Regulation of inflammatory response by LINC00346 via miR-25-3p-mediated modulation of the PTEN/PI3K/AKT/NF-κB pathway.

Long intergenic non-coding RNA 346 (LINC00346) has been reported to be involved in the development of atherosclerosis and specific cancers by affecting signaling pathways. However, its function in inflammation has not been thoroughly studied. Therefore, its expression pattern and function were determined in the human macrophage-like cell line THP-1. Lipopolysaccharide (LPS) treatment induced the expression of LINC00346. LPS-induced NF-κB activation and proinflammatory cytokine expression were suppressed or enhanced by the overexpression or knockdown of LINC00346, respectively. Analyses using dual luciferase assay and decoy RNAs that could block RNA-RNA interactions indicated that LINC00346 improves phosphatase and tensin homolog (PTEN) expression by sponging miR-25-3p. Subsequently, PTEN suppresses phosphoinositide-3 kinase (PI3K)-mediated conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3) as well as consequent activation of protein kinase B (AKT) and NF-κB. Interestingly, database analysis revealed that the expression levels of LINC00346 and PTEN were simultaneously decreased in breast cancer tissues. Further analyses conducted using a breast cancer cell line, MDA-MB-231, confirmed the functional relationship among LINC00346, miR-25-3p, and PTEN in LPS-induced activation of NF-κB. These results indicate that miR-25-3p-sponging activity of LINC00346 affects the balance between PTEN and PI3K as well as the downstream activation of AKT/NF-κB pathway in inflammatory conditions.

Extracellular vesicle-encapsulated miR-25-3p promotes epithelial-mesenchymal transition and migration of endometrial epithelial cells by inducing macrophage polarization.

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.

Cold exposure-induced plasma exosomes impair bone mass by inhibiting autophagy.

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.

Dysregulation of miR-25-3p in Diabetic Nephropathy and Its Role in Inflammatory Response.

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.

Early miR-320b and miR-25-3p miRNA levels correlate with multiple sclerosis severity at 10 years: a cohort study.

BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating autoimmune disorder which may cause long-term disability. MicroRNA (miRNA) are stable, non-coding molecules that have been identified in our Comprehensive Longitudinal Investigation of Multiple Sclerosis at the Brigham and Women's Hospital (CLIMB)-cohort, as well as other international cohorts, as potential disease biomarkers in MS. However, few studies have evaluated the association of miRNA expression early in the MS disease course with long-term outcomes. Therefore, we aimed to evaluate the potential role of three candidate serum miRNAs previously correlated with MS disability in patients with MS, miR-320b, miR-25-3p and miRNA 486-5p, as early biomarkers of MS disability at 10-year follow-up. MAIN BODY: We included 144 patients with serum obtained within three years of MS onset. miRNA expression was measured by RNA extraction followed by RT-PCR. Demographic, clinical, brain MRI and other biomarkers were collected. The primary outcome was the association between early miRNA expression and retaining benign MS, defined as EDSS ≤ 2 at 10-year follow-up. Among the 144 patients, 104 were benign and 40 were not benign at 10-year follow-up. 89 (62%) were women, with mean age at onset 37.7 (SD: 9.6) years. Patients who retained benign MS had lower values of miR-25-3p (p = 0.047) and higher miR-320b (p = 0.025) values. Development of SPMS was associated with higher miR-320b (p = 0.002) levels. Brain parenchymal fraction at year 10 was negatively correlated with miR-25-3p (p = 0.0004) and positively correlated with miR-320b (p = 0.006). No association was found between miR-486-5p and any outcome, and 10-year T2-lesion volume was not associated with any miRNA. CONCLUSIONS: Our results show that miR-320b and miR-25-3p expression are early biomarkers associated with MS severity and brain atrophy. This study provides class III evidence of that miR-320b and miR-25-3p are associated with long-term MS disability which may be a potential tool to risk-stratify patients with MS for early treatment decisions.

Mir-25-3p in extracellular vesicles from fibroblast-like synoviocytes alleviates pyroptosis of chondrocytes in knee osteoarthritis.

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-1ß 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.

Circ_PRDM5/miR-25-3p/ANKRD46 axis is associated with cell malignant behaviors in subjects with breast cancer evaluated by ultrasound.

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.

Lnc NR2F1-AS1 Promotes Breast Cancer Metastasis by Targeting the MiR-25-3p/ZEB2 Axis.

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.

Antidepressant-like effects of geniposide in chronic unpredictable mild stress-induced mice by regulating the circ_0008405/miR-25-3p/Gata2 and Oip5os1/miR-25-3p/Gata2 networks.

Evidence exists suggesting the anti-depressive activities of geniposide (GP), a major compound in Gardenia jasminoides Ellis. Accordingly, the present study attempts to explore the anti-depressive mechanism of GP in chronic unpredictable mild stress (CUMS)-induced depression-like behaviors of mice. CUMS-induced mice were given GP daily and subjected to behavioral tests to observe the effect of GP on the depression-like behaviors. It was noted that GP administration reduced depression-like behaviors in CUMS mice. Transcriptome sequencing was conducted in three control and three CUMS mice. Differentially expressed circRNAs, lncRNAs and mRNAs were then screened by bioinformatics analyses. Intersection analysis of the transcriptome sequencing results with the bioinformatics analysis results was followed to identify the candidate targets. We found that Gata2 alleviated depression-like behaviors via the metabolism- and synapse-related pathways. Gata2 was a target of miR-25-3p, which had binding sites to circ_0008405 and Oip5os1. circ_0008405 and Oip5os1 competitively bound to miR-25-3p to release the expression of Gata2. GP administration ameliorated depression-like behaviors in CUMS mice through regulation of the circ_0008405/miR-25-3p/Gata2 and Oip5os1/miR-25-3p/Gata2 crosstalk networks. Taken together, GP may exert a potential antidepressant-like effect on CUMS mice, which is ascribed to regulation of the circ_0008405/miR-25-3p/Gata2 and Oip5os1/miR-25-3p/Gata2 crosstalk networks.

Non-invasive diagnostic potential of salivary miR-25-3p for periodontal disease and osteoporosis among a cohort of elderly patients with type 2 diabetes mellitus.

OBJECTIVE: Osteoporosis (OP) and periodontal disease (PD) are two common health issues that threaten the older population and potentially connected each other in the context of type 2 diabetes mellitus (T2DM). Dysregulated expression of microRNAs (miRNAs) may contribute to the development and progression of both OP and PD among elderly T2DM patients. The present study aimed to evaluate the accuracy of miR-25-3p expression for the detection of OP and PD when compared to a mixed group of patients with T2DM. METHODS: The study recruited 45 T2DM patients with normal bone mineral density (BMD) and healthy periodontium, 40 type 2 diabetic osteoporosis patients coexistent with PD, 50 type 2 diabetic osteoporosis patients with healthy periodontium, and 52 periodontally healthy individuals. miRNA expression measurements in the saliva were determined by real-time PCR. RESULTS: The salivary expression of miR-25-3p was higher in type 2 diabetic osteoporosis patients than patients with T2DM only and healthy individuals (P < 0.05). Among type 2 diabetic osteoporosis patients, those with PD exhibited a higher salivary expression of miR-25-3p than those with healthy periodontium (P < 0.05). Among type 2 diabetic patients with healthy periodontium, a higher salivary expression of miR-25-3p was noted in those with OP than those without (P < 0.05). We also found a higher salivary expression of miR-25-3p in T2DM patients than healthy individuals (P < 0.05). It was revealed that the salivary expression of miR-25-3p was increased as the T scores of BMD of patients were lowered, the PPD and CAL values of patients were enhanced. The salivary expression of miR-25-3p used as a test to predict a diagnosis of PD among type 2 diabetic osteoporosis patients, a diagnosis of OP among type 2 diabetic patients, and a diagnosis of T2DM among healthy individuals produced AUC of 0.859. 0.824, and 0.886, respectively. CONCLUSION: The findings obtained from the study support salivary miR-25-3p confers non-invasive diagnostic potential for PD and OP among a cohort of elderly T2DM patients.

[Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway].

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-1ß(IL-1ß), 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-1ß, 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-1ß, 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-1ß. 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-1ß, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

miR-25-3p ameliorates SAE by targeting the TLR4/NLRP3 axis.

Sepsis-associated encephalopathy (SAE) is a severe complication of sepsis. It has been reported that miR-25-3p is closely related to the development of sepsis. However, the detailed mechanism of miR-25-3p in SAE requires further investigation. Caecum ligation and puncture (CLP) was performed to induce SAE in vivo. LPS stimulation was applied to mimic the in vitro inflammatory model. The expression levels of TLR4 and NLRP3 in the cerebral cortex were evaluated by immunofluorescence. The gene and protein expression levels were determined by qRT-PCR and a western blot analysis. ELISA was used to detect the levels of inflammatory cytokines. The interaction between miR-25-3p and TLR4 was validated by a dual luciferase reporter assay. TLR4 and NLRP3 were highly expressed in the cerebral cortex of SAE mice, while miR-25-3p was expressed at low levels. Activation of the inflammasome, increased release of cytokines and microglial activation were also observed in the SAE mouse model. The overexpression of miR-25-3p inhibited the expression of LPS-induced cytokines and microglial activation. Furthermore, miR-25-3p inhibited TLR4 expression by directly targeting TLR4. The anti-inflammatory effect of miR-25-3p in LPS-induced CHME5 was reversed by TLR4 overexpression. miR-25-3p overexpression attenuated the activation of microglia in SAE by inhibiting the NLRP3/IL-1ß/IL-18 axis by directly targeting TLR4, suggesting that miR-25-3p may be a potential target for SAE diagnosis and treatment.

Diagnostic value of serum miR-25-3p in hypertensive disorders in pregnancy.

Hypertensive disorders in pregnancy (HDIP) represent one of the leading causes of maternal and perinatal mortality. microRNA (miR)-25-3p plays roles in HDIP diagnosis. We explored miR-25-3p clinical roles in HDIP. HDIP patients [gestation hypertension (GH), mild preeclampsia (mPE), and severe preeclampsia (sPEz)], and normal pregnant women serving as the control were enrolled. Serum miR-25-3p expression patterns were detected by RT-qPCR. The diagnostic efficacy of miR-25-3p on HDIP was analyzed with a ROC curve. Patients were assigned to the high/low miR-25-3p expression groups according to the median value of miR-25-3p expression. All patients were followed up until delivery, and gestational weeks and pregnancy outcomes were recorded at delivery. The effects of miR-25-3p expression on pregnancy outcomes of GH, mPE, and sPEz patients were analyzed by Kaplan-Meier. miR-25-3p expression in GH, mPE, and sPEz patients was up-regulated. In sPEz patients, systolic and diastolic blood pressure, 24-h urine protein, AST, ALT, GGT, and SCr were increased, and PLT was decreased in the high expression group. High miR-25-3p expression was associated with an increased risk of adverse pregnancy outcomes in PE patients. Collectively, high miR-25-3p expression could aid HDIP diagnosis, and associated with an increased risk of adverse pregnancy outcomes in PE patients.

The Relationship and Expression of miR-451a, miR-25-3p and PTEN in Early Peritoneal Endometriotic Lesions and Their Modulation In Vitro.

BACKGROUND: miR-451a can function as a tumor suppresser and has been shown to be elevated in both endometriotic lesion tissue and serum from women with endometriosis. To further explore the role of miR-451a in the pathophysiology of endometriosis, specifically, further evaluating its association with the tumor suppressor, phosphatase and tensin homolog (PTEN), we examined their expression in individual endometriotic lesion tissue to gain insight into their relationship and further explore if miR-451a regulates PTEN expression. METHODS: A total of 55 red, peritoneal endometriotic lesions and matched eutopic endometrial specimens were obtained from 46 patients with endometriosis. miR-451a, miR-25-3p and PTEN mRNA levels were assessed by qRT-PCR and reported for each matched eutopic and ectopic sample. To evaluate miR-451a and miR-25-3p expression of miR-25-3p and PTEN, respectively, 12Z cells (endometriotic epithelial cell line) were transfected and miR-25-3p expression was assessed by qRT-PCR, while PTEN protein expression was assessed by Western blotting. RESULTS: PTEN and miR-25-3p expression exhibited an inverse relationship, as did miR-25-3p and miR-451a in individual lesions. Over-expression of miR-451a in 12Z cells resulted in down-regulation of miR-25-3p, while up-regulation of miR-25-3p resulted in down-regulation of PTEN protein expression. CONCLUSIONS: By assessing individual endometriotic lesion expression, we discovered an inverse relationship between miR-451a, miR-25-3p and PTEN, while in vitro cell transfection studies suggest that miR-451a may regulate PTEN expression via modulating miR-25-3p.

Cellular and Molecular Profiling of Tumor Microenvironment and Early-Stage Lung Cancer.

Lung cancers are broadly divided into two categories: non-small-cell lung carcinoma (NSCLC), which accounts for 80-85% of all cancer cases, and small-cell lung carcinoma (SCLC), which covers the remaining 10-15%. Recent advances in cancer biology and genomics research have allowed an in-depth characterization of lung cancers that have revealed new therapy targets (EGFR, ALK, ROS, and KRAS mutations) and have the potential of revealing even more biomarkers for diagnostic, prognostic, and targeted therapies. A new source of biomarkers is represented by non-coding RNAs, especially microRNAs (miRNAs). MiRNAs are short non-coding RNA sequences that have essential regulatory roles in multiple cancers. Therefore, we aim to investigate the tumor microenvironment (TME) and miRNA tumor profile in a subset of 51 early-stage lung cancer samples (T1 and T2) to better understand early tumor and TME organization and molecular dysregulation. We analyzed the immunohistochemistry expression of CD4 and CD8 as markers of the main TME immune populations, E-cadherin to evaluate early-stage epithelial-to-mesenchymal transition (EMT), and p53, the main altered tumor suppressor gene in lung cancer. Starting from these 4 markers, we identified and validated 4 miRNAs that target TP53 and regulate EMT that can be further investigated as potential early-stage lung cancer biomarkers.

CircRNA TGFBR2/MiR-25-3p/TWIST1 axis regulates osteoblast differentiation of human aortic valve interstitial cells.

INTRODUCTION: Calcified aortic valve disease (CAVD) is characterized by valve thickening and calcification. Osteoblast differentiation is one of the key steps of valve calcification. CircRNAs is involved in osteogenic differentiation of multiple mesenchymal cells. However, the function of circRNA TGFBR2 (TGFBR2) in CAVD remained unclear. We explored the effect and mechanism of TGFBR2 in modulating CAVD. MATERIALS AND METHODS: Human aortic valve interstitial cells (VICs) were subjected to osteogenic induction, and transfected with TGFBR2, miR-25-3p mimic and siTWIST1. The relationship between miR-25-3p and GFBR2 was predicted by starBase and confirmed by luciferase reporter and Person's correlation test. The relationship between miR-25-3p and TWIST1 was predicted by TargetScan and confirmed by luciferase reporter assay. The expressions of TGFBR2, miR-25-3p, TWIST1, osteoblast markers (RUNX2 and OPN) were detected by Western blot or/and qRT-PCR. Alkaline phosphatase (ALP) activity and calcium nodule was determined by colorimetric method and Alizarin Red S staining. RESULTS: The expression of TGFBR2 was down-regulated and that of miR-25-3p was up-regulated in calcific valves and osteogenic VICs. TGFBR2 was inversely correlated with miR-25-3p expression in calcific valves. TGFBR2 sponged miR-25-3p to regulate TWIST1 expression in osteogenic VICs. During osteogenic differentiation, ALP activity, calcium nodule, the levels of osteoblast markers were increased in VICs. MiR-25-3p overexpression or TWIST1 knockdown reversed the inhibitory effect of TGFBR2 overexpression on ALP activity, calcium nodule, the expressions of RUNX2 and OPN in osteogenic VICs. CONCLUSION: The findings indicated that TGFBR2/miR-25-3p/TWIST1 axis regulates osteoblast differentiation in VICs, supporting the fact that TGFBR2 is a miRNA sponge in CAVD.

Platelet microparticles-derived miR-25-3p promotes the hepatocyte proliferation and cell autophagy via reducing B-cell translocation gene 2.

Platelets are critical regulators of liver regeneration, but the mechanisms are still not fully understood. Platelets have been shown to contain a wide variety of microRNAs (miRNAs) and play an important role in many diseases. However, the mechanism that how the platelet microparticles (PMPs)-derived miRNA regulate the hepatocyte proliferation is not very clear. In this study, we have successfully isolated and identified PMPs. We also found that PMPs, which could be well integrated into the HHL-5 cells, could upregulate the level of miR-25-3p in HHL-5 cells. Meanwhile, we found that PMPs-derived miR-25-3p promoted HHL-5 cells proliferation by accelerating cells into the S phase, and enhanced the autophagy by increasing the LC3II expression and reducing the P62 expression. Then, we proved that the miR-25-3p could target the B-cell translocation gene 2 (BTG2) and downregulate the expression levels of the BTG2 gene in HHL-5 cells. In addition, the overexpression of BTG2 significantly inhibited the proliferation and autophagy abilities of HHL-5 cells, while cotransfected miR-25-3p mimics or PMPs could partially rescue HHL-5 cells proliferation and autophagy. Furthermore, we proved that PMPs accelerated hepatocyte proliferation by regulating autophagy pathways. Therefore, PMPs-derived miR-25-3p promoted HHL-5 cell proliferation and autophagy by targeting BTG2, which may be a new therapeutic method for liver regeneration.

M2 macrophage-derived exosomal miR-25-3p improves high glucose-induced podocytes injury through activation autophagy via inhibiting DUSP1 expression.

Diabetic nephropathy (DN) is the primary reason of chronic kidney disease. The aim of our study is to explore the role and action mechanism of M2 macrophage-derived exosomes in high glucose (HG)-induced podocytes injury. Here, 30 mmol/L of HG was used to induce podocytes injury. Annexin V-FITC/PI double staining was performed to measure podocytes apoptosis, and western blot was carried out to ensure proteins expression. The shape of exosomes was identified using TEM. Besides, the expression of miR-25-3p was determined by qRT-PCR, FAM-labeled miR-25-5p combined with DiI-labeled exosomes were utilized to explore the uptake of podocytes to exosomes. Relationship between miR-25-3p and DUSP family members was ensued by luciferase activity assay. In the beginning, we found that M2 macrophage ameliorated HG-induced podocytes apoptosis and epithelial-mesenchymal transition through secreting exosomes. Subsequently, highly expressed miR-25-3p was found in M2 macrophage-derived exosomes that effectively improved HG-induced podocytes injury. Furthermore, inhibition of miR-25-3p in M2 macrophage inefficiently repressed HG-induced podocytes injury, thus we proposed that M2 macrophage attenuated podocytes injury through secreting exosomal miR-25-3p. Then, we used an autophagy inhibitor to stimulate podocytes, and demonstrated that M2 macrophage-derived exosomal miR-25-3p improved HG-induced podocytes injury through activating autophagy. Finally, DUSP1 was proved to be a downstream target and mediated the inhibition of exosomal miR-25-3p to HG-induced podocytes injury. Our results indicated that M2 macrophage could improve HG-induced podocytes injury via secreting exosomal miR-25-3p to activate autophagy of the cells through suppressing DUSP1 expression. We proved a newly potential therapy strategy for DN treatment.

LncRNA JPX promotes cervical cancer progression by modulating miR-25-3p/SOX4 axis.

BACKGROUND: The long noncoding RNA (lncRNA) JPX is a molecular switch for X-chromosome inactivation. Accumulating studies have shown that the aberrant expression and function of lncRNAs are involved in the occurrence and development of tumors. However, the functional importance and mechanism of the action of lncRNA JPX in cervical cancer (CC) remain unknown. METHOD: In this study, qRT-PCR and western blotting were used to evaluate the mRNA or protein expression of JPX, miR-25-3p and SOX4 in CC tissues and cell lines. StarBase v2.0 database, luciferase reporter assay and RNA immunoprecipitation assay were used to explore the relationship between JPX and miR-25-3p. EdU assay, CCK-8 assay and transwell assay were utilized to evaluate the proliferation, migration and invasion of CC cells. The tumor xenograft assay in nude mice was performed to demonstrate the role of the JPX/miR-25-3p/SOX4 axis in CC. RESULTS: We found that JPX was markedly upregulated, whereas miR-25-3p was markedly downregulated in CC tissues and cell lines, and the expression of JPX was negatively correlated with miR-25-3p in CC tissues. Moreover, overexpression of JPX increased proliferation, migration and invasion of HeLa cells, whereas knockdown of JPX decreased proliferation, migration and invasion of HeLa cells. In contrast to JPX, overexpression of miR-25-3p decreased proliferation, migration and invasion of HeLa cells. In addition, knockdown of JPX was found to inhibit HeLa cell viability and tumor development via up-regulating the expression of miR-25-3p and inhibiting the expression of SOX4. CONCLUSIONS: Our study demonstrates that JPX promotes cervical cancer progression through modulating the miR-25-3p/SOX4 axis, and may serve as a potential target for CC therapy.

miR-25-3p promotes proliferation and inhibits autophagy of renal cells in polycystic kidney mice by regulating ATG14-Beclin 1.

MicroRNAs are involved in the regulation of the autophagy and proliferation in several diseases. This study aims to verify the role of miR-25-3p in the proliferation and autophagy of renal cells in polycystic kidney disease (PKD). We found that kidney to body weight and blood urea content were increased in PKD mice. Cystic dilations were increased in kidney tissue from PKD mice, and autophagy-related protein ULK1 and the ratio of LC3-II/LC3-I were decreased, indicating autophagy was inhibited in PKD mice. In addition, miR-25-3p was upregulated in PKD mice, and inhibition of miR-25-3p decreased cystic dilations in kidney tissues, increased ULK1 expression and the ratio of LC3-II/LC3-I, indicating inhibition of miR-25-3p enhanced the autophagy in PKD. Besides, inhibition of miR-25-3p suppressed the proliferation of renal cells and downregulated E2F-1 and PCNA expressions. Importantly, miR-25-3p targetedly suppressed ATG14 expression in PKD cells. Finally, silencing ATG14 abolished the inhibition effect of miR-25-3p inhibitor on renal cell proliferation, and reversed the inhibition effect of miR-25-3p inhibitor on E2F-1 and PCNA expressions in in vitro and in vivo experiments, which suggested that ATG14 was involved in the regulation of miR-25-3p-mediated kidney cell proliferation. Therefore, inhibition of miR-25-3p promoted cell autophagy and suppressed cell proliferation in PKD mice through regulating ATG14.