., 2011; Nahid et al., 2011; Qi and Zhang, 2014; Sipert et al., 2014). In the present study, we have focused on the role of the miR-29 family in the periodontal ECM under loading. The miR-29 family is a group of miRs that has recently emerged as a key modulator of ECM homeostasis (Villarreal et al., 2011). MiR-29 family members regulate a number of key ECM proteins, including collagens, elastin and several ECM enzymes and thus have the capacity to control ECM synthesis and remodeling (Luna et al., 2009). Here we decided to characterize the role of miR-29 family members in the expression of periodontal ECM genes and to determine the function of miR-29 in the stress-related expression changes that are part of the normal physiological environment in PDLCs. The present study for the first time reports stress-related changes in the expression of miR-29 family members and their potential stress-related effects on periodontal ECM gene expression.

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The data were presented as mean ± standard error for each group and analyzed using SPSS software (version 22, SPSS, Chicago, IL). Kruskal–Wallis non-parametric statistical analysis and a multiple comparison was analyzed using Mann–Whitney U test. Values of P b 0.05 were considered to be statistically significant.

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3. Results

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3.1. Isolation and phenotypes of periodontal ligament cells (PDLCs) and alveolar bone cells (ABCs)

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The luciferase reporter gene constructs containing miR-29 family targeting sites from the 3′-UTR of Col1a1, Col3a1 or Col5a1 mRNA were created as described previously (Jin et al., 2013) (Supplemental Fig. S1a,b,c). The corresponding mutant constructs were created by replacing the seed regions (positions 2–8) of the miR-29 family binding sites with 5′-TTTTTTT-3′ (Appendix Table 4). All constructs were verified by sequencing. The reporter constructs and the pRL-TK vector (Promega) were co-transfected using Lipofectamine 2000 (Invitrogen) in mPDLCs cells. The final concentration of 25 nM of either miR-29b mimic or negative control, 0.5 μg of either pGL3-WT or -mutant plasmid and 0.167 μg of pRL-TK were used for transfection into cells in a 6-well plate seeded with the mPDLCs at 1 × 105 cells/well on the previous day. After 48 h, the cells were collected, centrifuged (500 rpm, 5 min at 4 °C) and stored at −80 °C. The luciferase activities were determined with a dual luciferase reporter assay system following the manufacturer's (Promega) instruction using a TD-20/20 luminometer (Turner Designs). Experiments were performed in triplicate.

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Fig. S2b). Western blot analysis showed higher expression of secreted frizzled-related protein1 (sFRP1), POSTN and asporin (ASPN) in the cell lysates from PDLCs compared to those of ABCs and gingival fibroblasts (GFs) while s100A4 was markedly expressed in GFs (Supplemental Fig. S2c). When cultured in the mineralizing media, the mineralized nodules were detected in both PDLC and ABC cultures but undetected in GF culture (Supplemental Fig. S2d). Alkaline phosphatase activity was higher in PDLCs at early culture stage (Supplemental Fig. S2e).

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Fig. 1. Schematic illustrations of loading regimens on the human periodontal ligament cells (PDLCs). PDLCs were seeded in the uniflex silicone-base culture plate (A). The human periodontal ligament cells were stretched when the vacuum pressure was applied to the culture plate in the loading unit (B). The human periodontal ligament cells were compressed under the 2 g/cm2 force from the glass beads (C).

Periodontal ligament cells (PDLCs) have a capacity to form mineralized nodules in mineralizing media and express bone-associated markers (Marchesan et al., 2011). To determine whether the isolated human PDLCs possess PDLC phenotypes, expression of known PDL and bone markers was compared between PDLCs and ABCs (Supplemental Fig. S2a and b). Expression of bone markers such as runtrelated transcription factor 2 (RUNX2), osteocalcin (OCN) and integrinbinding sialoprotein (IBSP) was significantly higher in ABCs compared to PDLCs (P b 0.05) (Supplemental Fig. S2a) while other PDLC markers such as tenomodulin, periostin (POSTN) and S100A4 expression were significantly higher in PDLCs compared to ABCs (P b 0.05) (Supplemental

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3.2. MicroRNA microarray of PDLCs and ABCs

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To define miR PDLC and ABC expression profiles, the total RNA from each of these two cultured cells was subjected to miR microarrays. The heatmap of miR microarrays revealed differences in miR expression profiles of ABCs and PDLCs (Supplemental Fig. S3a). A total of 416 mature miRs were detected in ABCs while 273 mature miRs were detected in PDLCs (data not shown). All of the miRs detected in PDLCs were members of miRs detected in ABCs. After cyclic stretch application, the miR microarray of PDLCs exhibited different patterns between loaded and unloaded groups (Supplemental Fig. S3b). The list of selected miRs and their fold changes after cyclic stretch loading compared to the unloaded PDLCs is presented in Table 1.

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We further investigated the response of miR-29 family in the following experiments because of their significant response after cyclic stretch exposure (Table 1). The response of the miR-29 family in PDLCs after exposure to 24-h cyclic stretch and 24-h compression was investigated using Taqman quantitative RT-PCR (qRT-PCR) and specific miR PCR

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Table 1 t1:1 Selected miRNAs that were either up- or downregulated in PDLCs after cyclic stretch t1:2 application. t1:3 Annotation

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hsa-miR-29b-3p hsa-miR-193-3p hsa-miR-101-3p hsa-miR-27a-3p hsa-miR-33a-5p hsa-miR-29c-3p hsa-miR-337-5p hsa-miR-21-5p hsa-miR-29a-3p hsa-miR-27b-3p

−1.151 −1.147 −0.610 −0.583 −0.550 −0.527 −0.501 −0.446 −0.404 −0.399

hsa-miR-4419b hsa-miR-3960 hsa-miR-4497 hsa-miR-4708-3p hsa-miR-371b-5p hsa-miR-4787-5p hsa-miR-1469 hsa-miR-4285 hsa-miR-3178 hsa-miR-4795-3p

0.850 0.629 0.626 0.562 0.520 0.496 0.493 0.475 0.472 0.457

t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14

Please cite this article as: Chen, Y., et al., Cyclic stretch and compression forces alter microRNA-29 expression of human periodontal ligament cells, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.055

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regulated in the miR-29b inhibitor group (P b 0.05) compared to the 263 control group (Fig. 3A). 264

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assays (Appendix Table 3). Under cyclic stretch loading, miR-29 a, b, and c significantly decreased approximately 0.5 fold when compared to unloaded controls (P b 0.05) (Fig. 2A). The significantly increased (1.8–4 folds) expression of miR-29 family was detected when PDLCs were subjected to compression loading (P b 0.05) (Fig. 2A). The expression of major periodontal ECM genes such as COL1A1, COL3A1 and COL5A1 significantly increased under cyclic stretch loading but only COL1A1 and COL3A1 significantly decreased under compression loading (P b 0.05) (Fig. 2B). We also verified the response of other PDL markers such as POSTN and found no significant change after loading (Fig. 2B).

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To demonstrate the direct response of the major ECM genes to the altered level of miR-29b, the transient transfection of miR-29b mimic, inhibitor and negative control was performed in mPDLCs. After transient transfection for 72 h, Col1a1, Col3a1 and Col5a1 expression in mPDLCs was significantly down-regulated in the miR-29b mimic group (P b 0.05), while Col1a and Col5a1 were significantly up-

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To test whether Col1a1, Col3a1 and Col5a1 directly interact with miR-29b, a dual-luciferase reporter assay was performed in mPDLCs using constructs containing the 3′-UTR targeting sites (Appendix Table 4, Supplemental Fig. S1). When mPDLCs were transfected with miR-29b mimic, the luciferase activities of the construct containing wildtype (WT) targeting sites were significantly reduced as compared to the cells transfected with scramble control (Fig. 3B). When the seed regions of targeting sites were mutated (Mut), the miR-29b-mediated reduction in luciferase activity was not observed (Fig. 3B). These results

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Fig. 2. Quantitative real-time RT-PCR analysis of human miR-29 family (hsa-miR29 a, b and c) and major PDL ECM genes. Down-regulation of miR-29 a, b and c under cyclic stretch while up-regulation of miR-29 a, b and c under compression were found in PDLCs (A). Up-regulation of COL1A1, COL3A1 and COL5A1 under cyclic stretch while down-regulation of these ECM genes under compression were found in PDLCs (B). POSTN expression was not significantly changed under force loading (*P b 0.05).

Fig. 3. Major ECM gene expression in PDLCs after transfection with miR-29 mimic, inhibitor and scramble negative control. Down-regulation of Col1a1, Col3a1 and Col5a1 were observed in miR-29b mimic transfected group while up-regulation of Col1a1 and Col5a1 were observed in miR-29b inhibitor transfected group compared to the one transfected with negative control (*P b 0.05) (A). Decreased luciferase activity was observed when miR-29b mimic was transfected into the cells containing WT-3′-UTR targeting sequence while no change was observed into the cells containing Mut-3′-UTR targeting sequence as well as the scramble transfection (B).

Please cite this article as: Chen, Y., et al., Cyclic stretch and compression forces alter microRNA-29 expression of human periodontal ligament cells, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.055

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The authors declare that they have no conflict of interest.

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The present study was designed to determine whether changes of miR-29 family expression in PDLCs respond to forces and whether different types of forces affect the expression profile of miR-29 family members and possibly their target ECM genes. To address these questions, cyclic stretch and compression forces were loaded onto PDLCs, and the expression of miR-29 family members and target ECM genes was assessed. First we confirmed expression profiles of primary human PDLCs, which possessed several ligament and osteogenic markers and had a capacity to generate mineralized nodules when cultured in a mineralizing media as previously reported (Marchesan et al., 2011). After PDLCs were subjected to different types of forces, our data indicated that cyclic stretch up-regulated expression of miR-29 family members but compression forces down-regulated expression of these miRs. Few studies reported response of miR expression in PDLCs after loading (Qi and Zhang, 2014; Wei et al., 2014) however, we are the first to demonstrate the expression profile of miR-29 family to the orientation of loading forces and the association of miR-29 family with their target genes in PDLCs under stress loading. We investigated the expression of major periodontal ligament ECM genes, including collagen I, III and V, because these proteins are known to play important roles in periodontal ligament and alveolar bone remodeling (Xu et al., 2014). During orthodontic loading, increased expression of matrix anabolic genes such as Collagen I, III, V on the tension side and decreased expression of these matrix molecules on the compression side are essential for remodeling of alveolar bone resulting in tooth movement (Meikle, 2006; Xu et al., 2014). In our study, we also observed up-regulation of major periodontal ECM genes such as COL1A1, COL3A1 and COL5A1 in PDLCs under the cyclic stretch and down-regulation of these genes under compression loading which is similar to the findings in a previous report (Domon et al., 2001). Our study demonstrated that altered levels of miR-29b inversely affected the expression levels of these major PDL ECM genes. In addition, we demonstrated that Col1a1, Col3a1 and Col5a1 are direct targets of the miR-29 family in PDLCs using a dual-luciferase reporter assay. Overall, the miR-29 family is known to play an important role in ECM homeostasis (Kriegel et al., 2012; Mouw et al., 2014; Yang et al., 2013) by targeting several ECM genes (Kriegel et al., 2012), including collagen I, III and V (Luna et al., 2009; Sekiya et al., 2011). Taken together, these findings suggest possible roles of the miR-29 family in PDLCs as a modulator for ECM homeostasis in the periodontal ligament during masticatory function or orthodontic tooth movement. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.03.055.

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Wang et al., 2012 Acknowledgment This work was funded by a start-up grant from the College of Den-

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confirmed that miR-29b directly interacts with these targeting sites in Col1a1, Col3a1 and Col5a1 mRNA after transfection for 48 h (Fig. 3B).

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Please cite this article as: Chen, Y., et al., Cyclic stretch and compression forces alter microRNA-29 expression of human periodontal ligament cells, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.055

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