miR125a attenuates BMSCs apoptosis via the MAPK-ERK pathways in the setting of craniofacial defect reconstruction.

Bone mesenchymal stem cell (BMSC)-based regenerative therapy is critical for the craniofacial defect reconstruction. However, oxidative stress microenvironment after transplantation limits the therapeutic efficiency of BMSC. The miR-181c has been found to be associated with cell survival and proliferation. Herein, we investigated whether prior miR-181c treatment promoted BMSC proliferation and survival under oxidative stress injury. The results in our study demonstrated that hydrogen peroxide (H2 O2 ) treatment reduced BMSC viability and this effect could be reversed via additional supplementation of miR181-c. Mechanistically, oxidative stress increased cell apoptosis, augmented caspase-3 activity, promoted reactive oxygen species synthesis, impaired mitochondrial potential, and induced mitochondrial dynamics imbalance. However, miR-181c pretreatment reversed these effects of oxidative stress on BMSC. Moreover, miR-181c treatment improved BMSC proliferation, migration and paracrine, which are very important for craniofacial reconstruction. In addition, we identified that AMP-activated protein kinase (AMPK)-mitofusins-1 (Mfn1) axis was the direct targets of miR-181c in BMSC. Mfn1 silencing impaired the protective effects miR-181c on BMSC viability and proliferation under oxidative stress environment. Collectively, our results indicate that miR-181c participates in oxidative stress-mediated BMSC damage by modulating the AMPK-Mfn1 signaling pathway, suggesting miR-181c-AMPK-Mfn1 axis may serves as novel therapeutic targets to facilitate craniofacial defect reconstruction.

Pretreatment of bone mesenchymal stem cells with miR181-c facilitates craniofacial defect reconstruction via activating AMPK-Mfn1 signaling pathways.

Context: Bone mesenchymal stem cells (BMSC)-based regenerative therapy is critical for the craniofacial defect reconstruction. However, oxidative stress micro-environment after transplantation limits the therapeutic efficiency of BMSC. The miR-181c has been found to be associated with cell survival and proliferation. Objective: Herein, we investigated whether prior miR-181c treatment promoted BMSC proliferation and survival under oxidative stress injury. Materials and methods: Cells were treated with hydrogen peroxide (H2O2) and then cell viability was determined via MTT assay, TUNEL staining and ELISA. Western blotting and immunofluorescence assay were used to detect those alterations of mitochondrial function. Results: H2O2 treatment reduced BMSC viability and this effect could be reversed via additional supplementation of miR181-c. Mechanistically, oxidative stress increased cell apoptosis, augmented caspase-3 activity, promoted reactive oxygen species (ROS) synthesis, impaired mitochondrial potential, and induced mitochondrial dynamics imbalance. However, miR-181c pretreatment reversed these effects of oxidative stress on BMSC. Moreover, miR-181c treatment improved BMSC proliferation, migration and paracrine, which are very important for craniofacial reconstruction. In addition, we identified that AMPK-Mfn1 axis was the direct targets of miR-181c in BMSC. Mfn1 silencing impaired the protective effects miR-181c on BMSC viability and proliferation under oxidative stress environment. Conclusions: Collectively, our results indicate that miR-181c participates in oxidative stress-mediated BMSC damage by modulating the AMPK-Mfn1 signaling pathway, suggesting miR-181c-AMPK-Mfn1 axis may serves as novel therapeutic targets to facilitate craniofacial defect reconstruction.

Therapeutic effects of bone mesenchymal stem cells on oral and maxillofacial defects: a novel signaling pathway involving miR-31/CXCR4/Akt axis.

Context: Although bone mesenchymal stem cells (BMSCs) have been used for the treatment of oral and maxillofacial defects, the survival rate and limited proliferation reduces the therapeutic efficiency of BMSC.Objective: The aim of our study is to explore the role of miR-31 in regulating survival, proliferation, and migration of BMSC in vitro.Materials and methods: LPS was used in vitro to induce BMSC damage and then miR-31 was used to incubate with BMSC. Subsequently, BMSC proliferation, survival, and migration were determined via ELISA, qPCR, western blots, and immunofluorescence.Results: The expression of miR-31 was downregulated in response to LPS stress. Interestingly, supplementation of miR-31 could reverse the survival, proliferation and migration of BMSC under LPS. Mechanically, miR-31 treatment inhibited the activation of caspase, and thus promoted BMSC survival. Besides, miR-31 upregulated the genes related to cell proliferation, an effect that was followed by an increase in the levels of migratory factors. Further, we found that miR-31 treatment activated the CXCR4/Akt pathway and blockade of CXCR4/Akt could abolish the beneficial effects of miR-31 on BMSC proliferation, survival, and migration.Conclusions: miR-31 could increase the therapeutic efficiency of BMSC via the CXCR4/Akt pathway.

Inhibition of CD147 expression promotes chemosensitivity in HNSCC cells by deactivating MAPK/ERK signaling pathway.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world. CD147, a transmembrane glycoprotein, has been reported to be correlated with cancer progression, metastasis, and chemoresistance in various cancers. In this study, we aimed to investigate the mechanism of CD147 in regulating drug resistance in HNSCC cells. qRT-PCR were used to evaluated the expression of CD147 in 57 HNSCC tumorous tissues and 2 cell lines. Increased expression of CD147 was found in most HNSCC samples, and the expression level of CD147 was correlated with multidrug resistance. CD147 RNA silencing decreased the chemoresistance of HNSCC cells by deactivating MAPK/ERK signaling pathway. Further investigation revealed that either rescue expression of CD147 or treatment of MAPK/ERK activator phorbol 12-myristate 13-acetate (PMA) in CD147 knockdown CRC cell line attenuated the decreased chemoresistance in CD147 knockdown cells. Taken together, our results suggest that CD147 promotes chemoresistance by activating MAPK/ERK signaling pathway in HNSCC.

MiR-26a Rescues Bone Regeneration Deficiency of Mesenchymal Stem Cells Derived From Osteoporotic Mice.

Osteoporosis, caused by a relative increase of bone resorption over bone formation, is characterized by decreased bone mass and bone strength, resulting in an increased incidence of bone fractures, which often leads to further disability and early mortality in the elderly due to impaired bone healing ability. The majority of therapeutics currently used in clinics for the treatment of osteoporosis are antiresorptive agents that exert their clinical effect by decreasing the rate of bone resorption. However, strategies solely aimed at antiresorption have limited therapeutic efficacy in restoring bone remodeling balance and enhancing osteoporotic fracture healing. Here, we report that miR-26a plays a critical role in modulating bone formation during osteoporosis. We found that miR-26a treatment could effectively improve the osteogenic differentiation capability of mesenchymal stem cells isolated from littermate-derived ovariectomized osteoporotic mice both in vitro and in vivo. MiR-26a exerts its effect by directly targeting Tob1, the negative regulator of BMP/Smad signaling pathway by binding to the 3'-untranslated region and thus repressing Tob1 protein expression. Our findings indicate that miR-26a may be a promising therapeutic candidate to enhance bone formation in treatment of osteoporosis and to promote bone regeneration in osteoporotic fracture healing.

Knockdown of PDX1 enhances the osteogenic differentiation of ADSCs partly via activation of the PI3K/Akt signaling pathway.

BACKGROUND: Osteoporosis (OP) is a systemic bone disease manifested as low bone mass, destruction of bone microstructure, increased bone fragility and fracture risk. The purpose of this study was to explore the role and mechanism of PDX1 for osteogenic differentiation of adipose derived stem cells (ADSCs). METHODS: GSE37329 dataset was retrieved from NCBI Gene Expression Omnibus (GEO) database and performed bioinformatic analyses. ADSCs were incubated with normal medium, osteogenic induction medium (OIM) and OIM+si-PDX1. Then, alkaline phosphatase (ALP) staining and Alizarin Red Staining (ARS) were performed to assess the role of PDX1 for osteogenesis of ADSCs. PI3K inhibitor, LY294002 was then added to further explore the mechanism of PDX1 for osteogenic differentiation of ADSCs. Western blot assay was used to assess the osteogenic-related markers. Graphpad software was used to perform statistically analysis. RESULTS: A total of 285 DEGs were obtained from analysis of the dataset GSE37329, of which 145 were upregulated and 140 were downregulated genes. These differentially expressed genes mainly enriched in cell differentiation and PI3K/Akt signaling pathway. Moreover, PDX1 was decreased in osteogenic induced ADSCs. Knockdown of PDX1 significantly increased osteogenic differentiation capacity and p-PI3K and p-Akt protein levels. Administration with LY294002 could partially reversed the promotion effects of si-PDX1. CONCLUSION: In conclusion, knockdown of PDX1 promotes osteogenic differentiation of ADSCs through the PI3K/Akt signaling pathway.

Value of 3D Printed PLGA Scaffolds for Cartilage Defects in Terms of Repair.

Objective: To examine the poly (lactic-co-glycolic acid) and sodium alginate (SA) scaffolds produced by 3D printing technology, access the healing morphology of bones following PLGA/SA implantation within rat cartilage, and examine osteogenesis-related factors in rat serum to determine the efficacy of PLGA/SA scaffolds in healing animal cartilage injuries. To identify the potential of this material to repair a tissue engineering osteochondral injury. Methods: Polylactic acid-glycolic acid copolymer and sodium alginate were used as raw materials to create PLGA/SA scaffolds. We observed the scaffold's macrostructure and microstructure, and the scaffold's microstructure was observed through a scanning electron microscope (SEM). The mechanical toughness of a stent was assessed using a biomechanical device. Hematoxylin-eosin staining revealed immune rejection after embedding the scaffolds under the skin of SD rats. The CCK-8 cell proliferation test kit was used to measure cell proliferation. An experimental model of cartilage injury in the knee joint was created in rats. Rats were used to establish an experimental model of cartilage damage in the knee joint. 120 female rats aged 5 weeks were chosen at random from the pool and divided into the experimental and control groups. They were all completely anesthetized with an anesthetic before having the lateral skin of the knee articular cartilage incised. Implanted PLGA/SA scaffolds were not used in the control group and only in the experiment group. Both groups of rats had their muscles and skin sutured and covered in plaster bandages. On the third, seventh, fourteenth, twenty-first, twenty-eighth, and thirty-fifth days after the procedure, the two groups of rats were divided into groups. At various stages, bone tissue, blood samples, and cartilage were examined and evaluated. Immunohistochemistry was used to identify the local bone morphogenetic protein-2 (BMP2). Results: (1) PLGA/SA was successfully used to build an artificial cartilage scaffold. (2) Macroscopic and SEM observation results showed the material had increased density and numerous microvoids on the surface. (3) The result of the biomechanical test showed that the PLGA/SA scaffold had superior biomechanical characteristics. (4) The stent did not exhibit any noticeable immunological rejection, according to the results of the subcutaneous embedding experiment performed on rats. (5) The CCK-8 data demonstrated that as the cell development time rose, the number of cells gradually increased. However, there was not statistically significant difference between the growth of the cells in the scaffold extract and the control group (P > 0.05). (6) A successful rat model based on a cartilage defect of the medial knee joint has been built. (7) Observations of specimens revealed that the experimental group's bone tissue score was higher than that of the control group. (8) Using immunohistochemistry, it was found that the experimental group's BMP2 expression was higher on the 7th, 14th, and 28th days than it was in the control group (P < 0.05). Conclusion: Strong mechanical and biological properties are present in stable, biodegradable PLGA/SA scaffolds that mimic cartilage. We demonstrated that the cartilage biomimetic PLGA/SA scaffold may repair cartilage and prevent negative reactions such as osteoarthritis in rat knee cartilage, making it suitable as a cartilage scaffolding material for tissue engineering. The PLGA/SA scaffold was also able to promote BMP2 expression in the bone healing zone when inserted into a knee cartilage lesion. Improved cartilage damage is the outcome of BMP2's promotion of bone formation and restriction of bone resorption in the bone healing zone.

Hippo/Mst1 overexpression induces mitochondrial death in head and neck squamous cell carcinoma via activating ß-catenin/Drp1 pathway.

Mammalian Ste20-like kinase 1 (Mst1) is associated with cell apoptosis. In the current study, we explored the regulatory effects of Mst1 on squamous cell carcinoma of the head and neck (SCCHN) in vitro. SCCHN Cal27 cells and Tu686 cells were transfected with adenovirus-loaded Mst1 to detect the role of Mst1 in cell viability. Then, siRNA against Drp1 was transfected into cells to evaluate the influence of mitochondrial fission in cancer survival. Our data illustrated that Mst1 overexpression promoted SCCHN Cal27 cell and Tu686 cell death via activating mitochondria-related apoptosis. Cells transfected with adenovirus-loaded Mst1 have increased expression of DRP1 and higher DRP1 promoted mitochondrial fission. Active mitochondrial fission mediated mitochondrial damage, as evidenced by increased mitochondrial oxidative stress, decreased mitochondrial energy production, and reduced mitochondrial respiratory complex function. Moreover, Mst1 overexpression triggered mitochondria-dependent cell apoptosis via DRP1-related mitochondrial fission. Further, we found that Mst1 overexpression controlled mitochondrial fission via the ß-catenin/DRP1 pathways; inhibition of ß-catenin and/or knockdown of DRP1 abolished the pro-apoptotic effects of Mst1 overexpression on SCCHN Cal27 cells and Tu686 cells, leading to the survival of cancer cells in vitro. In sum, our results illustrate that Mst1/ß-catenin/DRP1 axis affects SCCHN Cal27 cell and Tu686 cell viability via controlling mitochondrial dynamics balance. This finding identifies Mst1 activation might be an effective therapeutic target for the treatment of SCCHN.

The promotion of bone regeneration through positive regulation of angiogenic-osteogenic coupling using microRNA-26a.

Bone is highly vascularized tissue reliant on coordinated coupling between angiogenesis and osteogenesis to regenerate. Delivery of a combination of growth factors involved in the coupling has to some extent enhanced bone regeneration. However, the stimulation may interrupt the balance of bone and vessel remodeling leading to the excessive bone formation or vascular leakage. MicroRNAs function as potent molecular managers that may simultaneously regulate multiple endogenous signaling pathways. Delivery of microRNA may provide a way to maximally mimic the native bone development environment. In this work, we identified an miRNA, miR-26a in vitro assays that positively regulates angiogenesis-osteogenesis coupling. This resulted in enhanced bone formation coordinated with vascularization in mouse subcutaneous assay. Furthermore, we constructed an miRNA enhancer delivery system to enhance miR-26a expression in a localized and sustained manner in vivo. We found that the system led to complete repair of the critical-size calvarial bone defect and increased vascularization accordingly. Host specific real-time PCR test of the neo-formed bone demonstrated that miR-26a optimized bone regeneration mainly due to simultaneously regulating endogenous angiogenesis-osteogenesis coupling. We anticipated our assay providing evidence that miRNA-based therapy can be a valuable tool to promote bone regeneration.