Recently, environmental temperature has been shown to regulate bone homeostasis. However, the mechanisms by which cold exposure affects bone mass remain unclear. In our present study, we observed that exposure to cold temperature (CT) decreased bone mass and quality in mice. Furthermore, a transplant of exosomes derived from the plasma of mice exposed to cold temperature (CT-EXO) can also impair the osteogenic differentiation of BMSCs and decrease bone mass by inhibiting autophagic activity. Rapamycin, a potent inducer of autophagy, can reverse cold exposure or CT-EXO-induced bone loss. Microarray sequencing revealed that cold exposure increases the miR-25-3p level in CT-EXO. Mechanistic studies showed that miR-25-3p can inhibit the osteogenic differentiation and autophagic activity of BMSCs. It is shown that inhibition of exosomes release or downregulation of miR-25-3p level can suppress CT-induced bone loss. This study identifies that CT-EXO mediates CT-induced osteoporotic effects through miR-25-3p by inhibiting autophagy via targeting SATB2, presenting a novel mechanism underlying the effect of cold temperature on bone mass.

To investigate the expression level of miR-25-3p in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN), and its effect on proliferation, apoptosis and inflammatory response of mesangial cells cultured with high glucose. Blood samples of all clinical subjects were collected for RT-qPCR analysis to detect serum miR-25-3p levels. Human mesangial cells (HMCs) cultured with high glucose were used to construct DN model in vitro. MTT assay, flow cytometry and ELISA were used to evaluate the effects of miR-25-3p on the proliferation, apoptosis, and inflammatory response of DN cell models. Serum miR-25-3p was decreased in both T2DM group and DN group, but more in DN group. Serum miR-25-3p was positively correlated with eGFR and negatively correlated with UAER. The expression of miR-25-3p was reduced in HMCs induced by high glucose. Transfection of miR-25-3p mimic could significantly up-regulate the miR-25-3p level in HMCs. Besides, high glucose culture resulted in abnormal proliferation of HMCs, reduced apoptotic cells, and increased inflammation. The addition of miR-25-3p mimic significantly inhibited cell proliferation and promoted cell apoptosis and reduced the production of inflammatory factors. The abnormal reduction of serum miR-25-3p in DN indicates that it may be a potential biomarker for clinical diagnosis of DN. In in vitro experiments, miR-25-3p was involved in the progression of DN by regulating cell proliferation, apoptosis, and inflammatory response.

Nerve injury often leads to severe dysfunction because of the lack of axon regeneration in adult mammal. Intriguingly a series of extracellular vesicles (EVs) have the obvious ability to accelerate the nerve repair. However, the detailed molecular mechanisms to describe that EVs switch neuron from a transmitter to a regenerative state have not been elucidated. This study elucidated the microRNA (miRNA) expression profiles of two types of EVs that promote nerve regeneration. The functions of these miRNAs were screened in vitro. Among the 12 overlapping miRNAs, miR-25-3p was selected for further analysis as it markedly promoted axon regeneration both in vivo and in vitro. Furthermore, knockdown experiments confirmed that PTEN and Klf4, which are the major inhibitors of axon regeneration, were the direct targets of miR-25-3p in dorsal root ganglion (DRG) neurons. The utilization of luciferase reporter assays and functional tests provided evidence that miR-25-3p enhances axon regeneration by targeting Tgif1. Additionally, miR-25-3p upregulated the phosphorylation of Erk. Furthermore, Rapamycin modulated the expression of miR-25-3p in DRG neurons. Finally, the pro-axon regeneration effects of EVs were confirmed by overexpressing miR-25-3p and Tgif1 knockdown in the optic nerve crush model. Thus, the enrichment of miR-25-3p in EVs suggests that it regulates axon regeneration, proving a potential cell-free treatment strategy for nerve injury.

The pathogenesis of adenomyosis is closely related to the epithelial-mesenchymal transition and macrophages. MicroRNAs have been extensively investigated in relation to the epithelial-mesenchymal transition in a range of malignancies. However, there is a paucity of research on extracellular vesicles derived from the eutopic endometrium of adenomyosis and their encapsulated microRNAs. In this study, we investigated the role of microRNA-25-3p derived from extracellular vesicles in inducing macrophage polarization and promoting the epithelial-mesenchymal transition in endometrial epithelial cells of patients with adenomyosis and controls. We obtained eutopic endometrial samples and isolated extracellular vesicles from the culture supernatant of primary endometrial cells. Real-time quantitative PCR analysis demonstrated that microRNA-25-3p was highly expressed in extracellular vesicles, as well as in macrophages stimulated by extracellular vesicles from eutopic endometrium of adenomyosis; and macrophages transfected with microRNA-25-3p exhibited elevated levels of M2 markers, while displaying reduced levels of M1 markers. After co-culture with the above polarized macrophages, endometrial epithelial cells expressed higher levels of N-cadherin and Vimentin, and lower protein levels of E-cadherin and Cytokeratin 7. It was revealed that microRNA-25-3p encapsulated in extracellular vesicles from eutopic endometrial cells could induce macrophage polarization toward M2, and the polarized macrophages promote epithelial-mesenchymal transition in epithelial cells. However, in vitro experiments revealed no significant disparity in the migratory capacity of endometrial epithelial cells between the adenomyosis group and the control group. Furthermore, it was observed that microRNA-25-3p-stimulated polarized macrophages also facilitated the epithelial-mesenchymal transition and migration of endometrial epithelial cells within the control group. Thus, the significance of microRNA-25-3p-induced polarized macrophages in promoting the development of adenomyosis is unclear, and macrophage infiltration alone may be adequate for this process. We emphasize the specificity of the local eutopic endometrial microenvironment and postulate its potential significance in the pathogenesis of adenomyosis.

暂无摘要。

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

Knee osteoarthritis (KOA) is defined as a joint disease that occurs mostly among elderly people. Fibroblast-like synoviocytes-derived extracellular vesicles (FLS-EVs) have impacts on the treatment of OA. This study elucidated the mechanism of miR-25-3p in pyroptosis of chondrocytes in KOA. FLSs and EVs were extracted from neonatal mice; destabilization of the medial meniscus (DMM) was used to simulate KOA in mice, followed by the evaluation of cartilage damage and the contents of MMP-3 and MMP-13 in KOA mice. Lipopolysaccharide (LPS) was used to induce inflammation damage in mouse chondrocytes ATDC5, and the cell viability and the expressions of NLRP3, Cleaved-Caspase-1, GSDMD-N, IL-18, and IL-1β were examined. We found that FLS-EV treatment mitigated the knee-joint damage and symptoms of KOA mice, decreased MMP-3 and MMP-13, and inhibited pyroptosis of chondrocytes in DMM mice and LPS-induced ATD5 cells. Then, Cy3-labeled miR-25-3p in mice chondrocytes was observed and the expressions and the binding relation of miR-25-3p and cytoplasmic polyadenylation element-binding protein 1 (CPEB1) were verified. It showed that FLS-EVs carried miR-25-3p into chondrocytes, and upregulated miR-25-3p expression while inhibited CPEB1 transcription, resulting in mitigation of pyroptosis of chondrocytes, and CPEB1 overexpression reversed the inhibition of FLS-EVs on pyroptosis of chondrocytes in KOA.

Background: Long noncoding RNAs (lncRNAs) substantially affect tumor metastasis and are aberrantly expressed in various cancers. However, its role in breast cancer (BC) remains unclear. Methods: A microarray assay of differentially expressed lncRNAs in epithelial-mesenchymal transition (EMT) and non-EMT cells was performed. The prognostic value of lnc NR2F1-AS1 expression in patients with BC was analyzed using The Cancer Genome Atlas database. Lnc NR2F1-AS1 expression levels in different BC cell lines were assessed using quantitative real-time PCR. The role of lnc NR2F1-AS1 in BC cell metastasis was investigated in vitro and in vivo. Dual luciferase reporter assay and RNA immunoprecipitation were performed to investigate the relationship between lnc NR2F1-AS1, miR-25-3p, and ZEB2. Results: High levels of lnc NR2F1-AS1 were observed in BC cells undergoing EMT and were closely correlated with adverse prognosis in patients with BC. Lnc NR2F1-AS1 knockdown significantly inhibited BC cell migration, invasiveness in vitro, and metastasis in vivo. Mechanistically, lnc NR2F1-AS1 competitively binds to miR-25-3p to impede ZEB2 degradation, a positive EMT transcription factor in BC. Conclusions: Our study revealed a novel lnc NR2F1-AS1/miR-25-3p/ZEB2 axis in BC metastasis and that lnc NR2F1-AS1 may serve as a potential therapeutic target for BC metastasis.

Circular RNAs (circRNAs) are key RNA molecules in cancer biology. CircRNA PR/SET domain 5 (circ_PRDM5, hsa_circ_0005654) was downregulated in breast cancer (BC) tissues. This study is designed to investigate the functional mechanism of circ_PRDM5 in BC. Ultrasound examinations were performed to evaluate BC patients and normal individuals. Circ_PRDM5, miR-25-3p, and Ankyrin repeat domain 46 (ANKRD46) level detection was carried out by reverse transcription-quantitative polymerase chain reaction. 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used for cell viability examination. Cell proliferation was evaluated by ethynyl-2\'-deoxyuridine assay and colony formation assay. The protein levels were examined using western blot. Cell migration and invasion abilities were assessed via transwell assay. Target interaction was analyzed via dual-luciferase reporter assay. The role of circ_PRDM5 in vivo was explored via xenograft tumor assay. Circ_PRDM5 expression was downregulated in BC tissues and cells. Overexpression of circ_PRDM5 suppressed proliferation and motility but enhanced apoptosis of BC cells. Circ_PRDM5 served as a sponge of miR-25-3p. Circ_PRDM5 impeded BC cell malignant development via sponging miR-25-3p. Circ_PRDM5 induced ANKRD46 upregulation by targeting miR-25-3p. Inhibition of miR-25-3p retarded BC progression by increasing the ANKRD46 level. Circ_PRDM5 repressed BC tumorigenesis in vivo through mediating the miR-25-3p/ANKRD46 axis. This study evidenced that circ_PRDM5 inhibited cell progression and tumor growth in BC via interacting with mir-25-3p/ANKRD46 network.

Ischemic stroke causes lethal damage to the brain. Identifying key regulators of OGD/R-induced cerebral injury is important for developing novel therapies for ischemic stroke. HMC3 and SH-SY5Y cells were treated with OGD/R as an in vitro ischemic stroke model. Cell viability and apoptosis were determined via CCK-8 assay and flow cytometry. Inflammatory cytokines were examined by ELISA. Luciferase activity was measured for evaluating the interaction of XIST, miR-25-3p, and TRAF3. Bcl-2, Bax, Bad, cleaved-caspase 3, total caspase 3, and TRAF3 were detected via western blotting. HMC3 and SH-SY5Y cells showed increased XIST expression and decreased miR-25-3p expression following OGD/R. Importantly, silencing of XIST and overexpression of miR-25-3p reduced apoptosis and inflammatory response following OGD/R. Furthermore, XIST worked as a miR-25-3p sponge, and miR-25-3p targeted TRAF3 to suppress its expression. Moreover, the knockdown of TRAF3 ameliorated OGD/R-induced injury. Loss of XIST-mediated protective effects was reversed by overexpression of TRAF3. LncRNA XIST exacerbates OGD/R-induced cerebral damage via sponging miR-25-3p and enhancing TRAF3 expression.