Original Article

MiR-335-5p Promotes Chondrogenesis in Mouse Mesenchymal Stem Cells and is Regulated Through Two Positive Feedback Loops†

Xiao Lin, Li Wu, Zhenming Zhang, Ruohuan Yang, Qian Guan, Xinfeng Hou, and Qiong Wu

MOE Key Lab. Bioinformatics, Center for Epigentics and Chromatin, Department of Biological Sciences and Biotechnology, School of Life Sciences, Tsinghua University, Beijing, 100084, China

*

Corresponding author:

Qiong Wu School of Life Sciences, Tsinghua University, Beijing 100084, China Tel: +86-10-6277-1664, Fax: +86-10-6279-4217 E-mail address: [email protected]

Disclosures: All authors state that they have no conflict of interest.

†

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jbmr.2163]

Additional Supporting Information may be found in the online version of this article.

Initial Date Submitted October 2, 2013; Date Revision Submitted December 2, 2013; Date Final Disposition Set December 11, 2013

Journal of Bone and Mineral Research © 2013 American Society for Bone and Mineral Research DOI 10.1002/jbmr.2163

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ABSTRACT Chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors and signal pathways, including transcription factors such as Sox9 and microRNAs. MiR-335-5p has been previously reported to regulate osteogenic and adipogenic differentiations of MSCs, but its role in chondrogenic differentiation of MSC remains unknown. In this study, we found that miR-335-5p and its host gene Mest are co-expressed and greatly upregulated during mouse MSCs (mMSCs) chondrogenesis. Overexpression of miR-335-5p in mMSCs increased expression of chondrogenic marker genes. Molecular mechanism explorations revealed that miR-335-5p targets Daam1 and ROCK1, a set of negative regulators of Sox9; Sox9 downregulates the expression of miR-29a and 29b, both negative regulators of Mest expression, thus forming a positive loop from miR-335-5p to Sox9 to Mest/miR-335-5p. In addition, miR-335-5p targets DKK1 during mMSC chondrogenic differentiation to increase -catenin/TCF activity, which leads to increased level of Mest transcription. These data showed miR-335-5p positively regulates MSC chondrogenesis and two positive feedback loops are identified for the expression of miR-335-5p and its host gene Mest during the early phase of mMSC chondrogenic differentiation.

KEY WORDS: miR-335-5p; Mest, stem cell; Sox9; Chondrogenic differentiation

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Introduction Bone marrow derived mesenchymal stem cells (BMSCs) have the characteristics of self-renewal and pluripotent differentiation. BMSCs have the potential to differentiate into chondrocytes, osteocytes, and adipocytes, thus they are considered as powerful seed cells in future clinical therapeutic applications to cure the disease caused by the lack of regeneration ability of the original cells.(1) Cartilage diseases are difficult to treat due to lack of regeneration capacity,(2) and the chondrogenic stem cells might could be a promising way to restore cartilage defects. Chondrogenesis takes place in endochondral bones where mature cartilage generates.(3) During chondrogenesis, cells produce cartilage-specific extracellular matrix such as type II collagen (Col21) and aggrecan. This process involves many signaling pathways, including p38 MAPK,(4) ERK1/2,(5) RhoA/ROCK signaling(6) and IGF-1.(7) MicroRNAs (miRNAs) are a class of non-coding RNAs which regulate gene expression at post-transcriptional level by directly targeting the 3’-untranslated region (3’-UTR) of genes.(8) They are important regulators for diverse biological progresses including cell proliferation, differentiation, migration, apoptosis and tumorigenesis.(9) Several miRNAs have been shown to regulate MSC chondrogenesis, such as miR-574-3p,(10) miR-23b(11) and miR-199a*.(9) MiR-335-5p was initially discovered as tumor metastasis suppressor targeting transcription factor SOX4 and tenascin C.(12) MiR-335-5p is encoded by intron 2 of Mest,(13) which is identified to be expressed in mesodermal tissues and known as a mesoderm-specific transcript.(14) MiR-335-5p was later found to be involved in many biological processes such as obesity(15) and cell differentiation. For instance, it could impair human MSCs osteogenic and adipogenic differentiation,(16) and modulate osteogenic differentiation through DKK1 (dickkopf-related protein 1), which is an inhibitor of Wnt signaling through binding to LRP5/6 receptor.(17) In mouse embryonic stem cells, miR-335-5p regulates Oct4 to integrate cell self-renewal and cell cycle.(18) However, the function of miR-335-5p in cell chondrogenic differentiation remains unknown. SRY-box containing gene 9 (Sox9) is expressed in chondrocytes and other tissues(19) and it plays essential 3   

 

roles in chondrogenic differentiation as a transcription factor.(20) Knocking out Sox9 would cause chondrocyte defect.(21) During chondrogenesis, Sox9 binds to the first intron of Col21 and regulates its expression;(22) Sox9 also regulates miRNAs, such as miR-29a and miR-29b.(23) On the other hand, Sox9 mRNA expression and glycosaminoglycan secretion are reported to be enhanced by inhibition of RhoA/ROCK signaling. In our preliminary experiments, we found miR-335-5p was upregulated during chondrogenesis of mMSC, and ROCK1 was a target of miR-335-5p, which was reported very recently,(24) Therefore we hypothesized that miR-335-5p could regulate MSC chondrogenesis through ROCK1. On the other hand, miR-335-5p was reported to co-express with Mest.(16) The promoter of Mest was predicted to be transcribed by -catenin/TCF, and -catenin was upregulated in TCF3 induced-chondrogenesis,(25) while miR-335-5p targeted DKK1, an inhibitor of Wnt/-catenin signaling.(17) Therefore, we also hypothesized that miR-335-5p might regulate Mest transcription through Wnt/-catenin/TCF in chondrogenesis. In addition, we predicted Mest was targeted by miR-29a and miR-29a, which were downregulated in chondrogenic mMSCs as downstream targets of Sox9. With these hypotheses, the present study was set up to elucidate the relationship between miR-335-5p and cell chondrogenic differentiation and how the expression of miR-335-5p and its host gene Mest were regulated during the process.

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Materials and Methods Cell culture Mouse MSCs (Cyagen, Guangdong, China) were cultured in OriCell

TM

Strain C57BL/6 Mouse

Mesenchymal Stem Cell Growth Medium (Cyagen ,Guangdong, China). Chondrocytes were obtained as previously described,(23) and cultured in DMEM supplemented with 10%FBS, 25 mg/L L-ascorbic acid-2 phosphate (Sigma Dorset, UK). Hela cells were purchased from ATCC (Manassas, USA) and cultured in DMEM supplemented with 10%FBS (Gibco, Carlsbad, USA) and antibiotics. Chondrogenesis of mMSCs was induced by condition medium as previously described.(26) Several chemicals including KCl (Sigma-Aldrich, MO, USA), LiCl (Sigma-Aldrich), CRTi3 (Millipore, Billerica, MA, USA) and Y27632 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added to expansion medium to when needed. Lentivirus infection The lentiviral particles for LV-GFP-miR-335-5p, LV-GFP-antagomiR-335-5p and LV-GFP-mock vectors respectively were purchased from Shanghai GenePharma Co.,Ltd (Shanghai China). Lentiviral particles were added to 5×105 mMSCs in T25 flash for 8 h, and expansion medium was changed. Transduced cells (GFP positive) were purified after 72 h transduction by FACSCalibur cell sorter (BD Bioecience, Franklin Lakes, NJ, USA). Cell transfection MiRNA mimics, miRNA inhibitor and plasmids were transfected using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instruction. MiRNA mimics and anti-miRNA oligos were synthesized by Shanghai GenePharma Co.,Ltd (Shanghai China). The pGL3 plasmid was a gift from Professor Jianzhong Xi (Peking University, Beijing, China). TOPflash plasmid was provided by Professer Wei Wu (Tsinghua University, Beijing, China).

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Real-Time RT-PCR Total RNAs were isolated from mMSCs by miRcute miRNA Isolation Kit (Tiangen, Beijing, China), and cDNA was synthesized using Fastquant RT Kit (Tiangen, Beijing, China) for mRNA analysis. The miRcute microRNA first-strand cDNA synthesis kit (Tiangen, Beijing, China) was used for miRNA analysis. Real-Time PCR assay for mRNA analysis was performed by SuperReal PreMix (SYBR Green) (Tiangen, Beijing, China) and miRNA analysis was performed by miRcute miRNA qPCR detection Kit (Tiangen, Beijing, China). Primers used for Real-Time PCR were listed in Table 1. Primers for miR-29a (CD201-0027) and miR-29b (CD201-0028) were from Tiangen (Beijing, China). U6 and GAPDH were used as control for miRNA and mRNA detection, respectively. Western blot analysis Whole-cell lysates for Western blot were extracted with PIPA Lysis Buffer (Beyotime, Jiangsu, China). Antibodies for DKK1 and ROCK1 were purchased from Abgent (San Diego, CA, USA), Mest and Col21 antibodies were obtained from Santa Cruz Bio Santa Cruz Biotechnology (Santa Cruz, CA, USA), Daam1 antibody was obtained from Abcam (Cambridge, UK), and Sox9 antibody was obtained from Millipore (Billerica, MA, USA). The secondary antibodies conjugated with horseradish peroxidase (HRP) (Sigma-Aldrich, MO, USA) were used for blotting. The blots were visualized by ECL chemiluminescence reagents from Pierce Biotechnology (Rockford, IL, USA). Dual-luciferase assay The 3’UTR fragments of target genes were cloned to pGL3 luciferase reporter vector. Site mutation of 3’UTR was performed by GENEWIZ (Beijing, China)(Table 2). Each reporter vector and miRNA mimics or antagonist were co-transfected into Hela cells. Luciferase activities were measured using Dual-Luciferase® Reporter Assay System (Promega BioSciences, LLC., San Luis Obispo, CA, USA). Analysis of cell cycle and cell proliferation MSCs were infected with lentivirus expressing miR-335-5p and antagomiR-335-5p respectively. For cell cycle and apoptosis assay, ~×105cells were fixed by ethanol, treated by Cell Cycle and Apoptosis Analysis Kit 6   

 

(Beyotime Institution of Biotechnology, Jiangsu, China) and analyzed with BD FACSCalibur. For cell proliferation assay, cells were seeded in 96-well plate. The analysis was performed by Cell Counting Kit-8 (Beyotime Institution of Biotechnology, Jiangsu, China). Alcain Blue Staining Treated cells were fixed in 4% paraformaldehyde for 5 min, and then incubated with 8X Alcian Blue (Cyagen, Guangdong, China) for 30 min. Then the excess stain was washed with double distilled water. Images were captured under light microscope. Immunohistochemistry Treated cells were fixed in 4% paraformaldehyde for 5 min. The analysis for Col21 was performed by DAB Horseradish Peroxidase Color Development Kit (Beyotime, Jiangsu, China). Statistical analysis Data were expressed as mean±SD. The student’s t test was performed for experiments for two groups. Values were considered statistically significant at **p