H-bond-type thermo-responsive schizophrenic copolymers: The phase transition correlation with their parent polymers and the improved protein co-assembly ability.

Schizophrenic copolymers are one type of the popular smart polymers that show invertible colloidal structures in response to temperature stimulus. However, the lack of principles to predict the phase transition temperature of a schizophrenic copolymer from its corresponding parent thermo-responsive polymers limits their development. Additionally, studies on their applications remain scarce. Herein, a series of schizophrenic copolymers were synthesized by polymerization of a RAFT-made polymer precursor poly(acrylamide-co-N-acryloxysuccinimide-co-acrylic acid) (P(AAm-co-NAS-co-AAc)) with the mixture of N-isopropylmethacrylamide (NIPAm) and acrylamide (AAm) in varying molar ratios. In aqueous solution, the block P(AAm-co-NAS-co-AAc) and the block poly(NIPAm-co-AAm) exhibited upper and lower critical solution temperature (UCST and LCST) behavior, respectively. The schizophrenic copolymers featured either UCST-LCST, LCST-UCST, or only LCST thermo-responsive transition. A preliminary correlation of phase transition between the schizophrenic copolymers and their parent polymers was summarized. Furthermore, the co-assembly of the schizophrenic copolymers and proteins were conducted and the kinetics of protein loading and protein activity were investigated, which showed that the schizophrenic copolymers were efficient platforms for protein co-assembly with ultra-high protein loading while preserving the protein bioactivities. Additionally, all the materials were non-toxic towards NIH 3T3 and MCF-7 cells. This work offers the prospects of the schizophrenic polymers in soft colloidal and assembly systems, particularly in guiding the design of new materials and their use in biomedical applications.

Design of a UCST Polymer with Strong Hydrogen Bonds and Reactive Moieties for Facile Polymer-Protein Hybridization.

Polymer-protein hybrids have been extensively used in biomedical fields. Polymers with upper critical solution temperature (UCST) behaviors can form a hydrated coacervate phase below the cloud point (Tcp), providing themselves the opportunity to directly capture hydrophilic proteins and form hybrids in aqueous solutions. However, it is always a challenge to obtain a UCST polymer that could aggregate at a high temperature at a relatively low concentration and also efficiently bind with proteins. In this work, a UCST polymer reactive with proteins was designed, and its temperature responsiveness and protein-capture ability were investigated in detail. The polymer was synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization of acrylamide (AAm) and N-acryloxysuccinimide (NAS). Interestingly, taking advantage of the partial hydrolysis of NAS into acrylic acid (AAc), the obtained P(AAm-co-NAS-co-AAc) polymer exhibited an excellent UCST behavior and possessed good protein-capture ability. It showed a relatively higher Tcp (81 °C) at a lower concentration (0.1 wt %) and quickly formed polymer-protein hybrids with high protein loading and without losing protein bioactivity, and both the polymer and polymer-protein nanoparticles showed good cytocompatibility. All the findings are attributed to the unique structure of the polymer, which provided not only the strong and stable hydrogen bonds but also the quick and mild reactivity. The work offers an easy and mild strategy for polymer-protein hybridization directly in aqueous solutions, which may find applications in biomedical fields.

Aqueous Synthesis of Upper Critical Solution Temperature and Lower Critical Solution Temperature Copolymers through Combination of Hydrogen-Donors and Hydrogen-Acceptors.

The synthesis of thermo-responsive polymers from non-responsive and water-soluble monomers has great practical advantages but significant challenges. Herein, the authors report a novel aqueous copolymerization strategy to prepare polymers with tunable upper critical solution temperature (UCST) or lower critical solution temperature (LCST) from non-responsive monomers. Acrylic acid (AAc), N-vinylpyrrolidone (NVP), and acrylamide (AAm) are copolymerized in water, yielding copolymers with UCST behavior. Interestingly, by simply replacing AAm with its methylated homologue, dimethyl acrylamide (DMA), the thermo-responsiveness of the copolymers is converted into LCST-type. The cloud points of the copolymers can be tuned rationally with their monomer ratios and the condition of the solvent. The UCST property of the poly(AAc-NVP-AAm) comes from the AAc-AAm and AAc-NVP hydrogen-bonds, while the LCST property of poly(AAc-NVP-DMA) originates from the hydrophobic aggregation of AAc-NVP complex and DMA, as indicated by temperature-dependent 1 H NMR and dynamic light scattering.

Clinical characteristics and the risk factors for severe events of elderly coronavirus disease 2019 patients. / 2019å† çŠ¶ç— æ¯’ç— è€å¹´æ‚£è€ çš„ä¸´åºŠç‰¹ç‚¹åŠé‡ç—‡çš„å±é™©å› ç´ .

OBJECTIVES: To investigate the clinical characteristics and risk factors for severe events of coronavirus disease 2019 (COVID-19) in elderly patients. METHODS: Retrospective analysis was performed on the clinical data of all elderly COVID- 19 patients treated in Changsha Public Health Treatment Center from January 17, 2020 to March 15, 2020, which included basic diseases, symptoms, test results, and other clinical characteristics, and prognostic indicators such as severity of illness, length of hospital stay, virus shedding time and mortality rate. The differences in clinical characteristics and prognostic indicators between elderly, middle-aged, and young COVID-19 patients were also analyzed. Logistic regression model was used to conduct univariate and multivariate analysis of risk factors for developing severe events in elderly COVID-19 patients; receiver operating characteristic (ROC) curve analysis was used to evaluate the prediction efficacy. RESULTS: Of the 230 COVID-19 adult patients, 34 were young patients (14.8%), 136 were middle-aged patients (59.1%), and 60 were elderly (26.1%). Among the 60 elderly patients, 23 were male (38.3%) and 37 were female (61.7%), with a medium age of 66 years old. Common symptoms were fever (66.7%), cough (50.0%), and fatigue (41.7%). C reactive protein (CRP) was increased significantly. The proportion of severe cases was 31.7%, and mortality was 1.7%. The median length of hospitalization and median virus shedding time were 18.5 days and 21 days, respectively. Compared with the young and the middle-aged patients, the elderly had a higher proportion of hypertension, diabetes, and cardiovascular diseases, more common shortness of breath, higher proportions of pneumonia and severe cases (all P<0.05), and the decreased lymphocyte count and lymphocyte percentage (both P<0.05), as well as higher CRP and erythrocyte sedimentation rate (ESR) levels (both P<0.05). Compared with non-severe cases, severe elderly patients demonstrated higher CRP and aspartate aminotransferase (AST) levels (all P<0.05), the reduced lymphocyte count (P<0.05), and the prolonged length of hospitalization and virus shedding duration (both P<0.05). Univariate logistic regression analysis indicated that the lymphocytes proportion, CRP and AST levels were significantly correlated with the risk for developing severe events in elderly COVID-19 patients (all P<0.05). Multivariate logistic regression found that severe events in elderly patients with COVID-19 were significantly correlated with CRP level (OR=1.041, P=0.013). ROC curve analysis revealed that the area under the curve (AUC) for CRP to diagnose severe events in elderly COVID 19 patients was 0.851. CONCLUSIONS: The proportion of severe cases in elderly COVID-19 patients is higher than that in young and middle-aged patients. CRP level has a good predictive value for the possibility of severe events in elderly COVID-19 patients.

Diagnostic Utility of Serum Golgi Phosphoprotein 3 in Bladder Cancer Patients.

BACKGROUND This study assessed whether serum Golgi phosphoprotein 3 (GOLPH3) could be used as a biomarker for detecting bladder cancer. MATERIAL AND METHODS Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) assay were performed to measure GOLPH3 expression in serum and tissue samples, respectively, of bladder cancer patients. The associations of serum GOLPH3 expression with clinicopathological factors and the diagnostic accuracy were statistically evaluated using the chi-square test and receiver operating characteristic (ROC) curve analysis. RESULTS Compared with the healthy control group, serum GOLPH3 level was distinctly enhanced in bladder cancer patients (P<0.001). Moreover, compared to the non-malignant tissues, GOLPH3 showed positive expression in bladder cancer tissues. The abnormal GOLPH3 levels were tightly related to grade (P=0.018), tumor stage (P=0.000), lymph node status (P=0.030), and muscle invasion (P=0.012). ROC analysis showed that serum GOLPH3 exhibited a high diagnostic value to distinguish bladder cancer patients from healthy persons. The area under the ROC curve (AUC) was 0.948. The specificity and sensitivity were 92.5% and 83.8%, respectively. CONCLUSIONS GOLPH3 was highly expressed in bladder cancer patients and could be used as a diagnostic tool.

GPR137 is a promising novel bio-marker for the prognosis of bladder cancer patients.

OBJECTIVES: G protein-coupled receptor 137 (GPR137) was reported to be associated with several cancers, but its role in bladder cancer has not been reported. The purpose of this study was to evaluate clinical significance of GPR137 in bladder cancer. METHODS: The expressions of GPR137 in pathological tissues and corresponding normal tissues from bladder cancer patients were detected via quantitative real time polymerase chain reaction (qRT-PCR). Western blot was performed to detect GPR137 expression in bladder cancer tissues and adjacent normal tissues. Chi-Squared test analyzed the relationship between GPR137 expression and clinical features of bladder cancer patients. Additionally, Kaplan-Meier method was adopted in estimating overall survival of bladder cancer patients. Prognostic value of GPR137 was evaluated through Cox regression analysis. RESULTS: The expression of GPR137 mRNA and protein in pathological tissues was significantly higher than that in adjacent normal tissues (P < .001). Moreover, similar result was found for bladder cancer patients and healthy controls (P < .001). And GPR137 expression was associated with tumor size (P = .006) and TNM stage (P = .012). The results of Kaplan-Meier analysis suggested that patients with high expression of GPR137 had shorter overall survival time than those with low expression (Log rank test, P = .001). Cox regression analysis indicated that GPR137 could act as an independent biomarker for bladder cancer prognosis (HR = 1.850, 95% CI = 1.272-2.689, P = .001). CONCLUSION: Abnormal expression of GPR137 is associated with bladder cancer and GPR137 is a potential biomarker for the therapy and prognosis of bladder cancer.

IFITM3 promotes bone metastasis of prostate cancer cells by mediating activation of the TGF-ß signaling pathway.

Advanced-stage prostate cancer (PCa) is often diagnosed with bone metastasis, for which there are limited therapies. Transforming growth factor ß (TGF-ß) is known to induce epithelial-mesenchymal transition (EMT), and abundance of TGF-ß in the bone matrix is one of the important growth factors contributing to bone metastasis. TGF-ß is reported as a key mediator of bone metastasis, but the underlying mechanism has not been elucidated. It was found in our study that Interferon-inducible Transmembrane Protein 3 (IFITM3) played a key role in the regulation of malignant tumor cell proliferation, invasion, and bone migration by binding to Smad4, thus activating the TGF-ß-Smads Signaling Pathway. Lentivirus-mediated short hairpin RNA (shRNA) knockdown of IFITM3 inhibited cell proliferation and colony formation, induced apoptosis and inhibited migration by reversing EMT and downregulating the expression of metastasis-related molecules including FGFs and PTHrP. Microarray analysis showed that IFITM3 knockdown could alter the MAPK pathway associated with TGF-ß-Smads signaling. By knocking down and overexpressing IFITM3, we demonstrated that IFITM3 expression level had an effect on MAPK pathway activation, and this change was more pronounced upon exogenous TGF-ß stimulation. These results suggest that IFITM3 played an oncogenic role in PCa progression and bone metastasis via a novel TGF-ß-Smads-MAPK pathway.

The value of FGF9 as a novel biomarker in the diagnosis of prostate cancer.

Background: Fibroblast growth factor 9 (FGF9) is reported to be associated with the pathogenesis of cancers. However, its clinic significance in prostate cancer (PCa) had not yet to be elucidated. The aim of this study was to investigate the diagnostic value of FGF9 in PCa. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of serum FGF9 at mRNA and protein level in 90 PCa patients, 48 prostatic benign diseases (PBD) patients and 30 normal individuals. The association between FGF9 and clinicopathological features was determined by Chi-square test. Receiver-operator characteristic (ROC) was established to evaluate the diagnostic performance of FGF9 and PSA. Results: Serum FGF9 expression was significantly elevated in PCa patients (p < .001) and was obviously decreased after surgery (p < .001). FGF9 expression was also associated with lymph node metastasis (p = .010). The diagnostic value of FGF9 was higher than the conventional tumor marker PSA with a AUC of 0.846 combined with a sensitivity of 83.3% and a specificity of 81.1%. Conclusions: Serum FGF9 may be employed as a potential diagnostic biomarker of PCa.

Upregulated osterix promotes invasion and bone metastasis and predicts for a poor prognosis in breast cancer.

Approximately 70% of patients with advanced breast cancer develop bone metastases, accompanied by complications, such as bone pain, fracture, and hypercalcemia. However, our understanding of the molecular mechanisms that govern this process remains fragmentary. Osterix (Osx) is a zinc finger-containing transcription factor essential for osteoblast differentiation and bone formation. Here, we identified the functional roles of Osx in facilitating breast cancer invasion and bone metastasis. Osx upregulation was associated with lymph node metastasis and was negatively prognostic for overall survival. Knockdown of Osx inhibited invasion of breast cancer and osteolytic metastasis by downregulating MMP9, MMP13, VEGF, IL-8, and PTHrP, which are involved in invasion, angiogenesis, and osteolysis; overexpression of Osx had the opposite effect. Moreover, MMP9 was a direct target of Osx and mediated the Osx-driven invasion of breast cancer cells. Together, our data showed that Osx facilitates bone metastasis of breast cancer by upregulating the expression of a cohort of genes that contribute to steps in the metastatic cascade. These findings suggest that Osx is an attractive target for the control of bone metastasis of breast cancers.

Oridonin induces G2/M cell cycle arrest and apoptosis via the PI3K/Akt signaling pathway in hormone-independent prostate cancer cells.

Oridonin is an active constituent isolated from the traditional Chinese herb Rabdosia rubescens, which exerts antitumor effects in experimental and clinical settings. However, its antitumor effects and underlying mechanisms on prostate cancer cells have not yet been clearly identified. In the present study, the androgen-independent prostate cancer PC3 and DU145 cell lines were used as models to investigate the effects and possible mechanisms of oridonin on cellular proliferation and apoptosis. Results demonstrated that oridonin inhibited cellular proliferation, and was able to significantly induce G2/M cell cycle arrest and apoptosis. Detailed signaling pathway analysis by western blotting demonstrated that the expression levels of p53 and p21 were upregulated, whereas the expression of cyclin-dependent kinase 1 was downregulated following oridonin treatment, which led to cell cycle arrest in the G2/M phase. Oridonin also upregulated the proteolytic cleaved forms of caspase-3, caspase-9 and poly (ADP-ribose) polymerase. Furthermore, the protein expression levels of B-cell lymphoma 2 were decreased and those of Bcl-2-associated X protein were increased following oridonin treatment. In addition, oridonin treatment significantly inhibited the expression of phosphoiniositide-3 kinase (PI3K) p85 subunit and the phosphorylation of Akt. The downstream gene murine double minute 2 was also downregulated, which may contribute to the elevated expression of p53 following oridonin treatment. In conclusion, the results of the present study suggested that oridonin is able to inactivate the PI3K/Akt pathway and activate p53 pathways in prostate cancer cells, resulting in the suppression of proliferation and the induction of caspase-mediated apoptosis.

Osterix acetylation at K307 and K312 enhances its transcriptional activity and is required for osteoblast differentiation.

Osterix (Osx) is an essential transcription factor involved in osteoblast differentiation and bone formation. The precise molecular mechanisms of the regulation of Osx expression are not fully understood. In the present study, we found that in cells, both endogenous and exogenous Osx protein increased after treatment with histone deacetylase inhibitors Trichostatin A and hydroxamic acid. Meanwhile, the results of immunoprecipitation indicated that Osx was an acetylated protein and that the CREB binding protein (CBP), and less efficiently p300, acetylated Osx. The interaction and colocalization of CBP and Osx were demonstrated by Co-immunoprecipitation and immunofluorescence, respectively. In addition, K307 and K312 were identified as the acetylated sites of Osx. By contrast, HDAC4, a histone deacetylase (HDAC), was observed to interact and co-localize with Osx. HDAC4 was demonstrated to mediate the deacetylation of Osx. Moreover, we found that acetylation of Osx enhanced its stability, DNA binding ability and transcriptional activity. Finally, we demonstrated that acetylation of Osx was required for the osteogenic differentiation of C2C12 cells. Taken together, our results provide evidence that CBP-mediated acetylation and HDAC4-mediated deacetylation have critical roles in the modification of Osx, and thus are important in osteoblast differentiation.

Overexpression of USP39 predicts poor prognosis and promotes tumorigenesis of prostate cancer via promoting EGFR mRNA maturation and transcription elongation.

Castration resistance is a serious problem facing clinical treatment of prostate cancer (PCa). The underlying molecular mechanisms of acquired proliferation ability of tumor cells upon androgen deprivation are largely undetermined. In the present study, we identified that ubiquitin specific peptidase 39 (USP39) was significantly upregulated in PCa samples and cell lines. Elevated USP39 expression was positively correlated with Gleason score, predicted a poor outcome, and functioned as an independent risk factor for biochemical recurrence (BCR) especially in patients with a Gleason score ≤7. Our cell-based study showed that the expression level of USP39 was the highest in AR-negative PCa cell lines. Knockdown of USP39 in PCa cells inhibited cancer colony formation and tumor cell growth, and induced G2/M arrest and cell apoptosis. Microarray analysis suggested that knockdown of USP39 caused a reduced expression of EGFR. Silencing of USP39 inhibited the expression of EGFR 3'-end, and presented a remarkable block to the maturation of EGFR mRNA, suggesting that silencing of USP39 decreased the transcriptional elongation and maturation of EGFR mRNA. Oncomine datasets analysis showed that USP39 expression was positively correlated with EGFR level. The above findings suggest that USP39 plays a vital oncogenic role in the tumorigenesis of PCa and may prove to be a potential biomarker for predicting the prognosis of PCa patients.

Low LKB1 Expression Results in Unfavorable Prognosis in Prostate Cancer Patients.

BACKGROUND The present study aimed to compare the expression of liver kinase B1 (LKB1) in prostate cancer (PCa) tissues and the paired adjacent tissues, then to evaluate the statistical relationship between LKB1 expression and prognosis of PCa patients. MATERIAL AND METHODS The relative expression of LKB1 at mRNA level was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of LKB1 at protein level was measured by immunohistochemistry (IHC) method. The relationship between LKB1 expression and clinicopathologic characteristics was estimated by chi-square test. Kaplan-Meier method was used to analyze the overall survival of PCa patients with different LKB1 expression. Cox regression analysis was performed to estimate the significance of LKB1 expression and clinicopathologic characteristics in the prognosis of PCa patients. RESULTS The relative expression of LKB1 at mRNA level was significantly lower in PCa tissues than in the normal tissues (P<0.001). The LKB1 expression was proved to be affected by clinical stage (P=0.019) and PSA concentration (P=0.031) of PCa patients. Moreover, patients with negative LKB1 expression had shorter survival than those with positive expression. Cox regression analysis confirmed that LKB1 could be regarded as a prognostic biomarker for PCa patients (P=0.001, HR=3.981, 95% CI=1.698-9.336). CONCLUSIONS The expression of LKB1 was lower in PCa tissues and might be a predictor for the prognosis of PCa patients.

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis.

Recent studies have revealed that osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, a traditional Chinese medicine, possesses anticancer activity. However, its effect on breast cancer cells so far has not been elucidated clearly. In the present study, we evaluated the effects of osthole on the proliferation, cell cycle and apoptosis of human breast cancer cells MDA-MB 435. We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells, The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole, as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation. The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression. Were observed taken together, these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.

Phosphorylation of Serine422 increases the stability and transactivation activities of human Osterix.

Osterix (Osx) is an essential regulator for osteoblast differentiation and bone formation. Although phosphorylation has been reported to be involved in the regulation of Osx activity, the precise underlying mechanisms remain to be elucidated. Here we identified S422 as a novel phosphorylation site of Osx and demonstrated that GSK-3ß interacted and co-localized with Osx. GSK-3ß increased the stability and transactivation activity of Osx through phosphorylation of the newly identified site. These findings expanded our understanding of the mechanisms of posttranslational regulation of Osx and the role of GSK-3ß in the control of Osx transactivation activity.

Oxidized low-density lipoprotein, OXPAPC, corrects defects in maturation and cytokine secretion of peripheral blood dendritic cells from sepsis patients.

OBJECTIVE: To investigate the expression differences in maturation and cytokine production of dendritic cells (DCs) from sepsis patients and the effect of oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OXPAPC) on DCs phenotypes. METHODS: Peripheral blood mononuclear cells from 50 sepsis patients and 50 controls were cultured in the presence of GM-CSF, IL-4 and TNF-α to induce DCs maturation. DCs from sepsis patients were also treated with three different concentrations of OXPAPC. Cells were characterized with optical and electron microscopy, FACS analysis for CD1α, HLA-DR and CD86 on cell surface and ELISA analysis of IL-12p70 in the supernatant. RESULTS: DCs from sepsis patients had smaller cell bodies and nucleus and had almost no surface projection. DCs had similar CD1α expression in sepsis patients (86.37 ± 17.24) and controls (88.58 ± 10.05). HLA-DR expression was dramatically reduced in sepsis patients (2.74 ± 5.15) compared to controls (198.35 ± 12.04). Similarly, CD86 expression was also drastically lower in sepsis patients (14.72 ± 4.83) than controls (154.56 ± 11.56). Furthermore, OXPAPC treatment of DCs from sepsis patients increased cell surface projection, HLA-DR and CD86 surface expression and IL-12p70 secretion in a dose-dependent manner. With 40 µg/ml of OXPAPC, DCs of sepsis patients have similar phenotypes observed in healthy controls. CONCLUSION: DCs from sepsis patients are defective in maturation and cytokine secretion and these defects can be corrected by OXPAPC treatment.

MDM2 promotes invasion and metastasis in invasive ductal breast carcinoma by inducing matrix metalloproteinase-9.

The molecular mechanisms that underpin invasive ductal breast cancer (IDC) invasion and metastasis are incompletely understood. The oncogene, mouse double minute 2 (MDM2), has been implicated in the pathogenesis of numerous cancers, where it stimulates the expression of matrix metalloproteinase 9 (MMP9), an important enzyme in the breakdown of the extracellular matrix. However, its role in breast cancer remains poorly understood. This study assessed the clinical significance of MDM2 expression in IDC and used in vitro expression assays to determine the molecular roles of MDM2. Immunohistochemical staining for MMP9 and MDM2 was performed using archived tumor blocks from 321 women who underwent surgical resection for IDC at the First Affiliated Hospital of Nanjing Medical University, China between January 2002 and December 2003. MCF-7 and MDA-MD-231 cell lines were transfected with siRNA targeted against MDM2, or MDM2 was overexpressed using transiently expressed vectors. The invasion, cell migration and proteolytic capabilities of cells that over- or underexpressed MDM2 was then assessed and compared against control cells, in addition to the consequent effects on MMP9 expression using RT-PCR. In vivo, 54.9% and 49.6% of samples were positive for MMP9 and MDM2 expression, respectively, and their expression was significantly correlated (r²â€Š= 0.171, P = 0.012). Moreover, MDM2 expression was markedly correlated with disease-free survival (HR 2.56, 95% CI 1.02-6.40, P = 0.038). In vitro, MDM2 overexpression significantly enhanced cell invasion, migration and proteolysis compared with control cells, and the converse effects were observed after MDM2-siRNA treatment. MDM2 overexpression induced MMP9 expression in a dose-dependent manner. Taken together, these results suggest that high levels of MDM2 are associated with a poorer prognosis in IDC. This might result from increased tumor invasiveness due to enhanced MMP9 expression causing increased extracellular matrix breakdown.

MicroRNA-214 suppresses osteogenic differentiation of C2C12 myoblast cells by targeting Osterix.

Osterix (Osx) is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Osx-null mice, which exhibit a complete absence of bone formation and arrested osteoblast differentiation, die immediately after birth. However, our understanding of the regulatory mechanism of Osx expression remains poor. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play pivotal roles in diverse biological processes, including the development, differentiation, proliferation, survival, and oncogenesis of cells and organisms. In this study, we aimed to investigate the impact of miRNAs on Osx expression. Bioinformatic analyses predicted that miR-214 would be a potential regulator of Osx. The direct binding of miR-214 to the Osx 3' untranslated region (3' UTR) was demonstrated by a luciferase reporter assay using a construct containing the Osx 3' UTR. Deletion mutant construction revealed that the Osx 3' UTR contained two miR-214 binding sites. MiR-214 expression was inversely correlated with Osx expression in Saos-2 and U2OS cells. The forced expression of miR-214 in Saos-2 cells led to a reduction in the level of Osx protein. Moreover, the role of miR-214 in the osteogenic differentiation of C2C12 cells was investigated. We found that the osteogenic differentiation of C2C12 cells was enhanced by the downregulation of miR-214 expression, as measured by increased alkaline phosphatase activity and matrix mineralization. Taken together, these results indicate that miR-214 is a novel regulator of Osx, and that it plays an important role in the osteogenic differentiation of C2C12 cells as a suppressor.