LncRNA AC005165.1 Alleviates IL-1ß-Induced Osteoarthritis via miR-199a-3p/TXNIP Axis.

Osteoarthritis (OA) is a chronic musculoskeletal disease and often causes impaired joint mobility and disability. Long noncoding RNAs (lncRNAs) play pivotal roles in OA development. This study was done to explore the role and mechanism of the lncRNA AC005165.1 in the cell model of interleukin-1ß (IL)-1ß-treated chondrocytes. This study recruited 20 surgically treated OA patients and 12 age- and gender-matched controls. Real-time reverse transcription quantitative polymerase chain reaction was used to examine the expression levels of AC005165.1, miR-199a-3p, and thioredoxin-interacting protein (TXNIP) in articular cartilage of patients and IL-1ß-treated human chondrocytes. Cell viability and apoptosis were evaluated by cell counting kit-8 and flow cytometry assays, respectively. The protein levels of inflammatory cytokines were assessed by western blotting. Enzyme-linked immunosorbent assay was conducted to detect the concentrations of the inflammatory cytokines in chondrocytes. Luciferase reporter assay and Pearson's correlation analysis were used for analyzing the interaction and the correlation among AC005165.1, miR-199a-3p, and TXNIP. AC005165.1 expression was downregulated in cartilage of OA patients and chondrocytes treated with IL-1ß, compared to that in the control groups. AC005165.1 knockdown increased apoptosis and aggravated inflammatory response in IL-1ß-treated chondrocytes. AC005165.1 interacted with miR-199a-3p, and TXNIP was targeted by miR-199a-3p. In rescue assay, miR-199a-3p knockdown and TXNIP overexpression significantly reduced apoptosis and mitigated inflammatory response in IL-1ß-treated chondrocytes with AC005165.1 knockdown. AC005165.1 knockdown promoted apoptosis and inflammatory response in IL-1ß-treated chondrocytes via the miR-199a-3p/TXNIP axis.

Inhibition of miR-199a-3p in a murine hypertrophic cardiomyopathy (HCM) model attenuates fibrotic remodeling.

Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ∼1: 200-500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

MiR-199a-3p Regulates the PTPRF/ß-Catenin Axis in Hair Follicle Development: Insights into the Pathogenic Mechanism of Alopecia Areata.

Alopecia areata is an autoimmune disease characterized by the immune system attacking self hair follicles, mainly in the scalp. There is no complete cure, and the pathogenesis is still not fully understood. Here, sequencing of skin tissues collected from 1-month-old coarse- and fine-wool lambs identified miR-199a-3p as the only small RNA significantly overexpressed in the fine-wool group, suggesting a role in hair follicle development. MiR-199a-3p expression was concentrated in the dermal papillae cells of sheep hair follicles, along with enhanced ß-catenin expression and the inhibition of PTPRF protein expression. We also successfully constructed a mouse model of alopecia areata by intracutaneous injection with an miR-199a-3p antagomir. Injection of the miR-199a-3p agomir resulted in hair growth and earlier anagen entry. Conversely, local injection with the miR-199a-3p antagomir resulted in suppressed hair growth at the injection site, upregulation of immune system-related genes, and downregulation of hair follicle development-related genes. In vivo and in vitro analyses demonstrated that miR-199a-3p regulates hair follicle development through the PTPRF/ß-catenin axis. In conclusion, a mouse model of alopecia areata was successfully established by downregulation of a small RNA, suggesting the potential value of miR-199a-3p in the study of alopecia diseases. The regulatory role of miR-199a-3p in the PTPRF/ß-catenin axis was confirmed, further demonstrating the link between alopecia areata and the Wnt-signaling pathway.

miR-199a-3p suppresses neuroinflammation by directly targeting MyD88 in a mouse model of bone cancer pain.

AIMS: Pain is a profoundly debilitating symptom in cancer patients, leading to disability, immobility, and a marked decline in their quality of life. This study aimed to investigate the potential roles of miR-199a-3p in a murine model of bone cancer pain induced by tumor cell implantation in the medullary cavity of the femur. MATERIALS AND METHODS: We assessed pain-related behaviors, including the paw withdrawal mechanical threshold (PWMT) and the number of spontaneous flinches (NSF). To investigate miRNA expression and its targets in astrocytes, we employed a combination of RNA-seq analysis, qRT-PCR, Western blotting, EdU, TUNEL, ChIP, ELISA, and luciferase reporter assays in mice (C3H/HeJ) with bone cancer pain and control groups. KEY FINDINGS: On days 10, 14, 21, and 28 post-surgery, we observed significant differences in PWTL, PWMT, and NSF when compared to the sham group (P < 0.001). qRT-PCR assays and miRNA sequencing results confirmed reduced miR-199a-3p expression in astrocytes of mice with bone cancer pain. Gain- and loss-of-function experiments demonstrated that miR-199a-3p suppressed astrocyte activation and the expression of inflammatory cytokines. In vitro investigations revealed that miR-199a-3p mimics reduced the levels of inflammatory factors in astrocytes and MyD88/NF-κB proteins. Furthermore, treatment with a miR-199a-3p agonist resulted in reduced expression of MyD88, TAK1, p-p65, and inflammatory mediators, along with decreased astrocyte activation in the spinal cord. SIGNIFICANCE: Collectively, these findings demonstrate that upregulation of miR-199a-3p may offer a therapeutic avenue for mitigating bone cancer pain in mice by suppressing neuroinflammation and inhibiting the MyD88/NF-κB signaling pathway.

Ultrasound-triggered release of miR-199a-3p from liposome nanobubbles for enhanced hepatocellular carcinoma treatment.

This study was aimed to develop an efficient tumour-targeted liposome nanobubbles (LNBs) system using ultrasound-targeted nanobubble destruction for enhanced release and transfection of miRNA-199a-3p in hepatocellular carcinoma (HCC) therapy. The prepared LNBs comprised a polyethylene glycol-modified liposome shell and a perfluoropentane (PFP) core. MiRNA-199a-3p was attached to the nanocomposite surface via electrostatic adsorption, while RGD peptide functionalized the LNBs surface for enhanced HCC cell targeting, namely PFP@miR-RGD-LNBs. The LNBs were spherical with a narrow size distribution. The gene-loaded LNBs effectively condensed miR-199a-3p and protected it from enzymatic degradation. Low-intensity focused ultrasound (LIFU) promoted a fast release of miR-199a-3p from the prepared LNBs, thereby enhancing therapeutic effects. The combined application of PFP@miR-RGD-LNBs and LIFU exhibited a more potent inhibitory effect on HepG2 cells than the other groups, potentially due to LIFU promoting rapid and efficient gene release at the target site and increasing cell membrane permeability. Quantitative reverse transcription-polymerase chain reaction analysis revealed significantly increased mRNA expression levels of key apoptosis markers (Bad, Bax, Caspase-9 and Caspase-3) in the PFP@miR-RGD-LNBs + LIFU group compared to other groups. These findings suggest that the prepared LNBs are highly likely to be promising candidates for further exploration of HCC gene delivery and therapy.

M2 Macrophage-Derived Exosomes Regulate miR-199a-3p Promoter Methylation Through the LINC00470-Mediated myc/DNMT3a Axis to Promote Breast Cancer Development.

Breast cancer (BC) is the most common invasive cancer in women. M2 macrophage exosomes promote cancer development and play multiple roles in the tumor microenvironment, but the mechanism of action by which M2 macrophage exosomes promote BC remains unclear. Therefore, the purpose of this study was to investigate the mechanism by which M2 macrophage-derived exosomes promote the development of breast cancer. We collected BC tissues and determined the expression of LINC00470, followed by the establishment of M2 macrophages in culture and the isolation and identification of M2 macrophage exosomes. Next, we investigated the effects of M2 macrophage exosomes on BC cell proliferation, invasion, miR-199a-3p promoter methylation, and the expression of LINC00470, myc, DNMT3A, and miR-199a-3p. Finally, LINC00470 expression was inhibited in M2 macrophage exosomes, while miR-199a-3p expression was inhibited in BC cells, and changes in BC cell proliferation, invasion, miR-199a-3p promoter methylation, and the expression of LINC00470, myc, DNMT3A, and miR-199a-3p were analyzed. We demonstrated that LINC00470 was highly expressed in BC tissues, M2-type macrophages were successfully induced in vitro, and Dil-labeled M2 macrophage exosomes could successfully enter MDA-MB-231 and MCF-7 cells. Coculture of M2 macrophage exosomes with MDA-MB-231 and MCF-7 cells significantly enhanced the proliferation and invasion of MDA-MB-231 and MCF-7 cells, upregulated the expression of LINC00470, myc, and DNMT3A and downregulated the expression of miR-199a-3p. Moreover, the inhibition of LINC00470 expression in M2 macrophage exosomes significantly downregulated the expression of LINC00470, myc, and DNMT3A in MDA-MB-231 and MCF-7 cells, upregulated the expression of miR-199a-3p, and hypomethylated the promoter of the miR-199a-3p locus. Moreover, inhibition of LINC00470 expression in M2 macrophage-derived exosomes significantly attenuated the proliferation and invasive ability of MDA-MB-231 and MCF-7 cells, while miR-199a-3p inhibitor transfection reversed this effect. Collectively, these findings indicated that M2-type macrophage-derived exosomes promote BC proliferation and migration by regulating miR-199a-3p promoter methylation through the LINC00470-mediated myc/DNMT3a axis.

Engineering exosomes derived from subcutaneous fat MSCs specially promote cartilage repair as miR-199a-3p delivery vehicles in Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease involving cartilage. Exosomes derived from Mesenchymal stem cells (MSCs) therapy improves articular cartilage repair, but subcutaneous fat (SC) stromal cells derived exosomes (MSCsSC-Exos), especially engineering MSCsSC-Exos for drug delivery have been rarely reported in OA therapy. This objective of this study was to clarify the underlying mechanism of MSCsSC-Exos on cartilage repair and therapy of engineering MSCsSC-Exos for drug delivery in OA. MSCsSC-Exos could ameliorate the pathological severity degree of cartilage via miR-199a-3p, a novel molecular highly enriched in MSCsSC-Exos, which could mediate the mTOR-autophagy pathway in OA rat model. Intra-articular injection of antagomiR-199a-3p dramatically attenuated the protective effect of MSCsSC-Exos-mediated on articular cartilage in vivo. Furthermore, to achieve the superior therapeutic effects of MSCsSC-Exos on injured cartilage, engineering exosomes derived from MSCsSC as the chondrocyte-targeting miR-199a-3p delivery vehicles were investigated in vitro and in vivo. The chondrocyte-binding peptide (CAP) binding MSCsSC-Exos could particularly deliver miR-199a-3p into the chondrocytes in vitro and into deep articular tissues in vivo, then exert the excellent protective effect on injured cartilage in DMM-induced OA mice. As it is feasible to obtain human subcutaneous fat from healthy donors by liposuction operation in clinic, meanwhile engineering MSCsSC-Exos to realize targeted delivery of miR-199a-3p into chondrocytes exerted excellent therapeutic effects in OA animal model in vivo. Through combining MSCsSC-Exos therapy and miRNA therapy via an engineering approach, we develop an efficient MSCsSC-Exos-based strategy for OA therapy and promote the application of targeted-MSCsSC-Exos for drug delivery in the future.

MicroRNA-199a-3p suppresses the invasion and metastasis of nasopharyngeal carcinoma through SCD1/PTEN/AKT signaling pathway.

MicroRNAs (miRs) are 18-25 nucleotides non-coding RNAs, which contribute to tumorigenesis. Previous studies have demonstrated that miR-199a-3p is dysregulated in human nasopharyngeal carcinoma (NPC), but its role in NPC progression still largely unknown. The current study aimed to determine the potential role of miR-199a-3p in NPC progression and the underlying mechanisms. In this study, miR-199a-3p was found to be prominently down-regulated in NPC tissues and cells. The cellular assay showed that transfection of miR-199a-3p markedly repressed the migration, invasion and induced epithelial-mesenchymal transition (EMT) in both 5-8F and CNE-2 cell lines. By dual-luciferase reporter, western blotting and gas chromatography assays, we found that SCD1 is not only highly expressed in NPC tissues and negatively associated with the prognosis of NPC patients but also can be apparently downregulated by miR-199a-3p in NPC cells, suggesting that SCD1 is a direct target gene of miR-199a-3p. Moreover, inhibition of miR-199a-3p expression activated PI3K/Akt signaling and up-regulated the expression of MMP-2. With tumor xenograft models in nude mice, we also showed that miR-199a-3p repressed tumor growth in vivo. Our study demonstrated that miR-199a-3p inhibited migration and invasion of NPC cells through downregulating SCD1 expression, thus providing a potential target for the treatment of NPC.

[Impacts of LncRNA NORAD on the Proliferation, Apoptosis, and Chemosensitivity of Non-small Cell Lung Cancer Cells by Regulating ZNF217 through MiR-199a-3p].

BACKGROUND: The treatment and diagnosis of non-small cell lung cancer (NSCLC) is still a difficult problem in the medical community, and exploring the molecular mechanism of the occurrence and development of NSCLC is a hot topic of the current research. Long non-coding RNA (lncRNA) NORAD is highly expressed in a variety of cancer cells. It may be a molecular target that promotes NSCLC. The aim of this study was to investigate the impacts of lncRNA NORAD on the proliferation, apoptosis, and chemosensitivity of NSCLC by regulating zinc finger protein 217 (ZNF217) through miR-199a-3p. METHODS: Real-time quantitative polymerase chain reaction (qRT-PCR) method was applied to detect the expressions of NORAD, miR-199a-3p and ZNF217 genes in normal lung epithelial cells BEAS-2B, lung cancer H460 cells, and Cisplatin (DDP) resistant cell lines H460/DDP. H460/DDP cells were devided into control group, si-NC group, si-NORAD group, miR-NC group, miR-199a-3p mimic group, si-NORAD+inhibitor NC group and si-NORAD+miR-199a-3p inhibitor group. Cell proliferation, apoptosis, the expression of NORAD, miR-199a-3p and ZNF217 genes of cells in each group were detected and the expression levels of Ki-67, caspase-9 and ZNF217 proteins in different cells were also observed. The relationship between miR-199a-3p, NORAD and ZNF217 was vefified. RESULTS: Compared with BEAS-2B cells, the expressions of NORAD, ZNF217 mRNA were significantly increased in H460 and H460/DDP cells (P<0.05) but the expression of miR-199a-3p was significantly reduced (P<0.05). Compared with H460 cells, the expression of NORAD and ZNF217 mRNA in H460/DDP cells was significantly increased (P<0.05) and the expression of miR-199a-3p was significantly reduced (P<0.05). Compared with the control group and si-NC group, the proliferation rate, NORAD and ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the si-NORAD group were significantly reduced (P<0.05), but the apoptosis rate, miR-199a-3p expression and caspase-9 expression were significantly increased (P<0.05). Compared with the miR-NC group, the proliferation rate, NORAD and ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the miR-199a-3p mimic group were significantly reduced (P<0.05), but the apoptosis rate, miR-199a-3p expression and caspase-9 expression were significantly increased (P<0.05). Compared with the si-NORAD+inhibitor NC group, the proliferation rate, ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the si-NORAD+miR-199a-3p inhibitor group were significantly increased (P<0.05), the apoptosis rate, miR-199a-3p expression and caspase-9 expression were obviously increased reduced (P<0.05). CONCLUSIONS: Down-regulating NORAD expression can enhance miR-199a-3p expression and inhibit ZNF217 expression, thereby inhibiting H460/DDP cell proliferation, promoting apoptosis and enhancing its DDP chemotherapy sensitivity.

Circulating miR-3613-5p but not miR-125b-5p, miR-199a-3p, and miR-451a are biomarkers of endometriosis.

OBJECTIVE: This study aimed to assess the utility of circulating miR-125b-5p, miR-199a-3p, miR-451a, and miR-3613-5p as biomarkers of endometriosis. STUDY DESIGN: Patients with stage III or IV of endometriosis according to the revised American Society of Reproductive Medicine (rASRM) staging classification, as well as control women, were recruited. We created a prospective study conducted on a group of 48 patients (n = 25 controls, n = 24 endometriosis) who had laparoscopic surgery. Blood samples were taken and plasma miRNA levels were measured by quantitative real-time polymerase chain reaction (RT-qPCR) and assessed with AUC and ROC curves. RESULTS: MiR-451a and miR-3613-5p were significantly decreased in the plasma of endometriosis patients. miR-451a had a receiver-operating characteristic (ROC) area under the curve 0.8283 and miR-3613-5p had a ROC area under the curve 0.7617. The concentration of circulating miR-125b-5p and miR-199-3p did not differ between endometriosis patients and controls. Plasma miRNA levels did not change with BMI, smoking status, fertility problems, or menstrual pain according to the VAS scale (p > 0.05). CONCLUSION: Circulating miR-451a and miR-3613-5p levels significantly differed between endometriosis and controls. However, the levels of miR-451a were discordant with previous studies. Therefore, miR-3613-5p may have better potential as the endometriosis biomarker. Circulating miR-125b-5p and miR-199a-3p cannot be used as reliable markers of endometriosis.

MiR-199a-3p promotes repair of myocardial infarction by targeting NACC2.

OBJECTIVE: Myocardial infarction (MI) has gained widespread interest due to its high death and disability rate worldwide. Some miRNAs are markers of heart disease. Therefore, it is necessary to understand the mechanism for repairing MI injury. METHODS: Here, we evaluated the relative expression levels of miR-199a-3p in mouse and human myocardial cell models of injury, and its effect on myocardial cells viability using Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridline (EdU) assay, and flow cytometry assay as well as western blot in vitro. Furthermore, we performed bioinformatic online analysis to investigate the role that miR-199a-3p plays in cardiomyocyte injury, measured by dual-luciferase reporter assay. RESULTS: The results showed that miR-199a-3p significantly increased the growth rate of cardiomyocytes after treating them with hydrogen peroxide (H2O2). miR-199a-3p also acted as an inhibitor that directly targeted NACC2, resulting in a higher NACC2 expression level in the injury model of cardiomyocytes than normal myocardial cells and thus preventing miR-199a-3p-induced proliferation promotion in model cardiomyocytes. CONCLUSION: Our results demonstrate that miR-199a-3p may be a prognostic biomarker in myocardial injury.

miR-199a-3p promotes gastric cancer progression by promoting its stemness potential via DDR2 mediation.

BACKGROUND: Peritoneal metastasis (PM) is an independent prognostic factor in gastric cancer (GC), however, the underlying mechanisms of PM occurrence remain unclear. METHOD: The roles of DDR2 were investigated in GC and its potential relationship to PM, and orthotopic implants into nude mice were performed to assess the biological effects of DDR2 on PM. RESULTS: Herein, DDR2 level is more significantly observed to elevate in PM lesion than the primary lesion. GC with DDR2-high expression evokes a worse overall survival (OS) in TCGA, similar results of the gloomy OS with high DDR2 levels are clarified via the stratifying stage of TNM. The conspicuously increased expression of DDR2 was found in GC cell lines, luciferase reporter assays verified that miR-199a-3p directly targeted DDR2 gene, which was correlated to tumor progression. We ulteriorly observed DDR2 participated in GC stemness maintenance via mediating pluripotency factor SOX2 expression and implicated in autophagy and DNA damage of cancer stem cells (CSCs). In particular, DDR2 dominated EMT programming through recruiting NFATc1-SOX2 complex to Snai1 in governing cell progression, controlling by DDR2-mTOR-SOX2 axis in SGC-7901 CSCs. Furthermore, DDR2 promoted the tumor peritoneal dissemination in gastric xenograft mouse model. CONCLUSION: Phenotype screens and disseminated verifications incriminating in GC exposit the miR-199a-3p-DDR2-mTOR-SOX2 axis as a clinically actionable target for tumor PM progression. The herein-reported DDR2-based underlying axis in GC represents novel and potent tools for studying the mechanisms of PM.

Mir-199a-3p Mediates Fluid Shear Stress-Induced Osteoblast Proliferation by Targeting CABLES-1 / 医用生物力学

Objective To explore the role of miR-199a-3p in osteoblast proliferation induced by fluid shear stress (FSS) and the potential molecular mechanism. Methods Osteoblast MC3T3-E1 was treated with 1. 2 Pa FSS with time gradients of 0, 15, 30, 45, 60, 75 and 90 min, respectively. MC3T3-E1 cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor. MC3T3-E1 cells were transfected with miR-199a-3p mimic and itsnegative control and then treated with 1. 2 Pa FSS for 45 min. The pc DNA NC, pc DNA-CABLES -1, si RNA NC and si RNA CABLES-1 were transfected into MC3T3-E1 cells. The pc DNA-CABLES-1 and mir-199a-3p mimic and SI NA-cables-1 and miR-199a-3p inhibitor were co-transfected, respectively. Cell activity was detected by CCK-8 assay. Real-time quantitative PCR (RT-qPCR) was used to detect expression levels of CABLES-1, miR-199a-3p, CDK 6, Cyclin D1 and PCNA. Luciferase reporting assay was used to detect targeting relationship between CABLES-1 and miR-199a-3p. Immunofluorescence was used to detect protein expression of CABLES-1.Western blot was used to detect protein expression of CABLES-1, CDK 6, PCNA and Cyclin D1. Results Mir- 199a-3p in MC3T3-E1 cells was significantly down-regulated by FSS. Over-expressed miR-199a-3p inhibitedosteoblast proliferation, and down-regulated miR-199a-3p expression promoted osteoblast proliferation. miR-199a- 3p could reverse the FSS-induced proliferation in osteoblasts. Dual luciferase assay showed that miR-199a-3p targeted to CABLES-1 and over-expressed miR-199a-3p inhibited expression of CBALES-1 protein. CABLES-1 could promote proliferation of osteoblasts. miR-199a-3p inhibited osteoblast proliferation induced by FSS through CABLES-1. Conclusions FSS-induced osteoblast proliferation can be realized by down-regulated miR-199a-3p expression via targeting CABLES-1. The findings in this study provide new direction for researches on mechanism of FSS-induced osteoblast proliferation, as well as new ideas for future research on clinical application of mechanical loading in the treatment of bone and joint diseases.

MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome.

Purpose: Acute respiratory distress syndrome (ARDS) is a prevalent illness in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) assist in ARDS lung injury and the inflammatory process through mechanisms that are unclear. This study investigated the role of AM-derived secretory autophagosomes (SAPs) in lung injury and microRNA (MiR)-199a-3p-regulated inflammation associated with ARDS in vitro and in a murine model. Methods: The ARDS model in mouse was established by intratracheal LPS lipopolysaccharide (LPS) injection. The agomirs or antagomirs of MiR-199a-3p were injected into the caudal vein to figure out whether MiR-199a-3p could influence ARDS inflammation and lung injury, whereas the mimics or inhibitors of MiR-199a-3p, siRNA of Rab8a, or PAK4 inhibitor were transfected or applied to RAW264.7 cells to evaluate the mechanism of SAP release. Culture supernatants of RAW264.7 cells treated with LPS or bronchoalveolar lavage fluid from mice were collected for the isolation of SAPs. Results: We found that MiR-199a-3p was over-expressed in the lungs of ARDS mice. The MiR-199a-3p antagomir alleviated, whereas the MiR-199a-3p agomir exacerbated LPS-induced inflammation in mice by promoting AM-derived SAP secretion. In addition, MiR-199a-3p over-expression exacerbated LPS-induced ARDS via activating Rab8a, and Rab8a silencing significantly suppressed the promoting influence of the MiR-199a-3p mimic on SAP secretion. Furthermore, MiR-199a-3p mimic activated Rab8a by directly inhibiting PAK4 expression. Conclusion: The novel finding of this study is that MiR-199a-3p participated in the regulation of SAP secretion and the inflammatory process via targeting of PAK4/Rab8a, and is a potential therapeutic candidate for ARDS treatment.

MiR-199a-3p Induces Mesenchymal to Epithelial Transition of Keratinocytes by Targeting RAP2B.

Cutaneous squamous cell carcinoma (CSCC) is an epidermal skin cancer that evolves from normal epidermis along several pre-malignant stages. Previously we found specific miRNAs alterations in each step along these stages. miR-199a-3p expression decreases at the transition to later stages. A crucial step for epithelial carcinoma cells to acquire invasive capacity is the disruption of cell-cell contacts and the gain of mesenchymal motile phenotype, a process known as epithelial-to-mesenchymal transition (EMT). This study aims to study the role of decreased expression of miR-199a-3p in keratinocytes' EMT towards carcinogenesis. First, we measured miR-199a-3p in different stages of epidermal carcinogenesis. Then, we applied Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) assay to search for possible biochemical targets of miR-199a-3p and verified that Ras-associated protein B2 (RAP2B) is a bona-fide target of miR-199a-3p. Next, we analyzed RAP2B expression, in CSCC biopsies. Last, we evaluated possible mechanisms leading to decreased miR-199a-3p expression. miR-199a-3p induces a mesenchymal to epithelial transition (MET) in CSSC cells. Many of the under-expressed genes in CSCC overexpressing miR-199a-3p, are possible targets of miR-199a-3p and play roles in EMT. RAP2B is a biochemical target of miR-199a-3p. Overexpression of miR-199a-3p in CSCC results in decreased phosphorylated focal adhesion kinase (FAK). In addition, inhibiting FAK phosphorylation inhibits EMT marker genes' expression. In addition, we proved that DNA methylation is part of the mechanism by which miR-199a-3p expression is inhibited. However, it is not by the methylation of miR-199a putative promoter. These findings suggest that miR-199a-3p inhibits the EMT process by targeting RAP2B. Inhibitors of RAP2B or FAK may be effective therapeutic agents for CSCC.

Plasma exosomal miR-199a-3p downregulates cell proliferation and migration in Hirschsprung's disease by targeting mTOR.

BACKGROUND: Plasma exosomal microRNAs have been suggested to be potential biomarkers of disease. However, the exosomal microRNAs in Hirschsprung's disease (HSCR) are still unclear. In this study, we analyzed the miRNA profiles of HSCR and elucidated the mechanism of the selected miR-199a-3p in the development of HSCR. METHODS: Plasma exosomes were isolated, and exosomal miRNA high-throughput sequencing was performed to obtain differentially expressed miRNAs. CCK-8 and Transwell assay were used to determine the function of the most differentially expressed miRNA, which was confirmed in tissue specimen. Thereafter, target genes of the selected miRNAs were predicted by the databases. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes Genomes (KEGG) analysis, and protein-protein interaction network (PPI) construction of possible target genes were used to perform enrichment analysis and interaction. Finally, the PCR, Western blot and recovery experiment were used to confirm the function of target gene, mammalian target of rapamycin (mTOR), in vitro. RESULTS: The expression of miR-199a-3p was upregulated in plasma exosomes and diseased colonic tissues of patients with HSCR. In vitro, miR-199a-3p can inhibit cell proliferation and migration. Bioinformatic analysis suggested that mTOR might be a potential target of miR-199a-3p in HSCR. mTOR was discovered to be downregulated by miR-199a-3p in vitro. The negative connection between mTOR and miR-199a-3p was confirmed in tissue samples. mTOR can partially reverse the effect of miR-199a-3p on cell proliferation and migration function in vitro. CONCLUSIONS: miR-199a-3p suppresses cell growth and motility, partially by targeting mTOR. Plasma exosomal miR-199a-3p, a diagnostic marker, is crucial for the development of HSCR.

Bta-miR-199a-3p Inhibits LPS-Induced Inflammation in Bovine Mammary Epithelial Cells via the PI3K/AKT/NF-κB Signaling Pathway.

Mastitis is characterized by inflammatory damage to mammary gland tissue, which could decline milk production and quality and significantly affect the economic benefits of ranching. MicroRNAs (miRNAs), such as miR-199a-3p, are novel therapeutic targets in inflammation, and their regulation is an effective strategy for inflammation control. Despite its importance in humans and animals, the molecular mechanism of bovine miR-199a-3p (bta-miR-199a-3p) in dairy cow mastitis and bovine mammary epithelial cell (bMEC) inflammation is unclear. In our study, a bovine mammary epithelial cell line (MAC-T) induced by lipopolysaccharide (LPS) was used as an inflammatory cell model to investigate the molecular mechanism of bta-miR-199a-3p in the MAC-T inflammatory response. bta-miR-199a-3p was up-regulated in the LPS-induced MAC-T cells, while CD2-associated protein (CD2AP) was revealed as its target gene in a double luciferase reporter gene experiment. In addition, the overexpression of bta-miR-199a-3p negatively regulated the expression of CD2AP and the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/nuclear factor kappa-B (NF-κB) signaling pathway. These subsequently inhibited the secretion of related inflammatory factors (TNF-α, IL-1ß, and IL-6) and the expression of apoptotic genes (CASP3 and CASP9), thereby alleviating the LPS-challenged inflammatory response in the MAC-T cells. Silencing of bta-miR-199a-3p, however, reversed the above effects. Thus, bta-miR-199a-3p inhibits LPS-induced inflammation in bMECs by directly targeting CD2AP and regulating the PI3K/AKT/NF-κB signaling pathway. This study reveals the potential regulatory mechanism of bta-miR-199a-3p in bMEC inflammatory immune response and may serve as a useful target for the treatment of mastitis.

RNA-seq reveals the role of miR-199a-3p in regulating inflammation of bovine mammary epithelial cells.

MicroRNAs (miRNAs) are involved in the regulation of a variety of biological processes. However, the research on the regulatory role of bovine mammary epithelial cells (bMECs) is scarce. To date, there are no reports about the role of miR-199a-3p in bMECs. In this study, RNA sequencing (RNA-seq) technology was used to detect the transcriptomes of the miR-199a-3p overexpression and negative control (NC) groups of bMECs. Then, the screening and functional annotation of differentially expressed genes (DEGs) were conducted. The results showed that there were 140 DEGs (109 up-regulated and 31 down-regulated) in the miR-199a-3p overexpression group. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that the DEGs might regulate the immune and inflammatory responses via the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, transforming growth factor-beta (TGF-ß) signaling pathway, and interleukin-17 (IL-17) signaling pathway, which revealed that miR-199a-3p might participate in regulating bMECs inflammation via affecting the expression of related genes and the above signaling pathways. This study may provide a new reference for potential therapeutic targets of cow mastitis.

miR-199a-3p increases the anti-tumor activity of palbociclib in liver cancer models.

Palbociclib is in early-stage clinical testing in advanced hepatocellular carcinoma (HCC). Here, we investigated whether the anti-tumor activity of palbociclib, which prevents the CDK4/6-mediated phosphorylation of RB1 but simultaneously activates AKT signaling, could be improved by its combination with a PI3K/AKT/mTOR inhibitor in liver cancer models. The selective pan-AKT inhibitor, MK-2206, or the microRNA-199a-3p were tested in combination with palbociclib in HCC cell lines and in the TG221 HCC transgenic mouse model. The combination palbociclib/MK-2206 was highly effective, but too toxic to be tolerated by mice. Conversely, the combination miR-199a-3p mimics/palbociclib not only induced a complete or partial regression of tumor lesions, but was also well tolerated. After 3 weeks of treatment, the combination produced a significant reduction in number and size of tumor nodules in comparison with palbociclib or miR-199a-3p mimics used as single agents. Moreover, we also reported the efficacy of this combination against sorafenib-resistant cells in vitro and in vivo. At the molecular level, the combination caused the simultaneous decrease of the phosphorylation of both RB1 and of AKT. Our findings provide pre-clinical evidence for the efficacy of the combination miR-199a-3p/palbociclib as anti-HCC treatment or as a new approach to overcome sorafenib resistance.

Effects of penehyclidine hydrochloride on myocardial ischaemia-reperfusion injury in rats by inhibiting TLR4/MyD88/NF-κB pathway via miR-199a-3p.

This study was to probe the role of penehyclidine hydrochloride (PHC) mediating the impact of toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) signalling pathway on myocardial ischaemia-reperfusion injury (MI/RI) in rats through miR-199a-3p. The rat MI/RI model was established through ligating left anterior descending (LAD) coronary artery. PHC was injected preoperatively into the model rats, and injected with miR-199a-3p lentiviral vector or TLR4 antagonist (TAK-242). Next, cardiac function of rats was examined by echocardiography, and rat serum indicators, oxidative stress levels and inflammatory factors were detected. HE staining was applied to detect pathological tissue structure, TUNEL staining to detect apoptosis rate, qRCR and western blot to detect miR-199a-3p and TLR4/MyD88/NF-κB expressions in rat myocardial tissues. Dual luciferase reporter experiment was conducted to confirm the relationship between miR-199a-3p and TLR4. In conclusion, PHC suppresses TLR4/MyD88/NF-κB signalling pathway through miR-199a-3p, thereby improving MI/RI in rats.