Platelet lysate induces chondrogenic differentiation of umbilical cord‐derived mesenchymal stem cells by regulating the lncRNA H19/miR‐29b‐3p/SOX9 axis

H19 expression is induced in platelet lysate‐stimulated human umbilical cord‐derived mesenchymal stem cells (hUCMSCs), and H19 acts as a sponge of miR‐29b‐3p to up‐regulate the expression of the master chondrocyte transcription factor SRY‐related high‐mobility‐group box 9, thereby promoting platelet lysate‐induced chondrogenic differentiation of hUCMSCs.

Platelet lysate (PL) has been shown to induce chondrogenic differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs). However, the underlying mechanism is still not clear. The aim of this study was to investigate whether long noncoding RNA H19 is involved in this process. hUCMSCs were isolated, identified and cultured in 5% PL-supplemented chondrogenic differentiation medium. Chondrogenic differentiation was assessed by Alcian blue staining. The expressions of H19, miR-29b-3p, SRY-related high-mobility-group box 9 (SOX9), collagen II and aggrecan were determined by quantitative real-time PCR and western blot. The interaction between miR-29b-3p and H19 or SOX9 was analyzed by luciferase reporter assay. During PL-induced chondrogenic differentiation of hUCMSCs, expressions of H19 and SOX9 were increased, whereas miR-29b-3p expression was decreased. H19 overexpression promoted, whereas H19 silencing attenuated the PL-induced chondrogenic differentiation of hUCMSCs. SOX9 was identified as a direct target of miR-29b-3p, and H19 was observed to act as a sponge of miR-29b-3p to up-regulate SOX9 expression. The chondrogenic differentiation-promoting effect of H19 overexpression was negated when miR-29b-3p expression was up-regulated by Lenti-miR-29b-3p infection. In conclusion, PL induced chondrogenic differentiation of hUCMSCs by regulating the H19/miR-29b-3p/SOX9 axis.
Osteoarthritis (OA) is one of the major joint diseases worldwide mainly characterized by articular cartilage degeneration [1]. Articular cartilage is an important component of the synovial joints and lacks the capacity for self-regeneration [2]. Mesenchymal stem cells (MSCs) have emerged as a relevant cell source for cartilage repair in recent years [3,4]. Zheng et al. [5] observed that human chondrocytes could be successfully induced by coculture of human umbilical cordderived mesenchymal stem cells (hUCMSCs) with rabbit chondrocytes.
chondrogenic differentiation of MSCs [11]. A previous study demonstrated that PL induced chondrogenic differentiation of hUCMSCs, concomitant with elevated expression of SRY-related high-mobility-group box 9 (SOX9), a cartilage-specific transcription factor that plays crucial roles in chondrocyte differentiation [12]. However, the underlying mechanism is still not clear.
Long noncoding RNAs (lncRNAs) are non-proteincoding transcripts longer than 200 nucleotides and play important regulatory roles in various physiological and disease processes [13]. Recent studies have suggested that lncRNA plays a critical role in chondrogenic differentiation of MSCs. For example, Barter et al. [14] reported that lncRNA regulator of chondrogenesis RNA (ROCR) induced chondrogenic differentiation of MSCs by up-regulating SOX9 expression. H19, a kind of lncRNA, has been shown to promote the osteogenic differentiation of MSCs [15,16] but inhibit the differentiation MSCs into adipocytes [17]. Existing evidence has demonstrated that H19 expression can be induced by TGF-β that presents in PL [10,18], suggesting that H19 might be involved in the PL-induced chondrogenic differentiation of hUCMSCs. In recent years, many studies have indicated that H19 is implicated in tenogenic differentiation [19], tumor cell epithelial-mesenchymal transition [20,21] and myocardial ischemia-reperfusion injury [22] by functioning as a miR-29b-3p sponge via competing endogenous RNA activity. It should be noted that miR-29b-3p is predicted to bind to the 3 0 -UTR of the master chondrocyte transcription factor SOX9 by bioinformatics software (http://starbase.sysu.edu.cn/). Thus, we hypothesized that H19 might be involved in PL-induced chondrogenic differentiation of hUCMSCs by regulating the miR-29b-3p/SOX9 axis.

Preparation of human PLs
Human PLs were prepared from PRP obtained from five donors as previously described [12]. In brief, 20 mL of peripheral blood was collected from each healthy volunteer and pooled. PRP was isolated from peripheral blood and then subjected to three cycles of freeze-thaw (frozen at −80°C and thawed at 37°C) to obtain PL. PL was centrifuged at 15 000 g for 20 min at 4°C, filtered through 0.22-μm filters and stored at −20°C until use. Written informed consent was obtained from all volunteers. This experiment was approved by the Ethics Committee of First Affiliated Hospital of Harbin Medical University. The study methodologies conformed to the standards set by the Declaration of Helsinki.

Isolation of hUCMSCs
hUCMSCs were isolated from umbilical cords of five cesareandelivered full-term neonates using the explant method [12]. Informed consent was obtained from the parent/legal guardian. In brief, umbilical cords were cut into smaller pieces of~5 × 5 cm, transferred to a 25-cm 2 flask and maintained in Dulbecco's modified Eagle's medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 5% PL, 100 μgÁmL −1 streptomycin and 100 UÁmL −1 penicillin at 37°C in a humidified atmosphere containing 5% CO 2 . The medium was replaced for the first time after 7 days of culture and thereafter every 3-4 days. The umbilical cord segments were removed on the 14th day, and adherent cells reaching 75-85% confluence were detached by 0.05% trypsin/EDTA and further cultivated until the third passage. hUCMSCs at passage 2 were used in the following experiments. Chondrogenic differentiation hUCMSCs at passage 2 (1 × 10 6 cells) were cultured in a chondrogenic differentiation medium supplemented with Dulbecco's modified Eagle's medium, 1 × insulin-transferrin-selenium, 50 mgÁL −1 ascorbic acid, 100 nM dexamethasone and 5% PL for 21 days. The culture medium was replaced twice a week.

Alcian blue staining
After infection with the indicated lentiviruses, hUCMSCs were cultured in a chondrogenic differentiation medium for 21 days as described earlier. Then chondrogenic differentiation was assessed by Alcian blue staining. In brief, the pellets were fixed, dehydrated, embedded in paraffin and sectioned at 5 μm thickness. For detection of proteoglycan and mucopolysaccharides, sections were deparaffinized, rehydrated and then stained with 1% Alcian blue (Sigma-Aldrich, St. Louis, MO, USA) for 30 min.

Statistical analysis
All statistical analyses were performed using SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). Statistical significance was analyzed using the Student's t-test between two groups and one-way ANOVA among three or more groups. A P value <0.05 was considered statistically significant.

H19 expression was increased during PL-induced chondrogenic differentiation of hUCMSCs
hUCMSCs were isolated from umbilical cords of cesarean-delivered full-term neonates and identified by flow cytometry. Data revealed that the obtained hUCMSCs were positive for CD105 and CD44, but negative for CD45 and CD34 (Fig. 1A), confirming that the cells were MSCs. hUCMSCs were then cultured in 5% PL-supplemented chondrogenic differentiation medium for 0, 7, 14 and 21 days. The results from qRT-PCR analysis demonstrated significant upregulation of H19 expression during PL-induced chondrogenic differentiation of hUCMSCs (Fig. 1B).

H19 promoted PL-induced chondrogenic differentiation of hUCMSCs
To determine the role of H19 in PL-induced chondrogenic differentiation of hUCMSCs, we overexpressed and silenced H19 in hUCMSCs, and then cultured them in 5% PL-supplemented chondrogenic differentiation medium for 21 days. The overexpression and silencing efficiencies of H19 were confirmed by qRT-PCR analysis (Figs 2A and S1A). Importantly, Alcian blue staining demonstrated that H19 overexpression led to increased production of proteoglycan in the differentiated cells, whereas H19 silencing yielded the opposite results (Fig. 2B). Furthermore, the protein levels of chondrogenic markers (collagen II and aggrecan) in the differentiated cells were significantly up- regulated by H19 overexpression but down-regulated by H19 silencing (Fig. 2C). These findings indicated that H19 promoted PL-induced chondrogenic differentiation of hUCMSCs.
H19 acted as a sponge of miR-29b-3p to upregulate SOX9 expression Next, we explored the molecular mechanism by which H19 promotes PL-induced chondrogenic differentiation of hUCMSCs. The results from qRT-PCR analysis revealed that miR-29b-3p expression was notably down-regulated, whereas SOX9 mRNA and protein levels were significantly up-regulated during PL-induced chondrogenic differentiation of hUCMSCs ( Fig. 3A-C). Furthermore, H19 overexpression led to a marked increase in SOX9 mRNA and protein levels. In contrast, H19 silencing significantly down-regulated SOX9 expression (Fig. 3D-E). In addition, the luciferase activity was significantly decreased in cells cotransfected with H19 WT reporter and miR-29b-3p mimic (Fig. 3F), indicating that H19 directly interacted with miR-29b-3p. We also observed decreased luciferase activity in cells cotransfected with SOX9 WT reporter and miR-29b-3p mimic (Fig. 3G), suggesting that 3 0 -UTR of SOX9 was directly targeted by miR-29b-3p. Moreover, transfection with miR-29b-3p mimic resulted in a significant decrease in mRNA and protein levels of SOX9. On the contrary, inhibition of miR-29b-3p noticeably increased SOX9 expression (Fig. 4A,B). Together, these results manifested that H19 might act as a sponge of miR-29b-3p to up-regulate SOX9 expression.

Discussion
MSCs are multipotent stem cells with the potential to differentiate into chondrocytes, and MSC-derived cartilage tissue engineering is a clinical method used for OA treatment [23]. PRP is a blood extraction product widely used in clinical practice to accelerate the wound healing process of some tissues, such as muscle [6], cartilage [7] and tendon [8]. Prepared from PRP, PL has been shown to reduce immunogenicity [10] and promote MSC chondrogenic differentiation, providing broad application prospects for allogeneic or xenogeneic transplantation in cartilagerelated diseases [24,25]. A previous study clearly showed that PL induced the differentiation of hUCMSCs into chondrocytes [12]. In this study, we demonstrated for the first time the molecular mechanism by which PL induces MSC chondrogenic differentiation; that is, PL induced chondrogenic differentiation of hUCMSCs by regulating the H19/ miR-29b-3p/SOX9 axis. A number of recent studies have suggested that lncRNA plays a critical role in chondrogenic differentiation of MSCs. An increasing number of lncRNAs have been identified with a potential role in chondrogenesis, such as ROCR [14], differentiation antagonizing non-protein-coding RNA [26], maternally expressed 3 [23] and ADAMTS9 antisense RNA 2 [27]. For instance, lncRNA urothelial cancer-associated 1 (UCA1) promotes chondrogenic differentiation of human bone marrow-derived MSCs via regulating the miR-145-5p/SMAD5 and miR-124-3p/SMAD4 axis [28]. lncRNA H19 has been shown to promote the osteogenic differentiation of MSCs [15,16] and inhibit the differentiation MSCs into adipocytes [17]. Yang et al. [18] demonstrated that in the bovine mammary alveolar cell-T cell line, TGF-β induced up-regulation of H19, which promoted epithelial-to-mesenchymal transition and contributed to mammary gland fibrosis. PL contains a relatively large number of growth factors, including TGF-β that is critical for chondrogenic differentiation [10]. Thus, we speculated that H19 might be involved in the PL-induced chondrogenic differentiation of hUCMSCs. To our knowledge, our findings in this study provided the first evidence that H19 overexpression promoted, whereas H19 silencing attenuated the chondrogenic differentiation of PL-treated MSCs. Evidence indicates that TGF-β1 up-regulated H19 expression through activating the phosphoinositide-3-kinase/AKT pathway in epithelial cells [29]. Whether the phosphoinositide-3-kinase/Akt pathway or other transcriptional regulation mechanisms are involved in the PL-induced H19 up-regulation remains to be further investigated.
Several studies have established that H19 regulates the expression and biological functions of certain miR-NAs by acting as a competing endogenous RNA or a miRNA sponge [30,31]. For example, H19 contributes to oxidative damage repair in the early age-related cataract by sponging miR-29a [32]. miR-29b-3p not only plays an important role in tumor initiation and progression [33] but also participates in DNA damage response [34], tissue fibrosis [35] and chondrogenic differentiation [36]. In this study, we performed bioinformatics analysis and luciferase reporter assay to analyze the interaction between miR-29b-3p and H19 or SOX9. Results showed that H19 can function as a sponge RNA for miR-29b-3p that negatively regulates the expression of target gene SOX9. SOX9 gene is a cartilage-specific transcription factor that controls the expression of numerous chondrocyte genes (e.g. collagen II and aggrecan) and plays essential roles in chondrocyte differentiation and cartilage formation [37,38]. Our results demonstrated that H19 positively regulated SOX9 expression and promoted MSC chondrogenic differentiation, and these effects were rescued by miR-29b-3p mimic.

Conclusion
The findings in this study demonstrate that H19 expression is induced in PL-stimulated hUCMSCs and that H19 acts as a sponge of miR-29b-3p to up-regulate the expression of the master chondrocyte transcription factor SOX9, thereby promoting PL-induced chondrogenic differentiation of hUCMSCs. These data provide new experimental evidence for elucidating the mechanism of MSC in cartilage repair and a novel therapeutic option for OA.