A unidirectional water-transport antibacterial bilayer nanofibrous dressing based on chitosan for accelerating wound healing.

Excessive accumulation of exudate from wounds often causes infection and hinders skin regeneration. To handle wound exudate quickly and prevent infection, we developed an antibacterial Janus nanofibrous dressing with a unidirectional water-transport function. The dressing consists of a hydrophilic chitosan aerogel (CS-A) as the outer layer and a hydrophobic laurylated chitosan (La-CS) nanofibrous membrane as the inner layer. These dressings achieved excellent liquid absorption performance (2987.8 ±â€¯123.5 %), air and moisture permeability (997.8 ±â€¯23.1 g/m2/day) and mechanical strength (5.1 ±â€¯2.6 MPa). This performance was obtained by adjusting the density of CS-A and the thickness of the La-CS membrane. Moreover, the dressing did not induce significant toxicity to cells and can prevent bacterial aggregation and infection at the wound site. Animal experiments showed that the dressing can shorten the inflammatory phase, enhance blood vessel generation, and accelerate collagen deposition, thus promoting wound healing. Overall, these results suggest that this Janus dressing is a promising material for clinical wound care.

A DNA-inspired injectable adhesive hydrogel with dual nitric oxide donors to promote angiogenesis for enhanced wound healing.

Chronic diabetic wounds are a severe complication of diabetes, often leading to high treatment costs and high amputation rates. Numerous studies have revealed that nitric oxide (NO) therapy is a promising option because it favours wound revascularization. Here, base-paired injectable adhesive hydrogels (CAT) were prepared using adenine- and thymine-modified chitosan (CSA and CST). By further introducing S-nitrosoglutathione (GSNO) and binary l-arginine (bArg), we obtained a NO sustained-release hydrogel (CAT/bArg/GSON) that was more suitable for the treatment of chronic wounds. The results showed that the expression of HIF-1α and VEGF was upregulated in the CAT/bArg/GSON group, and improved blood vessel regeneration was observed, indicating an important role of NO. In addition, the research findings revealed that following treatment with the CAT/bArg/GSON hydrogel, the viability of Staphylococcus aureus and Escherichia coli decreased to 14 ± 2 % and 6 ± 1 %, respectively. Moreover, the wound microenvironment was improved, as evidenced by a 60 ± 1 % clearance of DPPH. In particular, histological examination and immunohistochemical staining results showed that wounds treated with CAT/bArg/GSNO exhibited denser neovascularization, faster epithelial tissue regeneration, and thicker collagen deposition. Overall, this study proposes an effective strategy to prepare injectable hydrogel dressings with dual NO donors. The functionality of CAT/bArg/GSON has been thoroughly demonstrated in research on chronic wound vascular regeneration, indicating that CAT/bArg/GSON could be a potential option for promoting chronic wound healing. STATEMENT OF SIGNIFICANCE: This article prepares a chitosan hydrogel utilizing the principle of complementary base pairing, which offers several advantages, including good adhesion, biocompatibility, and flow properties, making it a good material for wound dressings. Loaded GSNO and bArg can steadily release NO and l-arginine through the degradation of the gel. Then, the released l-arginine not only possesses antioxidant properties but can also continue to generate a small amount of NO under the action of NOS. This design achieves a sustained and stable supply of NO at the wound site, maximizing the angiogenesis-promoting and antibacterial effects of NO. More neovascularization and abundant collagen were observed in the regenerated tissues. This study provides an effective repair hydrogel material for diabetic wound.

Salvianolic Acid B Suppresses ER Stress-Induced NLRP3 Inflammasome and Pyroptosis via the AMPK/FoxO4 and Syndecan-4/Rac1 Signaling Pathways in Human Endothelial Progenitor Cells.

Mounting evidence demonstrates uncontrolled endoplasmic reticulum (ER) stress responses can activate the inflammasome, which generally results in endothelial dysfunction, a major pathogenetic factor of chronic inflammatory diseases such as atherosclerosis. Salvianolic acid B (SalB), produced by Radix Salviae, exerts antioxidative and anti-inflammatory activities in multiple cell types. However, SalB's effects on ER stress-related inflammasome and endothelial dysfunction remain unknown. Here, we showed SalB substantially abrogated ER stress-induced cell death and reduction in capillary tube formation, with declined intracellular reactive oxygen species (ROS) amounts and restored mitochondrial membrane potential (MMP), as well as increased expression of HO-1 and SOD2 in bone marrow-derived endothelial progenitor cells (BM-EPCs). ER stress suppression by CHOP or caspase-4 siRNA transfection attenuated the protective effect of SalB. Additionally, SalB alleviated ER stress-mediated pyroptotic cell death via the suppression of TXNIP/NLRP3 inflammasome, as evidenced by reduced cleavage of caspase-1 and interleukin- (IL-) 1ß and IL-18 secretion levels. Furthermore, this study provided a mechanistic basis that AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 signaling pathway modulation by SalB substantially prevented BM-EPCs damage associated with ER stress by decreasing intracellular ROS amounts and inducing NLRP3-dependent pyroptosis. In summary, our findings identify that ER stress triggered mitochondrial ROS release and NLRP3 generation in BM-EPCs, while SalB inhibits NLRP3 inflammasome-mediated pyroptotic cell death by regulating the AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 pathways. The current findings reveal SalB as a potential new candidate for the treatment of atherosclerotic heart disease.

3D printed concentrated alginate/GelMA hollow-fibers-packed scaffolds with nano apatite coatings for bone tissue engineering.

Three-dimensional grafts/scaffolds with hierarchically biomimetic features from nano to macro scale and high porosity are required for bone tissue engineering. In this study, biomimetic organic/inorganic composite scaffolds with high porosity (78.7 ± 3.2%) and features from nano (nano apatite coatings) to macro (macro pores and hollow channels) scale were fabricated based on highly concentrated alginate/GelMA bioinks via co-axial 3D printing and in situ mineralization under mild conditions. Nano apatites were coated on both inner and outer surfaces of the hollow fiber scaffolds, homogeneously. Proteins were directly loaded in the bioinks achieving sustained release from the scaffolds over 28 days. The in vitro cell experiments showed that the scaffolds with good biocompatibility could support cells adhesion and proliferation. The nano apatite coatings presented remarkable osteogenic capability. The in vivo study indicated that the hollow fiber scaffolds with biomimetic nano apatite coatings showed the capability to enhance bone formation after 12 weeks of implantation. In conclusion, the prepared biomimetic organic/inorganic scaffolds with homogeneous nano apatite coatings and hollow channels structures might be potential candidates for bone tissue engineering.

Perillaldehyde inhibits bone metastasis and receptor activator of nuclear factor-κB ligand (RANKL) signaling-induced osteoclastogenesis in prostate cancer cell lines.

Perillaldehyde (PAH), one of the active ingredients of the traditional Chinese medicine (TCM) plant Perilla frutescens, is widely used and exerts crucial anti-cancer activities. The aim of current study is to illustrate the potential mechanisms of PAH-mediated regulation of bone metastasis and osteoclastogenesis in prostate cancer (PCa) cell lines. Effects of PAH on proliferation, invasion and migration of PC-3 cells were assessed with the Cell Counting Kit-8 (CCK-8) assay and Transwell assays, respectively. Effects of PAH on stem cell characteristics of PC-3 cells were evaluated by cell-matrix adhesion assay, colony formation assay, spheroid formation assay, as well as western blot . The anti-metastasis and anti-osteoclastogenesis activity of PAH in RAW264.7 cells was examined by osteoclast differentiation assay and western blot. The protein levels of CD133 and CD44 in PC-3 cells and the activity of nuclear factor kappa B (NF-κB) signaling pathway in RAW264.7 cells were measured by western blot. PAH suppressed proliferation, invasion and migration of PC-3 cells, prevented stem cell characteristics including cell-matrix adhesion, colony formation, spheroid formation as well as CD133 and CD44 expression. PAH inhibited bone metastasis and osteoclastogenesis via repressing the activation of NF-κB pathway as well as (RANKL) - and cancer cell-induced osteoclastogenesis in PCa cells. These findings suggested the potential therapeutic effects of PAH on the metastasis of patients with PCa.

Anticancer Activity of Sweroside Nanoparticles in Prostate Cancer Bone Metastasis in PC-3 Cells Involved in Wnt/ß-Catenin Signaling Pathway.

Bone metastasis is a significant cause of morbidity and mortality in patients with prostate cancer (PCa). This study is aimed at illustrating the mechanism of sweroside-mediated regulation in bone metastasis in PCa cells. Owing to the limitations of antitumor drugs in terms of their physical and chemical properties, making them into nanomaterials can effectively improve drug stability and bioavailability. Apoptosis was assessed with flow cytometry using the annexin V/propidium iodide binding assay; proteins, including p53, P21, Bcl-2, and Bax; and induction of intracellular reactive oxygen species (ROS). Using colony formation assay, sphere formation assay, and the expression changes in CD133 and CD44, stem cell characteristics were assessed. Epithelial-mesenchymal transition (EMT) activity was accessed by levels of the expression changes of EMT-related markers, vimentin and E-cadherin. Wnt/ß-catenin signaling pathway was examined to detect the levels of the expression changes of snail and ß-catenin. PC-3 cells were treated with lithium chloride (LiCl), which is an agonist of Wnt/ß-catenin signaling, and the levels of CD133, CD44, vimentin, E-cadherin, snail, and ß-catenin were detected. T-cell factor/lymphocyte enhancer factor (TCF/LEF) activity in cells overexpressing ß-catenin was used to detect the effects on ß-catenin transcription, and the expression of c-myc, Cyclin D1, Survivin, and MMP-7 were used to detect Wnt downstream target genes. Our results suggest that sweroside induces apoptosis and intracellular ROS; upregulates apoptotic proteins; and suppresses proliferation, invasion, and migration, preventing stem cell characteristics, including sphere formation, colony formation, and CD133 and CD44 expressions. Furthermore, sweroside nanoparticles exerts inhibitory effects on ß-catenin transcription by suppressing TTCF/LEF activity in cells overexpressing ß-catenin and downregulation of the expression of Wnt downstream target genes, including c-myc, Cyclin D1, Survivin, and MMP-7. The potential therapeutic effect of sweroside nanoparticles on bone metastatis of PCa was suggested, by these findings.

Circ_HECW2 regulates LPS-induced apoptosis of chondrocytes via miR-93 methylation.

INTRODUCTION: Circ_HECW2 plays a key role in lipopolysaccharide (LPS)-induced signal transduction, which is critical in osteoarthritis (OA). Thus, we analyzed the role of Circ_HECW2 in osteoarthritis. METHODS: The expression of Circ_HECW2 and miR-93 was examined using reverse-transcription polymerase chain reaction. Cell apoptosis was evaluated using Annexin V-FITC Apoptosis Detection Kit. RESULTS: Circ_HECW2 and miR-93 were inversely correlated, with Circ_HECW2 upregulated and miR-93 downregulated in OA and LPS-induced chondrocytes. Circ_HECW2 overexpression inhibited miR-93 expression and increased methylation of miR-93 coding gene. Cell apoptosis analysis showed that Circ_HECW2 overexpression increased LPS-induced chondrocyte apoptosis, while MiR-93 overexpression reversed the effects of Circ_HECW2 on chondrocyte apoptosis. CONCLUSION: In summary, our data revealed that the Circ_HECW2 is highly expressed in OA and might inhibit miR-93 expression through methylation to affect LPS-induced chondrocyte apoptosis.

miR-23a-3p regulated by LncRNA SNHG5 suppresses the chondrogenic differentiation of human adipose-derived stem cells via targeting SOX6/SOX5.

Cartilage generation and degradation are controlled by miRNAs. Our previous study showed miR-23a-3p was downregulated during chondrogenic differentiation in chondrogenic human adipose-derived mesenchymal stem cells (hADSCs). In the present study, we explored the function of miR-23a-3p in chondrogenesis differentiation. The role of miR-23a-3p in chondrogenic differentiation potential of hADSCs was assessed by Alcian blue staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. We show that miR-23a-3p suppressed the chondrogenic differentiation of hADSCs. LncRNA SNHG5 interacted with miR-23a-3p, and suppression or overexpression of SNHG5 correlates with inhibition and promotion of hADSC chondrogenic differentiation, respectively. We have determined that SNHG5 can sponge miR-23a-3p to regulate the expression of SOX6/SOX5, transcription factors that play essential roles in chondrocyte differentiation. Furthermore, the overexpression of SNHG5 activates the JNK/MAPK/ERK pathway. In conclusion, miR-23a-3p regulated by lncRNA SNHG5 suppresses the chondrogenic differentiation of human adipose-derived stem cells via targeting SOX6/SOX5.

Ectopic Expression of miR-532-3p Suppresses Bone Metastasis of Prostate Cancer Cells via Inactivating NF-κB Signaling.

miR-532-3p is a widely documented microRNA (miRNA) involved in multifaceted processes of cancer tumorigenesis and metastasis. However, the clinical significance and biological functions of miR-532-3p in bone metastasis of prostate cancer (PCa) remain largely unknown. Herein, we report that miR-532-3p was downregulated in PCa tissues with bone metastasis, and downexpression of miR-532-3p was significantly associated with Gleason grade and serum prostate-specific antigen (PSA) levels and predicted poor bone metastasis-free survival in PCa patients. Upregulating miR-532-3p inhibited invasion and migration abilities of PCa cells in vitro, while silencing miR-532-3p yielded an opposite effect on invasion and migration abilities of PCa cells. Importantly, upregulating miR-532-3p repressed bone metastasis of PCa cells in vivo. Our results further demonstrated that overexpression of miR-532-3p inhibited activation of nuclear facto κB (NF-κB) signaling via simultaneously targeting tumor necrosis factor receptor-associated factor 1 (TRAF1), TRAF2, and TRAF4, which further promoted invasion, migration, and bone metastasis of PCa cells. Therefore, our findings reveal a novel mechanism contributing to the sustained activity of NF-κB signaling underlying the bone metastasis of PCa.

miR-204-5p Represses Bone Metastasis via Inactivating NF-κB Signaling in Prostate Cancer.

The prime issue derived from prostate cancer (PCa) is its high prevalence to metastasize to bone. MicroRNA-204-5p (miR-204-5p) has been reported to be involved in the development and metastasis in a variety of cancers. However, the clinical significance and biological functions of miR-204-5p in bone metastasis of PCa are still not reported yet. In this study, we find that miR-204-5p expression is reduced in PCa tissues and serum sample with bone metastasis compared with that in PCa tissues and serum sample without bone metastasis, which is associated with advanced clinicopathological characteristics and poor bone metastasis-free survival in PCa patients. Moreover, upregulation of miR-204-5p inhibits the migration and invasion of PCa cells in vitro, and importantly, upregulating miR-204-5p represses bone metastasis of PCa cells in vivo. Our results further demonstrated that miR-204-5p suppresses invasion, migration, and bone metastasis of PCa cells via inactivating nuclear factor κB (NF-κB) signaling by simultaneously targeting TRAF1, TAB3, and MAP3K3. In clinical PCa samples, miR-204-5p expression negatively correlates with TRAF1, TAB3, and MAP3K3 expression and NF-κB signaling activity. Therefore, our findings reveal a new mechanism underpinning the bone metastasis of PCa, as well as provide evidence that miR-204-5p might serve as a novel serum biomarker in bone metastasis of PCa. This study identifies a novel functional role of miR-204-5p in bone metastasis of prostate cancer and supports the potential clinical value of miR-204-5p as a serum biomarker in bone metastasis of PCa.

miR-582-3p and miR-582-5p Suppress Prostate Cancer Metastasis to Bone by Repressing TGF-ß Signaling.

A number of studies have reported that aberrant expression of microRNAs (miRNAs) closely correlates with the bone metastasis of prostate cancer (PCa). However, clinical significance and functional roles of both strands of a single miRNA in bone metastasis of PCa remain undefined. Here, we reported that miR-582-3p and miR-582-5p expression were simultaneously reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Downexpression of miR-582-3p and miR-582-5p strongly and positively correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-582-3p and miR-582-5p inhibited invasion and migration abilities of PCa cells in vitro, as well as repressed bone metastasis in vivo. Our results further revealed that miR-582-3p and miR-582-5p attenuated bone metastasis of PCa via inhibiting transforming growth factor ß (TGF-ß) signaling by simultaneously targeting several components of TGF-ß signaling, including SMAD2, SMAD4, TGF-ß receptor I (TGFBRI), and TGFBRII. Moreover, deletion contributes to miR-582-3p and miR-582-5p downexpression in PCa tissues. Finally, clinical negative correlations of miR-582-3p and miR-582-5p with SMAD2, SMAD4, TGFBRI, and TGFBRII were demonstrated in PCa tissues. Thus, our findings explore a novel tumor-suppressive miRNA with its both strands implicated in bone metastasis of PCa, suggesting its potential therapeutic value in treatment of PCa bone metastasis.

miR-1307-3p suppresses the chondrogenic differentiation of human adipose-derived stem cells by targeting BMPR2.

MicroRNAs (miRs) are involved in several physiological processes, including chondrogenic differentiation, however, their expression and roles in the chondrogenic differentiation of human adipose­derived stem cells (hADSCs) remain to be fully elucidated to date. Our previous study showed that miR­1307­3p was significantly downregulated during chondrogenic differentiation by microarray and northern blot analysis. The present study aimed to investigate the effects of miR­1307­3p on chondrogenic differentiation and the underlying mechanisms. First, the decreased expression of miR­1307­3p was confirmed by reverse transcription­quantitative polymerase chain reaction analysis. Subsequently, gain­ and loss­of­function of miR­1307­3p experiments showed that the overexpression of miR­1307­3p suppressed the deposition of cartilage matrix proteoglycans and decreased the expression of cartilage­related markers, including sex determining region Y­box 9, collagen type II α1 chain and aggrecan, whereas the knockdown of miR­1307­3p had the opposite effect. In addition, bone morphogenetic protein receptor type 2 (BMPR2) was identified as a target of miR­1307­3p. Further mechanistic investigations showed that miR­1307­3p attenuated the chondrogenic differentiation of hADSCs at least partly by inhibiting BMPR2­mothers against decapentaplegic signaling pathways. In conclusion, the findings revealed that miR­1307­3p inhibited the chondrogenic differentiation of hADSCs by targeting BMPR2 and its downstream signaling pathway, which may provide novel therapeutic clues for the treatment of cartilage injury.

Downregulation of miR-133a-3p promotes prostate cancer bone metastasis via activating PI3K/AKT signaling.

BACKGROUND: Bone metastasis is a leading cause of morbidity and mortality in advanced prostate cancer (PCa). Downexpression of miR-133a-3p has been found to contribute to the progression, recurrence and distant metastasis in PCa. However, clinical significance of miR-133a-3p in bone metastasis of PCa, and the biological role of miR-133a-3p and its molecular mechanisms underlying bone metastasis of PCa remain unclear. METHODS: miR-133a-3p expression was evaluated in 245 clinical PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-133a-3p expression and clinicopathological features, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-133a-3p in the bone metastasis of PCa were investigated both in vitro and in vivo. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to demonstrate the relationship between miR-133a-3p and its potential targets. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the anti-tumor role of miR-133a-3p. Clinical correlation of miR-133a-3p with its targets was verified in human PCa tissues. RESULTS: miR-133a-3p expression is reduced in PCa tissues compared with the adjacent normal tissues and benign prostate lesion tissues, particularly in bone metastatic PCa tissues. Low expression of miR-133a-3p is significantly correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients by statistical analysis. Moreover, upregulating miR-133a-3p inhibits cancer stem cell-like phenotypes in vitro and in vivo, as well as attenuates anoikis resistance in vitro in PCa cells. Importantly, administration of agomir-133a-3p greatly suppresses the incidence of PCa bone metastasis in vivo. Our results further demonstrate that miR-133a-3p suppresses bone metastasis of PCa via inhibiting PI3K/AKT signaling by directly targeting multiple cytokine receptors, including EGFR, FGFR1, IGF1R and MET. The negative clinical correlation of miR-133a-3p with EGFR, FGFR1, IGF1R, MET and PI3K/AKT signaling activity is determined in clinical PCa tissues. CONCLUSION: Our results unveil a novel mechanism by which miR-133a-3p inhibits bone metastasis of PCa, providing the evidence that miR-133a-3p may serve as a potential bone metastasis marker in PCa, and delivery of agomir-133a-3p may be an effective anti-bone metastasis therapeutic strategy in PCa.

Transcriptional downregulation of miR-133b by REST promotes prostate cancer metastasis to bone via activating TGF-ß signaling.

High avidity of bone metastasis is an important characteristic in prostate cancer (PCa). Downexpression of miR-133b has been reported to be implicated in the development, progression and recurrence in PCa. However, clinical significance and biological roles of miR-133b in bone metastasis of PCa remain unclear. Here we report that miR-133b is downregulated in PCa tissues and further decreased in bone metastatic PCa tissues. Downexpression of miR-133b positively correlates with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-133b inhibits invasion, migration in vitro and bone metastasis in vivo in PCa cells. Mechanistically, we find that miR-133b suppresses activity of TGF-ß signaling via directly targeting TGF-ß receptor I and II, which further inhibits bone metastasis of PCa cells. Our results further reveal that overexpression of REST contributes to miR-133b downexpression via transcriptional repression in PCa tissues. Importantly, silencing miR-133b enhances invasion and migration abilities in vitro and bone metastasis ability in vivo in REST-silenced PCa cells. The clinical correlation of miR-133b with TGFBRI, TGFBRII, REST and TGF-ß signaling activity is verified in PCa tissues. Therefore, our results uncover a novel mechanism of miR-133b downexpression that REST transcriptionally inhibits miR-133b expression in PCa cells, and meanwhile support the notion that administration of miR-133b may serve as a rational regimen in the treatment of PCa bone metastasis.

Downregulation of miR­19a­3p promotes invasion, migration and bone metastasis via activating TGF­ß signaling in prostate cancer.

Constitutive activation of TGF­ß signaling pathway is a well-documented mechanism responsible for the bone metastasis of prostate cancer (PCa). MicroRNAs (miRNAs) have been reported to be crucial for the activation of TGF­ß signaling via targeting downstream components of TGF­ß signaling pathway. Here, we report that miR­19a­3p is downregulated in bone metastatic PCa tissues and cells. Upregulation of miR­19a­3p suppresses invasion, migration in vitro and inhibits bone metastasis in vivo in PCa cells. Conversely, silencing miR­19a­3p yields the opposite effect. Our results further demonstrate that miR­19a­3p inhibits invasion and migration abilities of PCa cells via targeting downstream effectors of TGF­ß signaling, SMAD2 and SMAD4, resulting in the inactivation of TGF­ß signaling. Therefore, our results uncover a novel mechanistic understanding of miR­19a­3p-induced suppressive role in bone metastasis of PCa, which will facilitate the development of effective cancer therapy methods against PCa.

miR­328­3p enhances the radiosensitivity of osteosarcoma and regulates apoptosis and cell viability via H2AX.

Osteosarcoma is a kind of high-risk sarcoma of the skeleton typically observed in people under 25 years old. Currently, radiotherapy is widely applied in cancer treatment. However, osteosarcoma is radioresistant and accordingly new, more effective radiosensitizers are needed. miRNAs have been reported to play an important role in osteosarcoma radiosensitivity. We examined the modulating effect of miR­328­3p in vivo and in vitro. miR­328­3p was downregulated in HOS­2R cells. The overexpression of miR­328­3p enhanced the radiosensitivity of osteosarcoma cells. miR­328­3p inhibited proliferation and promoted apoptosis in osteosarcoma cells under radiation conditions. In cells overexpressing miR­328­3p, H2AX expression was downregulated. We found that miR­328­3p targets H2AX and inhibits its expression. It was concluded, that miR­328­3p enhances the radiosensitization of osteosarcoma following X-ray irradiation, and determined that it directly targets H2AX to regulate radiosensitization.

Downregulation of miR-141-3p promotes bone metastasis via activating NF-κB signaling in prostate cancer.

BACKGROUND: Clinically, prostate cancer (PCa) exhibits a high avidity to metastasize to bone. miR-141-3p is an extensively studied miRNA in cancers and downregulation of miR-141-3p has been widely reported to be involved in the progression and metastasis of several human cancer types. However, the clinical significance and biological roles of miR-141-3p in bone metastasis of PCa are still unclear. METHODS: miR-141-3p expression was examined in 89 non-bone metastatic and 52 bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to investigate the clinical correlation between miR-141-3p expression levels and clinicopathological characteristics in PCa patients. The biological roles of miR-141-3p in bone metastasis of PCa were evaluated both in vitro and a mouse intracardial model in vivo. Bioinformatics analysis, Western blot, luciferase reporter and miRNA immunoprecipitation assays were performed to explore and examine the relationship between miR-141-3p and its potential targets. Clinical correlation of miR-141-3p with its targets was examined in clinical PCa tissues. RESULTS: miR-141-3p expression is reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Low expression of miR-141-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Furthermore, upregulating miR-141-3p suppresses the EMT, invasion and migration of PCa cells in vitro. Conversely, silencing miR-141-3p yields an opposite effect. Importantly, upregulating miR-141-3p dramatically reduces bone metastasis of PC-3 cells in vivo. Our results further show that miR-141-3p inhibits the activation of NF-κB signaling via directly targeting tumor necrosis factor receptor-associated factor 5(TRAF5) and 6 (TRAF6), which further suppresses invasion, migration and bone metastasis of PCa cells. The clinical negative correlation of miR-141-3p expression with TRAF5, TRAF6 and NF-κB signaling activity is demonstrated in PCa tissues. CONCLUSION: Our findings unravel a novel mechanism underlying the bone metastasis of PCa, suggesting that miR-141-3p mimics might represent a potential therapeutic avenue for the treatment of PCa bone metastasis.

miR-30b regulates chondrogenic differentiation of mouse embryo-derived stem cells by targeting SOX9.

The present study aimed to investigate the mechanisms underlying microRNA (miRNA)-mediated regulation of chondrogenic differentiation. Mouse embryo-derived stem cells C3H10T1/2 were cultured and chondrogenic differentiation was induced using transforming growth factor-ß3 (TGF-ß3). In addition, miRNA expression profiles were detected via miRNA array analysis, and quantitative polymerase chain reaction was performed to verify the differentially expressed miRNAs. Furthermore, bioinformatics software was used to predict the putative targets and the prediction was validated by dual-luciferase reporter assays and western blot analysis. In addition, cell proliferation and glycosaminoglycans were measured by a direct cell count method and alcian blue staining, respectively. Compared with the control group, 86 miRNAs were identified as differentially expressed in TGF-ß3-induced cells and the expression levels of 28 miRNAs were increased while the remaining 58 miRNAs exhibited a decline in expression. Amongst the differentially expressed miRNAs, miR-30b expression was observed to have significantly decreased during chondrogenic differentiation. SOX9 is a target gene of miR-30b, and miR-30b inhibits SOX9 expression during chondrogenic differentiation. Furthermore, the alcian blue staining results demonstrated that miR-30b inhibited early chondrogenic differentiation. However, the data of the present study indicated that miR-30b had no influence on C3H10T1/2 cell line proliferation. In conclusion, miR-30b is a key negative regulator of TGF-ß3-induced C3H10T1/2 cell chondrogenic differentiation, which functions by directly targeting SOX9.

miRNA-140 inhibits C3H10T1/2 mesenchymal stem cell proliferation by targeting CXCL12 during transforming growth factor-ß3-induced chondrogenic differentiation.

The aim of the present study was to investigate the role of microRNA (miRNA or miR)-140 in C3H10T1/2 mesenchymal stem cells (MSCs). Cluster analysis was used to evaluate the miRNA expression profile. The expression level of miRNA­140 was validated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). TargetScan and microRNA.org databases were used to predict target miRNAs and cartilage­associated target genes. Binding sites between miR­140 and the target gene were predicted by bioinformatics software. A dual­luciferase reporter assay was performed to determine whether miR­140 could target C­X­C motif chemokine ligand 12 (CXCL12). Following the promotion/inhibition of miR­140, 1, 7 and 14 days following transforming growth factor­ß3 (TGF­ß3)­induction, western blotting was utilized to evaluate CXCL12 protein levels. MTT assays and alcian blue staining were applied to assess C3H10T1/2 MSC viability and chondrogenic differentiation, respectively. In the TGF­ß3­induced group, RT­qPCR verified that the mRNA level of Mus musculus (mmu)­miR­140 was significantly elevated when compared with the control group. miR­140 was predicted to recognize and interact with CXCL12­3'UTR and the dual luciferase reporter assay further validated that miR­140 targeted the predicted region of CXCL12. CXCL12 was markedly decreased following miR­140 overexpression and visibly increased following miR­140 inhibition. In addition, the level of CXCL12 expression declined as the duration of induction increased. Following the promotion/inhibition of miR­140, at 1 and 7 days following TGF­ß3­induction, C3H10T1/2 MSCs inhibited or promoted cell viability, respectively, when compared with the control groups. In addition, in pellets achieved by chondrogenic differentiation following the induction of C3H10T1/2 MSCs for 7 days, alcian blue staining revealed no significant difference in characteristic extracellular matrix glycosaminoglycans between the miR­140 up and downregulated groups, and their respective control groups. The present study concludes that miRNA­140 inhibition promoted C3H10T1/2 MSC viability however, not C3H10T1/2 MSC differentiation by targeting and reducing CXCL12 protein levels during the process of TGF­ß3­induced chondrogenic differentiation. In conclusion, the present study provided a potential target for the treatment of cartilage defection.