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ORIGINAL ARTICLE
Year : 2018  |  Volume : 3  |  Issue : 1  |  Page : 6-11

MicroRNA-564 promotes the differentiation and proliferation of synovial mesenchymal stem cells into chondrocytes by targeting transforming growth factor beta 1


1 Department of Orthopedics, Jinling Hospital, Nanjing University, School of Medicine, Nanjing, Jiangsu, China
2 Department of Orthopedics, Jinling Hospital, School of Medicine, Nanjing Medical University, Nanjing, Jiangsu, China

Date of Submission29-Nov-2017
Date of Acceptance08-Feb-2018
Date of Web Publication22-Mar-2018

Correspondence Address:
Jianning Zhao
Department of Orthopedics, Jinling Hospital, Nanjing University, School of Medicine, 305 East Zhongshan Road, Nanjing 210002, Jiangsu
China
Lei Zhang
Department of Orthopedics, Jinling Hospital, Nanjing University, School of Medicine, 305 East Zhongshan Road, Nanjing 210002, Jiangsu
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ts.ts_23_17

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  Abstract 


Aim: To investigate the role of miR-564 in promoting the proliferation and differentiation of synovial mesenchymal stem cells (SMSCs) to chondrocytes. Methods: Third-generation SMSCs were used, and the experiments involved untreated SMSCs (control; Group A), SMSCs transfected with Hsa-miR-564 inhibitor NC (inhibitor blank; Group B), and SMSCs transfected with Hsa-miR-564 inhibitor (Group C). The expression of miR-564 in SMSCs was determined by real-time quantitative polymerase chain reaction. The SMSCs were induced to form cartilage for 3 weeks. The morphology of the induced chondrocytes was observed by hematoxylin and eosin and toluidine blue staining and cell viability recorded. Chondrocyte differentiation of SMSCs related to genes and proteins (COL2A1, Aggrecan, SOX9, transforming growth factor beta 1 [TGF-β1], and Smad4) was assessed. The chondrogenic effect of miR-564 was examined after blocking the target gene TGF-β1. Results: The morphology and characteristics of the induced cells were consistent with those of chondrocytes. The cell proliferative rate of Group C (miR-564 downregulation) was significantly higher than that of other groups. The expression of genes and proteins related to chondrocyte differentiation was significantly decreased in Group C. The relative expression of genes related to cartilage differentiation decreased after blocking TGF-β1. Conclusion: The downregulation mediated by miR-564 can promote the differentiation and proliferation of SMSCs into chondrocytes by targeting TGF-β1.

Keywords: Chondrocytes, miR-564, synovial mesenchymal stem cells, transforming growth factor beta 1


How to cite this article:
Sun X, Zhang P, Zhang L, Zhao J, Zhou L. MicroRNA-564 promotes the differentiation and proliferation of synovial mesenchymal stem cells into chondrocytes by targeting transforming growth factor beta 1. Transl Surg 2018;3:6-11

How to cite this URL:
Sun X, Zhang P, Zhang L, Zhao J, Zhou L. MicroRNA-564 promotes the differentiation and proliferation of synovial mesenchymal stem cells into chondrocytes by targeting transforming growth factor beta 1. Transl Surg [serial online] 2018 [cited 2018 Apr 25];3:6-11. Available from: http://www.translsurg.com/text.asp?2018/3/1/6/228311




  Introduction Top


Osteoarthritis (OA) is a common asymmetric and progressive inflammatory joint disease characterized by degeneration and loss of articular cartilage, with regeneration of marginal and subchondral bone.[1] Fifty percent of the population over the age of 60 years have evidence of OA and 35%-50% have clinical manifestations.[2] Patients with terminal OA may need to choose an artificial joint replacement, which exacts a burden on the patient's health and the national economy. However, if the articular cartilage could be repaired in the early stage of OA at the time of articular cartilage damage, the occurrence of terminal OA could be avoided. Many clinical scholars have proposed a variety of mechanism for the repair of cartilage damage, including microfracture, mosaic transplantation, autologous chondrocyte transplantation, and tissue engineering technology. None has achieved the desired clinical effect.[3],[4],[5] The primary explanation appears to be the failure of the experimental process to simulate the process required for self-healing of articular cartilage. The transplantation or formation of cartilage tissue that fails to integrate with native cartilage will change the mechanical properties, such as the ability to rebound after compression, or the misdistribution of stress forces, that can injure articular cartilage. Thus, the recovery of damaged cartilage requires a different approach.[6] Recent clinical studies have indicated the potential therapeutic value of enhancing the constitutive healing capacity of cartilage.[7]

When injury to the articular cartilage occurs, synovial mesenchymal stem cells (SMSCs) migrate to the site of injury to repair the cartilage damage in concert with other local cells.[8] SMSCs have high chondrogenic differentiation ability allows for more facile harvest compared to traditional bone marrow mesenchymal stem cells, and thus highlights this cell type as ideal for the study of articular cartilage repair.[9] A variety of signaling pathways are involved in the process of chondrocyte proliferation and differentiation from SMSCs. These pathways include the transforming growth factor-bone morphogenetic protein (TGF-BMP), Wnt, fibroblast growth factor (FGF), and mitogen-activated protein kinase as several examples. A variety of cytokines and microRNAs (miRNAs) play important roles in these pathways.[10]

MiRNAs are noncoding, endogenous, small RNAs (approximately 18–24 nucleotides in length) are ubiquitous in eukaryotes. MiRNAs can regulate messenger RNA (mRNA) degradation or inhibit mRNA translation by binding to the 3' untranslated region (3'UTR) and participating in the posttranscriptional regulation of gene expression.[11] At least 2600 miRNAs have been identified in the human genome, and 60% of these are predicted to involve genome regulation and play an important role in many biological processes [12] including cell proliferation, differentiation, apoptosis, metabolism, and signal transduction.[13] Many studies have shown that one miRNA, miR-564, is widely involved in the regulation of gene expression in organisms. MiR-564 was first investigated by Lai, and subsequent research has indicated the value of miR-564 and six other miRNAs as biomarkers in patients with latent schizophrenia.[14] A recent study reported that miR-564 can inhibit the proliferation and differentiation of glioblastoma by targeting transforming growth factor beta 1 (TGF-β1) in the TGF-BMP pathway.[15]

Given that the TGF-BMP pathway is also a key path for chondrogenesis, we hypothesized that miR-564 is also likely to promote the differentiation of SMSCs into chondrocytes through this pathway. To explore this, we detected the expression of miRNAs in the process of differentiation of SMSCs into chondrocytes using microarray analysis. Forty-four miRNAs were downregulated and the downregulation of miR-564 was significant. The finding indicated that miR-564 can regulate the differentiation of SMSCs to chondrocytes. We further explored the potential function of miR-564 in the process of chondrogenic differentiation and the possible mechanism of this process. The results indicate that the downregulation of miR-564 can promote the proliferation and differentiation of SMSCs into chondrocytes through the downstream cascade of the TGF-BMP pathway, which can ultimately repair the articular cartilage damage.


  Methods Top


Experimental time and experimental site

This study was conducted at Jinling Hospital, Medical School of Nanjing University from October 2016 to June 2017.

Isolation of synovial mesenchymal stem cells

Human SMSCs were originally provided by Jiangsu Kaiji Biotechnology Co. Ltd. (Nanjing, China) and were confirmed using flow cytometry. The primary SMSCs were removed from the liquid nitrogen aliquot and resuspended. After centrifugation, 1 × 10[4] nucleated cells were plated in 25 cm [2] flasks. The cells were cultured in basal medium (high-glucose Dulbecco's modified Eagle's medium [HG-DMEM], 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin; Gibco, Carlsbad, CA, USA). The culture medium and unattached cells were simultaneously removed after 24 h. The third-generation of cells was used as the experimental subset following 1 week of culture.

Transfection of miRNA inhibitor

An appropriate number of SMSCs was dispensed into wells of a cell culture plate, followed by the addition of antibiotic-free culture medium to dilute the cell density to approximately 80% of the original density. Five microliters of 20 μM miRNA inhibitor stock solution was added to 250 μL of serum-free Opti-MEM and mixed gently, followed by gentle mixture with 250 μL of serum-free Opti-MEM diluted with 5 μL of lipofectamine 2000 (Kaiji Biotechnology, Nanjing, China) and incubated at room temperature for 20 min. The miRNA inhibitor lipofectamine 2000 (Kaiji Biotechnology) was added to the specified wells and cultured for 4–6 h. This was followed by incubation at 37°C for 48 h. The experiment involved three groups. Group A was an untreated SMSC blank control group. Group B was the miRNA inhibitor blank control group, in which SMSCs were transfected with Hsa-miR-564 inhibitor NC as described above). Group C was the miR-564 inhibitor treatment group, in which SMSCs were transfected with Hsa-miR-564 inhibitor.

Real-time quantitative polymerase chain reaction

(qRT-PCR) analysis of miRNA


After transfecting SMSCs, the expression of miR-564 was detected using the miRcute miRNA Isolation Kit (DP501; TIANGEN Biotech [Beijing] Co., Ltd., Beijing, China). The first cDNA strand was synthesized using the miRcute miRNA cDNA Synthesis Kit (KR201; TIANGEN Biotech [Beijing] Co., Ltd.) according to the manufacturer's instructions. qRT-PCR was conducted with the ABI Stepone Plus real-time PCR platform using SYBR Green I PCR reagents (Toyobo, Osaka, Japan). U6 was used as the normalization control. The relative expression level of miR-564 was calculated with the 2^-ΔΔCt method. Primer sequences are listed in [Table 1].
Table 1: Primer sequences of each gene

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Chondrogenic differentiation of synovial mesenchymal stem cells

The chondrogenic induction medium (HG-DMEM containing 10% fetal bovine serum, 10 μg/L TGF-β3, penicillin, and streptomycin [each 100 U/mL; Saiye Biotechnology, Co., Guangzhou, China]) was dispensed into  Petri dish More Detailses containing SMSCs. The concentration of TGF-β3 was maintained at 10 μg/L, and the solution was changed every 3 days. After 3 weeks of induction, the cells were stained with hematoxylin and eosin (H and E) and toluidine blue and observed under an inverted microscope.

Cell proliferation assays

SMSCs were formulated into cell suspensions at a concentration of 3 × 10[4] cells/mL and 100 μL of each cell suspension added to a 96-well cell culture plate. The cells were cultured for 1–7 days in chondrogenic induction medium. Cell viability was determined using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based assay.

QRT-PCR analysis the mRNA expression level

Total RNA was extracted with TRIzol reagent (Invitrogen). Reverse transcription was performed with the PrimeScript First Strand cDNA Synthesis Kit according to the manufacturer's instructions. The qRT-PCR was performed using the ABI 7500 System (Applied Biosystems, Franklin Lakes, NJ, USA) as previously described. GAPDH was used as the endogenous control. The primers are listed in [Table 1].

Western blotting

Total protein was extracted from frozen tissues or cells using a Total Protein Extraction Kit (Kaiji Biotechnology) according to the manufacturer's instructions. Protein concentration was confirmed by the Bradford assay. Protein was separated by 10% SDS-PAGE, and then transferred to PVDF membranes using standard procedures. The membrane was incubated with the primary antibody for TGF-β1 (Sigma-Aldrich, St. Louis, MO, USA) or GAPDH (Cell Signaling Technology, Beverly, MA, USA), washed, and incubated with horseradish peroxidase-conjugated secondary antibody. The intensity of the bands was visualized by enhanced chemiluminescence system (Amersham Pharmacia Biotech Ltd., Little Chalfont, UK).

Blocking transforming growth factor beta 1 by short hairpin RNA

A constructed shRNA plasmid expression vector was used to transfect SMSCs. TGF-β1 expression was detected by qRT-PCR to confirm that the TGF-β1 gene was successfully blocked. Then, SMSCs were induced to differentiate to cartilage for 3 weeks. Finally, the expression of downstream cartilage-related genes was detected by qRT-PCR.

Statistical analyses

Numerical data are presented as mean ± standard deviation. All statistical analyses were performed using SPSS 21.0 software (IBM Corp., Armonk, NY, USA). The significant differences between the two study groups were assessed using Student's t-test or ANOVA test. The P < 0.05 was considered as statistically significant.


  Results Top


Synovial mesenchymal stem cell morphology and proliferation

SMSCs began to grow 2 days of inoculation. The primary cultured SMSCs proliferated well. The cells were long-spindle or spindle-shaped [Figure 1]a. The cell density had increased by day 4 of culture and the morphology had changed to a rounded spindle shape [Figure 1]b. The expression of miR-564 seen in Group C was significantly lower than that in Group A and B [Figure 1]c. After 3 weeks of chondrocyte induction, all groups of SMSCs were identified as chondrocytes by H and E staining and toluidine blue staining. The H and E staining revealed spindle-shaped cartilage cells with each cell displaying a purple-blue nucleus and purple cytoplasm. The number of chondrocytes in Group C was significantly increased compared with Group A and B [Figure 1]d. Toluidine blue staining revealed dark blue nuclei with light blue heterochromatic granules visible in the cells. Group C cells contained more light blue heterochromatic granules than the cells in the control group [Figure 1]e. The plot of cell viability with time produced an expected and typical growth curve, with the logarithmic phase beginning on day 3 of culture, with cells entering a rapid proliferative growth cycle beginning on day 3 of culture. The proliferation rate of the experimental group beginning at day 3 was significantly greater than that of the control group [Figure 1]f, indicating that the downregulation of miR-564 can promote the proliferation of the chondrocytes.
Figure 1: SMSC morphology and proliferation. (a) The morphology of SMSCs after cultured 2 days; (b) The morphology of SMSCs after cultured 4 days; (c) The relative expression of miR-564 was detected with qRT-PCR assay, the significance (P < 0.01) was determined by ANOVA test; (d) The H and E staining of chondrocytes in three groups; (e) The toluidine blue staining of chondrocytes in three groups; (f) The cell proliferation curve of SMSCs, P < 0.01, ANOVA test; (g) Alignment analysis showed that the 3'UTR of TGF-β1 has putative binding site for miR-564, located at 93-99 nt. SMSC: Synovial mesenchymal stem cell, qRT-PCR: Real-time quantitative-polymerase chain reaction, TGF-β1: Transforming growth factor beta 1, 3'UTR: 3' untranslated region

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Expression of chondrocyte differentiation-related genes and proteins, and the chondrogenic effect after blocking TGF-β1

After chondrogenic induction, the expressions of COL2A1, Aggrecan, and SOX9 in the chondrocytes in the three groups were detected by qRT-PCR. COL2A1, Aggrecan, and SOX9 expression within Group C (miR-564 downregulation group) was significantly higher than that of Group A and Group B [Figure 2]a, indictating more chondrogenic differentiation in Group C. Concurrent western blot analysis revealed significantly higher expression of COL2A1, Aggrecan, and SOX9 protein in group C compared to the other two groups [Figure 2]b. In further experiments, we detected the gene expressions of TGF-β1 and smad4 in the downstream TGF-BMP pathway by qRT-PCR. The gene expressions in Group C were significantly higher than those in the control group [Figure 2]c. Similarly, we also detected the expression of the TGF-β1 and Smad4 proteins in the downstream TGF-BMP pathway. The expression of both proteins was increased in Group C compared with Group A and B [Figure 2]d. To further verify whether miR-564 acts on the target gene TGF-β1, we further blocked the TGF-β1 gene expression after miR-564 transfection of SMSCs followed by cartilage induction for 3 weeks, and then detected the expression of cartilage-related genes by qRT-PCR. A significant decrease in TGF-β1 gene expression in shRNA transfected cells confirmed successful transfection [Figure 2]e. The qRT-PCR analysis revealed that the expression of cartilage-related genes in the shRNA transfected group was lower than that in miR-564 downregulated group but was higher than that in blank control group [Figure 2]f.
Figure 2: Expression of chondrocyte differentiation-related genes and proteins, and the chondrogenic effect after blocking TGF-β1. (a) The expression of chondrocyte differentiation-related genes, P < 0.01, P < 0.05, ANOVA test; (b) The expression of chondrocyte differentiation-related proteins; (c) The expression of TGF-BMP pathway related genes, P < 0.01, P < 0.05, ANOVA test; (d) The expression of TGF-BMP pathway-related proteins. (e) The knockdown efficiency of TGF-β1; P < 0.01, Student's t-test; (f) The expression of chondrocyte differentiation related genes after blocking TGF-β1, P < 0.01, ANOVA test. TGF-β1: Transforming growth factor beta 1, TGF-BMP: Transforming growth factor-bone morphogenetic protein

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  Discussion Top


Cartilage lesions often fail to heal well because of the poor regenerative capacity of cartilage. Conversely, SMSCs are highly capable of forming cartilage and are expected to repair cartilage injury.[16] MiRNAs regulate target gene expression by inducing mRNA to degrade or suppress mRNA translation by binding to the 3'UTR. Accumulating evidence has illustrated the essential roles of miRNA in the differentiation and development of bone and cartilage.[17] Recent studies have revealed the upregulated expression of miR-410, miR-99a, and miR-140 during chondrogenesis.[18],[19],[20] Using miRNA microarray analysis, we demonstrated that the expression level of miR-564 was significantly decreased during the process of differentiation of SMSCs. MiR-564 can inhibit the proliferation and differentiation of glioblastoma by acting on the TGF-BMP pathway ultimately targeting TGF-β1.[15] TGF-β, BMP, insulin-like growth factor, and other factors are closely related to the repair of cartilage damage in the process of articular cartilage metabolism.[21] It is believed that the TGF-BMP pathway plays a key role in the process of cartilage damage repair. We speculate that miR-564 can also promote the differentiation of SMSCs into chondrocytes by acting on the TGF-BMP pathway. Future studies will seek to confirm this idea.

Our further experiments showed several differences between the miR-564 downregulation group (Group C) and the control group. H and E staining and toluidine blue staining indicated higher chondrocyte differentiation level in the experimental group. Cells in the experimental group displayed significantly greater expression of chondrocyte-related genes and protein (COL2A1, Aggrecan, and SOX9) compared with the control group. The proliferation of cartilage cells in the experimental group was more robust. The results support the idea that the downregulation of miR-564 can promote SMSC chondrocyte proliferation and differentiation. To further explore whether miR-564 achieves this by acting on the target protein TGF-β1, we conducted an experiment that revealed the significantly increased gene and protein expression of TGF-β1 in the miR-564 downregulated group compared with the control group, as well as the increased expressions of the downstream smad4 gene and protein. The targeting of TGF-β1 by miR-564 was supported by an mRNA alignment analysis, which showed that the 3'UTR of TGF-β1 contains one binding site for miR-564, located at 93-99 nt [Figure 1]g. Furthermore, when the expression of TGF-β1 was blocked by shRNA, the downstream cartilage differentiation-related genes were downregulated compared to the miR-564 downregulation group. Therefore, we conclude that miR-564 can act on the target protein TGF-β1, causing a cascade reaction downstream of the TGF-BMP pathway to promote the proliferation and differentiation of SMSCs into chondrocytes.

In conclusion, the downregulation of miR-564 can promote the differentiation of SMSCs into chondrocytes through the action of TGF-β1, which is a target protein in the TGF-BMP pathway. However, the present results were derived from in vitro cell experiments. Further experiments will need to be conducted to determine whether miR-564 exhibits the same effect in vivo. The repair of injured articular cartilage involves a variety of miRNAs. The results lay the foundation for the future studies that could someday provide a new clinical regimen for patients with OA.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (Grant No. 81702170), China Postdoctoral Science Foundation (Grant No. 2017T100826), and Jiangsu Province Natural Science Foundation (Grant No. BK20170624).

Conflicts of interest

There are no conflicts of interest.



 
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