The Protective Role of CD44 and microRNA-146a in Tendinopathy: A Signaling Axis Targets Apoptosis

doi:10.21203/rs.3.rs-4702424/v1

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The Protective Role of CD44 and microRNA-146a in Tendinopathy: A Signaling Axis Targets Apoptosis

https://doi.org/10.21203/rs.3.rs-4702424/v1

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Objective

Tendinopathy refers to the clinical condition characterized by pain in a tendon accompanied by significant dysfunction, with preference given to pathological terms such as tendinitis and tendinosis. It is influenced by multiple factors, including chronic inflammation, senescence, and apoptosis. CD44 serves as a principal cell-surface receptor for hyaluronan (HA), a constituent of the extracellular matrix. Previous studies have shown that inhibition of the CD44 signaling pathway induces apoptosis, inflammation, and expression of matrix metalloproteinases in tendinopathic tenocytes. Furthermore, microRNA-146a (miR-146a) blocks interleukin-1β (IL)-1β-induced senescence in tendinopathic tenocytes. Therefore, the aim of this study is to investigate the signaling axis between CD44 and miR-146a in tendinopathic tenocytes and tendons, which may finely regulate apoptosis.

Methods

Overexpression of CD44 and miR-146a in rat primary tendinopathic tenocytes and tendons were achieved through lentiviral vector-mediated transfer of CD44 cDNA (LVCD44) and precursor miR-146a (LVmiR-146a). TUNEL staining was performed to evaluate apoptosis. Additionally, LY294002, a PI3K/AKT inhibitor, and OX-50, an antagonizing antibody that blocks CD44 activity, in situ hybridization (ISH), and immunohistochemistry were used to examine the CD44-AKT-miR-146a signaling axis targeting Smad4 in tendinopathic tenocytes and tendons.

Results

Tendinopathic tenocyte transfectants overexpressing CD44 and miR-146a exhibited lower apoptotic cell numbers compared to transfectants treated with control vectors. Furthermore, the CD44-AKT-miR-146a signaling axis was shown to alleviate apoptosis in IL-1β-stimulated tendinopathic tenocytes and rat collagenase-induced Achilles tendons by inhibiting Smad4 expression.

Conclusions

We demonstrate that overexpression of CD44 and miR-146a protects tendinopathic tenocytes and tendons from apoptosis through the AKT/miR-146a/Smad4 signaling pathway.

Tendinopathy

CD44

microRNA-146a

lentiviral vector

AKT

Smad4

Tendinopathy manifests as pain in a tendon along with significant dysfunction, with the preferred pathological terms being tendinitis and tendinosis(1). The condition is thought to arise from a combination of various factors including overuse(2), chronic inflammation(3), apoptosis(4), and aging(5, 6), all of which contribute to its pathogenesis. Consequently, recent translational studies suggest that targeting apoptosis(7), inflammation(8), and senescence(9) in tenocytes could offer potential therapeutic avenues for managing this tendon disorder.

The apoptosis theory in tendinopathy links high levels of cyclic strain with oxidative stress, the acquisition of a cartilage phenotype, and the activation of matrix metalloproteinases, leading to the development of degenerative injuries(10, 11). Moreover, the gradual loss of tendon cells due to apoptosis further compromises the tissue's ability to repair itself(10). Samples obtained from patients with degenerative tendinopathy, as well as those from a rat model of overuse tendinopathy, exhibited increased expression of caspase 3 and caspase 8, both pivotal mediators of apoptosis(11, 12). Additionally, tenocytes play a crucial role in maintaining extracellular matrix (ECM) homeostasis, and a pivotal link between changes in ECM composition and heightened tenocyte apoptosis has been well-documented(13, 14). Therefore, the identification of molecular mechanisms underlying dysregulated tenocyte apoptosis is essential for understanding the pathophysiology of tendinopathy.

Hyaluronan (HA) is a prevalent extracellular glycosaminoglycan within mammalian ECM and CD44 serves as a HA-binding cell surface receptor(15). Our prior research has unequivocally established the connection between CD44 and apoptosis in tenocytes(4). When rat tendinopathic tenocytes were treated with an antagonistic antibody targeting CD44, they exhibited increased tenocyte apoptosis, decreased cell viability and AKT inactivation, and down-regulation of ECM-related protein expression(4). Additionally, these cells displayed senescence-associated secretory phenotypes (SASPs)(4). Furthermore, we have shown that cross-linked HA exerts anti-apoptotic and anti-senescent effects on human tendinopathic tenocytes and rat tendinopathic tendons(16), suggesting that the CD44 signaling pathway is implicated in improving tenocyte apoptosis.

MicroRNA-146a (miR-146a) is among the earliest identified differentially expressed miRNAs in osteoarthritis (OA). Studies have revealed that miR-146a-deficient mice display an early onset of OA characteristics, such as synovitis, cartilage degeneration, and osteophytes(17). Conversely, mice overexpressing miR-146a in chondrogenic cells demonstrate resistance to aging-related and post-traumatic OA(17). This protective effect is attributed to the molecular mechanism involving the inhibition of IL-1β-mediated catabolism(17). Notably, decreased expression of miR-146a correlates with declining levels of type 2 collagen and increasing MMP-13 expression(18). Reduction in miR-146a expression can be induced by IL-1β in primary chondrocytes in an NF-κB-dependent manner(19). Moreover, miR-146a overexpression leads to a reduced cellular response to TGF-β in chondrocytes by targeting Smad4, a crucial intracellular adaptor for the TGF-β pathway(19). IL-1β can also induce TGF-β expression via ERK1/2 activation in tendon-derived cells(20), and during repetitive mechanical loading in tendon fibroblasts, enhanced apoptosis accounts for the decreased fibroblast numbers(21). This phenomenon can be reversed by exogenous addition of an anti-TGFβ-1 antibody or siRNA targeting TGFβ-1(21). Collectively, IL-1β facilitates catabolism in OA articular cartilage and tendon-derived cells. The underlying mechanisms may involve the elevation of apoptosis-related molecules via TGF-β signaling, with miR-146a potentially impeding this process.

Our prior research has indicated that over-expression of miR-146a in IL-1β-stimulated rat tendinopathic tenocytes can diminish senescence and SASPs by inactivating NF-κB(22). However, the upstream regulator responsible for this mechanism remains unknown. In this study, our aim is to explore the signaling relationship between CD44 and miR-146a in tendinopathic tenocytes, which could finely modulate apoptosis. We hypothesize that CD44 may exert anti-apoptotic effects through the PI3K/AKT pathway, thereby stimulating miR-146a expression in tenocytes and attenuating IL-1β-induced apoptosis.

Ethics statement

The animal experiments conducted in this study adhered to stringent ethical guidelines and were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of National Cheng Kung University (approval numbers: 107–165 and 112–135).

Generation of lentiviral vectors that stably expressing CD44 and miR-146a

To generate lentiviral vectors ensuring stable expression of miR-146a and CD44, we conducted transient transfection of 293T cells with PMIRH146a PA 1 2 and pLenti-GIII-EF1α-CD44, in conjunction with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G encoding vesicular stomatitis virus G glycoprotein (VSVG). This was carried out using calcium phosphate precipitation methods as previously detailed(6, 22).

Primary culture of rat tendinopathic tenocytes

For the primary culture of rat tendinopathic tenocytes, male Sprague-Dawley rats aged 4–6 weeks were sourced from LASCO, Taiwan. They were housed in the Laboratory Animal Center, College of Medicine, National Cheng Kung University, and the Taiwan Animal Consortium (with AAALAC International Full Accreditation). Rigorous health monitoring was conducted by our center's administrators. The rats underwent intra-tendinous injection of collagenase I (0.015 mg/µL, 10 µL injection/rat, Sigma-Aldrich, St. Louis, MO, USA). One week following the initial procedure, Achilles tendons were harvested post-euthanasia of the rats via an overdose of isoflurane. The procedures for tendon sample preparation and tenocyte culture remained consistent with those outlined in our previous studies.(4, 6).

Generation of rat tenocytes stably expressing CD44 and miR146a and IL-1β stimulated apoptosis

To establish rat tenocytes expressing CD44 and miR146a stably, and to induce IL-1β-stimulated apoptosis, rat tendinopathic tenocytes were exposed to lentiviral vectors encoding CD44 cDNA and precursor miR-146a, or empty and scramble sequences (LVSin and LVmiR-scramble served as control vectors) for a duration of 72 h. MiR-146a expression was assessed via qRT-PCR using a TaqMan MicroRNA Assay kit and TaqMan Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA), with U6 small RNA as the internal control, and employing the 2^−∆∆CT method(22). CD44 expression was determined by immunoblotting(6). Subsequently, LVCD44, LVSin, LVmiR-146a, and LVmiR-scramble-transduced rat tenocytes were stimulated with IL-1β (10 ng/mL) for 120h, and apoptosis was evaluated using the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA).

Determination of CD44-AKT -miR-146a signaling axis in tenocytes

To explore the CD44-AKT-miR-146a signaling axis in tenocytes, we conducted the following: Tendinopathic tenocytes were infected with LVSin and LVCD44 for 72h. Stable transfectants overexpressing CD44 were subjected to treatment with the PI3K/AKT inhibitor (50 µM of LY294002, Selleckchem) for 24h. In a parallel experiment, tenocytes were treated with OX-50, an antagonistic antibody targeting CD44 (at a concentration of 10 µg/mL, GeneTex) for 24h. Subsequently, miR-146a expressions were quantified using qRT-PCR. Cell lysates from the aforementioned experiments were subjected to immunoblot analyses with antibodies against phosphorylated AKT (1:1000, Cell Signaling Technology) and total AKT (1:1000, Cell Signaling), along with a horseradish peroxidase–conjugated secondary antibody (1:10,000; Jackson ImmunoResearch) and quantitative control anti-actin antibodies (1:10,000, SigmaAldrich). The signal intensity in immunoblot analyses was further quantitated using ImageJ software (National Institutes of Health)(4).

Determination of Smad4 expression and apoptosis in miR-146a over-expressed tenocytes.

Tendinopathic tenocytes were infected with LVmiR-scramble and LVmiR-146a for 72h and stimulated with IL-1β for 120h. Cell lysates were subjected to immunoblot analyses with antibodies against Smad4 (1:1000, Abcam), cleaved caspase 3 (c-Cas3, 1:1000, Cell Signaling), and procaspase 3 (p-Cas3, 1:1000, Cell Signaling), along with a horseradish peroxidase–conjugated secondary antibody (1:10,000, Jackson ImmunoResearch) and quantitative control anti-actin antibodies (1:10,000, SigmaAldrich).

AntagomiR-146a transfer in CD44 over-expressed tenocytes and IL-1β-stimulated apoptosis.

The antagomiR-146a used in this study was synthesized by MD Bio. Inc. (Taipei City, Taiwan), following a previously published method(23). Briefly, the sequence for antagomiR-146a employed was as follows: 5’-a_sa_scccauggaauucaguuc_su_sc_sa_s-Chol-3’. Here, the lower case letters denote 2’-OMe-modified nucleotides; subscript ‘s’ signifies a phosphorothioate linkage; and ‘Chol’ denotes a cholesterol group linked through a hydroxyprolinol linkage. Tendinopathic tenocytes were infected with LVSin and LVCD44 for 72h and subsequently treated with antagomiR-146a at serial concentrations of 10, 20, and 40 µM for 72h. The expression of miR-146a was assessed using qRT-PCR. In a separate experiment, cells treated as described above were stimulated with IL-1β (10 ng/mL) for 120h and then subjected to TUNEL assay.

Lentiviral vector-mediated gene transfer in Achilles tendons from rats with tendinopathy

In our previous study, we established a rat collagenase-induced tendinopathy model(6, 24, 25). Briefly, male Sprague-Dawley rats aged 8 weeks and weighing between 250–300 g were intratendinously injected with 10 µL of bacterial collagenase I (0.015 mg/µL, 10 µL injection per rat) into their right Achilles tendons to induce tendinopathy. By week 8 post the initial procedure, the rat tendinopathy model had matured(24, 25). All rats were randomly allocated into two groups: LVSin and LVCD44 (n = 6 each). Three days following the index procedure on their right hindlimbs, three consecutive intratendinous injections of LVSin or LVCD44 (4×10⁷ lentiviral vectors) were administered into their Achilles tendons at a 2-week interval. After eight weeks post-collagenase injection, the rats were euthanized for histological examination and further analyses(6).

Histopathological, immunohistochemical (IHC), in situ hybridization (ISH), immunoblotting, and TUNEL analyses

The rat specimens were fixed in fresh 4% paraformaldehyde for 16–24 hours at 4°C, followed by dehydration, paraffin embedding, and longitudinal sectioning. Sequential 4-µM sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope to assess changes in tenocyte morphology and collagen-bundle characteristics. To evaluate rat Achilles tendinopathy, we employed a 4-point system based on eight parameters, as previously described(6, 24, 25). The maximum total possible score was 24. For IHC staining, sections were deparaffinized in xylene, dehydrated in alcohol, and underwent epitope unmasking by heating. Subsequently, they were immersed in H₂O₂ and stained with antibodies against Smad4 (1:100, Abcam), in conjunction with the chromogen 3-amino-9-ethylcarbazole (Zymed Laboratories). Signal intensity was quantitated using ImageJ 1.42q in three randomly chosen high-power fields (200×)(6). For ISH and TUNEL analyses, sections were deparaffinized in xylene, dehydrated in alcohol, and processed following the published protocol for ISH(22), along with the DeadEnd™ Colorimetric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Tendon tissue lysates underwent immunoblot analysis with antibodies against c-Cas3 and p-Cas3, along with a horseradish-peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) and quantitative control anti-β-actin antibodies (Sigma-Aldrich), as previously described(6).

Statistical analysis.

The data are presented as the mean ± SEM. Normality was confirmed for each data point using the Shapiro-Wilk test. Differences between two groups and among groups were analyzed using Student’s t-test and one-way analysis of variance, respectively, followed by Dunnett’s multiple comparison test (Prism 5.0). A p-value less than 0.05 was considered statistically significant.

CD44 and miR-146a overexpression ameliorate apoptosis in IL-1β-stimulated tendinopathic tenocytes

Based on our prior research findings, inhibition of the CD44 signaling pathway triggers apoptosis in rat tendinopathic tenocytes(4), while overexpression of miR-146a in these cells mitigates senescence and SASPs under IL-1β-stimulated conditions(22). Consequently, we plan to investigate whether CD44 and miR-146a collectively regulate apoptosis in tendinopathic tenocytes. To assess this, cells were exposed to lentiviral vectors carrying CD44 cDNA (LVCD44) and precursor miR-146a (LVmiR-146a) following IL-1β stimulation to evaluate apoptosis levels. The percentage of TUNEL-positive cells was significantly lower in LVCD44 and LVmiR-146a-transduced tenocyte transfectants compared to control counterparts treated with LVSin and LVmiR-scramble (Fig. 1).

The CD44-AKT-miR-146a axis targets Smad4 to resolve apoptosis in IL-1β-stimulated tendinopathic tenocytes.

As previously reported, AKT activation is implicated in the signaling pathway mediated by CD44 in tenocytes(4). Accordingly, we observed a higher phospho-AKT/AKT ratio in LVCD44-transduced tenocyte transfectants compared to control LVSin-transduced ones (Fig. 2A). Moreover, this ratio decreased in tendinopathic tenocytes treated with OX-50 (an antagonizing antibody targeting CD44) compared to untreated control cells (Fig. 2B), as determined by immunoblotting. Under these conditions, miR-146a expression increased in CD44-overexpressing tendinopathic tenocytes (Fig. 2C) and decreased in both PI3 kinase inhibitor (LY2940002) and OX-50-treated cells (Fig. 2C and 2D), suggesting that CD44 functions through PI3K/AKT pathway to modulate miR-146a expression in tenocytes.

To further address whether up-regulated miR-146a expression regulates apoptosis, LVmiR-146a-transduced tenocyte transfectants were subjected to IL-1β stimulation. Tenocytes with overexpressed miR-146a exhibited nearly a 150-fold increase in miR-146a levels compared to control transfectants (LVmiR-scramble), as determined by qRT-PCR (Fig. 2E). Furthermore, LVmiR-146a-transduced tenocyte transfectants displayed decreased Smad4 protein levels and c-Cas3/p-Cas3 protein ratios compared to control transfectants in response to IL-1β stimulation (Fig. 2F).

Since CD44 upregulates miR-146a expression to mediate its anti-apoptotic effect, we further introduced antagomir-146a to CD44-overexpressed tenocytes to assess apoptosis. The expression of miR-146a increased dose-dependently in LVCD44-transduced tenocytes compared to LVSin-transduced counterparts (Fig. 3A). TUNEL-positive cells decreased in LVCD44-transduced tenocyte transfectants under IL-1β-stimulated conditions (Figs. 3B, lane F); however, the number increased after antagomir-146a transfer under the same conditions (Fig. 3B, lane H, p < 0.05). In summary, our in vitro findings propose a novel CD44-AKT-miR-146a signaling axis that inhibits IL-1β-induced apoptosis in tenocytes by targeting Smad4 expression.

CD44 mitigates apoptosis and tendinopathic characteristics by up-regulating miR-146a expression in vivo.

To further investigate whether this axis could modulate apoptosis in vivo, rat tendinopathic tendons transduced with LVCD44 and LVSin were subjected to H&E and TUNEL stainings. Consistent with previous findings(6), tendons treated with LVCD44 exhibited significantly lower histological scores compared to those treated with LVSin (Fig. 4B, p < 0.05). Reduced percentages of TUNEL-positive cells were also observed in LVCD44-injected tendinopathic tendons (Figs. 4A and 4C). Additionally, the in vivo protein ratios of c-Cas3/p-Cas3 were markedly lower in the LVCD44 group compared to the LVSin group (Fig. 4D). Furthermore, up-regulated miR-146a and down-regulated Smad4 expressions were observed in LVCD44-treated tendinopathic tendons compared to their LVSin-treated counterparts, as determined by ISH and IHC staining (Fig. 4E and 4F).

In this study, we elucidated a novel CD44-AKT-miR-146a signaling axis in tendinopathic tenocytes and tendons, which attenuates apoptosis both in vitro and in vivo. This axis targets Smad4, thereby reducing IL-1β-stimulated apoptosis in tenocytes (Fig. 5). Consistent with our previous findings, overexpression of CD44 and engagement with HA trigger anti-senescent and apoptotic effects on IL-1β-stimulated tendinopathic tenocytes and rat tendinopathic tendons(6, 16). Additionally, gene transfer of miR-146a diminishes senescence in IL-1β-stimulated tendinopathic tenocytes(22). In summary, our current findings establish a causal link between CD44 and miR-146a in mitigating tenocyte apoptosis through the PI3K/AKT signaling pathway, which integrate our previous research findings.

The PI3K/AKT pathway has been implicated in the tendon healing process. Curcumin, a natural polyphenolic compound with anti-inflammatory and antioxidant activities(26), has been shown to enhance tendon rupture healing by stimulating tenogenesis through PI3K/AKT signaling in tendon stem/progenitor cells (TSPCs)(27). Additionally, TSPC senescence induced by botulinum toxin is associated with AKT inactivation(28). Moreover, deficiency in the long noncoding RNA FOXD2-AS1 accelerates Achilles tendon injury healing and ameliorates tendon degeneration by promoting PI3K/AKT signaling pathway activation(29). Taken together, we have proved that this pathway can mediate CD44-induced miR-146a expression and is involved in the anti-apoptotic effects in tendinopathic tenocytes and tendons.

The relationship between CD44-related miRNAs and tendinopathy was substantiated by a study demonstrating an altered miRNA profile in the synovial fluid of knee OA patients(30). The pathogenic mechanism of tendinopathy may mirror that of OA in several respects, notably in the role of IL-1β as a disease initiator, inducing the expression of matrix-degrading enzymes while diminishing the production of ECM(31, 32). The expression levels of three miRNAs, namely miR-146a, miR-181a, and miR-155, were down-regulated in OA prior to HA treatment compared to the healthy control group(30). However, following HA treatment, their levels in the synovial fluid of OA patients were up-regulated(30). Interestingly, our unpublished data indicate that HA treatment can increase the levels of miR-146a in rat tendinopathic tenocytes when stimulated with IL-1β. Therefore, we suggest that the expression of specific miRNAs may be down-regulated in tendinopathy. Restoring their levels, either through extracellular stimuli or intracellular over-expression of CD44 or miRNA, could represent a potential therapeutic strategy for treating tendon disorders. This approach would involve precision targeting of their responsive pathways.

This study possesses several limitations. Firstly, our primary focus lies in elucidating the CD44-AKT-miR-146a signaling axis through animal models and in vitro experiments. However, the generalizability of these findings to human tendinopathy may be restricted, necessitating further validation in clinical settings. Secondly, although the study sheds light on the molecular mechanisms underlying tendinopathy, the precise interplay between CD44, miR-146a, and the PI3K/AKT pathway might not be fully understood. Further mechanistic studies are imperative to comprehensively grasp the intricacies of this signaling axis. Thirdly, the in vivo effects of miR-146a require evaluation. Our in vitro analysis and previous findings have demonstrated protective effects of miR-146a against IL-1β-induced tenocyte senescence(22) and apoptosis (Fig. 2). We posit that miR-146a may exhibit therapeutic effects on rats with collagenase-induced Achilles tendinopathy, a hypothesis to be explored in subsequent investigations.

This study sheds light on the causal roles of two protective factors in tendinopathy, CD44 and miR-146a, which exhibit anti-apoptotic and anti-senescent effects in tendinopathic tenocytes and tendons. Moving forward, understanding the CD44-AKT-miR-146a signaling axis could offer novel therapeutic avenues for managing tendinopathy by precisely modulating apoptosis in IL-1β responsive cells, potentially through targeted interventions aimed at the TGF-β-Smad4 pathway.

Hyaluronan

Interleukin

SASPs

Senescence-associated secretory phenotypes

ECM

Extracellular matrix

MiR-146a

MicroRNA-146a

osteoarthritis

VSVG

vesicular stomatitis virus G glycoprotein

LVCD44

lentiviral vector encoding CD44 cDNA

LVmiR-146a

Lentiviral vector encoding precursor miR-146a

LVmiR-scramble

lentiviral vector endoding precursor miR-scramble

LVSin

lentiviral vector encoding no transgene

Immunohistochemical (IHC)

In situ hybridization (ISH).

Funding: The present study received support from the National Science and Technology Council, Taiwan (MOST 109-2622-E-006-029-CC2, NSTC 112-2622-E-006-013, NSTC 111-2314-B-006-054, and NSTC 112-2314-B-006-065), National Cheng Kung University Hospital (NCKUH11204035 and NCKUH11302049), and National Cheng Kung University (NCKUEDA11212 and NCKUEDA11307).

Author Contributions:

Conception and design of study: Che-Chia Hsu, Shih-Yao Chen, Po-Ting Wu

Acquisition of data: Che-Chia Hsu, Po-Yen Ko, I-Ming Jou, Po-Ting Wu

Analysis and/or interpretation of data: Che-Chia Hsu, Shih-Yao Chen, Hung-Wei Yang, I-Ming Jou, Po-Ting Wu

Drafting the manuscript: Che-Chia Hsu, Shih-Yao Chen, Po-Yen Ko, Po-Ting Wu

Revising the manuscript critically for important intellectual content: Che-Chia Hsu, Shih-Yao Chen, Po-Yen Ko, Woei-Jer Chung, Po-Ting Wu

Approval of the version of the manuscript to be published: Che-Chia Hsu, Shih-Yao Chen, Po-Yen Ko, I-Ming Jou, Hung-Wei Yang, Woei-Jer Chung, Po-Ting Wu

Acknowledgments: We extend our gratitude to the Skeleton Materials and Biocompatibility Core Lab at the Clinical Medicine Research Center, National Cheng Kung University Hospital, for their invaluable assistance with this study. Additionally, we would like to express our appreciation to Ms. Yu-Ying Chen for her valuable support.

Ethics approval and consent to participate

Consent for publication

Not applicable.

Data Availability Statement: The data supporting the findings of this study are available upon request from the corresponding author.

Conflict of interests: All authors declare no competing interests.

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No competing interests reported.

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The Protective Role of CD44 and microRNA-146a in Tendinopathy: A Signaling Axis Targets Apoptosis

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Version 1

Abstract

Objective

Methods

Results

Conclusions

Figures

Introduction

Materials and Methods

Ethics statement

Generation of lentiviral vectors that stably expressing CD44 and miR-146a

Primary culture of rat tendinopathic tenocytes

Generation of rat tenocytes stably expressing CD44 and miR146a and IL-1β stimulated apoptosis

Determination of CD44-AKT -miR-146a signaling axis in tenocytes

Lentiviral vector-mediated gene transfer in Achilles tendons from rats with tendinopathy

Histopathological, immunohistochemical (IHC), in situ hybridization (ISH), immunoblotting, and TUNEL analyses

Statistical analysis.

Results

CD44 and miR-146a overexpression ameliorate apoptosis in IL-1β-stimulated tendinopathic tenocytes

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1