Vascular smooth muscle cell-specific miRNA-214 deficiency alleviates simulated microgravity-induced vascular remodeling.

The human cardiovascular system has evolved to accommodate the gravity of Earth. Microgravity during spaceflight has been shown to induce vascular remodeling, leading to a decline in vascular function. The underlying mechanisms are not yet fully understood. Our previous study demonstrated that miR-214 plays a critical role in angiotensin II-induced vascular remodeling by reducing the levels of Smad7 and increasing the phosphorylation of Smad3. However, its role in vascular remodeling evoked by microgravity is not yet known. This study aimed to determine the contribution of miR-214 to the regulation of microgravity-induced vascular remodeling. The results of our study revealed that miR-214 expression was increased in the forebody arteries of both mice and monkeys after simulated microgravity treatment. In vitro, rotation-simulated microgravity-induced VSMC migration, hypertrophy, fibrosis, and inflammation were repressed by miR-214 knockout (KO) in VSMCs. Additionally, miR-214 KO increased the level of Smad7 and decreased the phosphorylation of Smad3, leading to a decrease in downstream gene expression. Furthermore, miR-214 cKO protected against simulated microgravity induced the decline in aorta function and the increase in stiffness. Histological analysis showed that miR-214 cKO inhibited the increases in vascular medial thickness that occurred after simulated microgravity treatment. Altogether, these results demonstrate that miR-214 has potential as a therapeutic target for the treatment of vascular remodeling caused by simulated microgravity.

The novel circRNA circ_0045881 inhibits cell proliferation and invasion by targeting mir-214-3p in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer (BC). The circRNA-miRNA‒mRNA axis is a promising biomarker for the early diagnosis and prognosis of BC. However, the critical circRNA mediators involved in TNBC progression and the underlying regulatory mechanism involved remain largely unclear. METHODS: In this study, we carried out a circRNA microarray analysis of 6 TNBC patients and performed a gene ontology (GO) analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to characterize important circRNAs involved in TNBC progression. The interaction between circRNAs and miRNAs was determined by dual luciferase and RNA immunoprecipitation (RIP) assays. Moreover, Transwell, wound healing and Cell Counting Kit-8 (CCK8) assays were performed with altered circRNA or miRNA expression in MDA-MB-231 and BT-549 cells to investigate the roles of these genes in cell invasion, migration and proliferation. RESULTS: A total of 78 circRNAs were differentially expressed in TNBC tissues, and the hsa_circ_0045881 level was significantly decreased in TNBC tissues and cells. Lentivirus-mediated hsa_circ_0045881 overexpression in MDA-MB-231 and BT-549 cells significantly reduced cell invasion and migration capacity. Additionally, hsa_circ_0045881 interacted with miR-214-3p in MDA-MB-231 cells. miR-214-3p mimics in MDA-MB-231 and BT-549 cells significantly enhanced cell invasion, migration and proliferation, but the other combinations of inhibitors had opposite effects on cell activity. CONCLUSIONS: Our data indicated that the circRNA has_circ_0045881 plays key roles in TNBC progression and that hsa_circ_0045881 might act as a sponge for miR-214-3p to modulate its levels in TNBC cells, thereby regulating cell invasion, metastasis and proliferation. hsa_circ_004588 might be a potential prognostic marker and therapeutic target for TNBC.

MiR-214_L-1R+4 regulate gossypol-induced immune response through MyD88-dependent signaling pathway in Cyprinus carpio.

MicroRNAs (miRNAs) have been demonstrated to act as crucial modulators with considerable impacts on the immune system. Cottonseed meal is often used as a protein source in aqua feed, cottonseed meal contains gossypol, which is harmful to animals. However, there is a lack of research on the role of miRNAs in fish exposed to gossypol stress. To determine the regulatory effects of miRNAs on gossypol toxicity, Cyprinus carpio were given to oral administration of 20 mg/kg gossypol for 7 days, and the gossypol concentration in the tissues was tested. Then, we detected spleen index, histology, immune enzyme activities of fish induced by gossypol. The results of miRNA sequencing revealed 8 differentially expressed miRNAs in gossypol group, and miR-214_L-1R+4 was found involved in immune response induced by gossypol. The potential targets of miR-214_L-1R+4 were predicted, and found a putative miR-214_L-1R+4 binding site in the 3'UTR of MyD88a. Furthermore, dual-luciferase reporter assays displayed miR-214_L-1R+4 decreased MyD88a expression through binding to the 3'UTR of MyD88a. Moreover, miR-214_L-1R+4 antagomir were intraperitoneally administered to C. carpio, down-regulated miR-214_L-1R+4 could increase MyD88a expression, as well as inflammatory cytokines and anti-inflammatory cytokines expression. These findings revealed that miR-214_L-1R+4 via the MyD88-dependent signaling pathway modulate the immune response to gossypol in C. carpio spleen.

The lncRNA lnc_AABR07044470.1 promotes the mitochondrial-damaged inflammatory response to neuronal injury via miR-214-3p/PERM1 axis in acute ischemic stroke.

PURPOSE: We investigated the role of lnc_AABR07044470.1 on the occurrence and development of acute ischemic stroke (AIS) and neuronal injury by targeting the miR-214-3p/PERM1 axis to find a novel clinical drug target and prediction and treatment of AIS. METHODS: The mouse AIS animal model was used in vivo experiments and hypoxia/reoxygenation cell model in vitro was established. Firstly, infarction volume and pathological changes of mouse hippocampal neurons were detected using HE staining. Secondly, rat primary neuron apoptosis was detected by flow cytometry assay. The numbers of neuron, microglia and astrocytes were detected using immunofluorescence (IF). Furthermore, binding detection was performed by bioinformatics database and double luciferase reporter assay. Lnc_AABR07044470.1 localization was performed using fluorescence in situ hybridization (FISH).Lnc_AABR07044470.1, miR-214-3pand PERM1mRNA expression was performed using RT-qPCR. NLRP3, ASC, Caspase-1 and PERM1 protein expression was performed using Western blotting. IL-1ß was detected by ELISA assay. RESULTS: Mouse four-vessel occlusion could easily establish the animal model, and AIS animal model had an obvious time-dependence. HE staining showed that, compared with the sham group, infarction volume and pathological changes of mouse hippocampal neurons were deteriorated in the model group. Furthermore, compared with the sham group, neurons were significantly reduced, while microglia and astrocytes were significantly activated. Moreover, the bioinformatics prediction and detection of double luciferase reporter confirmed the binding site of lnc_AABR07044470.1 to miR-214-3p and miR-214-3p to Perm1. lnc_AABR07044470.1 and PERM1 expression was significantly down-regulated and miR-214-3pexpression was significantly up-regulated in AIS animal model in vivo. At the same time, the expression of inflammasome NLRP3, ASC, Caspase-1 and pro-inflammatory factor IL-1ß was significantly up-regulated in vivo and in vitro. The over-expression of lnc_AABR07044470.1 and miR-214-3p inhibitor could inhibit the neuron apoptosis and the expression of inflammasome NLRP3, ASC, Caspase-1 and pro-inflammatory factor IL-1ß and up-regulate the expression of PERM1 in vitro. Finally, over-expression of lnc_AABR07044470.1 and miR-214-3p inhibitor transfected cell model was significant in relieving the AIS and neuronal injury. CONCLUSION: Lnc_AABR07044470.1 promotes inflammatory response to neuronal injury via miR-214-3p/PERM1 axis in AIS.

Depression of LncRNA DANCR alleviates tubular injury in diabetic nephropathy by regulating KLF5 through sponge miR-214-5p.

OBJECTIVE: Diabetic nephropathy (DN) manifests a critical aspect in the form of renal tubular injury. The current research aimed to determine the function and mechanism of long non-coding ribonucleic acid (LncRNA) differentiation antagonising non-protein coding RNA (DANCR), with a focus on its impact on renal tubular injury. METHODS: Quantitative reverse transcription polymerase chain reaction was employed to analyze the RNA levels of DANCR in the serum of patients with DN or human proximal tubular epithelial cells (human kidney 2 [HK2]). The diagnostic significance of DANCR was assessed using a receiver operating characteristic curve. A DN model was established by inducing HK-2 cells with high glucose (HG). Cell proliferation, apoptosis, and the levels of inflammatory factors, reactive oxygen species (ROS), and malondialdehyde (MDA) were detected using the Cell Counting Kit - 8, flow cytometry, and enzyme-linked immunosorbent assay. The interaction between microRNA (miR)-214-5p and DANCR or Krüppel-like factor 5 (KLF5) was investigated using RNA immunoprecipitation and dual-luciferase reporter assays. RESULTS: Elevated levels of DANCR were observed in the serum of patients with DN and HG-inducted HK-2 cells (P < 0.05). DANCR levels effectively identified patients with DN from patients with type 2 diabetes mellitus. Silencing of DANCR protected against HG-induced tubular injury by restoring cell proliferation, inhibiting apoptosis, and reducing the secretion of inflammatory factors and oxidative stress production (P < 0.05). DANCR functions as a sponge for miR-214-5p, and the mitigation of DANCR silencing on HG-induced renal tubular injury was partially attenuated with reduced miR-214-5p (P < 0.05). Additionally, KLF5 was identified as the target of miR-214-5p. CONCLUSION: DANCR was identified as diagnostic potential for DN and the alleviation of renal tubular injury via the miR-214-5p/KLF5 axis, following DANCR silencing, introduces a novel perspective and approach to mitigating DN.

Abnormal expression and role of MicroRNA-214-3p/SLC8A1 in neonatal Hypoxic-Ischaemic encephalopathy.

Neonatal hypoxic-ischaemic encephalopathy (HIE) refers to brain damage caused by intra-uterine distress and asphyxia/hypoxia during the perinatal and neonatal periods. MicroRNA (MiR)-214-3p plays a critical role in cell growth and apoptosis. The aim of this study was to investigate the expression and role of miR-214-3p in neonatal HIE development, and to explore the underlying mechanisms. The expression of miR-214-3p was significantly down-regulated, while that of Slc8a1, a direct target of miR-214-3p, was significantly up-regulated, in the brain tissue of neonatal HIE rats. The over-expression of miR-214-3p promoted the proliferation and inhibited the apoptosis of neurones, while its down-regulation had the opposite effect. Our results indicate that miR-214-3p expression was down-regulated in neonatal HIE rats, and the up-regulation of miR-214-3p expression protected against HIE development by inhibiting neuronal apoptosis.

Exosomes from chondrocytes overexpressing miR-214-3p facilitate M2 macrophage polarization and angiogenesis to relieve Legg Calvé-Perthes disease.

OBJECTIVE: Legg-Calvé-Perthes disease (LCPD) is a partial or total necrosis of femoral head bone caused by blood supply disorder and its etiology is not clear. Studies have revealed that microRNA-214-3p (miR-214-3p) plays a vital role in LCPD, however, its exact mechanism is still unclear. In this study, we investigated the potential role of chondrocytes-derived exosomes carrying miR-214-3p (exos-miR-214-3p) in the pathogenesis of LCPD. METHODS: RT-qPCR was performed to evaluate miR-214-3p expression level in femoral head cartilage, serum and chondrocytes of patients with LCPD, as well as dexamethasone (DEX)-exposed TC28 cells. Effects of exos-miR-214-3p on the proliferation and apoptosis were verified via MTT assay, TUNEL staining and caspase3 activity assay. The M2 macrophage markers were assessed by flow cytometry, RT-qPCR and Western blot. Moreover, angiogenic effects of human umbilical vein endothelial cells (HUVECs) were tested using CCK-8 and tube formation assays. Bioinformatics prediction, luciferase assay and ChIP were applied to verify the association between ATF7, RUNX1 and miR-214-3p. RESULTS: miR-214-3p was found to be decreased in patients with LCPD and DEX-treated TC28 cells, of which overexpression promoted cell proliferation and suppressed apoptosis. Mechanistically, exos-miR-214-3p facilitated M2 polarization by ATF7/TLR4 axis and HUVECs angiogenesis via RUNX1/VEGFA axis. CONCLUSION: miR-214-3p alleviates LCPD by promoting M2 polarization of macrophages and angiogenesis.

Synovial fibroblast-miR-214-3p-derived exosomes inhibit inflammation and degeneration of cartilage tissues of osteoarthritis rats.

MicroRNAs (miRs) are regulators of number of cellular process. miRs enclosed within exosomes can be crucial regulators of intercellular signalling and could be an important biomarker of various age-associated disorders. Role of exosomal enclosed miRs in osteoarthritis (OA) chondrocytes and synovial fibroblasts (SFBs) remains poorly studied. Here, we profiled and studied the effect of synovial fluid-derived exosomal miRs on inflammation, survival, proliferation of chondrocyte in correlation with cartilage degeneration. Exosomes were isolated from synovial fluid collected from OA subjects and were analysed by transmission electron microscopy. miRs were isolated and were submitted to microarray profiling. Web-based PCR analysis was done. Chondrocyte proliferation and colony formation assay were performed. Apoptosis study was done by flow cytometer. Gene expression was done by qRT-PCR analysis and protein expression by western blot assay. Rat model of OA was created by operating the knee by anterior cruciate ligament and resection of medial menisci (ACLT + MMx) method. Micro-CT analysis, histological analysis, immunohistochemical staining, and TUNEL assay were also performed. About 17 miRs were found to be expressed differentially in the synovial fluid collected from the control and OA subjects. Microarray analysis confirmed, expression of miR-214-3p was significantly downregulated in the synovial fluid exosome of OA subjects. miR-214-3p mimic promoted proliferation of chondrocyte and suppressed apoptosis. Treatment also inhibited the levels of TNF-α, IL-1ß and IL-6. SFB-miR-214-3p exosomes suppressed apoptosis and also inflammation in chondrocytes. In vivo study suggested that SFB-exosomal miR-214-3p from rats suppressed the formation of osteophytes, prevented degeneration of cartilage and exerted anti-inflammatory and anti-apoptotic effect in articular cartilage tissue. The findings suggested that SFB-miR-214-3p exosomes can ameliorate chondrocyte inflammation and degeneration of cartilage tissues. The study confirms therapeutic potential of SFB-miR-214-3p exosomes in treating OA.

Transcriptome sequencing to explore the effect of miR-214 on chicken primary myoblasts.

MicroRNAs are involved in a series of biological processes, such as proliferation, differentiation and apoptosis of primary myoblasts. The research group found that miR-214 is highly expressed in chicken primary myoblasts (CPMs), so we used miR-214 as a starting point to explore the biological function of miR-214 in skeletal muscle growth and development. In this experiment, CPMs were cultured in vitro; miR-214 was overexpressed in CPMs; and cell samples were collected for subsequent transcriptome sequencing (RNA-seq). After miR-214 overexpression, we identified 97 differentially expressed genes (DEGs), of which 21 DEGs were up-regulated and 76 DEGs were down-regulated. After bioinformatics analysis, these DEGs were found to be significantly enriched in myofibrils, muscle system processes, myofibril assembly and other biological processes related to muscle development. The significantly enriched KEGGs include focal adhesion and type II diabetes mellitus. The protein network of DEGs was drawn by STRING and Cytoscape software, and 5 DEGs were randomly selected to verify the sequencing results by real-time fluorescence quantification. CAV3 is not only an important node protein in the protein network but also a member of the focal adhesion signaling pathway. It is speculated that miR-214 may regulate muscle development through the focal adhesion signaling pathway.

LINC00536 Promotes Breast Cancer Progression by Regulating ROCK1 via Sponging of miR-214-5p.

Accumulating evidence has shown that long noncoding RNAs (lncRNAs) play a significant role in regulating gene expression and participating in the progression of various malignancies. In our study, by analyzing data from The Cancer Genome Atlas (TCGA), LINC00536 was found to be highly expressed in breast cancer (BC) tissues, but its function and clinical significance in BC are still unknown. Therefore, we aimed to explore the role and molecular mechanism of LINC00536 in BC. We collected human BC tissue specimens and validated that LINC00536 was overexpressed in BC tissues. Increased LINC00536 expression was associated with advanced TNM stage, larger tumor diameter, lymph node metastasis and poor prognosis in patients with BC. Univariate and multivariate Cox regression analyses showed that high LINC00536 expression was an independent prognostic risk factor for overall survival in BC patients. Furthermore, quantitative reverse transcription PCR (qRT-PCR) showed that LINC00536 was upregulated in BC cell lines. Then, we confirmed that LINC00536 silencing-inhibited BC cell proliferation, migration, and invasion and led to cell cycle arrest in vitro. Animal experiments showed that knockdown of LINC00536 expression suppressed tumorigenesis in vivo. Mechanistically, LINC00536 serves as a ceRNA for miR-214-5p, increasing the expression of ROCK1, which acts as a tumor promoter in BC. Rescue assays revealed that miR-214-5p inhibition or ROCK1 overexpression could neutralize the suppressive effects of LINC00536 knockdown on cell proliferation, migration and invasion. Our data indicated that LINC00536 accelerates BC progression by regulating the miR-214-5p/ROCK1 pathway, which might provide a new perspective to investigate the development process of BC.

miR-125b-5p, miR-155-3p, and miR-214-5p and Target E2F2 Gene in Oral Squamous Cell Carcinoma.

It is known that E2F2 (E2F transcription factor 2) plays an important role as controller in the cell cycle. This study aimed to analyse the expression of the E2F2 gene and E2F2 protein and demonstrate E2F2 target microRNAs (miRNAs) candidates (miR-125b-5p, miR-155-3p, and miR-214-5p) in oral squamous cell carcinoma tumour and margin samples. The study group consisted 50 patients. The E2F2 gene and miRNAs expression levels were assessed by qPCR, while the E2F2 protein was assessed by ELISA. When analysing the effect of miRNAs expression on E2F2 gene expression and E2F2 protein level, we observed no statistically significant correlations. miR-125b-5p was downregulated, while miR-155-3p, and miR-214-5p were upregulated in tumour samples compared to margin. We observed a difference between the miR-125b-5p expression level in smokers and non-smokers in margin samples. Furthermore, HPV-positive individuals had a significantly higher miR-125b-5p and miR-214-5p expression level compared to HPV-negative patients in tumour samples. The study result showed that the E2F2 gene is not the target for analysed miRNAs in OSCC. Moreover, miR-155-3p and miR-125b-5p could play roles in the pathogenesis of OSCC. A differential expression of the analysed miRNAs was observed in response to tobacco smoke and HPV status.

Circular RNA circFOXO3 regulates KDM2A by targeting miR-214 to promote tumor growth and metastasis in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a pathological type of oral cancer, which accounts for over 90% of oral cancers. It has been widely shown that circRNA is involved in the regulation of multiple malignant oral diseases including OSCC. However, the mechanism underlying how circRNA regulates OSCC is still not clearly elucidated. In this article, we report circFOXO3 promotes tumor growth and invasion of OSCC by targeting miR-214 which specifically degrades the lysine demethylase 2A (KDM2A). CircRNA sequencing was conducted in OSCC tumor and tumor-side tissues, and the expression of circFOXO3 is found to be markedly increased in tumor tissues. CircFOXO3 is also highly expressed in several OSCC cell lines compared with human oral keratinocytes. Transwell assay and colony formation showed that knockdown of circFOXO3 prevents the invasion and proliferation of oral cancer cells. Via bioinformatic research, miR-214 was found to be the target of circFOXO3 and correlate well with circFOXO3 both in vitro and in vivo. KDM2A was then validated by database analysis and luciferase assay to be the direct target of miR-214. KDM2A helps to promote tumor invasiveness and proliferation of OSCC. Collectively, our results proved that circFOXO3 sponges miR-214 to up-regulate the expression of KDM2A, thus promotes tumor progression in OSCC.

Study on the Role of MicroRNA-214 in the Rehabilitation of Cartilage in Mice with Exercise-Induced Traumatic Osteoarthritis.

This study aimed to explore the possible relationship between the expression of Micro RNA-214 (miR-214) and the pathogenesis and recovery in mice with post-traumatic osteoarthritis (PTOA). In this study, 40 male C57BL/6 mice were randomly divided into five groups: model control (MC) group, model (M) group, rehabilitation control (RC) group, model + rehabilitation (M + R) group, and model + convalescent (M + C) group. Four weeks of high-intensity treadmill exercise (HITE) and 4 weeks of moderate-intensity treadmill exercise (MITE) were implemented for PTOA modeling and rehabilitation, respectively. In vitro, 10% elongation mechanical strain was used for IL-1ß stimulated chondrocytes. We found that compared with the MC group, there was a significant increase in the aspect of inflammation and catabolism while a dramatic fall in miR-214 expression was observed in the M group. After the 4 weeks of MITE, the level of inflammation and metabolism, as well as miR-214 expression, was partially reversed in the M + R group compared with the M + C group. The expression of miR-214 decreased dramatically after chondrocyte stimulation by IL-1ß and then increased significantly after 10% strain was applied to IL-1ß-treated cells. These results suggest that a suitable mechanical load can increase the expression of miR-214, and that miR-214 may play a chondroprotective effect in the development of OA.

ATF1/miR-214-5p/ITGA7 axis promotes osteoclastogenesis to alter OVX-induced bone absorption.

BACKGROUND: The dynamic balance of osteoblast and osteoclast is critical for bone homeostasis and overactive osteoclastic function may lead to osteoporosis. Activating transcription factor 1 (ATF1) is involved in osteoclastogenesis. However, the detailed mechanisms remain to be explored. METHODS: RAW264.7 cells were used and induced toward osteoclast by RANKL administration. We performed flow cytometry, CCK-8 assay and tartrate-resistant acid phosphatase (TRAP) staining to examine cell apoptosis, proliferation and differentiation of RAW264.7 cells, respectively. Mice were subjected to ovariectomy to induce osteoporosis. Micro CT, HE staining and TRAP staining were performed to evaluate bone loss in the OVX mouse model. Bioinformatics methods, luciferase assays and Chromatin Immunoprecipitation (ChIP) were used to predict and validate the interaction among ATF1, miR-214-5p, and ITGA7. RESULTS: ATF1 and miR-214-5p were up-regulated while ITGA7 was inhibited in RANKL-induced osteoclasts. MiR-214-5p was transcriptionally activated by ATF1. ATF1 knockdown suppressed osteoclast formation by miR-214-5p inhibition. ITGA7 was the direct target of miR-214-5p. Knockdown of miR-214-5p abolished osteoclastogenesis, which was reversed by ITGA7 knockdown. In OVX model, miR-214-5p knockdown suppressed osteoclast differentiation and prevented bone loss. CONCLUSION: ATF1/miR-214-5p/ITGA7 axis regulated osteoclast formation both in vivo and in vitro, thereby affecting OVX-induced bone resorption in mice. Knockdown of ATF1 might be a promising strategy to manage osteoporosis.

The hsa-miR-214-3p/ATGL axis regulates aberrant lipolysis to promote acute myeloid leukemia progression via PPARα in vitro.

Aberrant lipid metabolism is a hallmark of malignant cancers. Recent studies have shown that abnormal activation of the lipolysis pathway might contribute to acute myeloid leukemia (AML) progression. However, the molecular mechanism through which lipid metabolism mediates AML progression is unknown. RNA-sequencing was used to screen out the target gene pnpla2/ATGL(adipose triglyceride lipase), which showed differential expression in AML. A comparison was made of ATGL mRNA levels in different AML cell lines by real-time PCR. ATGL expression was blocked using siRNAs, and then ATGL expression, proliferation, apoptosis, and cell cycle progression of si-ATGL AML cell lines and si-control AML cell lines were respectively tested. Online tools were used to analyze the potential target microRNAs of ATGL. The mechanism through which hsa-miR-214-3p regulates ATGL was detected by western blotting, proliferation assays, flow cytometry, and dual-luciferase reporter assays. Our results showed that ATGL was overexpressed in AML cell lines. Moreover, ATGL promoted the growth of AML cells. Additionally, hsa-miR-214-3p could suppress ATGL. Finally, we show that hsa-miR-214-3p regulates ATGL through the hsa-miR-214-3p/ATGL/PPARα pathway. This study showed that hsa-miR-214-3p-regulates aberrant lipolysis by promoting ATGL expression, which causes AML progression through the PPARα pathway.

Vascular smooth muscle cell-specific miRNA-214 knockout inhibits angiotensin II-induced hypertension through upregulation of Smad7.

Vascular remodeling is a prominent trait during the development of hypertension, attributable to the phenotypic transition of vascular smooth muscle cells (VSMCs). Increasing studies demonstrate that microRNA plays an important role in this process. Here, we surprisingly found that smooth muscle cell-specific miR-214 knockout (miR-214 cKO) significantly alleviates angiotensin II (Ang II)-induced hypertension, which has the same effect as that of miR-214 global knockout mice in response to Ang II stimulation. Under the treatment of Ang II, miR-214 cKO mice exhibit substantially reduced systolic blood pressure. The vascular medial thickness and area in miR-214 cKO blood vessels were obviously reduced, the expression of collagen I and proinflammatory factors were also inhibited. VSMC-specific deletion of miR-214 blunts the response of blood vessels to the stimulation of endothelium-dependent and -independent vasorelaxation and phenylephrine and 5-HT induced vasocontraction. In vitro, Ang II-induced VSMC proliferation, migration, contraction, hypertrophy, and stiffness were all repressed with miR-214 KO in VSMC. To further explore the mechanism of miR-214 in the regulation of the VSMC function, it is very interesting to find that the TGF-ß signaling pathway is mostly enriched in miR-214 KO VSMC. Smad7, the potent negative regulator of the TGF-ß/Smad pathway, is identified to be the target of miR-214 in VSMC. By which, miR-214 KO sharply enhances Smad7 levels and decreases the phosphorylation of Smad3, and accordingly alleviates the downstream gene expression. Further, Ang II-induced hypertension and vascular dysfunction were reversed by antagomir-214. These results indicate that miR-214 in VSMC established a crosstalk between Ang II-induced AT1R signaling and TGF-ß induced TßRI /Smad signaling, by which it exerts a pivotal role in vascular remodeling and hypertension and imply that miR-214 has the potential as a therapeutic target for the treatment of hypertension.

Novel long non-coding RNA lncAMPC downregulates PTEN via miR-214 to promote nasopharyngeal carcinoma progression.

Long non-coding RNAs (lncRNAs) are a type of non-coding RNAs with transcript lengths exceeding 200 nt. lncRNAs suppress or promote cancer mainly by regulating gene expression. The aim of this study was to explore the role of lncRNAs activated in metastatic PCa (lncAMPC) in nasopharyngeal carcinoma (NPC). Total RNAs were isolated from NPC and adjacent non-tumor tissues from 60 NPC patients and subjected to RT-qPCR to analyze differential expression of lncAMPC and miR-214. The roles of lncAMPC and miR-214 in regulating PTEN expression were analyzed using RT-qPCR and Western blot. Cell proliferation was analyzed using the BrdU assay. The results showed that lncAMPC was overexpressed in NPC tissues and was localized in both nuclei and cytoplasms of NPC cells. MiR-214 was positively correlated with lncAMPC. LncAMPC overexpression upregulated miR-214 and indirectly downregulated PTEN via miR-214. BrdU incorporation assay showed that lncAMPC and miR-214 overexpression increased cell proliferation. PTEN overexpression completely reversed the promoting effects of lncAMPC and miR-214 overexpression on cell proliferation. Therefore, lncAMPC might downregulate PTEN via miR-214 to increase NPC cell proliferation.

lncRNAs MALAT1 and LINC00657 upstream to miR-214-3p/BMP2 regulate osteogenic differentiation of human mesenchymal stem cells.

BACKGROUND: Osteogenic differentiation of human mesenchymal stem cells (hMSCs) holds significant clinical implications for patients with bone diseases. LncRNAs are an emerging group of epigenetic modulators involved in the osteogenesis of hMSCs. In this study, we explored lncRNA profiles that are upstream to the hsa-miR-214-3p/BMP2 axis in osteogenic differentiation of hMSCs. METHOD: HMSCs were induced toward osteogenesis for 14 days. Between day 1 and day 14, qRT-PCR was conducted to compare the expressions of BMP2, Runx2, hsa-miR-214-3p, and biochemical assays to compare alkaline phosphatase and Alizarin Red S activities. 145 lncRNAs, which were experimentally confirmed upstream to hsa-miR-214-3p were examined. Five significantly upregulated lncRNAs, MEG3, SNHG16, FAM83H-AS1, MALAT1 and LINC00657 were downregulated in differentiated hMSCs and their impact on osteogenic differentiation were examined. Hsa-miR-214-3p was silenced in lncRNAs-downregulated hMSCs to further examine the association between lncRNAs and hsa-miR-214-3p/BMP2 axis. RESULTS: From day 1 to day 14, hMSCs underwent significant osteogenic differentiation, and KCNQ1OT1, MEG3, SNHG16, FAM83H-AS1, MALAT1 and LINC00657 were significantly upregulated. Downregulations of MEG3, SNHG16, FAM83H-AS1, MALAT1 and LINC00657 all suppressed osteogenic differentiation. However, qRT-PCR and RIP assay demonstrated that only MALAT1 and LINC00657 acted through hsa-miR-214-3p/BMP2 to regulate osteogenic differentiation. Furthermore, silencing hsa-miR-214-3p only rescued osteogenic differentiation in MALAT1- or LINC00657- downregulated hMSCs. CONCLUSIONS: Our data strongly indicated that lncRNAs MALAT1 and LINC00657 acted through miR-214-3p/BMP2 axis to regulate osteogenic differentiation of hMSCs.

The lncRNA DANCR promotes development of atherosclerosis by regulating the miR-214-5p/COX20 signaling pathway.

BACKGROUND: Although long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) has been reported to be involved in atherosclerosis (AS) development, its specific mechanism remains unclear. METHODS: DANCR expression levels in blood samples of AS patients and oxidized low-density lipoprotein (ox-LDL) treated vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The small interfering RNA targeting DANCR (si-DANCR) was used to silence DANCR expression. Cell viability was assessed by CCK-8 assay. Cell apoptosis was evaluated by flow cytometry. Levels of inflammatory cytokines, anti-oxidative enzyme superoxide dismutase (SOD) activity, and malonaldehyde (MDA) were detected by specific commercial kits. An animal AS model was established to confirm the role of DANCR/microR-214-5p/COX20 (the chaperone of cytochrome c oxidase subunit II COX2) in AS development. RESULTS: DANCR was significantly increased in the blood samples of AS patients and ox-LDL treated VSMCs and HUVECs. DANCR downregulation obviously increased viability and reduced apoptosis of ox-LDL-treated VSMCs and HUVECs. Meanwhile, DANCR downregulation reduced the levels of inflammatory cytokines, including interleukin (IL)-6 (IL-6), IL-1beta (IL-1ß), IL-6 and tumor necrosis factor (TNF)-alpha (TNF-α) and MDA while increasing the SOD level in ox-LDL-treated VSMCs and HUVECs. DANCR regulated COX20 expression by acting as a competing endogenous RNA (ceRNA) of miR-214-5p. Rescue experiments demonstrated that miR-214-5p downregulation obviously attenuated si-DANCR-induced protective effects on ox-LDL-caused endothelial injury. CONCLUSIONS: Our results revealed that DANCR promoted AS progression by targeting the miR-214-5p/COX20 axis, suggesting that DANCR might be a potential therapeutic target for AS.

CircFNDC3B knockdown restrains the progression of oesophageal squamous cell carcinoma through miR-214-3p/CDC25A axis.

Circular RNA (circRNAs) Fibronectin Type III Domain Containing 3B (FNDC3B) (circFNDC3B) has been revealed to be involved in the progression of oesophageal squamous cell carcinoma (ESCC). Hence, the potential regulatory network of circFNDC3B in ESCC was further investigated. Levels of genes and proteins were examined by qRT-PCR and Western blot. In vitro assays were performed using colony formation assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, wound healing assay, and transwell assay. The target relationship between miR-214-3p and circFNDC3B or cell division cycle 25 homologue A (CDC25A) was verified by dual-luciferase reporter and RIP assays. In vivo assay was carried out using the xenograft nude mice model. CircFNDC3B was highly expressed in ESCC, and high circFNDC3B expression was tightly associated with poor prognosis in ESCC patients. Functionally, circFNDC3B knockdown not only suppressed ESCC cell growth, migration and invasion in vitro, but hindered ESCC tumour growth in vivo. Mechanistically, circFNDC3B acted as a sponge for miR-214-3p to up-regulate the expression of its target CDC25A. Rescue experiments showed that miR-214-3p inhibitor reversed the anticancer effects of circFNDC3B knockdown. Moreover, forced expression of miR-214-3p suppressed the malignant phenotypes mentioned above, while this condition was abolished by CDC25A overexpression. CircFNDC3B silencing restrains the tumorigenesis of oesophageal squamous cell carcinoma through miR-214-3p/CDC25A axis, which opens a new window to the development of novel therapeutic strategy for ESCC patients.