MiR-143-3p regulates chondrogenic differentiation of synovium derived mesenchymal stem cells under mechanical stress through the BMPR2-Smad signalling pathway by targeting BMPR2.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood. OBJECTIVE: This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress. METHODS: The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot. RESULTS: High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway. CONCLUSION: This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.

An integrated liposome-based microfluidic strategy for rapid colorimetric analysis: A case study of microRNA-21 detection.

In this study, a novel integrated liposome-based microfluidic platform combined with a smartphone was designed for the rapid colorimetric detection of microRNA-21 (miRNA-21) in real samples. The flowing surface-functionalized liposomes were first captured by nucleic acid-functionalized Au nanoparticles in the microfluidic chip. In the presence of miRNA-21, the DNA strand modified on the surface of Au nanoparticles hybridized with the target to form double-stranded products and was cleaved by duplex-specific nuclease (DSN) enzyme, causing the liposomes to be re-released. Then, as the liposomes in the colorimetric module were lysed and the "cellular" contents were released, a step-by-step "glucose-glucose oxidase-3,3',5,5'-tetramethylbenzidine (TMB)" colorimetric reaction process catalyzed by the G-quadruplex/hemin was triggered. The grayscale values were recorded and recognized by the smartphone camera for miRNA-21 analysis. The advantages of the present strategy included the portability of smartphone-based colorimetric assay, the encapsulation and transport of reactants by liposomes and the low solvent usage of microfluidic chip. Under optimal conditions, this assay exhibited a wide linear range from 1 pM to 1 nM (r2 = 0.9981), and the limit of detection of miRNA-21 was as low as 0.27 pM. Moreover, the high specificity of this strategy allowed its successful application to the rapid analysis of miRNA-21 in real blood serum samples of people with type 2 diabetes.

Alteration in cartilage matrix stiffness as an indicator and modulator of osteoarthritis.

Osteoarthritis (OA) is characterized by cartilage degeneration and destruction, leading to joint ankylosis and disability. The major challenge in diagnosing OA at early stage is not only lack of clinical symptoms but also the insufficient histological and immunohistochemical signs. Alteration in cartilage stiffness during OA progression, especially at OA initiation, has been confirmed by growing evidences. Moreover, the stiffness of cartilage extracellular matrix (ECM), pericellular matrix (PCM) and chondrocytes during OA development are dynamically changed in unique and distinct fashions, revealing possibly inconsistent conclusions when detecting cartilage matrix stiffness at different locations and scales. In addition, it will be discussed regarding the mechanisms through which OA-related cartilage degenerations exhibit stiffened or softened matrix, highlighting some critical events that generally incurred to cartilage stiffness alteration, as well as some typical molecules that participated in constituting the mechanical properties of cartilage. Finally, in vitro culturing chondrocytes in various stiffness-tunable scaffolds provided a reliable method to explore the matrix stiffness-dependent modulation of chondrocyte metabolism, which offers valuable information on optimizing implant scaffolds to maximally promote cartilage repair and regeneration during OA. Overall, this review systematically and comprehensively elucidated the current progresses in the relationship between cartilage stiffness alteration and OA progression. We hope that deeper attention and understanding in this researching field will not only develop more innovative methods in OA early detection and diagnose but also provide promising ideas in OA therapy and prognosis.

Oral microbiome-driven virulence factors: A novel approach to pancreatic cancer diagnosis.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, often associated with a poor prognosis for patients. One of the major challenges in managing PDAC is the difficulty in early diagnosis, owing to the limited and invasive nature of current diagnostic methods. Recent studies have identified the oral microbiome as a potential source of non-invasive biomarkers for diseases, including PDAC. In this study, we focused on leveraging the differential expression of virulence factors (VFs) encoded by the oral microbiome to create a diagnostic tool for PDAC. We observed a higher alpha diversity in VF categories among PDAC patients compared to healthy controls. We then identified a panel of VF categories that were significantly upregulated in PDAC patients, these being associated with bacterial adherence, exoenzyme production, and nutritional/metabolic processes. Moreover, Streptococcus-derived VFs were notably enriched in PDAC patients. We developed a diagnostic model using random forest analysis based on the levels of these VFs. The model's diagnostic accuracy was evaluated using receiver operating characteristic (ROC) curve analysis, with an area under the curve (AUC) of 0.88, indicating high accuracy in differentiating PDAC patients from healthy controls. Our findings suggest that VFs encoded by the oral microbiome hold potential as diagnostic tools for PDAC, offering a non-invasive approach that could significantly enhance early detection and prognosis, ultimately leading to improved patient outcomes.

Dual role of endoplasmic reticulum stress-ATF-6 activation in autophagy and apoptosis induced by cyclic stretch in myoblast.

OBJECTIVE: Mandibular growth that is induced by functional appliances is closely associated with skeletal and neuromuscular adaptation. Accumulating evidence has proved that apoptosis and autophagy have a vital role in adaptation process. However, little is known about the underlying mechanisms. This study sought to determine whether ATF-6 is involved in stretch-induced apoptosis and autophagy in myoblast. The study also sought to uncover the potential molecular mechanism. MATERIALS AND METHODS: Apoptosis was assessed by TUNEL and Annexin V and PI staining. Autophagy was detected by transmission electron microscopy (TEM) analysis and immunofluorescent staining for autophagy-related protein light chain 3 (LC3). Real time-PCR and western blot were performed to evaluate the expression level of mRNA and proteins that were associated with endoplasmic reticulum stress (ERS), autophagy and apoptosis. RESULTS: Cyclic stretch significantly decreased the cell viability and induced apoptosis and autophagy of myoblasts time-dependently. Stretching stimuli activated ATF-6 pathway and induced ERS-mediated apoptosis. Moreover, using 4-PBA significantly inhibited ERS-related apoptosis, as well as partially decreasing autophagy. In addition, inhibition of autophagy by 3-MA enhanced apoptosis by affecting the expression of CHOP and Bcl-2. However, it had no obvious effects on ERS-related proteins of GRP78 and ATF-6. More importantly, knockdown ATF-6 effectively weakened apoptosis and autophagy. It did so by regulating the expression of Bcl-2, Beclin1 and CHOP, but not cleaved Caspase-12, LC3II and p62 in stretched myoblast. CONCLUSION: ATF-6 pathway was activated in myoblast by mechanical stretch. ATF-6 may regulate the process of stretch-induced myoblast apoptosis and autophagy via CHOP, Bcl-2 and Beclin1 signaling.

Hydro-mechanical coupling characteristics and weakening mechanisms of filling joint resulting from water injection.

The injection of fluids into fault gouges in rock formations disturbs the in situ stress conditions, leading to fault slip and increasing the risk of inducing earthquakes. The weakening effect and the permeation of the injected fluid can be influenced significantly by the presence of fault gouges. To investigate this issue, the hydro-mechanical characteristics of fault gouges were evaluated using physical tests to study the combined effects of coupling injecting water and shear deformation. We propose a new experimental apparatus that allows us to measure the spatial distribution of the thickness of a gouge layer sample under combined conditions of shearing and water injection, using 3D scanning technology to evaluate the primary flow path. The test results showed that injecting water had a significant effect in reducing the maximum shear strength, but the degree to which the strength was affected varied with the gouge fill material. The effect of shear deformation is that it will increase the inhomogeneity of the thickness distribution and therefore the distribution of injected water along the fault is not uniformly radial. Additionally, the properties of gouge fill material have an important influence on flow characteristics during fault slipping.

Influence of CaO on Physical and Environmental Properties of Granulated Copper Slag: Melting Behavior, Grindability and Leaching Behavior.

Due to its potential pozzolanic activity, granulated copper slag (GCS) has been proven to act as a supplementary cementitious material (SCM) after thermochemical modification with CaO. This modification method reduces cement consumption and CO2 emissions; however, the additional energy consumption and environmental properties are also not negligible. This paper aims to evaluate the economics and environmental properties of thermochemically modified GCS with CaO through the melting temperature, grindability, and heavy metal leaching characteristics. The X-ray fluorescence spectroscopy (XRF) results indicated that the composition of the modified GCS shifted to the field close to that of class C fly ash (FA-C) in the CaO-SiO2-Al2O3 ternary phase diagram, demonstrating higher pozzolanic activity. The test results on melting behavior and grindability revealed that adding CaO in amounts ranging from 5 wt% to 20 wt% decreased the melting temperature while increasing the BET surface area, thus significantly improving the thermochemical modification's economics. The unconfined compressive strength (UCS) of the cement paste blended with 20 wt% CaO added to the modified GCS after curing reached 17.3, 33.6, and 42.9 MPa after curing for 7, 28, and 90 d, respectively. It even exceeded that of Portland cement paste at 28 d and 90 d curings. The leaching results of blended cement proved that the heavy metal elements showed different trends with increased CaO content in modified GCS, but none exceeded the limit values. This paper provides a valuable reference for evaluating thermochemically modified GCS's economics and environmental properties for use as SCM.

Can Direct-Immersion Aqueous-Aqueous Microextraction Be Achieved When Using a Single-Drop System?

For the analysis of biological analytes in complex matrices, it is difficult to achieve extraction of analytes and enrichment in an aqueous-aqueous single-drop microextraction system. In this study, we proposed a pH-dependent polydopamine (PDA)-coated vesicle/Fe3O4 magnetic aqueous-aqueous in a single-drop microreactor (SDMR) for the direct fluorescence detection of glutathione S-transferase (GST), a metabolic enzyme involved with crucial biological processes, in biological samples. After extracting and enriching the GST target from an aqueous-aqueous single-drop interface, the extraction process was conducted rapidly in 6 s in the SDMR system. The GST was first extracted from the sample solution via the GST-Aptamer on the polydopamine-coated vesicle/Fe3O4 nanospheres (Fe3O4@PDA@GST-Aptamer). Then, as the pH changed from weakly acidic to weakly alkaline in the SDMR system, the GST and GST-Aptamer were released from Fe3O4@PDA@GST-Aptamer nanospheres and captured by polydiacetylene vesicles via the capture probe. These changes altered the effective conjugation length and angle of the vesicle trunk, generating a highly enhanced fluorescence signal. This not only achieved the purpose of target enrichment but also reduced interferences posed by matrix effects. The approach can be used for the direct detection of GST in genuine urine and blood without any sample pretreatment. The linear range was 0.005 to 0.5 µg/mL, and the limit of detection was 0.834 ng/mL. The recoveries of GST in genuine blood samples ranged from 90.8 to 108.0% and in urine from 91.6 to 102.8%. The method has the capability of handling complex samples directly by enabling microextraction in an aqueous-aqueous single-drop system.

Intensive stretch-activated CRT-PMCA1 feedback loop promoted apoptosis of myoblasts through Ca2+ overloading.

Mechanical stretch exerted pro-apoptotic effect on myoblasts, the mechanism of which is currently unknown. Intracellular Ca2+ accumulation has been implicated in stretch-induced apoptosis. calreticulin (CRT) and plasma membrane Ca2+ transporting ATPase 1 (PMCA1) are two critical components of Ca2+ signaling system participating in intracellular Ca2+ homeostasis. In this study, we explored the contribution of CRT and PMCA1 in mediating stretch-induced Ca2+ accumulation and apoptosis of myoblasts. Stretching stimuli elevated level of CRT while inhibited activity of PMCA1. Moreover, there were bidirectional regulations between CRT and PMCA1, which formed the positive feedback loop leading to continuous increment of CRT level and repression of PMCA1 activity, in stretched myoblasts. Specifically, increased CRT level inhibited PMCA1 activity via suppressing Calmodulin (CaM), while reduced PMCA1 activity promoted CRT expression through activating p38MAPK pathway. Thus, the CRT-CaM-PMCA1 and PMCA1-p38MAPK-CRT pathways constituted a close cycle comprising CRT, PMCA1, CaM and p38MAPK. Inhibition of both CaM and p38MAPK affected the other three factors in stretched myoblasts. Circulation of the vicious cycle resulted in escalated Ca2+ overloading in myoblasts under continuous stretching stimuli. CRT knock-down, PMCA1 overexpression, and p38MAPK inhibition all attenuated the raised intracellular Ca2+ level and ameliorated myoblast apoptosis in the stretching environment. Conversely, CRT overexpression, PMCA1 knock-down, and CaM inhibition all aggravated stretch-induced Ca2+ overloading and myoblast apoptosis. A positive feedback loop between CRT and PMCA1 was activated in stretched myoblasts, which contributed to intracellular Ca2+ accumulation and resultant myoblast apoptosis.

Enhanced Sunscreen Effects via Layer-By-Layer Self-Assembly of Chitosan/Sodium Alginate/Calcium Chloride/EHA.

The sunscreen nanocapsules were successfully synthesized by the way of layer-by-layer self-assembly using charged droplets (prepared by emulsification of LAD-30, Tween-80 and EHA (2-Ethylhexyl-4-dimethylaminobenzoate)) as templates. Chitosan/sodium alginate/calcium chloride were selected as wall materials to wrap EHA. The emulsions with the ratio of Tween-80 to EHA (1:1) were stable. A stable NEI negative emulsion can be obtained when the ratio of Tween-80 and LAD-30 was 9:1. Chitosan solutions (50 kDa, 0.25 mg/mL) and sodium alginate solutions (0.5 mg/mL) were selected to prepare nanocapsules. The nanocapsules were characterized via some physico-chemical methods. Based on the synergistic effects of the electrostatic interaction between wall materials and emulsifiers, EHA was effectively encapsulated. DLS and TEM showed that the sunscreen nanocapsules were dispersed in a spherical shape with nano-size, with the increasing number of assembly layers, the size increased from 155 nm (NEI) to 189 nm (NEII) to 201 nm (NEIII) and 205 nm after solidification. The release studies in vitro showed sustained release behavior of the nanocapsules were observed with the increase of the number of deposition layers, implying a good coating effect. The sunscreen nanocapsules could control less than 50% the release of EHA after crosslinking of calcium chloride and sodium alginate, which also could effectively avoid the stimulation of the sun protection agent on the skin.

Molecular and Biomechanical Adaptations to Mechanical Stretch in Cultured Myotubes.

Myotubes are mature muscle cells that form the basic structural element of skeletal muscle. When stretching skeletal muscles, myotubes are subjected to passive tension as well. This lead to alterations in myotube cytophysiology, which could be related with muscular biomechanics. During the past decades, much progresses have been made in exploring biomechanical properties of myotubes in vitro. In this review, we integrated the studies focusing on cultured myotubes being mechanically stretched, and classified these studies into several categories: amino acid and glucose uptake, protein turnover, myotube hypertrophy and atrophy, maturation, alignment, secretion of cytokines, cytoskeleton adaption, myotube damage, ion channel activation, and oxidative stress in myotubes. These biomechanical adaptions do not occur independently, but interconnect with each other as part of the systematic mechanoresponse of myotubes. The purpose of this review is to broaden our comprehensions of stretch-induced muscular alterations in cellular and molecular scales, and to point out future challenges and directions in investigating myotube biomechanical manifestations.

Sunscreen Enhancement of Octyl Methoxycinnamate Microcapsules by Using Two Biopolymers as Wall Materials.

Octyl methoxycinnamate (OMC) is widely used as a chemical sunscreen in sunscreen cosmetics. However, its direct contact with the skin would bring certain risks, such as skin photosensitive reaction. How to improve the effect of skin photodamage protection has become a current research hotspot. Encapsulating ultraviolet (UV) filters into microcapsules is an interesting method to increase the photostability of filters. In this study, sodium caseinate (SC) and arabic gum (GA) are chosen as wall materials to prepare synergistic sunscreen microcapsules by complex coacervation technology. A series of experiments are conducted to investigate the effects of pH, wall material concentration, and wall/core ratio on the formation of OMC microcapsules. The morphology, composition, and stability of OMC microcapsules are characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). The OMC microcapsule is uniform in size distribution, smooth in surface morphology, and has good thermal stability. The results show that the ultraviolet absorption of the OMC microcapsules is better than that of the uncoated OMC for the ultraviolet-B (280-320 nm). Moreover, the OMC microcapsule released 40% in 12 h, while OMC released 65%, but the sun protection factor (SPF) of the OMC microcapsule sunscreen is 18.75% higher than that of OMC. This phenomenon may be attributed to the hydrophobic interaction between SC and OMC and the electrostatic interaction between SC and GA.

Morbidity and All-Cause Mortality Following Radical Prostatectomy Compared with Observation for Localized Prostate Cancer in Chinese Men: A Non-Randomized Retrospective Study.

BACKGROUND The Chinese 2018 guidelines and the current 2014 Chinese Urological Association guidelines for prostate cancer recommend radical prostatectomy for Chinese men with localized prostate cancer as the first choice, but it has treatment-related adverse effects. This study aimed to study morbidity and all-cause mortality following radical prostatectomy compared with observation for localized prostate cancer in Chinese men from a single center. MATERIAL AND METHODS Men diagnosed (histologically) as stage T1-T2N×M0 prostate cancer of any grade with 1-year history were included in the analysis. A total of 201 men underwent radical prostatectomy (RP cohort) and 209 men did not undergo radical prostatectomy (OS cohort). RESULTS During follow-up (17-24 years), 135 (67%) men died in the RP cohort and 156 (75%) men died in the OS cohort (P=0.103). All-cause mortality was lower for men with prostate-specific antigen level >10 ng/mL (P<0.0001), Gleason score ≥7 (P=0.004), and high D'Amico tumor risk scores (P=0.007) if they underwent radical prostatectomy. Age ≥65 years (P=0.041), Gleason score ≥7 (P=0.049), and tumor stage ≥2c (P=0.045) were associated with all-cause mortality. CONCLUSIONS The findings from this study showed that radical prostatectomy has no significant beneficial effects when compared with observation for Chinese men with localized prostate cancer, unless they had a prostate-specific antigen level >10 ng/mL, Gleason score ≥7, and high D'Amico tumor risk scores.

Biofunctional Extracellular Matrix-Polycaprolactone-Hydroxyapatite Scaffold and Synovium Mesenchymal Stem Cells/Chondrocytes for Repairing Cartilage Defects.

Articular cartilage defects and degeneration can be caused by multiple factors, and the current clinical treatment schemes for pathological changes are relatively limited. Engineered cartilage tissue represents an alternative therapy for repairing cartilage defects in regenerative medicine. The scaffold material is considered the framework of tissue engineering; thus, scaffold material selection plays a crucial role in the therapy outcome. Polycaprolactone (PCL)-hydroxyapatite (HA) has been applied as a scaffold material for bone and cartilage tissue engineering with nontoxic, harmless metabolites and proper physical properties. The extracellular matrix (ECM) is mainly composed of collagen and proteoglycan, as well as a large number of growth factors and cytokines, which provide a tissue-specific microenvironment for host cells. Adipose-derived stem cells are pluripotent stem cells, and transforming growth factor-ß3 (TGF-ß3) enables mesenchymal stem cells to promote ECM production. This study, via in vitro and in vivo experiments, elucidated that the synovium mesenchymal stem cells (SMSCs) + chondrocytes + ECM-PCL-HA repair system, which is constructed upon the ECM-PCL-HA scaffold material, exhibits an adequate chondrogenic ability and reparatory effect. Overall, ECM-PCL-HA can be defined as a biofunctional scaffold material. The SMSCs + chondrocytes + ECM-PCL-HA repair system showed good confluency between the new cartilage and the surface, as well as the interface of the adjacent host cartilage. Furthermore, the structure of new cartilage tissue is consistent with adjacency. Thus, it can be used as a preferred plan for articular cartilage defect repair. Impact statement Studies investigating the chondrogenic ability and reparatory effect of the synovium mesenchymal stem cells (SMSCs) + chondrocytes + extracellular matrix (ECM)-polycaprolactone (PCL)-hydroxyapatite (HA) repair system provided a theoretical and practical basis for choosing ECM-PCL-HA as the scaffold material for cartilage tissue engineering. In this study, the transforming growth factor-ß3 (TGF-ß3) gene was introduced into adipose-derived stem cells (ADSCs) using a lentiviral vector to enhance ECM production. The decellularized ADSCs-ECM-PCL-HA acted as a biofunctional scaffold material with suitable physicochemical properties, which was advantageous for SMSC and chondrocyte adhesion and growth. Lastly, the SMSCs + chondrocytes + ECM-PCL-HA repair system showed excellent capability in the flush fusion state of the prosthetic surface and interface.

Nanosilver/poly (dl-lactic-co-glycolic acid) on titanium implant surfaces for the enhancement of antibacterial properties and osteoinductivity.

Background: Despite titanium (Ti) implants have been commonly used in the medical device field due to their superior biocompatibility, implant-associated bacterial infection remains a major clinical complication. Nanosilver, an effective antibacterial agent against a wide spectrum of bacterial strains, with a low-resistance potential, has attracted much interest too. Incorporation of nanosilver on Ti implants may be a promising approach to prevent biofilm formation. Purpose: The objective of the study was to investigate the antibacterial effects and osteoinductive properties of nanosilver/poly (dl-lactic-co-glycolic acid)-coated titanium (NSPTi). Methods: Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and the Gram-negative opportunistic pathogen Pseudomonas aeruginosa (PAO-1) were used to evaluate the antibacterial activity of NSPTi implants through the analysis of bacterial colonization in vitro and in vivo. Furthermore, we examined the osteoinductive potential of NSPTi implants by investigating the proliferation and differentiation of MC3T3-E1 preosteoblast cells. In vivo, the osteoinductive properties of NSPTi implants were assessed by radiographic evaluation, H&E staining, and Masson's trichrome staining. Results: In vitro, bacterial adhesion to the 2% NSPTi was significantly inhibited and <1% of adhered bacteria survived after 24 hours. In vitro, the average colony-forming units (CFU)/g ratios in the 2% NSPTi with 103 CFU MRSA and PAO-1 were 1.50±0.68 and 1.75±0.6, respectively. In the uncoated Ti groups, the ratios were 1.03±0.82×103 and 0.94±0.49×103, respectively. These results demonstrated that NSPTi implants had prominent antibacterial properties. Proliferation of MC3T3-E1 cells on the 2% NSPTi sample was 1.51, 1.78, and 2.22 times that on the uncoated Ti control after 3, 5, and 7 days' incubation, respectively. Furthermore, NSPTi implants promoted the maturation and differentiation of MC3T3-E1 cells. In vivo, NSPTi accelerated the formation of new bone while suppressing bacterial survival. Conclusion: NSPTi implants have simultaneous antibacterial and osteoinductive activities and therefore have the potential in clinical applications.

MiRNA-218 regulates osteoclast differentiation and inflammation response in periodontitis rats through Mmp9.

Periodontitis is a multiple infection and inflammatory disease featured by connective tissue homeostasis loss, periodontal inflammation, and alveolar bone resorption. MicroRNAs (miRNAs) are involved in the mediation of a large scale of pathological processes. Here, we show that miRNA-218 provides protective effect on periodontitis via regulation of matrix metalloproteinase-9 (Mmp9). This pathway is aberrant in periodontium from rats with periodontitis and human periodontal ligament progenitor cells stimulated by lipopolysaccharide, with downregulation of miR-218 and higher levels of Mmp9 compared with periodontium from healthy rats and cells without stimulation. Overexpression of miR-218 can suppress the degradation of Collagen Types I and IV and dentin sialoprotein (DSP), attenuate osteoclast formation, and inhibit the secretion of proinflammatory cytokines. On the other hand, overexpression of Mmp9 promotes the degradation of Collagen Types I and IV and DSP as well as RANKL-induced osteoclast formation and elevates inflammatory factors levels. Furthermore, the inhibitory effect of miR-218 was prevented by rescuing the Mmp9 expression. In addition, we also have showed that miR-218 was able to attenuate bone resorption and inflammation in a periodontitis rat model. Collectively, our findings therefore suggest that miR-218 acts as a protective role in periodontitis through the regulation of Mmp9.

Upregulation of long noncoding RNA MEG3 inhibits the osteogenic differentiation of periodontal ligament cells.

OBJECTIVE: This study aims to discuss long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) function of regulating osteogenesis in human periodontal ligament cells (hPDLCs). METHODS: First, use of a mineralizing solution induced osteogenic differentiation of hPDLCs to establish a differentiated cell model. Through microarray analysis, we selected a lncRNA MEG3 with marked changes between differentiated and undifferentiated cells. The quantitative polymerase chain reaction was used to detect the MEG3 content and an enzyme-linked immunosorbent assay was used to detect changes in related proteins. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptosis was measured by flow cytometry. Alizarin red staining was also used to evaluate cells' osteogenic level. Finally, RNA-binding protein immunoprecipitation assays were conducted to further clarify the endogenous relationship between MEG3 and bone morphogenetic protein 2 ( BMP2) in hPDLCs. RESULTS: MEG3 was downregulated in osteogenic differentiation hPDLCs induced by mineralizing solution. Overexpression of MEG3 inhibited cell viability and increased cell apoptosis. MEG3 overexpression can reverse osteogenic differentiation induced by mineralizing solution. MEG3 can suppress BMP2 through interaction with heterogeneous nuclear ribonucleoprotein I. CONCLUSION: Upregulation of MEG3 inhibits the osteogenic differentiation of periodontal ligament cells by downregulating BMP2 expression.

MicroRNA-223 Suppresses Osteoblast Differentiation by Inhibiting DHRS3.

BACKGROUND/AIMS: In this study, we aimed to use bioinformatics tools to identify the specific miRNAs and mRNAs involved in osteogenic differentiation and to further explore the way in which miRNA regulates osteogenic differentiation. METHODS: The microarray GSE80614, which includes data from 3 human mesenchymal stromal cells (hMSCs) and 3 hMSCs after 72 hours (hr) of osteogenic differentiation, was used to screen for differentially expressed mRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these mRNAs were conducted using Gene Set Enrichment Analysis (GSEA). Then, the miRanda website was employed to detect the binding sites of DHRS3. In vitro experiments, including RT-PCR and western blotting, were used to detect miR-233 and DHRS3 expression levels 7 and 14 days (d) after the induction of osteogenic differentiation using human bone marrow-derived mesenchymal stem cells (hBMSCs). The target relationship between miR-223 and DHRS3 was confirmed by a dual luciferase assay. ALP (alkaline phosphatase) staining, ARS (Alizarin Red S) staining and western blotting (Runx2, OPN, OCN) were used to detect the level of osteogenic differentiation after transfection with miR-223 mimics and DHRS3 cDNA. RESULTS: In this study, 127 mRNAs differentially expressed during osteogenic differentiation were identified in GSE80614. GO term and KEGG pathway enrichment analyses found that the retinol metabolism pathway was activated during osteogenic differentiation and that DHRS3, which is involved in the pathway, was upregulated. During osteogenic differentiation in hBMSCs, miR-223 was gradually downregulated, while DHRS3 was upregulated. After 14 days of osteogenic differentiation, ALP and ARS staining assay results showed strong ALP activity and extracellular matrix calcification with the inhibition of miR-223 or the overexpression of DHRS3. Furthermore, the expression levels of Runx2, OPN, and OCN were upregulated with the knockdown of miR-223 or the overexpression of DHRS3, while the simultaneous transfection of a miR-223 agomir and DHRS3 cDNA resulted in no significant difference from the negative control (NC) group. CONCLUSION: The inhibition of miR-223 promotes the osteogenic differentiation of hBMSCs via the upregulation of DHRS3.