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.

Ceramide Ehux-C22 Targets the miR-199a-3p/mTOR Signaling Pathway to Regulate Melanosomal Autophagy in Mouse B16 Cells.

Melanosomes are specialized membrane-bound organelles where melanin is synthesized and stored. The levels of melanin can be effectively reduced by inhibiting melanin synthesis or promoting melanosome degradation via autophagy. Ceramide, a key component in the metabolism of sphingolipids, is crucial for preserving the skin barrier, keeping it hydrated, and warding off the signs of aging. Our preliminary study indicated that a long-chain C22-ceramide compound (Ehux-C22) isolated from the marine microalga Emiliania huxleyi, reduced melanin levels via melanosomal autophagy in B16 cells. Recently, microRNAs (miRNAs) were shown to act as melanogenesis-regulating molecules in melanocytes. However, whether the ceramide Ehux-C22 can induce melanosome autophagy at the post-transcriptional level, and which potential autophagy-dependent mechanisms are involved, remains unknown. Here, miR-199a-3p was screened and identified as a novel upregulated miRNA in Ehux-C22-treated B16 cells. An in vitro high melanin expression model in cultured mouse melanoma cells (B16 cells) was established by using 0.2 µM alpha-melanocyte-stimulating hormone(α-MSH) and used for subsequent analyses. miR-199a-3p overexpression significantly enhanced melanin degradation, as indicated by a reduction in the melanin level and an increase in melanosome autophagy. Further investigation demonstrated that in B16 cells, Ehux-C22 activated miR-199a-3p and inhibited mammalian target of rapamycin(mTOR) level, thus activating the mTOR-ULK1 signaling pathway by promoting the expression of unc-51-like autophagy activating kinase 1 (ULK1), B-cell lymphoma-2 (Bcl-2), Beclin-1, autophagy-related gene 5 (ATG5), and microtubule-associated protein light chain 3 (LC3-II) and degrading p62. Therefore, the roles of Ehux-C22-regulated miR-199a-3p and the mTOR pathway in melanosomal autophagy were elucidated. This research may provide novel perspectives on the post-translational regulation of melanin metabolism, which involves the coordinated control of melanosomes.

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.

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.

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.

Circ-ZEB1 promotes PIK3CA expression by silencing miR-199a-3p and affects the proliferation and apoptosis of hepatocellular carcinoma.

BACKGROUND: Although the prognostic outcomes of liver cancer (LC) cases have improved with the advancement in diagnostic technology and treatment methods, the transferability and recurrence of HCC and the 5-year and 10-year survival rates of patients have remained unsatisfactory. As a result, there is a need for more accurate diagnostic indicators that can detect liver cancer early, effectively improving the prognosis of patients. Whole-genome sequencing (WGS) revealed that circ-ZEB1 and PIK3CA are highly expressed in HCC tissues, whereas miR-199a-3p is significantly downregulated in HCC. Multiple databases search and biological analysis revealed that elevated expression of circ-ZEB1 and PIK3CA was related to poor prognosis of HCC. In vitro and in vivo studies revealed that upregulated levels of PIK3CA and circ-ZEB1 were closely associated with HCC proliferation and apoptosis. Based on these results, we believe that circ-ZEB1 and PIK3CA could be used as biomarkers to diagnose and treat patients with HCC. More importantly, circ-ZEB1 can promotes the expression of PIK3CA by silencing miR-199a-3p and affecting the progression of HCC. METHODS AND RESULTS: Postoperative specimens from 56 patients with HCC who had not undergone chemotherapy from 2015 to 2018 were collected from the Department of Hepatobiliary Surgery, Second Affiliated Hospital of Nanchang University. WGS revealed differential expression of genes in HCC. Furthermore, RT-qPCR detected the expression of circ-ZEB1, miR-199a-3p, and PIK3CA in HCC tissues. MTT, EdU, and plate cloning experiments were conducted to detect cell proliferation, whereas flow cytometry analysis was used to detect apoptosis. FISH was used to co-localize circ-ZEB1 and miR-199a-3p, and biotin-coupled probe pull-down assay was used to detect the specific binding of circ-ZEB1 and miR-199a-3p. The dual-luciferase report assay detected the association of miR-199a-3p with PIK3CA. Western blotting was used to study the expression of PIK3CA protein. Circ-ZEB1 and PIK3CA were upregulated in HCC and predicted a poor prognosis. MiR-199a-3p showed low expression in HCC, whereas downregulation of circ-ZEB1 reduced HCC cell proliferation and promoted cell apoptosis. MiR-199a-3p blocked the effect of circ-ZEB1 on HCC. Circ-ZEB1 served as a biomarker of HCC. Circ-ZEB1 promoted the expression of PIK3CA by silencing miR-199a-3p to affect the progress of HCC. CONCLUSIONS: Circ-ZEB1 promoted the expression of PIK3CA by depleting miR-199a-3p, thereby affecting HCC proliferation and apoptosis.

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.

MiR-199a-3p Overexpression Suppressed Cell Proliferation and Sensitized Chronic Myeloid Leukaemia Cells to Imatinib by Inhibiting mTOR Signalling.

INTRODUCTION: Chronic myeloid leukaemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Drug resistance remains one of the major challenges in CML therapy. MicroRNA (miR)-199a-3p plays an important role in many tumours but has rarely been investigated in CML. We aimed to analyse the role and mechanism of miR-199a-3p in regulating imatinib resistance in CML. METHODS: The expression of miR-199a-3p and mammalian target of rapamycin (mTOR) in the serum of CML patients and CML cells was examined by quantitative real-time polymerase chain reaction. The levels of apoptosis-related proteins were determined using western blot. The relative cell survival rate and cell proliferation were determined using a CCK-8 assay and a bromodeoxyuridine (BrdU) assay, respectively. Cell cycle and apoptosis were analysed using flow cytometry. Moreover, a dual-luciferase reporter assay was performed to verify the correlation between miR-199a-3p and mTOR. RESULTS: MiR-199a-3p was downregulated in the serum of CML patients and in CML cells, while mTOR was upregulated. Both miR-199a-3p overexpression and mTOR silencing inhibited CML cell proliferation, promoted CML cell apoptosis, and sensitized these cells to imatinib. mTOR silencing reversed the promoting effect of miR-199a-3p inhibition on the proliferation of CML cells and the inhibitory effects on cell apoptosis and sensitivity to imatinib. MiR-199a-3p directly targeted mTOR. CONCLUSION: MiR-199a-3p suppressed cell propagation, facilitated apoptosis of CML cells, and sensitized CML cells to imatinib by downregulating mTOR signalling.

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.

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.

Circ003429 Regulates Unsaturated Fatty Acid Synthesis in the Dairy Goat Mammary Gland by Interacting with miR-199a-3p, Targeting the YAP1 Gene.

Fatty acid composition is a key factor affecting the flavor and quality of goat milk. CircRNAs are now recognized as important regulators of transcription, and they play an important role in the control of fatty acid synthesis. Thus, understanding the regulatory mechanisms controlling this process in ruminant mammary glands is of great significance. In the present study, mammary tissue from dairy goats during early lactation and the dry period (nonlactating) were collected and used for high-throughput sequencing. Compared to levels during the dry period, the expression level of circ003429 during early lactation was lower (12.68-fold downregulated). In isolated goat mammary epithelial cells, circ003429 inhibited the synthesis of triglycerides (TAG) and decreased the content of unsaturated fatty acids (C16:1, C18:1, and C18:2), indicating that this circRNA plays an important role in regulating lipid synthesis. A binding site for miR-199a-3p in the circ003429 sequence was detected, and a dual-luciferase reporter system revealed that circ003429 targets miR-199a-3p. Overexpression of circ003429 (pcDNA-circ003429) downregulated the abundance of miR-199a-3p. In contrast, overexpression of miR-199a-3p increased TAG content and decreased mRNA abundance of Yes-associated protein 1 (YAP1) (a target gene of miR-199a-3p), and TAG content was decreased and mRNA abundance was increased in response to overexpression of circ003429. These results indicate that circ003429 alleviates the inhibitory effect of miR-199a-3p on the mRNA abundance of YAP1 by binding miR-199a-3p, resulting in subsequent regulation of the synthesis of TAG and unsaturated fatty acids.

miR-199a-3p may be an early warning marker for acute rejection after liver transplantation in rats.

BACKGROUND: Prevention of acute rejection is the key of the success of liver transplantation. However, there are no specific indicators available for prediction of acute rejection after liver transplantation. MicroRNAs (miRNAs) are highly conserved and small noncoding RNA molecules that can be detected in peripheral blood. Here, we evaluated the potential of circulating miRNAs to serve as noninvasive biomarkers for acute rejection after liver transplantation in rats. METHODS: The liver grafts retrieved from Lewis rats were orthotopically transplanted into BN rats or Lewis rats in the acute rejection and immune tolerance group respectively, and the BN rats in the immune intervention group was intraperitoneally injected with transforming growth factor-ß1 overexpressed immature dendritic cells to suppress acute rejection before orthotopically transplanted with livers from Lewis rats. MiRNAs profiling studies were used to determine the regulation of circulating miRNAs in plasma samples of rats. Candidate miRNA was verified by quantitative reverse transcriptase polymerase chain reaction. Furthermore, the relationship between candidate miRNA and acute rejection was also evaluated. RESULTS: Microarray analysis revealed that miR-199a-3p was the mostly differentially regulated miRNAs in plasma samples among the three groups. The plasmid PCDH-CMV-EGFP-hTGF-ß1 was identified by PCR and DNA sequencing, and successfully expressed in imDCs. There were differences in the expression of miR-199a-3p in the liver tissues of the AR group on the 3rd, 7th and 10th day after liver transplantation (all p < 0.01). With time, the RAI score increased gradually, and the difference of miR-199a-3p expression gradually increased (rs = 0.92, p < 0.001), suggesting that it may be related to acute rejection. The expression of miR-199a-3p in the serum of the AR and TGF-ß1-imDCs groups gradually increased, reaching a peak at day 7 and then decreasing. There was positive relationship between the expression of miR-199a-3p and RAIs within 7 days post operation. (rs = 0.942, p < 0.05). CONCLUSION: miR-199a-3p might be an early warning marker for acute rejection after liver transplantation in rats.

MiR-199a-3p inhibits the proliferation, migration, and invasion of endothelial cells and retinal pericytes of diabetic retinopathy rats through regulating FGF7 via EGFR/PI3K/AKT pathway.

PURPOSE: MiR-199a-3p is low expressed in diabetic retinopathy (DR). In the current study, we investigated the effects of miR-199a-3p on DR and the potential mechanisms. METHODS: A DR rat model was established, and endothelial cells (ECs) and retinal pericytes (RPs) were extracted from the DR model rats to detect miR-199a-3p expression. Bioinformatics analysis predicted that fibroblast growth factor 7 (FGF7) was a target gene for miR-199a-3p, which was confirmed by dual-luciferase assay. Cell proliferation, migration, and invasion were detected by cell counting kit-8 (CCK-8), colony formation assay, wound-healing, and Transwell assay. Quantitative real-time polymerase chain reaction (q-PCR) and Western blot were performed to detect the expressions of mRNAs and proteins. RESULTS: MiR-199a-3p was low expressed and FGF7 was high-expressed in ECs and RPs. Overexpressed miR-199a-3p suppressed the proliferation, migration, and invasion, and FGF7 expression of ECs and RPs. However, overexpression of FGF7 effectively eliminated the suppressive effects of miR-199a-3p overexpression malignant behaviors of the cells. Meanwhile, up-regulation of FGF7 noticeably reversed the phosphorylation of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) and the expression of epidermal growth factor receptor (EGFR) reduced by miR-199a-3p. CONCLUSION: Our findings revealed that in the DR rat model, miR-199a-3p inhibited cell proliferation, migration, and invasion of EC and RP through targeting FGF7 and inhibiting the activation of the EGFR/PI3K/AKT pathway. This study may provide a new direction for the search for the treatment of DR.

Exploring the potential of engineered exosomes as delivery systems for tumor-suppressor microRNA replacement therapy in ovarian cancer.

MicroRNA (miRNA) plays a pivotal role in cancer biology. Therefore, tumor suppressor (TS) miRNAs are an attractive target for cancer therapy. However, clinical trials have failed due to the difficulties in miRNA delivery, warranting the development of a novel drug delivery system (DDS). Exosomes are stable in circulation and selectively picked up by cancer cells, indicating that they can serve as a miRNA carrier. The aim of this study was to explore the possibility of exosomes as a carrier for miRNA replacement therapy for ovarian cancer (OC). First, exosomes were purified from primary-cultured omental fibroblasts of OC patients. miR-199a-3p was selected as a TS miRNA, and the synthesized miR-199a-3p was loaded into exosomes by electroporation. Treatment with miR199a-3p-loaded-exosomes (miR-199a-3p-Exo) drastically increased miR-199a-3p expression level in OC cell lines (CaOV3; 8592-, SKOV3; 67188-, and OVCAR3; 2280-fold). miR-199a-3p-Exo suppressed c-Met expression, a direct target of miR-199a-3p, and thereby inhibited cell proliferation and invasion. In a xenograft study, miR-199a-3p-Exo also drastically inhibited peritoneal dissemination in OC mice model, and diminished c-Met expression, ERK phosphorylation, and MMP2 expression in tumors. These results suggest that miRNA replacement therapy using exosomes shows promise for treatment of OC. Given that omental fibroblasts can be obtained from most OC patients, patient-derived exosomes can be utilized as a DDS for future molecular-targeted therapies.

MiR-199a-3p inhibition facilitates cardiomyocyte differentiation of embryonic stem cell through promotion of MEF2C.

MicroRNAs (miRNAs) is a small molecule (19-25 nucleotide) noncoding RNA that inhibits the expression of target messenger RNA (mRNA) at the posttranscriptional level as an endogenous regulator. There is an increasing evidence that miR-199a-3p has a significant effect on the development of multiple tumors. However, the specific roles of miR-199a-3p in myocardial differentiation of embryonic stem cell still need to be investigated. Method of the hanging drop was used to build the model of cardiomyocyte differentiation of stem cell and beating rate of embryoid bodies (EBs) was calculated. The levels of intracellular MEF2C, a-MHC, GATA4, Nkx2.5, and cTnT mRNA were measured by real-time quantitative polymerase chain reaction, while the expressions of miR-199a-3p were detected simultaneously. Protein levels of MEF2C, a-MHC, GATA4, Nkx2.5, and cTnT were quantified by western blot analysis. Immunoreactivities of MEF2C and cTnT were analyzed by immunofluorescence. The interaction between miR-199a-3p and its predicted target (3'-untranslated region of MEF2C mRNA) was verified by luciferase assay. MiR-199a-3p levels increased during cardiogenesis. MiR-199a-3p inhibitor increased the beating rate of EBs and promoted expressions of cardiac-specific markers (GATA4, Nkx2.5, cTnT, and a-MHC). Notably, miR-199a-3p inhibition brought upregulation of MEF2C, which is the target of miR-199a-3p that we predicted and verified experimentally. In addition, MEF2C siRNA decreased miR-199a-3p inhibitor promoted EBs beating and attenuated miR-199a-3p inhibitor-induced cTnT and MEF2C expressions. The results above showed that MEF2C was involved in the process of promoting the differentiation of stem cells into cardiac myocytes by miR-199a-3p inhibitors.

Silence of lncRNA HEIH suppressed liver cancer cell growth and metastasis through miR-199a-3p/mTOR axis.

BACKGROUND/AIMS: High expression in hepatocellular carcinoma (HEIH) is an long noncoding RNA (lncRNA) which is highly expressed in hepatocellular carcinoma (HCC). Aberrant expression of HEIH is implicated in regulating HCC cells growth and metastasis. This study attempted to illustrate the effects of HEIH on HCC cell lines. METHODS: The expression changes of HEIH in HCC tumor tissues and the paracancerous tissues derived from 20 patients with HCC were tested. Effects of HEIH on Huh7 and Hep3B cells viability, apoptosis, migration, and invasion were assessed by silencing HEIH in vitro. Furthermore, downstream effector and signaling of HEIH were studied. RESULTS: As compared with the paracancerous tissues, the HEIH expression was highly expressed in tumor tissues. Silence of HEIH significantly reduced Huh7 and Hep3B cells viability, migration, and invasion, but induced apoptosis. It was coupled with the downregulated CyclinD1, Bcl-2, MMP-2, MMP-8, Vimentin, the upregulated p53, Bax, as well as the cleaved caspase-3. MicroRNA (miR)-199a-3p was identified as a downstream effector of HEIH, as its expression was upregulated by HEIH silence, and the functional effects of HEIH on Huh7 and Hep3B cells were all attenuated when miR-199a-3p expression was suppressed. Furthermore, HEIH silence suppressed the activation of mTOR signaling via upregulating miR-199a-3p. CONCLUSION: HEIH silence might be a promising target for suppressing HCC cells growth and metastasis. Silence of HEIH exerted its antitumor properties possibly through upregulating miR-199a-3p, and thereby blockage of mTOR signaling.

p53 induces miR-199a-3p to suppress mechanistic target of rapamycin activation in cisplatin-induced acute kidney injury.

How p53 participates in acute kidney injury (AKI) progress and what are the underlying mechanisms remain illusive. For this issue, it is important to probe into the role of p53 in cisplatin-induced AKI. We find that p53 was upregulated in cisplatin-induced AKI, yet, pifithrin-α inhibites the p53 expression to attenuated renal injury and cell apoptosis both in vivo cisplatin-induced AKI mice and in vitro HK-2 human renal tubular epithelial cells. To knock down p53 by siRNA significantly decreased the miRNA, miR-199a-3p, expression in HK-2 cells. Blockade of miR-199a-3p significantly reduced cisplatin-induced cell apoptosis and inhibited caspase-3 activity. Mechanistically, we identified that miR-199a-3p directly bound to mechanistic target of rapamycin (mTOR) 3'-untranslated region and overexpressed miR-199a-3p reduce the expression and phosphorylation of mTOR. Furthermore, we demonstrated that p53 inhibited mTOR activation through activating miR-199a-3p. In conclusion, our findings reveal that p53, upregulating the expression of miR-199a-3p affects the progress of cisplatin-induced AKI, which might provide a promising therapeutic target of AKI.

miR-199a-3p promotes cardiomyocyte proliferation by inhibiting Cd151 expression.

Adult mammalian cardiomyocytes have extremely limited capacity to regenerate, and it is believed that a strong intrinsic mechanism is prohibiting the cardiomyocytes from entering the cell cycle. microRNAs that promote proliferation in cardiomyocyte can be used as probes to identify novel genes suppressing cardiomyocytes proliferation, thus dissecting the mechanism(s) preventing cardiomyocytes from duplication. In particular, miR-199a-3p has been found as a potent activator of proliferation in rodent cardiomyocyte, although its molecular targets remain elusive. Here, we identified Cd151 as a direct target of miR-199a-3p, and its expression is greatly suppressed by miR-199a-3p. Cd151 gain-of-function reduced cardiomyocyte proliferation, conversely Cd151 loss-of-function increased cardiomyocytes proliferation. Overexpression of Cd151 blocks the activating effect of miR-199a-3p on cardiomyocyte proliferation, suggesting Cd151 is a functional target of miR-199a-3p in cardiomyocytes. Mechanistically, we found that Cd151 induces p38 expression, a known negative regulator of cardiomyocyte proliferation, and pharmacological inhibition of p38 rescued the inhibitory effect of Cd151 on proliferation. Together, this work proposes Cd151 as a novel suppressor of cardiomyocyte proliferation, which may provide a new molecular target for developing therapies to promote cardiac regeneration.

Long noncoding RNA NORAD regulates cancer cell proliferation and migration in human osteosarcoma by endogenously competing with miR-199a-3p.

In this study, we evaluated the expressions and functions of a long noncoding RNA (lncRNA), Noncoding RNA activated by DNA damage (NORAD) in human osteosarcoma. NORAD expressions were evaluated by qRT-PCR in in vitro osteosarcoma cell lines and in vivo clinical specimens. SiRNA-induced NORAD downregulation was conducted in Saos-2 and 143B cells, and the functional effects of NORAD downregulation on osteosarcoma cells were evaluated by CCK-8 proliferation assay, 24-well transwell invasion assay and in vivo tumor explant assay, respectively. The possibility of NORAD endogenously competing microRNA target, hsa-miR-199a-3p was examined by dual-luciferase reporter assay and qRT-PCR. Then, hsa-miR-199a-3p was downregulated in NORAD-inhibited osteosarcoma cells to examine its role in regulating NORAD inhibition induced cancer suppression in osteosarcoma cells. NORAD was found to be significantly overexpressed in both osteosarcoma cells and osteosarcoma tumors. In Saos-2 and 143B cells transfected with NORAD-specific siRNA, their proliferation, invasion, and in vivo explant growth were all markedly suppressed. Hsa-miR-199a-3p was confirmed to be the competing target of NORAD. Its downregulation in Saos-2 and 143B cells inversely augment proliferation and invasion that were initially suppressed by NORAD-downregulation. The results of our study show that NORAD plays an important role in regulating cancer cell functions of osteosarcoma, possibly through endogenously competing with hsa-miR-199a-3p.

Small RNA and transcriptome sequencing reveal the role of miR-199a-3p in inflammatory processes in cystic fibrosis airways.

Cystic fibrosis (CF) is the most common lethal genetic disease, caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. CF is characterized by an ionic imbalance and thickened mucus, which impair mucociliary clearance and promote bacterial colonization and the establishment of infection/inflammation cycles. However, the origin of this inflammation remains unclear, although microRNAs (miRNAs) are suspected to be involved. MiRNAs are small non-coding RNAs that bind to the 3'-untranslated regions (UTRs) of target gene mRNA, thereby repressing their translation and/or inducing their degradation. The goal of this study was to investigate the role of microRNAs associated with pulmonary inflammation in CF patients. Through the analysis of all miRNAs (miRNome) in human primary air-liquid interface cultures, we demonstrated that miR-199a-3p is the only miRNA downregulated in CF patients compared to controls. Moreover, through RNA sequencing (transcriptome) analysis, we showed that 50% of all deregulated mRNAs are linked directly or indirectly to the NF-κB pathway. To identify a specific target, we used bioinformatics analysis to predict whether miR-199a-3p targets the 3'-UTR of IKBKB, which encodes IKKß, a major protein in the NF-κB pathway. Subsequently, we used bronchial explants from CF patients to show that miR-199a-3p expression is downregulated compared to controls and inversely correlated with increases in expression of IKKß and IL-8. Through functional studies, we showed that miR-199a-3p modulates the expression of IKBKB through a direct interaction at its 3'-UTR in bronchial epithelial cells from CF patients. In miR-199a-3p overexpression experiments, we demonstrated that for CF cells, miR-199a-3p reduced IKKß protein expression, NF-κB activity, and IL-8 secretion. Taken together, our findings show that miR-199a-3p plays a negative regulatory role in the NF-κB signalling pathway and that its low expression in CF patients contributes to chronic pulmonary inflammation. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.