miR-199a-3p mediates bone cancer pain through upregulation of dnmt3a expression in spinal dorsal horn neurons.

Due to its complex pathological mechanisms, bone cancer pain (BCP) has become an increasingly challenging clinical issue, there is an urgent need to identify the underlying mechanisms of BCP. In our present study, we found that decreased expression of miR-199a-3p in spinal dorsal horn (SDH) neurons contributed to BCP hypersensitivity. Intrathecal administration of miR-199a-3p agomir alleviated the initiation of tumor inoculation induced pain hypersensitivity and suppressed the expression of DNMT3A. Subsequently, luciferase assays confirmed direct binding between miR-199a-3p and Dnmt3a mRNA. AAV-DNMT3A-shRNA microinjection relieved mechanical hyperalgesia and upregulated the expression of Nrf2 levels in BCP. In naïve rats, Overexpression of DNMT3A yielded the opposite effects. Finally, increase of DNMT3A by lentiviral vector abolished miR-199a-3p-mediated alleviation hypersensitivity effects on BCP progression. Taken these together, our findings highlighted a novel contribution of miR-199a-3p to BCP and provided a fresh outlook on potential mechanism research for BCP.

Down-regulated microRNA-199a-3p enhances osteogenic differentiation of bone marrow mesenchymal stem cells by targeting Kdm3a in ovariectomized rats.

Osteoporosis is a prevalent systemic skeletal disorder entailing bone fragility and increased fracture risk, often emerging in post-menopausal life. Emerging evidence implicates the dysregulation of microRNAs (miRNAs or miRs) in the progression of osteoporosis. This study investigated the effect of miR-199a-3p on osteoporosis and its underlying mechanism. We first examplished an ovariectomized (OVX)-induced rat osteoporosis model, and then isolated mesenchymal stem cells (MSCs) from bone marrow of the model rats. The overexpression and knock down of miR-199a-3p were conducted in OVX rats and MSCs to verify the role of miR-199a-3p on MSC differentiation. Calcium nodules were measured using alizarin red S (ARS) staining. RT-qPCR and Western blot assay were performed to measure the expression of miR-199a-3p, Kdm3a and osteogenic differentiation-related markers in rat tissues and cells. The correlation between miR-199a-3p and Kdm3a was confirmed using dual-luciferase reporter assay. The enrichment of Kdm3a at the Erk2 and Klf2 promoter was assessed using chromatin immunoprecipitation (ChIP) assay. Isolated MSCs were positive for CD29, CD44, CD90, and CD45, suggesting successful isolation of MSCs. There was increased expression of miR-199a-3p and inhibited osteogenic differentiation in OVX rats. Kdm3a was negatively targeted by miR-199a-3p. Our results also demonstrated that Kdm3a elevated the expression of Erk2 and Erk2 by promoting Erk2 and Klf2 demethylation, which further contributed to osteogenic differentiation. Overall, our results revealed a regulatory network of miR-199a-3p in osteogenic differentiation, highlighting miR-199a-3p as a potential target for therapeutic interventions in osteoporosis.

MicroRNA-199a-3p inhibits angiogenesis by targeting the VEGF/PI3K/AKT signalling pathway in an in vitro model of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a major inducer of blindness and visual impairment. As a critical cause for DR, hyperglycaemia is able to trigger multiple biochemical alterations. MiRNAs, which contain various functions, can effectively regulate blood glucose levels. This research aims to confirm the roles of miRNA-199a-3p in the progression of angiogenesis in an in vitro model of DR. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried to determine the expression levels of miR-199a-3p and VEGF in both hRMECs and APRE-19 cells. The luciferase reporter assay was used to study the interaction between miR-199a-3p and VEGF. Western blot assay was conducted to examine the expression levels of VEGF and the PI3K/AKT signalling pathway. The cell proliferation capacity was detected via the CCK-8 test. The impact of miR-199a-3p on migration was determined using Transwell and wound healing assays. A Matrigel tube formation assay was employed to determine the vascular formation of hRMECs. Flow cytometry was used to determine cell apoptosis in the presence of LY294002 as a PI3K inhibitor. RESULTS: Our results showed that high glucose (HG) decreased the relative expression level of miR-199a-3p but increased VEGF expression in hRMECs and APRE-19 cells. MiR-199a-3p inhibitor augmented cell growth, migration and angiogenesis of hRMECs. Moreover, upregulation of miR-199a-3p evidently alleviated the increases in cell proliferation, migration and angiogenesis caused by HG. In addition, the luciferase reporter assay indicated that miR-199a-3p directly targeted VEGF. The overexpression of miR-199a-3p obviously restrained the HG-stimulated PI3K/AKT signalling pathway and angiogenesis, which could be further inhibited by LY294002. Moreover, LY294002 could slightly ameliorate the miR-199a-3p inhibitor-stimulated PI3K/AKT signalling pathway and angiogenesis. CONCLUSION: MiR-199a-3p upregulation ameliorated HG-stimulated angiogenesis of hRMECs by modulating the PI3K/AKT pathway through inhibiting VEGF. Although retinal neovascularization in vivo has not been studied, these in vitro findings provide more evidence for the role of miR-199a-3p upregulation against HG-induced angiogenesis.

[Retracted] MicroRNA­199a­3p inhibits ovarian cancer cell viability by targeting the oncogene YAP1.

Following the publication of the above article, the authors have submitted a request that it be retracted on account of the fact that, when requested to do so, the first author was unable to provide the original data for this article. The Editor of Molecular Medicine Reports has agreed with the request that this article be retracted. Note that all the authors agree with the decision to retract this article. The Editor and the authors regret any inconvenience that this retraction will cause to the readership of the Journal. [Molecular Medicine Reports 23: 237, 2021; DOI: 10.3892/mmr.2021.11876].

MicroRNA-199a-3p regulates proliferation and milk fat synthesis of ovine mammary epithelial cells by targeting VLDLR.

In our previous study, microRNA (miR)-199a-3p was found to be the most upregulated miRNA in mammary gland tissue during the non-lactation period compared with the peak-lactation period. However, there have been no reports describing the function of miR-199a-3p in ovine mammary epithelial cells (OMECs) and the biological mechanisms by which the miRNA affects cell proliferation and milk fat synthesis in sheep. In this study, the effect of miR-199a-3p on viability, proliferation, and milk fat synthesis of OMECs was investigated, and the target relationship of the miRNA with very low-density lipoprotein receptor (VLDLR) was also verified. Transfection with a miR-199a-3p mimic increased the viability of OMECs and the number of Edu-labeled positive OMECs. In contrast, a miR-199-3p inhibitor had the opposite effect with the miR-199a-3p mimic. The expression levels of three marker genes were also regulated by both the miR-199a-3p mimic and miR-199-3p inhibitor in OMECs. Together, these results suggest that miR-199a-3p promotes the viability and proliferation of OMECs. A dual luciferase assay confirmed that miR-199a-3p can target VLDLR by binding to the 3'-untranslated regions (3'UTR) of the gene. Further studies found a negative correlation in the expression of miR-199a-3p with VLDLR. The miR-199a-3p mimic decreased the content of triglycerides, as well as the expression levels of six milk fat synthesis marker genes in OMECs, namely, lipoprotein lipase gene (LPL), acetyl-CoA carboxylase alpha gene (ACACA), fatty acid binding protein 3 gene (FABP3), CD36, stearoyl-CoA desaturase gene (SCD), and fatty acid synthase gene (FASN). The inhibition of miR-199a-3p increased the level of triglycerides and the expression of LPL, ACACA, FABP3, SCD, and FASN in OMECs. These findings suggest that miR-199a-3p inhibited milk fat synthesis of OMECs. This is the first study to reveal the molecular mechanisms by which miR-199a-3p regulates the proliferation and milk fat synthesis of OMECs in sheep.

Diverging targets mediate the pathological roleof miR-199a-5p and miR-199a-3p by promoting cardiac hypertrophy and fibrosis.

MicroRNA-199a-5p (miR-199a-5p) and -3p are enriched in the myocardium, but it is unknown whether miR-199a-5p and -3p are co-expressed in cardiac remodeling and what roles they have in cardiac hypertrophy and fibrosis. We show that miR-199a-5p and -3p are co-upregulated in the mouse and human myocardium with cardiac remodeling and in Ang-II-treated neonatal mouse ventricular cardiomyocytes (NMVCs) and cardiac fibroblasts (CFs). miR-199a-5p and -3p could aggravate cardiac hypertrophy and fibrosis in vivo and in vitro. PPAR gamma coactivator 1 alpha (Ppargc1a) and sirtuin 1 (Sirt1) were identified as target genes to mediate miR-199a-5p in promoting both cardiac hypertrophy and fibrosis. However, miR-199a-3p aggravated cardiac hypertrophy and fibrosis through targeting RB transcriptional corepressor 1 (Rb1) and Smad1, respectively. Serum response factor and nuclear factor κB p65 participated in the upregulation of miR-199a-5p and -3p in Ang-II-treated NMVCs and mouse CFs, and could be conversely elevated by miR-199a-5p and -3p. Together, Ppargc1a and Sirt1, Rb1 and Smad1 mediated the pathological effect of miR-199a-5p and -3p by promoting cardiac hypertrophy and fibrosis, respectively. This study suggests a possible new strategy for cardiac remodeling therapy by inhibiting miR-199a-5p and -3p.

MicroRNA-199a-3p Exhibits Beneficial Effects in Asymptomatic Atherosclerosis by Inhibiting Vascular Smooth Muscle Cell Proliferation and Migration.

Atherosclerosis (AS) is a serious healthy burden worldwide, it occurs accompany with the disfunction of vascular smooth muscle cells (VSMCs). MicroRNAs play pivotal role in the pathogenesis of various diseases. This study aimed to investigate the expression and clinical value of miR-199a-3p in patients with asymptomatic AS, and further explore its regulatory role on VSMCs biological function. Quantitative real-time PCR was used to estimate the expression of miR-199a-3p. Correlation of miR-199a-3p with carotid intima-media thickness (CIMT) and C-reactive protein (CRP) was evaluated by Pearson correlation coefficient. A receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of miR-199a-3p. Effects of miR-199a-3p on cell proliferation and migration in VSMCs were analyzed using cell-counting method and Transwell assay. Luciferase reporter assay was performed for the target gene analysis. Serum expression of miR-199a-3p was decreased in asymptomatic AS patients compared with the healthy controls. The negative correlations of miR-199a-3p with CIMT and CRP were obtained. The decreased miR-199a-3p was proved to have diagnostic accuracy with an area under the ROC curve (AUC) of 0.912, and was an independent predictor for the occurrence of asymptomatic AS. In VSMCs, overexpression of miR-199a-3p led to inhibited cell proliferation and migration, while the knockdown of miR-199a-3p resulted in the opposite results. SP1 was proved to be the target gene of miR-199a-3p. Taken together, downregulated expression of miR-199a-3p is a candidate diagnostic biomarker in the patients with asymptomatic AS. Overexpression of miR-199a-3p exists suppressive effects on VSMC proliferation and migration, indicating that miR-199a-3p may be a potential therapeutic target for AS treatment.

MicroRNA­199a­3p inhibits ovarian cancer cell viability by targeting the oncogene YAP1.

MicroRNA­199a­3p (miR­199a­3p) is aberrantly expressed in various types of cancer where it exhibits a tumor suppressive role. However, the biological role of miR­199a­3p in ovarian cancer (OC) remains unclear. The present study aimed to investigate whether miR­199a­3p was a tumor suppressor in OC and to identify the possible mechanisms. It was found that miR­199a­3p expression was significantly downregulated in the tumor tissues and blood samples of patients with OC, as well as in three OC cell lines. In addition, its low expression was closely associated with International Federation of Gynecology and Obstetrics disease stage, histological grade and lymph node metastasis. It was demonstrated that overexpression of miR­199a­3p inhibited the viability and promoted apoptosis of OV90 and SKOV­3 cells. In addition, Yes­associated protein 1 (YAP1), a well­known oncogene, was identified as a direct target of miR­199a­3p in OC cells. Additionally, it was observed that YAP1 was significantly increased and inversely correlated with miR­199a­3p expression in OC tissues. Notably, YAP1 overexpression abrogated the tumor suppressive effects of miR­199a­3p in vitro. Collectively, the present results indicated that miR­199a­3p suppressed viability in OC cells, at least partly via inhibiting the YAP1 oncogene, suggesting that miR­199a­3p may act as a biomarker and therapeutic target for patients with OC.

Physcion 8-O-ß-glucopyranoside inhibits testicular germ cell tumors through regulating microRNA-199a.

Testicular germ cell tumors (TGCTs) are frequently diagnosed solid tumors in young adult males. The present study aimed to investigate the anti-tumor function of Physcion 8-O-ß-Glucopyranoside (PG) in TGCTs, and to explore the underlying anti-tumor mechanism of PG in TGCTs. Cell viability was measured by a cell counting kit-8 (CCK-8) assay, and the cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was measured using TUNEL staining and flow cytometry. The lactate dehydrogenase (LDH) release, glucose consumption, lactate production, and ATP generation were determined using a Flex Bioanalyzer. The expression of miR-199a was measured by qRT-PCR, and the expression of marker proteins were analyzed by western blotting. Moreover, a xenograft mouse model was used for investigating PG's antitumor effect in vivo. The results showed that PG significantly inhibited NTERA2 and NCCIT cell proliferation, blocked the cell cycle, and induced cell apoptosis. Furthermore, PG suppressed LDH release, glucose consumption, lactate production, and ATP generation in NTERA2 and NCCIT cells. The results of investigating the molecular mechanism of PG in TGCTs showed that miR-199a was decreased in TGCTs but was increased by PG in a dose-dependent manner. MiR-199a knockdown significantly increased NTERA2 and NCCIT cell proliferation, decreased the apoptotic cell rates, and promoted LDH release, glucose consumption, lactate production, and ATP generation. The effects of PG on TGCTs were reversed by miR-199a silencing. In addition, we found that PG significantly inhibited tumor growth in vivo. In conclusion, our study revealed that PG modulated cell proliferation, the cell cycle, apoptosis, and glucose metabolism in TGCTs through regulating miR-199a. These findings indicate a novel therapeutic strategy and a potential biomarker for TGCT treatment.

miR­199a­3p suppresses cervical epithelial cell inflammation by inhibiting the HMGB1/TLR4/NF­κB pathway in preterm birth.

Preterm birth (PTB) is the primary cause of neonatal mortality worldwide. Infection and inflammation are considered to be the primary causes of PTB. Cervical remodeling is an important step in the process of preterm delivery, and the destruction of the cervical epithelial barrier and inflammation are important triggers of cervical remodeling. The aim of the present study was to determine the effect and underlying mechanism of microRNA (miR)­199a­3p/high­mobility group box 1 protein (HMGB1) signaling in cervical epithelial inflammation in PTB. The results of this study revealed that miR­199a­3p was significantly decreased in cervical epithelial tissue samples from patients in both the preterm labor and preterm premature rupture of membrane groups. This decrease was also observed in tissue samples from a lipopolysaccharide (LPS)­induced PTB mouse model and in LPS­induced ectocervical and endocervical cells. Whereas, the expression of HMGB1 and toll­like receptor 4 (TLR4) was significantly increased, which was associated with the upregulation of interleukin (IL)­1ß and tumor necrosis factor (TNF)­α expression. Furthermore, overexpression of miR­199a­3p significantly suppressed the expression and activation of HMGB1 and TLR4/NF­κB signaling, and decreased the levels of IL­1ß and TNF­α in vitro and in vivo. Additionally, overexpression of HMGB1 and/or TLR4 reversed the anti­inflammatory effects of miR­199a­3p mimics in vitro and in vivo. These results indicate that miR­199a­3p acts as a negative inflammatory regulator in PTB by targeting HMGB1 to regulate the TLR4/NF­κB pathway.

Reduced expression of microRNA-199a-3p is associated with vascular endothelial cell injury induced by type 2 diabetes mellitus.

The aim of the present study was to investigate the function and mechanism of action of microRNA (miRNA or miR)-199a-3p in vascular endothelial cell injury induced by type 2 diabetes mellitus (T2DM). A total of 36 patients with T2DM (26 males and 10 females; mean age, 52.5±7.0 years) and 20 healthy subjects (10 males and 10 females; mean age, 55.6±4.5 years) were included in the present study. Peripheral blood samples were obtained from all participants and total RNA was extracted Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression of miR-199a-3p. Following the transfection of human umbilical vein endothelial cells (HUVECs) with a negative control (NC) miRNA or miR-199a-3p mimics, cell proliferation was assessed using a Cell Counting kit-8 assay. Cell migration was investigated using Transwell assays and flow cytometry was performed to detect the apoptosis of HUVECs. HUVECs were infected with Ad-GFP-LC3B and laser-scanning confocal microscopy was performed to observe autophagosomes in HUVECs. Western blotting was used to measure the expression of proteins associated with autophagy and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor (NF)-κB signaling pathway. MiR-199a-3p was downregulated in peripheral blood from patients with T2DM compared with healthy subjects. Transfection with miR-199a-3p mimics promoted the proliferation and migration of HUVECs. However, miR-199a-3p overexpression inhibited the apoptosis of HUVECs. MiR-199a-3p facilitated HUVEC autophagy by affecting autophagy-associated signaling pathways. Furthermore, miR-199a-3p regulated the biological functions of HUVECs via the PI3K/AKT/NF-κB signaling pathway. The results of the present study suggest that miR-199a-3p expression was reduced in patients with T2DM compared with healthy subjects and may be associated with vascular endothelial cell injury. In addition, miR-199a-3p promoted the proliferation, migration and autophagy of HUVECs, potentially by regulating the PI3K/AKT/NF-κB signaling pathway. Therefore, miR-199a-3p may function as protector of vascular endothelia.

MicroRNA-199a-3p attenuates hepatic lipogenesis by targeting Sp1.

Emerging studies have demonstrated that microRNAs (miRs) are profoundly involved in non-alcoholic fatty liver disease (NAFLD) and related metabolic diseases. Previously, we revealed a repertoire of miRs dysregulated in NAFLD by high-throughput sequencing. Here, we showed that microRNA-199a-3p was down-regulated in the livers of C57BL/6J mice fed a high-fat-diet (HFD) and oleic acid/palmitic acid-induced Hepa1-6 cells. Gain-of-function and loss-of-function studies demonstrated that microRNA-199a-3p exhibited a suppressive role in hepatic lipogenesis. Adenoviral mediated microRNA-199a-3p expression in C57BL/6J mice largely attenuated triglyceride (TG) accumulation and expression of lipogenic genes. Furthermore, we identified Specificity Protein 1 (Sp1) as the functional target of miR-124. Restoration of Sp1 expression largely compromised the effect of microRNA-199a-3p on hepatic TG metabolism. Taken together, our findings uncover a novel function of microRNA-199a-3p/Sp1 axis in NAFLD and provide a mechanism underlying perturbations of hepatic TG homeostasis.