MiR-199a-5p aggravates against renal ischemia-reperfusion and transplant injury by targeting AKAP1 to disrupt mitochondrial dynamics.

Renal ischemia-reperfusion injury (IRI) is a major cause of delayed graft function (DGF) after transplantation. Currently, a targeted therapy for this important clinical disorder is still lacking. MicroRNA (miRNA) has important roles in the pathogenesis of IRI and may therapeutic approaches to mitigate renal IRI. METHODS: Small RNA sequencing was performed to profile microRNA expression in mouse kidneys after transplantation. Lentivirus incorporating a miR-199a-5p modulator was injected into mouse kidney in situ before unilateral IRI and syngenetic transplantation, to determine the effect of miR-199a-5p in vivo. miR-199a-5p mimic or inhibitor was transfected cultured tubular cells before renal tubular ATP depletion recovery treatment to the examine the role of miR-199a-5p in vitro. RESULTS: Sequencing showed upregulation of miR-199a-5p in post-transplantation mouse kidney following renal IRI was localized to renal tubular epithelial cells. Lentivirus incorporating a miR-199a-5p mimic aggravated renal IRI and opposing effects were obtained with a miR-199a-5p inhibitor. Treatment with the miR-199a-5p inhibitor ameliorated graft function loss, tubular injury and immune response after cold storage transplantation. In vitro experiments demonstrated aggravation of cell death caused by ATP depletion and repletion when the miR-199a-5p mimic was present while the miR-199a-5p inhibitor reduced cell death. miR-199a-5p was shown to target a-kinase anchoring protein 1(AKAP1) by double luciferase assay and miR-199a-5p activation reduced dynamin related protein 1 (Drp1)-s637 phosphorylation and mitochondrial length. Overexpression of AKAP1 preserved Drp1-s637 phosphorylation and reduced mitochondrial fission. CONCLUSION: MiR-199a-5p activation reduced AKAP1 expression, promoted Drp1-s637 dephosphorylation, aggravated the disruption of mitochondrial dynamics and contributed to ischemic kidney injury.

Alternative splicing-related long noncoding RNA ANRIL facilitates hepatocellular carcinoma by targeting the miR-199a-5p/SRSF1 axis and impacting Anillin.

Hepatocellular carcinoma (HCC) is characterized by aberrant alternative splicing (AS), which plays an important part in the pathological process of this disease. However, available reports about genes and mechanisms involved in AS process are limited. Our previous research has identified ANRIL as a long noncoding RNA related to the AS process of HCC. Here, we investigated the exact effect and the mechanism of ANRIL on HCC progress. The ANRIL expression profile was validated using the real-time quantitative polymerase chain reaction assay. The western blot analysis and IHC assay were conducted on candidate targets, including SRSF1 and Anillin. The clinicopathological features of 97 patients were collected and analyzed. Loss-of and gain-of-function experiments were conducted. The dual-luciferase reporter assay was applied to verify the interaction between ANRIL, miR-199a-5p, and SRSF1. Anomalous upregulation of ANRIL in HCC was observed, correlating with worse clinicopathological features of HCC. HCC cell proliferation, mobility, tumorigenesis, and metastasis were impaired by depleting ANRIL. We found that ANRIL acts as a sponger of miRNA-199a-5p, resulting in an elevated level of its target protein SRSF1. The phenotypes induced by ANRIL/miR-199a-5p/SRSF1 alteration are associated with Anillin, a validated HCC promoter. ANRIL is an AS-related lncRNA promoting HCC progress by modulating the miR-199a-5p/SRSF1 axis. The downstream effector of this axis in the development of HCC is Anillin.

ALKBH5-mediated m6A demethylation of pri-miR-199a-5p exacerbates myocardial ischemia/reperfusion injury by regulating TRAF3-mediated pyroptosis.

Myocardial ischemia‒reperfusion injury (MI/RI) is closely related to pyroptosis. alkB homolog 5 (ALKBH5) is abnormally expressed in the MI/RI models. However, the detailed molecular mechanism of ALKBH5 in MI/RI has not been elucidated. In this study, rats and H9C2 cells served as experimental subjects and received MI/R induction and H/R induction, respectively. The abundance of the targeted molecules was evaluated using RT-qPCR, Western blotting, immunohistochemistry, immunofluorescence, and enzyme-linked immunosorbent assay. The heart functions of the rats were evaluated using echocardiography, and heart injury was evaluated. Cell viability and pyroptosis were determined using cell counting Kit-8 and flow cytometry, respectively. Total m6A modification was measured using a commercial kit, and pri-miR-199a-5p m6A modification was detected by Me-RNA immunoprecipitation (RIP) assay. The interactions among the molecules were validated using RIP and luciferase experiments. ALKBH5 was abnormally highly expressed in H/R-induced H9C2 cells and MI/RI rats. ALKBH5 silencing improved injury and inhibited pyroptosis. ALKBH5 reduced pri-miR-199a-5p m6A methylation to block miR-199a-5p maturation and inhibit its expression. TNF receptor-associated Factor 3 (TRAF3) is a downstream gene of miR-199a-5p. Furthermore, in H/R-induced H9C2 cells, the miR-199a-5p inhibitor-mediated promotion of pyroptosis was reversed by ALKBH5 silencing, and the TRAF3 overexpression-mediated promotion of pyroptosis was offset by miR-199a-5p upregulation. ALKBH5 silencing inhibited pri-miR-199a-5p expression and enhanced pri-miR-199a-5p m6A modification to promote miR-199a-5p maturation and enhance its expression, thereby suppressing pyroptosis to alleviate MI/RI through decreasing TRAF3 expression.

The miR-199a-5p/HIF1α dual-regulatory axis participates in hypoxia-induced aggressive phenotypes of oral squamous cell carcinoma (OSCC) cells.

BACKGROUND: The late-stage diagnosis and distant metastasis of oral squamous cell carcinoma (OSCC) remain a huge challenge to clinical treatment for OSCC. During the past decades, targeting glycolysis-inducing factors becomes an attractive new strategy in OSCC therapies. METHODS: OSCC cells were stimulated with hypoxia or transfected with agomir-199a-5p, antagomir-199a-5p, and siRNA for HIF1A, cell proliferation was detected by CCK-8 assay; HIF1α, GLUT1, HK2 and LDHA expression levels were examined with western blot; miR-199 expression was determined with RT-PCR; cell migratory and invasive abilities were examined using wound healing and transwell assays; the lactate and glucose in culture medium were also determined. Luciferase assay or CHIP assay was applied for confirm the binding between miR-199a-5p and HIF1A 3'UTR, or between HIF1α and miR-199a promoter. RESULTS: HIF1α showed to be abnormally up-regulated, and miR-199a-5p showed to be abnormally down-regulated within OSCC under hypoxia. Hypoxia considerably enhanced OSCC cell proliferation, glycolysis, migratory ability, and invasive ability. MiR-199a-5p bound to HIF1A 3'-UTR and suppressed HIF1A expression; HIF1α targeted miR-199a-5p promoter region and downregulated miR-199a-5p expression. Under hypoxia, miR-199a-5p overexpression significantly repressed HIF1α up-regulation inresponse to hypoxia, OSCC cell proliferation, glycolysis, migratory ability, and invasive ability. CONCLUSION: miR-199a-5p and HIF1α form a dual-regulatory axis in OSCC cells; the miR-199a-5p/HIF1α dual-regulatory axis contributes to hypoxia-induced aggressive OSCC phenotypes.

MiR-199a-5p-containing macrophage-derived extracellular vesicles inhibit SMARCA4 and alleviate atherosclerosis by reducing endothelial cell pyroptosis.

BACKGROUND: Endothelial cell disturbance underpins a role in pathogenesis of atherosclerosis. Notably, accumulating studies indicate the substantial role of microRNAs (miRs) in atherosclerosis, and miR-199a-5p dysregulation has been associated with atherosclerosis and other cardiovascular disorders. However, the effect of miR-199a-5p on the phenotypes of endothelial cells and atherosclerosis remains largely unknown. METHODS: ApoE-/- male mice were fed with high-fat diet for detection of inflammation and aorta plaque area. Extracellular vesicles (EVs) were separated from THP-1-derived macrophage (THP-1-DM) that was treated by oxidized low-density lipoprotein, followed by co-culture with human aortic endothelial cells (HAECs). Ectopic expression and downregulation of miR-199a-5p were done in THP-1-DM-derived EVs to assess pyroptosis and lactate dehydrogenase (LDH) of HAECs. Binding relationship between miR-199a-5p and SMARCA4 was evaluated by luciferase activity assay. RESULTS: EVs derived from ox-LDL-induced THP-1-DM expedited inflammation and aorta plaque area in atherosclerotic mice. Besides, miR-199a-5p expression was reduced in EVs from ox-LDL-induced THP-1-DM, and miR-199a-5p inhibition facilitated HAEC pyroptosis and LDH activity. Moreover, miR-199a-5p targeted and restricted SMARCA4, and then SMARCA4 activated the NF-κB pathway by increasing PODXL expression in HAECs. CONCLUSION: EV-packaged inhibited miR-199a-5p from macrophages expedites endothelial cell pyroptosis and further accelerates atherosclerosis through the SMARCA4/PODXL/NF-κB axis, providing promising targets and strategies for the prevention and treatment of atherosclerosis.

Atherosclerosis-associated endothelial dysfunction is promoted by miR-199a-5p/SIRT1 axis regulated by circHIF1ɑ.

BACKGROUND AND AIMS: Atherosclerosis (AS) is a chronic inflammatory disease that damages the arterial wall as a result of hyperlipidemia and causes endothelial cell dysfunction, which increases the risk of atherothrombotic events. Multiple pathological conditions have shown ectopic miR-199a-5p levels to cause endothelial injury, but its role in the AS competitive endogenous RNA (CeRNA) network is still unknown. METHODS AND RESULTS: The high-fat diet (HFD) apoE-/- mouse model was constructed in vivo, and ECs were cultured under ox-LDL treatment to induce EC injury in vitro. Immunohistochemistry and immunofluorescence staining were used to assess the effect of miR-199a-5p on the macrophage, SMC, collagen content, and endothelial coverage in the artery wall of mouse model. miR-199a-5p level was validated to be overexpression in the aorta tissue of HFD apoE-/- mice and in the ox-LDL-treated ECs, and even in the plasma EVs of the patients with cerebral AS. Silencing of miR-199a-5p significantly attenuated atherosclerotic progress in HFD apoE-/- mice, and the gain/loss-of-function assay indicated that miR-199a-5p overexpression aggravated ox-LDL-induced disabilities of endothelial proliferation, motility, and neovascularization based on cell counting kit-8 assay, transwell assay and matrigel assay. Mechanistically, miR-199a-5p prevented EC activation by activating the FOXO signaling pathway by targeting SIRT1. Additionally, circular RNA (circRNA) circHIF1ɑ was identified as having a low expression in the ox-LDL-treated EC and mediated SIRT1 expression via sponging miR-199a-5p to rescue ox-LDL-induced EC injury. CONCLUSIONS: Our study demonstrated the vital role of miR-199a-5p/SIRT1 axis regulated by circHIF1ɑ in AS pathogenesis and provided novel effective targets for AS treatment.

miR-199a-5p from bone marrow mesenchymal stem cell exosomes promotes the proliferation of neural stem cells by targeting GSK-3ß.

Bone marrow mesenchymal stem cell (BMSC)-derived exosomes are a promising therapeutic agent for human disease, but their effects on neural stem cells (NSCs) subject to spinal cord ischaemia-reperfusion injury (SCIRI) remain unknown. Here, we examine the impact of miR-199a-5p-enriched exosomes derived from BMSCs on NSC proliferation. We establish a rat model of aortic cross-clamping to induce SCIRI in vivo and a primary NSC model of oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate SCIRI in vitro. CCK8, EdU, and BrdU assays are performed to evaluate the proliferation of NSCs. Hematoxylin and eosin (H&E) staining is used to determine the number of surviving neurons. The Basso, Beattie, and Bresnahan (BBB) scale and inclined plane test (IPT) are used to evaluate hind limb motor function. DiO-labelled exosomes are efficiently internalized by NSCs and increase ectopic amounts of miR-199a-5p, which promotes the proliferation of NSCs. In contrast, exosomes derived from miR-199a-5p-depleted BMSCs exert fewer beneficial effects. MiR-199a-5p targets and negatively regulates glycogen synthase kinase 3ß (GSK-3ß) and increases nuclear ß-catenin and cyclin D1 levels. miR-199a-5p inhibition reduces the total number of EdU-positive NSCs after OGD/R, but the GSK-3ß inhibitor CHIR-99021 reverses this effect. In vivo, intrathecal injection of BMSC-derived exosomes increases the proliferation of endogenous spinal cord NSCs after SCIRI. In addition, more proliferating NSCs are found in rats intrathecally injected with exosomes overexpressing miR-199a-5p. In summary, miR-199a-5p in BMSC-derived exosomes promotes NSC proliferation via GSK-3ß/ß-catenin signaling.

Sulforaphane suppresses skin squamous cell carcinoma cells proliferation through miR-199a-5p/Sirt1/CD44ICD signaling pathway.

BACKGROUND: The present study aimed to explore the impact of sulforaphane on the growth of sSCC cells, and the activation of miR-199a-5p/Sirt1 and CD44ICD signaling pathways. METHODS: Cell viability, count, apoptosis, and invasion assays were performed in the sSCC cell line (SCC-13) in which miR-199a-5p was over-expressed or under-expressed. The expression levels of miR-199a-5p, Sirt1 and CD44ICD mRNA were measured by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Sulforaphane significantly inhibited the cell growth and invasion of SCC-13 cells, and dramatically induced cell apoptosis. Additionally, sulforaphane also greatly increased miR-199a-5p expression and suppressed Sirt1 and CD44ICD mRNA levels. Moreover, miR-199a-5p overexpression considerably down-regulated the expressions of Sirt1 and CD44ICD mRNA, and promoted the ability of sulforaphane to represses cell growth and invasion, and to induce cell apoptosis. However, miR-199a-5p underexpression has the opposite effects. CONCLUSIONS: Sulforaphane appears to inhibit sCC progression by impacting its growth and invasion ability, and regulates miR-199a-5p/Sirt1 and CD44ICD signaling pathways, and may be utilized to develop a curative approach for sSCC.

MiR-199a-5p-Regulated SMARCA4 Promotes Oral Squamous Cell Carcinoma Tumorigenesis.

SWI/SNF related, matrix associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4, also known as BRG1), an ATPase subunit of the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex, plays an important regulatory role in many cytogenetic and cytological processes during cancer development. However, the biological function and mechanism of SMARCA4 in oral squamous cell carcinoma (OSCC) remain unclear. The present study aimed to investigate the role of SMARCA4 in OSCC and its potential mechanism. Using a tissue microarray, SMARCA4 expression was found to be highly upregulated in OSCC tissues. In addition, SMARCA4 upregulate expression led to increased migration and invasion of OSCC cells in vitro, as well as tumor growth and invasion in vivo. These events were associated with the promotion of epithelial-mesenchymal transition (EMT). Bioinformatic analysis and luciferase reporter assay confirmed that SMARCA4 is a target gene of microRNA miR-199a-5p. Further mechanistic studies showed that the miR-199a-5p regulated SMARCA4 can promote the invasion and metastasis of tumor cells through EMT. These findings indicate that the miR-199a-5p- SMARCA4 axis plays a role in tumorigenesis by promoting OSCC cell invasion and metastasis through EMT regulation. Our findings provide insights into the role of SMARCA4 in OSCC and the mechanism involved, which may have important implications for therapeutic purposes.

Diagnostic Significance of hsa-miR-21-5p, hsa-miR-192-5p, hsa-miR-155-5p, hsa-miR-199a-5p Panel and Ratios in Hepatocellular Carcinoma on Top of Liver Cirrhosis in HCV-Infected Patients.

Early hepatocellular carcinoma (HCC) diagnosis is challenging. Moreover, for patients with alpha-fetoprotein (AFP)-negative HCC, this challenge is augmented. MicroRNAs (miRs) profiles may serve as potential HCC molecular markers. We aimed to assess plasma homo sapiens-(hsa)-miR-21-5p, hsa-miR-155-5p, hsa-miR-192-5p, and hsa-miR-199a-5p-expression levels as a panel of biomarkers for HCC in chronic hepatitis C virus (CHCV) patients with liver cirrhosis (LC), especially AFP-negative HCC cases, as a step toward non-protein coding (nc) RNA precision medicine. SUBJECTS AND METHODS: 79 patients enrolled with CHCV infection with LC, subclassified into an LC group without HCC (n = 40) and LC with HCC (n = 39). Real-time quantitative PCR was used to measure plasma hsa-miR-21-5p, hsa-miR-155-5p, hsa-miR-192-5p, and hsa-miR-199a-5p. RESULTS: Plasma hsa-miR-21-5p and hsa-miR-155-5p demonstrated significant upregulation, while hsa-miR-199a-5p demonstrated significant downregulation in the HCC group (n = 39) when compared to the LC group (n = 40). hsa-miR-21-5p expression was positively correlated with serum AFP, insulin, and insulin resistance (r = 0.5, p < 0.001, r = 0.334, p = 0.01, and r = 0.303, p = 0.02, respectively). According to the ROC curves, for differentiating HCC from LC, combining AFP with each of hsa-miR-21-5p, hsa-miR-155-5p, and miR199a-5p improved the diagnostic sensitivity to 87%, 82%, and 84%, respectively, vs. 69% for AFP alone, with acceptable specificities of 77.5%, 77.5%, and 80%, respectively, and AUC = 0.89, 0.85, and 0.90, respectively vs. 0.85 for AFP alone. hsa-miR-21-5p/hsa-miR-199a-5p and hsa-miR-155-5p/hsa-miR-199a-5p ratios discriminated HCC from LC at AUC = 0.76 and 0.71, respectively, with sensitivities = 94% and 92% and specificities = 48% and 53%, respectively. Upregulation of plasma hsa-miR-21-5p was considered as an independent risk factor for HCC development [OR = 1.198(1.063-1.329), p = 0.002]. CONCLUSIONS: Combining each of hsa-miR-21-5p, hsa-miR-155-5p, and hsa-miR-199a-5p with AFP made it possible to identify HCC development in the LC patients' cohort with higher sensitivity than using AFP alone. hsa-miR-21-5p/hsa-miR-199a-5p and hsa-miR-155-5p/hsa-miR-199a-5p ratios are potential HCC molecular markers for AFP-negative HCC patients. hsa-miR-21-5p was linked, clinically and via in silico proof, to insulin metabolism, inflammation, dyslipidemia, and tumorigenesis in the HCC patients' group as well as for an upregulated independent risk factor for the emergence of HCC from LC in the CHCV patients.

LncRNA CDKN2B-AS1 contributes to glioma development by regulating the miR-199a-5p/DDR1 axis.

BACKGROUND: Although cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) is upregulated in glioma, its function and potential mechanism in glioma remain unclear. METHODS: CDKN2B-AS1 level in glioma tissues and cell lines LN229, U251, and U87 was measured by qRT-PCR. Loss-of-function assays using short hairpin RNA for CDKN2B-AS1 (sh-CDKN2B-AS1) were performed to evaluate the effect of CDKN2B-AS1 on cell invasion, migration, proliferation, and apoptosis. The relationship among CDKN2B-AS1, miR-199a-5p, and DDR1 was determined by bioinformatics analysis and luciferase reporter assay. Rescue experiments were conducted to explore the function of CDKN2B-AS1 and miR-199a-5p in glioma. An in vivo animal model of lentivirally transduced U87 glioma xenografts in mice was established to confirm the role of CDKN2B-AS1. RESULTS: CDKN2B-AS1 is significantly upregulated in glioma tissues and cell lines. CDKN2B-AS1 knockdown significantly inhibits cell proliferation, invasion, and migration, while promoting apoptosis of glioma cell lines U251 and U87. Further, a miR-199a-5p inhibitor attenuates the inhibitory effects of sh-CDKN2B-AS1 on these cell phenotypes. CDKN2B-AS1 positively regulates DDR1 expression by directly sponging miR-199a-5p. Moreover, CDKN2B-AS1 knockdown efficiently inhibits U87 tumor xenograft growth in mice. CONCLUSION: Our study reveals that CDKN2B-AS1 promotes glioma development by regulating the miR-199a-5p/DDR1 axis, suggesting that this lncRNA might be a potential therapeutic target.

The regulation of autophagy by the miR-199a-5p/p62 axis was a potential mechanism of small cell lung cancer cisplatin resistance.

BACKGROUND: Autophagy has been found to be involved in the multidrug resistance (MDR) of cancers, but whether it is associated with resistance of small cell lung cancer (SCLC) has not been studied. Here, we hypothesized that a potential autophagy-regulating miRNA, miR-199a-5p, regulated cisplatin-resistant SCLC. METHODS: We validated the MDR of H446/EP using CCK-8 and LDH. We tested the binding of miR-199a-5p to p62 using the Dual-Luciferase assay and validated the association of miR-199a-5p and p62 in SCLC samples. We overexpressed (OE) and knocked down (KD) miR-199a-5p in H446 and H446/EP and determined the expression of miR-199a-5p, autophagy-related proteins, and the formation of autophagolysosomes using QPCR, western blotting, and MDC staining respectively. These results were validated in an orthotopic H446 mouse model of SCLC. RESULTS: H446/EP was resistant to cisplatin, etoposide, paclitexal, epirubicin, irinotecan, and vinorelbine. Exposure of cisplatin at 5 µg/ml for 24 h increased LC3II/LC3I, ATG5, p62, and the formation of autophagolysosomes in H446 cells, but not in H446/EP cells. The expression of miR-199a-5p was up-regulated in H446/EP compared to H446. MiR-199a-5p directly targeted the p62 gene. The expression of miR-199a-5p and p62 were correlated in SCLC samples. In H446 and H69PR, the OE of miR-199a-5p increased LC3II/LC3I, p62, and the formation of autophagolysosomes, but not ATG5, while the KD of miR-199a-5p decreased p62, but did not affect LC3II/LC3I, ATG5, and the formation of autophagolysosomes. In H446/EP, the OE of miR-199a-5p decreased p62 only. These results were generally consistent to results in the animal tumor samples. CONCLUSIONS: The regulation of autophagy by the miR-199a-5p/p62 axis was a potential mechanism of small cell lung cancer cisplatin resistance.

The miR-199a-5p/PD-L1 axis regulates cell proliferation, migration and invasion in follicular thyroid carcinoma.

BACKGROUND: Follicular thyroid carcinoma (FTC) is the second most common cancer of the thyroid and easily develops into distant metastasis. PD-L1 is known to be associated with the carcinogenesis and progression of thyroid carcinoma. Our study aimed to investigate the biological functions of PD-L1 and to identify miRNAs that were responsible for modulating the activity of PD-L1. METHODS: A total of 72 patients with FTC at The Second Affiliated Hospital of Fujian Medical University were enrolled in this retrospective study. Immunohistochemical (IHC) assay was used to measure PD-L1 expression in FTC. The association between PD-L1 expression and clinicopathologic characteristics was evaluated. Bioinformatics analysis, RT-qPCR and western blotting were used to examine the relationships between miR-199a-5p, PD-L1 and Claudin-1. Cell proliferation, migration and invasion were evaluated by using CCK8 and Transwell migration and invasion assays. Target prediction and luciferase reporter assays were performed to verify the binding between miR-199a-5p and PD-L1. Rescue assay was performed to confirm whether PD-L1 downregulation abolished the inhibitory effect of miR-199a-5p. RESULTS: Among 72 pairs of tumor and normal specimens, the proportion of PD-L1 positive samples was higher in FTC tissues than in normal tissues. The results of ESTIMATE and CIBERSORT illustrated that there was a positive correlation between PD-L1 expression and immune infiltration, especially regulatory T cells and M1 macrophages. Prediction of immunotherapy revealed that patients with high PD-L1 expression might benefit from immune checkpoint inhibitors. Transwell migration and invasion assays showed that PD-L1 downregulation in FTC cells could significantly inhibit cell migration and invasion. The bioinformatics analysis and luciferase activity results indicated that PD-L1 was a potential target of miR-199a-5p. Knockdown of PD-L1 reversed the miR-199a-5p inhibitor mediated promotion effect. In addition, we found that PD-L1 expression was positively correlated with Claudin-1 expression and that miR-199a-5p affected the progression of FTC cells through the negative regulation of PD-L1 and Claudin-1. CONCLUSIONS: Our study revealed that PD-L1 expression was elevated in FTC and was closely associated with tumor aggressiveness and progression. MiR-199a-5p has a functional role in the progression and metastasis of FTC by regulating PD-L1 and Claudin-1 expression.

6-O-desulfated heparin attenuates myocardial ischemia/reperfusion injury in mice through the regulation of miR-199a-5p/klotho axis.

Heparin has been documented to reduce myocardial injury caused by ischemia/reperfusion (I/R), but its clinical application is limited due to its strong intrinsic anticoagulant property. Some desulfated derivatives of heparin display low anticoagulant activity and may have potential value as therapeutic agents for myocardial I/R injury. In this study, we observed that 6-O-desulfated heparin, a desulfated derivative of heparin, shortened the activated partial thromboplastin time and exhibited lower anticoagulant activity compared with heparin or 2-O-desulfated heparin (another desulfated derivative of heparin). Then, we explored whether 6-O-desulfated heparin could protect against myocardial I/R injury, and elucidated its possible mechanisms. Administration of 6-O-desulfated heparin significantly reduced creatine kinase activity, myocardial infarct size and cell apoptosis in mice subjected to 30 min of myocardial ischemia following 2 h of reperfusion, accompanied by a reverse in miR-199a-5p elevation, klotho downregulation and reactive oxygen species (ROS) accumulation. In cultured H9c2 cells, the mechanism of 6-O-desulfated heparin against myocardial I/R injury was further explored. Consistent with the results in vivo, 6-O-desulfated heparin significantly ameliorated hypoxia/reoxygenation-induced injury, upregulated klotho and decreased miR-199a-5p levels and ROS accumulation, and these effects were reversed by miR-199a-5p mimics. In conclusion, these results suggested that 6-O-desulfated heparin with lower anticoagulant activity attenuated myocardial I/R injury through miR-199a-5p/klotho and ROS signaling. Our study may also indicate that 6-O-desulfated heparin, as an excellent heparin derivative, is a potential therapeutic agent for myocardial I/R injury.

MicroRNA-199a-5p aggravates angiotensin II-induced vascular smooth muscle cell senescence by targeting Sirtuin-1 in abdominal aortic aneurysm.

Vascular smooth muscle cells (VSMCs) senescence contributes to abdominal aortic aneurysm (AAA) formation although the underlying mechanisms remain unclear. This study aimed to investigate the role of miR-199a-5p in regulating VSMC senescence in AAA. VSMC senescence was determined by a senescence-associated ß-galactosidase (SA-ß-gal) assay. RT-PCR and Western blotting were performed to measure miRNA and protein level, respectively. The generation of reactive oxygen species (ROS) was evaluated by H2DCFDA staining. Dual-luciferase reporter assay was used to validate the target gene of miR-199a-5p. VSMCs exhibited increased senescence in AAA tissue relative to healthy aortic tissue from control donors. Compared with VSMCs isolated from control donors (control-VSMCs), those derived from patients with AAA (AAA-VSMCs) exhibited increased cellular senescence and ROS production. Angiotensin II (Ang II) induced VSMC senescence by promoting ROS generation. The level of miR-199a-5p expression was upregulated in the plasma from AAA patients and Ang II-treated VSMCs. Mechanistically, Ang II treatment significantly elevated miR-199a-5p level, thereby stimulating ROS generation by repressing Sirt1 and consequent VSMC senescence. Nevertheless, Ang II-induced VSMC senescence was partially attenuated by a miR-199a-5p inhibitor or Sirt1 activator. Our study revealed that miR-199a-5p aggravates Ang II-induced VSMC senescence by targeting Sirt1 and that miR-199a-5p is a potential therapeutic target for AAA.

miR-199a-5p inhibits the proliferation of hepatocellular carcinoma cells by regulating CDC25A to induce cell cycle arrest.

BACKGROUND: Hepatocellular carcinoma (HCC) has been verified as a really common cancer worldwide. Several studies have suggested that the suppression of malignancy growth can be traced to miR-199a-5p. Even though CDC25A could activate the tumorigenesis of various cancer by modulating cell cycle, the modulation of the miR-199a-5p/CDC25A axis is still not clear in HCC. Our aim is to identify the modulation of the miR-199a-5p/CDC25A axis in HCC. METHODS: The expression of CDC25A and miR-199a-5p in HCC cells and tissues was assessed using qRT-PCR. After using western blot assay to confirm the protein level, luciferase reporter and RNA pull-down assays were performed to explore the relation between CDC25A and miR-199a-5p. Functional assays such as CCK8 assay, BrdU proliferation assay and flow cytometry analysis identified the cell progression. RESULTS: Experimental findings indicated the downregulation of miR-199a-5p in HCC samples. It was also found that miR-199a-5p overexpression declined the development of the cells with HCC and that it could bind to CDC25A to suppress the progression of HCC. CONCLUSION: Research suggested that miR-199a-5p could restrain the proliferation ability of HCC cells by regulating CDC25A, thus inducing cell-cycle arrest.

Circular RNA circVMA21 ameliorates lipopolysaccharide (LPS)-induced acute kidney injury by targeting the miR-199a-5p/NRP1 axis in sepsis.

BACKGROUND: Sepsis is a serious and elusive syndrome caused by infection, with high mortality worldwide. Circular RNAs vacuolar ATPase assembly factor (circVMA21) has been reported to be related to the inflammatory damages in sepsis. This study is designed to explore the role and mechanism of circVMA21 in the lipopolysaccharide (LPS)-induced cell injury in sepsis. METHODS: Cell viability and apoptosis were detected by CCK-8, and flow cytometry assays. CircVMA21, microRNA-199a-5p (miR-199a-5p), and Neuropilin-1 (NRP1) level were determined by RT-qPCR. Protein levels of Bcl-2, Bax, cleaved-caspase 3, and NRP1 were examined by Western blot assay. IL-1ß, IL-6, and TNF-α were detected using ELISA. Superoxide Dismutase (SOD) and glutathione (GSH) were measured by the special kits. The binding relationship between miR-199a-5p and circVMA21 or NRP1 was predicted by Starbase 3.0 and then verified by a dual-luciferase reporter and RIP assays. RESULTS: CircVMA21 and NRP1 were decreased, and miR-199a-5p was increased in LPS-induced THP-1 cells. Moreover, circVMA21 overexpression could repress LPS-mediated cell viability, apoptosis, inflammation, and oxidative stress in THP-1 cells. The mechanical analysis suggested that circVMA21 regulated NRP1 expression through sponging miR-199a-5p. CONCLUSION: CircVMA21 upregulation could attenuate LPS-triggered THP-1 cell injury through modulating the miR-199a-5p/NRP1 axis, hinting an underlying therapeutic strategy for sepsis patients.

Down-Regulation of ID2-AS1 Alleviates the Neuronal Injury Induced by 1-Methy1-4-Phenylpyridinium in Human Neuroblastoma Cell Line SH-SY5Y Cells Through Regulating miR-199a-5p/IFNAR1/JAK2/STAT1 Axis.

We aimed to illustrate the roles and molecular mechanisms of ID2-AS1 in parkinson's disease (PD). Methods: qRT-PCR detected the expression of ID2-AS1. CCK-8, LDH release assays the effect of ID2-AS1 knockdown on PD cells. Flow cytometry and Western Blot were used to detect the effect of ID2-AS1 inhibition on PD cell apoptosis. ELISA analysis showed that ID2-AS1 inhibition can reduce the inflammation of PD cells. ROS activity assay showed that inhibiting ID2-AS1 attenuated the oxidative stress induced by 1-methy1-4-phenylpyridinium (MPP+). RNA binding protein immunoprecipitation assay showed that ID2-AS1 is mainly located in the cytoplasm. The luciferase reporter assay is used to verify the interaction. In our study, ID2-AS1 was concentration-dependently and time-dependently up-regulated in MPP+ -treated human neuroblastoma cell line SH-SY5Y. ID2-AS1 knockdown enhanced cell proliferation and decreased cell death in PD cells. Knockdown of ID2-AS1 attenuates MPP+ -induced cytotoxicity in SH-SY5Y cells. ID2-AS1 is a sponge of miR-199a-5p. IFNAR1 is a target of miR-199a-5p. Inhibition of miR-199a-5p and overexpression of IFNAR1 alleviate the inhibitory effect of ID2-AS1 knockdown on MPP+ triggered neuronal injury. Inhibition of miR-199a-5p and overexpression of IFNAR1 alleviate the inhibitory effect of ID2-AS1 knockdown on MPP+ -triggered JAK2/STAT1 activation. Overall, down-regulation of ID2-AS1 alleviated the neuronal injury in PD through regulating miR-199a-5p/IFNAR1/JAK2/STAT1 axis.

mmu-miR-199a-5p regulates CYP2B10 through repression of E4BP4 in mouse AML-12 hepatocytes.

miR-199a-5p is an important regulator of many biological processes. However, whether and how CYP enzymes are regulated by miR-199a-5p are unknown. Here, we aimed to investigate the potential role of mmu-miR-199a-5p in regulating CYP2 enzymes.Regulatory effects of mmu-miR-199a-5p on CYP expression were assessed in mouse AML-12 hepatocytes. The metabolic activity of CYP2B10 was probed using cyclophosphamide (CPA) as a specific substrate. The regulatory mechanism was investigated using combined luciferase reporter assays and chromatin immunoprecipitation.Of several important drug-metabolizing CYPs, mmu-miR-199a-5p significantly increased the mRNA levels of Cyp2a10, Cyp2c29, and Cyp2j5 in AML-12 cells with Cyp2a10 altered the most. Consistently, mmu-miR-199a-5p enhanced the expression of CYP2B10 protein and cellular metabolism of CPA. Based on database analysis, Cyp2b10 was not a direct target gene of mmu-miR-199a-5p. Thus, a mediator is necessary for the miRNA regulation of CYP2B10. We found that E4BP4 repressed Cyp2b10 transcription and expression through specific binding to a D-box element in the gene promoter. Moreover, mmu-miR-199a-5p inhibited the expression of E4bp4 at the posttranscriptional level by directly targeting the 59-65 nt segment in its 3'-UTR.In conclusion, mmu-miR-199a-5p positively regulates CYP2B10 expression through inhibiting its repressor E4BP4. Our findings may provide an increased understanding of the complex regulatory pathways for CYP2B10.

[Chikusetsu saponin â £a ameliorates myocardial hypertrophy of rats through regulating expression of miR199a-5p/Atg5].

The present study investigated the effects of chikusetsu saponin Ⅳa(CHS Ⅳa) on isoproterenol(ISO)-induced myocardial hypertrophy in rats and explored the underlying molecular mechanism. ISO was applied to establish a rat model of myocardial hypertrophy, and CHS Ⅳa(5 and 15 mg·kg~(-1)·d~(-1)) was used for intervention. The tail artery blood pressure was measured. Cardiac ultrasound examination was performed. The ratio of heart weight to body weight(HW/BW) was calculated. Morphological changes in the myocardial tissue were observed by HE staining. Collagen deposition in the myocardial tissue was observed by Masson staining. The mRNA expression of myocardial hypertrophy indicators(ANP and BNP), autophagy-related genes(Atg5, P62 and beclin1), and miR199 a-5 p was detected by qRT-PCR. Atg5 protein expression was detected by Western blot. The results showed that the model group exhibited increased tail artery blood pressure and HW/BW ratio, thickened left ventricular myocardium, enlarged myocardial cells, disordered myocardial fibers with widened interstitium, and a large amount of collagen aggregating around the extracellular matrix and blood vessels. ANP and BNP were largely expressed. Moreover, P62 expression was up-regulated, while beclin1 expression was down-regulated. After intervention by CHS Ⅳa at different doses, myocardial hypertrophy was ameliorated and autophagy activity in the myocardial tissue was enhanced. Meanwhile, miR199 a-5 p expression declined and Atg5 expression increased. As predicted by bioinformatics, Atg5 was a target gene of miR199 a-5 p. CHS Ⅳa was capable of preventing myocardial hypertrophy by regulating autophagy of myocardial cells through the miR-199 a-5 p/Atg5 signaling pathway.