LncRNA-NEAT1 blocks the Wnt/ß-catenin signaling pathway by targeting miR-217 to inhibit trophoblast cell migration and invasion.

OBJECTIVE: This study aimed to study the correlation between preeclampsia (PE) and lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), and to examine the molecular mechanisms behind the development of PE. METHODS: 30 PE and 30 normal pregnant women placental samples were assessed the levels of NEAT1 and miR-217 by quantitative real-time PCR (qRT-PCR). The trophoblast cell line HTR8/SVneo was used for silencing NEAT1 or miR-217 inhibitor in the absence or presence of an inhibitor and H2O2. Cell counting Kit 8 (CCK-8), flow cytometry, and Transwell were used to detect cell proliferation, apoptosis, migration, and invasion. Luciferase reporter gene assay was utilized to verify the binding between miR-217 and Wnt family member 3 (Wnt3), and between the miR-217 and NEAT1. Proteins related to the Wnt/ß-catenin signaling pathway were detected using western blotting. RESULTS: The PE group exhibited a significantly downregulated expression of miR-217 and a significantly upregulated expression of NEAT1. NEAT1 targeted miR-217, and Wnt is a miR-217 target gene. siRNA-NEAT1 inhibited the apoptosis of trophoblast cells, but promoted their invasion, migration, and proliferation. MiR-217 inhibitor could partially reverse the effects of siRNA-NEAT1. The expression of the Wnt/ß-catenin signaling pathway-related proteins, WNT signaling pathway inhibitor 1 (DKK1), cyclin-D1 and ß-catenin, was significantly increased after siRNA-NEAT1. CONCLUSIONS: NEAT1 could reduce trophoblast cell invasion and migration by suppressing miR-217/Wnt signaling pathway, leading to PE.

Extracellular vesicles derived from mesenchymal stem cells confer protection against intervertebral disc degeneration through a microRNA-217-dependent mechanism.

OBJECTIVE: Extracellular vesicles released by mesenchymal stem cells (MSC-EVs) can be applied to alleviate intervertebral disc degeneration (IVDD) by curbing apoptosis of nucleus pulposus cells (NPCs). The current study aims to evaluate the effect of MSC-EVs on NPC apoptosis and IVDD and the related regulatory mechanisms involving microRNA (miR)-217. METHOD: Expression of miR-217 was examined in tumor necrosis factor-α (TNF-α)-induced NPCs and MSC-EVs, followed by identification in the relationship between miR-217, enhancer of zeste homolog 2 (EZH2) and forkhead box O-3 (FOXO3). After isolation of EVs from MSCs and subsequent co-culture with NPCs, we assessed effects of miR-217 on NPC viability, autophagy, senescence and apoptosis along with extracellular matrix (ECM) degradation. Further in vivo experiments were conducted in rat models of IVDD to substantiate the effect of miR-217 on IVDD. RESULTS: Poor miR-217 expression was found in TNF-α-induced NPCs, while high miR-217 expression was identified in MSC-EVs (P < 0.05). MSC-EVs transferred miR-217 to NPCs and increased its expression, thus attenuating NPC apoptosis and ECM degradation (elevated collagen II and aggrecan but reduced MMP13 and ADAMTS5) (P < 0.05). miR-217 targeted EZH2, and EZH2 bound to the FOXO3 promoter and consequently downregulated its expression. FOXO3 restrained NPC apoptosis and ECM degradation by stimulating cell autophagy (P < 0.05). Furthermore, in vivo experimental results confirmed the suppressive role of miR-217 shuttled by MSC-EVs in IVDD. CONCLUSION: Overall, the delivery of miR-217 may be a novel mechanism underlying the effect of MSC-EVs on NPC apoptosis and ECM degradation following IVDD.

Baicalin augments the differentiation of osteoblasts via enhancement of microRNA-217.

Baicalin (BAI), a sort of flavonoid monomer, acquires from Scutellaria baicalensis Georgi, which was forcefully reported in diversified ailments due to the pleiotropic properties. But, the functions of BAI in osteoblast differentiation have not been addressed. The intentions of this study are to attest the influences of BAI in the differentiation of osteoblasts. MC3T3-E1 cells or rat primary osteoblasts were exposed to BAI, and then cell viability, ALP activity, mineralization process, and Runx2 and Ocn expression were appraised through implementing CCK-8, p-nitrophenyl phosphate (pNPP), Alizarin red staining, western blot, and RT-qPCR assays. The microRNA-217 (miR-217) expression was evaluated in MC3T3-E1 cells or rat primary osteoblasts after BAI disposition; meanwhile, the functions of miR-217 in BAI-administrated MC3T3-E1 cells were estimated after miR-217 inhibitor transfection. The impacts of BAI and miR-217 inhibition on Wnt/ß-catenin and MEK/ERK pathways were probed to verify the involvements in BAI-regulated the differentiation of osteoblasts. BAI accelerated cell viability, osteoblast activity, and Runx2 and Ocn expression in MC3T3-E1 cells or rat primary osteoblasts, and the phenomena were mediated via activations of Wnt/ß-catenin and MEK/ERK pathways. Elevation of miR-217 was observed in BAI-disposed MC3T3-E1 cells or rat primary osteoblasts, and miR-217 repression annulled the functions of BAI in MC3T3-E1 cell viability and differentiation. Additionally, the activations of Wnt/ß-catenin and MEK/ERK pathways evoked by BAI were both restrained by repression of miR-217. These explorations uncovered that BAI augmented the differentiation of osteoblasts via activations of Wnt/ß-catenin and MEK/ERK pathways by ascending miR-217 expression.

MicroRNA-217 ameliorates inflammatory damage of endothelial cells induced by oxidized LDL by targeting EGR1.

Oxidized low-density lipoprotein (ox-LDL) modulates gene transcription and expression and induces the development of endothelium inflammation and endothelial dysfunction, in which microRNAs (miRNAs) play a crucial role. However, the mechanism of ox-LDL in inflammatory damage of endothelial cells still remains elusive. Herein, we focused on the effect of hsa-miR-217-5p (miR-217) on endothelial dysfunction induced by ox-LDL by targeting early growth response protein-1 (EGR1). In the present study, 31 upregulated miRNAs and 59 downregulated miRNAs (Fold Change > 2, P value < 0.05) were identified after 6 h of 80 µg/mL ox-LDL exposure in human aortic endothelial cells (HAECs) by small RNA sequencing, including miR-217 that was significantly decreased (FC = 0.2787, P value = 5.22E-16). MiR-217 knockdown inhibited cell proliferation and increased level of IL-6, IL-1ß, ICAM-1 and TNF-α, while overexpression of miR-217 relieved the growth inhibition induced by ox-LDL and demonstrated anti-inflammatory effect in HAECs. EGR1 was predicted as a potential candidate target gene of miR-217 by TargetScan. The subsequent dual-luciferase reporter assay confirmed the direct binding of miR-217 to 3'UTR of EGR1. And EGR1 expression was negatively correlated with the level of miRNA-217 in HAECs after exposure to ox-LDL. Overexpression of EGR1 recapitulated the effects of miR-217 knockdown on cell proliferation inhibition and inflammation in HAECs, while knockdown EGR1 relieved the proliferative inhibition and demonstrated anti-inflammatory effect in ox-LDL-induced HAECs. The present study confirmed miR-217 ameliorates inflammatory damage of endothelial cells induced by oxidized LDL by targeting EGR1.

Effects of microRNA-217 on proliferation, apoptosis, and autophagy of hepatocytes in rat models of CCL4-induced liver injury by targeting NAT2.

Liver injury is an important cause of serious liver disease. This study aims to explore the effects of miR-217 targeting NAT2 on hepatocyte proliferation, apoptosis, and autophagy following carbon tetrachloride (CCL4)-induced liver injury. Rat models of CCL4-induced liver injury were established. Healthy Wistar rats were randomized into the normal, blank, negative control (NC), microRNA-217 (miR-217) mimic, miR-217 inhibitor, small interfering RNA (siRNA)-N-acetyltransferase 2 (NAT2), and miR-217 inhibitor + siRNA-NAT2 groups. NAT2 activity was evaluated with reversed-phase high-performance liquid chromatographic method. Immunohistochemistry was used to detect NAT2 protein positive rate. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to examine expressions of miR-217, NAT2, Bcl-2, Bax, p35, LC3-II, Becline-1, and the ratio of caspase-3/cleaved caspase-3. Autophagy, proliferation, and cell cycle distribution were determined by electron microscope, CCK-8, and flow cytometry. NAT2 protein positive rate and miR-217, NAT2, Bcl-2, and p35 expressions were higher and Bax, LC3-II, and Becline-1 expressions and the ratio of caspase-3/cleaved caspase-3 lower in the normal group than the other six groups. Compared with the blank and NC groups, in the miR-217 mimic and siRNA-NAT2 groups, Bax, LC3-II, and Becline-1 expressions and the ratio of caspase-3/cleaved caspase-3, and hepatocyte apoptosis and autophagy increased, while NAT2, Bcl-2, and p35 expressions and hepatocyte proliferation decreased; opposite results were observed in the miR-217 inhibitor group. Collectively, miR-217 targeting NAT2 inhibits proliferation and promotes apoptosis and autophagy of hepatocytes in CCL4-induced liver injury.

MicroRNA-217 relieved neuropathic pain through targeting toll-like receptor 5 expression.

Neuropathic pain is the most common chronic pain that is caused by nerve injury or disease that influences the nervous system. Increasing evidence suggested that microRNAs (miRNAs) play a crucial role in neuropathic pain and neuroinflammation development. However, the functional role of miR-217 in the development of neuropathic pain remains unknown. In this study, we used rats to establish a neuropathic pain model and showed that the miR-217 expression level was upregulated in the spinal dorsal horn of bilateral sciatic nerve chronic constriction injury (bCCI). However, the expression of miR-217 was not changed in the anterior cingulated cortex (ACC), hippocampus, and dorsal root ganglion (DRG) of bCCI rats. Ectopic expression of miR-217 attenuated neuropathic pain and suppressed neuroinflammation expression in vivo. We identified toll-like receptor 5 (TLR5) as a direct target gene of miR-217 in the PC12 cell. In addition, we demonstrated that the expression level of TLR5 was upregulated in bCCI rats. Moreover, restoration of TLR5 rescued the inhibitory roles induced by miR-217 overexpression on neuropathic pain and neuroinflammation development. These data suggested that miR-217 played a pivotal role in the development of neuropathic pain partly through regulating TLR5 expression.

Chemerin-induced angiogenesis and adipogenesis in 3 T3-L1 preadipocytes is mediated by lncRNA Meg3 through regulating Dickkopf-3 by sponging miR-217.

PURPOSE: Obesity is often caused by the excess adipogenesis and regulated by long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). We performed this study to investigate the influence of Meg3 expression on adipogenesis and also the Meg3/miR-217/Dkk3 axis-mediated molecular mechanism in adipogenesis and angiogenesis. METHODS: 3 T3-L1 preadipocytes were incubated with chemerin and transfected with Meg3-overexpressing (OE-Meg3) and Dkk3-overexpressing (OE-Dkk3) plasmids, siRNAs, and miR-217 mimics, inhibitor and scrambled sequences for 48 h or 72 h. The changes in cell proliferation, adipogenesis and angiogenesis ability in 3 T3-L1 preadipocytes was detected by using the corresponding assay. The expressions of related proteins were detected via western blot. RESULTS: Chemerin decreased miR-217 expression and increased Meg3 expression, meanwhile promoted the proliferation, adipogenesis and angiogenesis in 3 T3-L1 preadipocytes. Besides, OE-Meg3 exerted the synergistic effect on 3 T3-L1 preadipocytes when co-treated with chemerin. The target interactions between Meg3 and miR-217 as well as between miR-217 and Dkk3 were validated using dual-luciferase reporter system. SiMeg3 antagonized chemerin-induced changes, while the addition of miR-217 inhibitor attenuated siMeg3-induced changes in 3 T3-L1 preadipocytes. The proliferation, adipogenesis and angiogenesis in 3 T3-L1 preadipocytes were suppressed by miR-217 mimics, while promoted by the OE-Dkk3 Chemerin promoted the expression of fatty acid binding protein 4 and vascular endothelial growth factor (VEGF) proteins, and decreased the expression of cyclin D1, c-Myc, and ß-catenin proteins. Meanwhile, these effects were further enhanced by OE-Meg3 or OE-Dkk3. However, the transfection of siMeg3, or miR-217 mimics, or siDkk3 reversed the previous changes. CONCLUSIONS: Meg3/miR-217/Dkk3 induced adipogenesis and angiogenesis in 3 T3-L1 preadipocytes via activating VEGF signaling pathway and inhibiting Wnt/ß-catenin signaling pathway.

MicroRNA-217 attenuates intima-media complex thickness of ascending aorta measured by ultrasound bio-microscopy and inhibits inflammation and lipid metabolism in atherosclerotic models of ApoE-/- mice.

BACKGROUND: Little investigation was done to test the efficiency of microRNA-217 (miR-217) on atherosclerosis in vivo. METHODS: ApoE-/- mice were used to construct atherosclerotic models and ultrasound bio-microscopy (UBM) was applied to detect the intima-media thickness (IMT) of the ascending aorta. The serum level of miR-217 and correlation with IMT was investigated. After miR-217 mimic administration, the IMT, inflammation, and lipid-associated molecules were assayed. RESULTS: The serum level of miR-217 was reduced in ApoE-/- mice and showed a negative correlation with the IMT of the ascending aorta (r2 = 0.5899, p < 0.0001). miR-217 mimic administration attenuated IMT and down-regulated the level of serum triglyceride (TG), total cholesterol (TC), and low-density-lipoprotein cholesterol (LDL-C), while it could up-regulate high-density lipoprotein cholesterol (HDL-C). Inflammation relevant genes, such as F4/80, tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, and monocyte chemoattractant protein (MCP)-1, and lipid metabolism associated gene, such as LDL receptor, class A scavenger receptors (SR-A), scavenger receptor class B type I (SR-BI), CD36, ATP binding cassette subfamily A member 1 (ABCA1), and ATP binding cassette subfamily G member 1 (ABCG1) in the aorta were significantly down-regulated in miR-217 group when compared with atherosclerosis group. CONCLUSION: miR-217 could down-regulate IMT and modulate the inflammation and lipid metabolism process, which indicates that miR-217 could be a potential treatment option.

Mir-217 promotes inflammation and fibrosis in high glucose cultured rat glomerular mesangial cells via Sirt1/HIF-1α signaling pathway.

BACKGROUND: Silent information regulator 1 (Sirt1) plays a protective role in kidney. Sirt1 suppresses activation of hypoxia-inducible factor-1 alpha (HIF-1α), with MircroRNA-217 (Mir-217) being closely related to Sirt1. The relationship of Sirt1, HIF-1α and Mir-217, however, has never been reported in high glucose cultured rat glomerular mesangial cells (RMCs). Thus, we explored the role of Mir-217 on inflammation and fibrosis in RMCs cultured with high glucose in vitro through Sirt1/HIF-1α signaling pathway. METHODS: Rat glomerular mesangial cells were pre-incubated with Sirt1 activator Resveratrol prior to high glucose treatment. Furthermore the cells were transiently transfected with Sirt1 small interfering RNA (siRNA), HIF-1α siRNA and Mir-217 inhibitor using Lipofectamine 2000. Real-time PCR was used to analyse the expression of Mir-217, Sirt1 mRNA and HIF-1α mRNA; Western Blot was used to observe protein expression of Sirt1, HIF-1α, connective tissue growth factor, endothelin-1 and fibronectin; enzyme-linked immunosorbent assay was used to detect protein expression of transforming growth factor-ß1 and vascular endothelial growth factor. RESULTS: High glucose increased Mir-217 expression. High glucose decreased Sirt1 expression, accompanied by the increased HIF-1α expression and then promoted inflammation and fibrosis. In addition, Mir-217 gene silencing or Resveratrol could suppress the expression of HIF-1α, which in turn restrained inflammation and fibrosis in rat glomerular mesangial cells cultured with high glucose. CONCLUSION: This study clarified the role of Mir-217 in high glucose cultured rat glomerular mesangial cells through Sirt1/HIF-1α signaling pathway and provided new therapeutic targets for diabetic nephropathy. Copyright © 2016 John Wiley & Sons, Ltd.

MicroRNA-217 suppresses homocysteine-induced proliferation and migration of vascular smooth muscle cells via N-methyl-D-aspartic acid receptor inhibition.

Hyperhomocysteine has become a critical risk for atherosclerosis and can stimulate proliferation and migration of vascular smooth muscle cells (VSMCs). N-methyl-D-aspartic acid receptor (NMDAR) is a receptor of homocysteine and mediates the effects of homocysteine on VSMCs. Bioinformatics analysis has shown NMDAR is a potential target of microRNA-217 (miR-217), which exerts multiple functions in cancer tumorigenesis and carotid plaque progression. In this study, we sought to investigate the role of miR-217 in VSMCs phenotype transition under homocysteine exposure and elucidate its effect on atherosclerotic plaque formation. After treating with several doses of homocysteine (0-8 × 10(-4)  mol/L) for 24 hours, the expression of miR-217 in HA-VSMCs and rat aortic VSMCs was not altered. Intriguingly, the expression of NMDAR mRNA and protein was reduced by homocysteine in a dose-dependent manner. Transfection of miR-217 mimic significantly inhibited the proliferation and migration of VSMCs with homocysteine treatment, while transfection of miR-217 inhibitor promoted VSMCs migration. Moreover, miR-217 mimic down-regulated while miR-217 inhibitor up-regulated NMDAR protein expression but not NMDAR mRNA expression. Through luciferase reporter assay, we showed that miR-217 could directly bind to the 3'-UTR of NMDAR. MiR-217 mimic transfection also released the inhibition of cAMP-response element-binding protein (CREB)-PGC-1α signalling induced by homocysteine. Additionally, restoration of PGC-1α expression via AdPGC-1α infection markedly suppressed VSMCs proliferation through the degradation of NADPH oxidase (NOX1) and reduction of reactive oxygen species (ROS). Collectively, our study identified the role of miR-217 in regulating VSMCs proliferation and migration, which might serve as a target for atherosclerosis therapy.

MicroRNA-217 aggravates breast cancer through activation of NF1-mediated HSF1/ATG7 axis and c-Jun/ATF3/MMP13 axis.

Application of microRNA-mediated mRNA expression in treatment of diverse cancers has been documented. The current study was explored to study the role of miR-217 in breast cancer (BC) progression and the related downstream factors. Clinical tissue samples, BC cell lines and the established xenograft models were prepared for ectopic expression and depletion experiments to discern the regulatory roles of miR-217-mediated NF1 in BC cell proliferation, metastasis and chemoresistance as well as tumorigenic ability of BC cells in nude mice. miR-217 was upregulated in BC, which was a predictor of poor prognosis of BC patients. NF1 could be targeted by miR-217. miR-217 promoted malignant characteristics of BC cells through enhancing ATF3-MMP13 interaction by inhibiting NF1. miR-217 repressed sensitivity against anti-cancer drugs by inducing autophagy of BC cells through the NF1/HSF1/ATG7 axis. Also, miR-217 could inhibit NF1 to facilitate tumorigenic ability of BC cells in vivo. Our study emphasized that miR-217 could potentially inhibit NF1 expression to activate the c-Jun, thus enhancing the expression and interaction of ATF3/MMP13 and promoting the malignant features of BC cells. Furthermore, miR-217 conferred chemoresistance on BC by enhancing BC cell autophagy, which was achieved by limiting NF1 expression to induce the HSF1/ATG7 pathway.

miR-217-5p suppresses epithelial-mesenchymal transition and the NF-κB signaling pathway in breast cancer via targeting of metadherin.

MicroRNAs (miRNAs) have been associated with a number of human malignancies, including breast cancer (BC). However, the expression, biological function and fundamental underlying mechanism of miR-217-5p in BC remain unclear. Therefore, in the present study, the expression levels of miR-217-5p and metadherin (MTDH) were examined in BC tissues and BC cell lines using reverse transcription-quantitative PCR. Cell Counting Kit-8 assays, cell proliferation, wound healing assays, Transwell assays and western blotting were used to examine the effects of miR-217-5p on cell proliferation, migration, the epithelial-mesenchymal transition (EMT) and NF-κB signaling pathway expression. The direct relationship between miR-217-5p and MTDH was assessed using a dual-luciferase reporter assay. The results demonstrated that significantly reduced expression levels of miR-217-5p but significantly increased mRNA expression levels of MTDH were observed in BC tissues from 35 patients with BC compared with non-tumor breast tissues. Furthermore, BC cell lines SK-BR3 and BT549 expressed miR-217-5p at markedly lower levels and MTDH at markedly higher levels compared with the breast epithelial MCF10A cell line. miR-217-5p overexpression significantly inhibited cell proliferation, invasion and migration and suppressed the EMT in BC cells. miR-217-5p overexpression also inhibited the NF-κB signaling pathway by markedly decreasing p65 mRNA and protein expression levels but significantly increasing IκBα expression levels. Furthermore, miR-217-5p knockdown markedly increased MTDH mRNA and protein expression levels. The expression levels of miR-217-5p were negatively correlated with those of MTDH in BC tissues. These results suggested that restoration of MTDH expression levels could potentially attenuate the inhibitory effects of miR-217-5p overexpression on BC cell proliferation. Therefore, in conclusion miR-217-5p overexpression may inhibit cell migration, invasion, the EMT and NF-κB signaling pathway in BC via targeting of MTDH. miR-217-5p may serve as an important potential target in BC therapy.

MicroRNA-217 modulates inflammation, oxidative stress, and lung injury in septic mice via SIRT1.

Inflammation and oxidative stress contribute to the initiation and progression of septic lung injury. MicroRNA-217 (miR-217) is proved to be involved in controlling inflammatory response and oxidative stress, yet its role and underlying mechanism in the pathogenesis of septic lung injury remain elusive. Caecal ligation and puncture surgery were performed to generate sepsis in vivo and mice were kept for 12 h to imitate septic lung injury. Next, mice were administrated with miR-217 antagomir or agomir to decrease or increase the expression of miR-217 in lung tissue. Moreover, primary peritoneal macrophages were separated and incubated with lipopolysaccharide (LPS) to further verify the role of miR-217 in vitro. miR-217 was upregulated in septic lungs and primary macrophages. miR-217 antagomir alleviated, whereas miR-217 agomir aggravated inflammation and oxidative stress in septic mice and LPS-stimulated macrophages. Further detection identified SIRT1 was responsible for miR-217 antagomir-mediated anti-inflammatory and anti-oxidant effects, and SIRT1 inhibition abolished the beneficial effects of miR-217 antagomir in vivo and in vitro. Our data defined miR-217 as a therapeutic target for treating septic lung injury.

miR-217 through SIRT1 regulates the immunotoxicity of cadmium in Cyprinus carpio.

MicroRNAs (miRNAs) play a critical role in regulating the response of animals exposed to heavy metal stress. As a globally dispersed heavy metal in aquatic ecosystems, cadmium (Cd) is highly toxic to many aquatic species. However, little is known about the miRNA response to Cd stress in fish. To investigate the regulatory effect of miRNAs in response to Cd, common carp (Cyprinus carpio) were exposed to Cd2+-containing water (0.005 mg/L, 0.05 mg/L, 0.5 mg/L) for 30 days. After exposure, Cd2+ contents were significantly higher in the kidneys of C. carpio compared to other tissues, when exposed to 0.5 mg/L Cd2+. Hematoxylin and eosin staining images revealed that elevated Cd induced inflammatory damage in the kidneys of C.carpio. Further, miRNA sequencing revealed nine differentially expressed miRNAs (miR-217, miR-205 and seven novel miRNAs) in the kidneys, between 0.5 mg/L Cd2+ exposure and control groups. Potential target mRNAs of miRNAs suggest that miR-217 is involved in immunotoxicity. miR-217 agomir was intraperitoneally administered to C. carpio and RT-PCR revealed that the expression of IL-8 and SIRT1 decreased, while TLR-4, TRAF6, NF-kB, TNF-α, IL-1ß, and TGF-ß increased in the kidneys of C.carpio. Additionally, the expression of SIRT1 decreased, while the expression of other mRNAs increased in kidneys of C. carpio exposed to Cd. According to mRNAs expression in the agomir and Cd treatment, miRNAs inhibit the expressions of target mRNAs. These results demonstrate that miR-217 via SIRT1 plays a regulatory role in the immunotoxicity of Cd to C. carpio.

Knockdown of Circ_0000144 Suppresses Cell Proliferation, Migration and Invasion in Gastric Cancer Via Sponging MiR-217.

Previous studies have uncovered the role of circ_0000144 in various tumors. Here, we investigated the function and mechanism of circ_0000144 in gastric cancer (GC) progression. The expression of circ_0000144 in GC tissues and cells was detected through quantitative real-time polymerase chain reaction (qRT-PCR) method. Gain- and loss-of-function experiments including colony formation, wound healing and transwell assays were performed to examine the role of circ_0000144 in GC cells. Furthermore, western blot was conducted to determine the expressions of epithelial mesenchymal transition (EMT)-related proteins. The interaction between circ_0000144 and miR-217 was analyzed by bioinformatic analysis and luciferase reporter assays. The circ_0000144 expression was obviously upregulated in GC tissues and cells. Silencing of circ_0000144 inhibited cell proliferation, migration and invasion of GC cells, but ectopic expression of circ_0000144 showed the opposite results. Moreover, circ_0000144 sponged miR-217, and rescue assays revealed that silencing miR-217 expression reversed the inhibitory effect of circ_0000144 knockdown on the progress of GC. Our findings reveal that circ_0000144 inhibition suppresses GC cell proliferation, migration and invasion via absorbing miR-217, providing a new biomarker and potential therapeutic target for treatment of GC.

CircRNA-100284 activates aurora kinase B by inducing methylation of HSP70 via microRNA-217 to promote proliferation of bladder cancer cells.

OBJECTIVE: The malignant transformation of normal bladder cells (SV-HUC-1) was induced by arsenite to explore the possible mechanism of circRNA-100284 influencing bladder cancer cell proliferation. METHODS: Normal bladder SV-HUC-1 cells were cultured with 2 µM arsenite to induce malignant transformation. After 0, 3, 6, 12, and 24 h of culture, the expression level of circRNA-100284 in cells was detected by quantitative real-time PCR. Western blotting assays were used to detect the expression levels of EZH2 and cyclin-D1 proteins in cells treated with different media. Cell cycle was analyzed by flow cytometry. In addition, through cell transfection and CCK-8 experiments, the effect and mechanism of circRNA-100284 targeting microRNA-217 on proliferation was determined. The interaction between HSP70 methylation and Aurora-B was determined by Western blotting and immunoprecipitation experiments. RESULTS: With prolonged contact time with arsenite, the expression level of circRNA-100284 in cells increased continuously (P < 0.05). Western blotting assays showed that the expression levels of EZH2 and cyclin-D1 proteins in arsenite-transformed cells increased. Flow cytometry and CCK-8 showed that circRNA-100284 accelerated cell cycle transition and cell proliferation through miR-217. Finally, after culturing human bladder cancer T24 cells, combined with immunoprecipitation and in vitro kinase experiments, it was found that K561- dimethyl HSP70 activated Aurora-B, thus promoting the proliferation of bladder cancer cells. CONCLUSION: CircRNA-100284 activates aurora kinase B by inducing methylation of HSP70 via microRNA-217 to promote the proliferation of bladder cancer cells.

Cullin 4B regulates cell survival and apoptosis in clear cell renal cell carcinoma as a target of microRNA-217.

Cullin 4B (CUL4B) was reported to be closely related to the progression of some tumors, but its function in clear cell renal cell carcinoma (ccRCC) has not been reported. Our present study found CUL4B was upregulated in ccRCC, and CUL4B knockdown markedly inhibited ccRCC cell growth and induced apoptosis. In addition, CUL4B knockdown markedly inhibited antiapoptotic proteins' expression in ccRCC cells, including Mcl-1 and Bcl-2, and silenced CUL4B also induced the cleavages of PARP, an important index of apoptosis. We also confirmed microRNA-217 (miR-217) was downregulated in ccRCC tumor tissues, and negatively correlated with CUL4B expression. Further investigations revealed miR-217 targeted CUL4B and markedly inhibited its expression in ccRCC cells. In addition, overexpression of miR-217 by mimics significantly suppressed ccRCC cell growth. In contrast, enforced expression of CUL4B significantly abolished miR-217-induced cell survival inhibition in ccRCC cells. In conclusion, our present results suggested targeting miR-217-CUL4B axis would be a promising strategy for ccRCC treatment.

LINC00467 facilitates osteosarcoma progression by sponging miR­217 to regulate KPNA4 expression.

Osteosarcoma (OS) is a musculoskeletal malignancy that originates from interstitial cells. An increasing number of studies have verified that long non­coding RNAs (lncRNAs) participate in the progression of numerous types of cancer. It has been reported that LINC00467 is a cancer­promoting gene in some types of cancer; however, the regulatory mechanism of LINC00467 in OS remains unknown. In the present study, reverse transcription-quantitative PCR was used to determine LINC00467 expression in OS tissues and cells. Additionally, the impact of LINC00467­knockdown on OS cell proliferation, migration and invasion was analyzed using Cell Counting Kit­8, colony formation and Transwell assays, as well as western blot analysis. RNA pulldown and luciferase reporter assays were conducted to investigate the regulatory mechanism of LINC00467 in OS. The results delineated that LINC00467 expression was elevated in OS tissues and cells, and that high LINC00467 expression was associated with a poor prognosis in patients with OS. LINC00467 inhibition suppressed OS progression by inhibiting cell proliferation, migration, invasion and epithelial­mesenchymal transition. LINC00467 served as a molecular sponge for microRNA (miR)­217, while karyopherin subunit α4 (KPNA4) was a downstream target gene of miR­217. Moreover, the overexpression of KPNA4 reversed the inhibitory effects of LINC00467 inhibition on OS progression. Therefore, the present study elucidated the potential mechanism of LINC00467 in OS and indicated that LINC00467 exerted its carcinogenic effects on OS through the miR­217/KPNA4 axis, implying that LINC00467 may be a novel potential therapeutic target for OS.

microRNA-217 suppressed epithelial-to-mesenchymal transition through targeting PTPN14 in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the one of most common malignancies and its mechanism of metastasis remains unclear. The study was designed to investigate the effects of microRNA-217 on epithelial-to-mesenchymal transition. METHODS: The expression levels of miR-217 in GC were assayed by real-time qPCR. Metastasis and invasion of cancer cell were assayed by transwell chamber. Double luciferase reporter gene was used to verify the target regulatory relationship between microRNA-217 and tyrosine-protein phosphatase non-receptor type 14 (PTPN14) on gastric cell lines. Epithelial-to-mesenchymal transition (EMT) markers were assayed by Western blot. RESULTS: We found that miR-217 had a low level expression in gastric tumor tissues of 40 patients with GC, and a lower expression in the gastric tumor tissues of the patients with GC metastasis. Moreover, miR-217 markedly suppressed the metastasis and invasion of gastric cancer cell line in vitro. Furthermore, miR-217 inhibited the expression of PTPN14 by directly targeting its 3'UTR. Moreover, the down-regulation of PTPN14 reduced the metastasis and invasion, whereas up-regulation of PTPN14 led to the enhanced metastases and invasion of gastric cells. miR-217 induced the down-regulation of PTPN14 and inhibited the EMT in gastric cancer cells. CONCLUSION: miR-217 inhibited the EMT through directly targeting to the 3'UTR of PTPN14.

miR­217 and miR­543 downregulation mitigates inflammatory response and myocardial injury in children with viral myocarditis by regulating the SIRT1/AMPK/NF­κB signaling pathway.

The aim of the present study was to investigate the expression levels and roles of microRNA (miR)­217 and miR­543 in viral myocarditis, and to examine their underlying mechanisms. Coxsackievirus B3 (CVB3) was used to establish in vivo and in vitro models of viral myocarditis. The levels of miR­217 and miR­543 were detected using reverse transcription­quantitative PCR. The association between miR­217 and miR­543 and sirtuin­1 (SIRT1) was predicted and confirmed by TargetScan and dual­luciferase reporter assay. Cell viability was detected using Cell Counting Kit­8 assay, and cell apoptosis was measured by analyzing the expression levels of Bcl­2 and Bax, and by flow cytometry. In addition, the synthesis of various pro­inflammatory factors was determined by ELISA. In addition, superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were measured in cardiomyocytes following transfection and CVB infection. miR­217 and miR­543 were found to be highly expressed in the peripheral blood of pediatric patients with viral myocarditis, in the peripheral blood and myocardial tissues of viral myocarditis mice and in CVB3­infected cardiomyocytes. SIRT1 was found to be a target of both miR­217 and miR­543, and SIRT1 expression level was downregulated in viral myocarditis. Further analysis indicated that the reduced cell viability, increased cell apoptosis, enhanced synthesis of inflammatory factors, increased MDA content and decreased SOD activity associated with myocarditis were significantly reversed after inhibition of miR­217 or miR­543. Importantly, the present results showed that all the effects of miR­217 and miR­543 inhibition on cardiomyocytes were significantly suppressed following SIRT1 knockdown. Collectively, the present data indicated that miR­217 and miR­543 were significantly upregulated in viral myocarditis, and downregulation of miR­217 and miR­543 attenuated CVB3 infection­induced cardiomyocyte injury by targeting SIRT1. miR­217 and miR­543 may be potential therapeutic targets for developing novel viral myocarditis treatments in the future.