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ABSTRACT Background: Ovarian cancer is a fatal gynecologic malignancy. Long non-coding RNA (lncRNA) has been verified to serve as key regulator in ovarian cancer tumorigenesis. Objective: The aim of the study was to study the functions and mechanism of lncRNA PITPNA-AS1 in ovarian cancer cellular process. Methods: Clinical ovarian cancer samples were collected and stored at an academic medical center. Cellular fractionation assays and fluorescence in situ hybridization were conducted to locate PITPNA-AS1 in OC cells. TUNEL staining, colony-forming assays, and Transwell assays were performed for evaluating cell apoptosis as well as proliferative and migratory abilities. Western blot was conducted for quantifying protein levels of epithelial-mesenchymal transition markers. The binding relation between genes was verified by RNA pulldown, RNA immunoprecipitation, and luciferase reporter assays. Gene expression levels in ovarian cancer tissues and cells were subjected to RT-qPCR. Results: PITPNA-AS1 level was downregulated in ovarian cancer samples and cells. PITPNA-AS1 overexpression contributed to the accelerated ovarian cancer cell apoptosis and inhibited cell migration, proliferation, and epithelial-mesenchymal transition process. In addition, PITPNA-AS1 interacted with miR-223-3p to regulate RHOB. RHOB knockdown partially counteracted the repressive impact of PITPNA-AS1 on ovarian cancer cell activities. Conclusion: PITPNA-AS1 inhibited ovarian cancer cellular behaviors by targeting miR-223-3p and regulating RHOB. (Rev Invest Clin. 2024;76(2):103-15)
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ObjectiveTo investigate the effect of modified Simiaosan on miR-223-3p and NOD-like receptor thermal protein domain associated protein 3 （NLRP3）/interleukin-1β （IL-1β） signaling pathway in rat model with acute gouty arthritis （AGA） and explore the anti-inflammatory mechanism of modified Simiaosan on AGA. MethodA total of 72 8-week-old male SD rats were selected. They were divided into blank group， model group， colchicine group （0.3 mg·kg-1）， high-dose modified Simiaosan group （31.75 g·kg-1）， medium-dose modified Simiaosan group （15.75 g·kg-1）， and low-dose modified Simiaosan group （7.875 g·kg-1） according to random number table method， with 12 rats in each group. Except for the blank group， monosodium urate （MSU） crystal suspension was injected into the right ankle joint of rats by the Coderre method in other groups to replicate the rat model with AGA. The drug administration groups were given the corresponding drug solution by gavage， and the model group and the blank group were given an equal volume of sterile sodium chloride solution by gavage for one week. The circumference of the rats' ankle joint was measured， and the swelling degree of the ankle joint was calculated. Hematoxylin-eosin （HE） staining was used to detect the pathological morphological changes in the synovial tissue of the ankle joint. The levels of IL-1β， tumor necrosis factor-α （TNF-α）， and interleukin-6 （IL-6） in the serum of rats in each group were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the protein expressions of NLRP3， Caspase-1， and apoptosis-related spot-like protein （ASC） in synovial tissue of rats in each group， and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expressions of NLRP3， Caspase-1， and ASC and the expression of miR-223-3p in synovial tissue of rats. ResultCompared with that in the normal group， the swelling degree of the ankle joint in the model group was higher （P<0.01）， and the synovial tissue structure was disordered. Synovial cells proliferated obviously， and a large number of inflammatory cells were infiltrated. The levels of IL-1β， IL-6， and TNF-α in the serum of the model group increased significantly （P<0.01）， and the protein and mRNA expressions of NLRP3， Caspase-1， and ASC increased， while expression of miR-223-3 decreased. Compared with the model group， the swelling degree of ankle joint in the colchicine group and high-dose and medium-dose modified Simiaosan groups was lower （P<0.05）. Synovial cell proliferation and inflammatory cell infiltration of the colchicine group and high-dose， medium-dose， and low-dose modified Simiaosan groups were reduced to varying degrees， among which the colchicine group and high-dose modified Simiaosan group improved most obviously. The levels of IL-1β， IL-6， and TNF-α in the serum of rats in different dose groups of modified Simiaosan and colchicine group decreased significantly （P<0.01）， while the protein and mRNA expressions of NLRP3， Caspase-1， and ASC increased （P<0.01）. The expression of miR-223-3p in synovial tissue of the medium-dose and high-dose modified Simiaosan groups and colchicine group increased significantly （P<0.01）. Compared with those in the colchicine group， the levels of IL-1β， IL-6， and TNF-α in the low-dose modified Simiaosan group increased greatly （P<0.01）. In the medium-dose modified Simiaosan group， the protein expressions of NLRP3， Caspase-1， and ASC increased， and the expression of miR-223-3p decreased （P<0.05）. In the low-dose modified Simiaosan group， the levels of IL-1β， IL-6， and TNF-α increased greatly （P<0.01）， as well as the protein and mRNA expressions of NLRP3， Caspase-1， and ASC， while the expression of miR-223-3p was decreased （P<0.01）. ConclusionModified Simiaosan may play an anti-inflammatory role by intervening in the NLRP3/IL-1β signaling pathway via regulating miR-223-3p.
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Objective To investigate the biological function of long non-coding RNA(LncRNA)LINC01137 in immune escape of non-small cell lung cancer(NSCLC)cells and its potential regulatory mechanisms.Methods The blood samples of 24 healthy volunteers and 24 NSCLC patients were collected.The tumor tissues and paracancerous tissues of 24 NSCLC patients were collected,and the levels of LINC01137 were detected.The binding sites of LINC01137 and miR-22-3p were predicted by Starbase database and verified by the luciferase reporter gene analysis.A549 cells were transfected with exosomes derived from A549 cells and/or sh-LINC01137 interference sequence to detect cell proliferation and invasion.The supernatant of A549 cells were collected to culture CD8+T cells,and the levels of CD8+T cell exhaustion markers,including interfereron-γ(IFN-γ),tumor necrosis factor-α(TNF-α),granzyme B and interleukin-2(IL-2),and the percentage of PD-1+Tim3+CD8+T cells were detected.CD8+T cells were transfected with exosomes and/or miR-22-3p mimics to detect the protein level of PD-1.Results The expression of LINC01137 in tumor tissues of patients with NSCLC was increased compared with paracancerous tissues(3.357±0.548 vs 1.011±0.371),while the expression of LINC01137 in peripheral blood of patients with NSCLC was increased compared with healthy volunteers(3.216±0.342 vs 1.007±0.313),with statistically significant differences(t=-17.367,-17.147,all P<0.001).There was a positive correlation between the expression of LINC01137 in tumor tissue and peripheral blood(r=0.755,P<0.05).LINC01137 was significantly enriched in exosomes derived from A549 cells.Compared with Exo+sh-NC group,the cell viability(65.85%±4.71%vs 100.15%±11.93%)and cell invasion(21.46%±3.48%vs 43.12%±1.44%)in Exo+sh-LINC01137 group were decreased,and the differences were statistically significant(t=4.630,9.953,all P<0.01).The expression of LINC01137 in peripheral blood of NSCLC patients was negatively correlated with the percentage of CD8+T cells(r=-0.520,P<0.05).Compared with Exo+sh-NC group,the IFN-γ(3 865.31±543.85 pg/ml vs 1 786±105.98 pg/ml),TNF-α(4 631.93±510.71 pg/ml vs 1 973.24±379.62 pg/ml),Granzyme B(3 876.49±312.43 pg/ml vs 1 879.43±287.58 pg/ml),and IL-2 mRNA levels(3.286±0.437 vs 1.015±0.314)were increased,and the percentage of PD-1+Tim3+CD8+T cells(7.68%±2.18%vs 18.95%±3.21%)was decreased in Exo+sh-LINC01137 group,with statistical significances(t=-6.497,-7.237,-8.146,-7.310,5.021,all P<0.01).Our results showed that miR-22-3p was the target gene of LINC01137.Compared with Exo+NC mimic group,the level of PD-1 protein in Exo+miR-22-3p group(0.384±0.087 vs 1.003±0.147)was significantly decreased,and the difference was statistically significant(t=6.277,P<0.01).Conclusion The expression of LINC01137 was significantly up-regulated in tumor tissues and plasma of NSCLC patients.Exosomes LINC01137 derived NSCLC cell induces CD8+T cell exhaustion by targeting miR-22-3p and inhibiting its expression,and thus promoting NSCLC cell immune escape.
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@#Objective To use the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)to authenticate the chemical composition of Morinda citrifolia,and further experiments verify that Morinda citrifolia regulate miR-223-3p/NLRP3 signaling pathway to inhibit the inflammatory response in rats with polycystic ovary syndrome(PCOS).Methods Use UPLC-MS/MS analyze the chemical composition of Morinda citrifolia.SD female rats were randomly divided into three groups:normal control group(n=5),PCOS model group(n=5)and Morinda citrifolia gavage group(n=5).HE staining observe the morphology of ovarian histology,ELISA method detect the expression levels of testosterone(T),luteinizing hormone(LH),follicle stimulating hormone(FSH),interleukin(IL)-1βand IL-18,RT-qPCR detect the mRNA expression of miR-223-3p and NLRP3,and Western blot detect the protein expression of NLRP3.Results UPLC-MS/MS identify 641 chemical composition of Morinda citrifolia,including luteolin,Apigenin,emodin and other composition.Compared with the normal control group,the number of cell layers of ovarian granule in the PCOS model group is reduced,and follicular cystic dilation,atresia follicles increased,T and LH levels increased(P<0.05),FSH levels decreased(P<0.05),IL-18 and IL-1β levels increased(P<0.05),and ovarian tissue miR-223-3p mRNA expression decreased(P<0.01),NLRP3 mRNA and protein expression increased(P<0.01)in PCOS model group.Compared with the PCOS model group,the proportion of follicles at all levels is normal,the number of granule cell layers increased,T and LH levels decreased(P<0.05),FSH levels increased(P<0.05),IL-18 and IL-1β levels decreased(P<0.05),miR-223-3p mRNA expression in ovarian tissue increased((P<0.05),NLRP3 mRNA and protein expression decreased(P<0.05)in Morinda citrifolia gavage group.Conclusion Morinda citrifolia can improve the ovary pathological state of PCOS rats,change the abnormal sex hormones and reduce the levels of inflammatory factors IL-18 and IL-1β.The mechanism maybe regulate the miR-223-3p/NLRP3 signaling pathway to inhibit the inflammatory response in PCOS rats.
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Objective:To explore the expression of long non-coding RNA (lncRNA) CDK5RAP3 in gastric cancer tissue and its regulatory effect on gastric cancer cell proliferation and invasion.Methods:The expression differences of CDK5RAP3 in gastric cancer tissues and adjacent tissues were analyzed by TCGA database. By transfecting the pcDNA3.1-CDK5RAP3 plasmid into Hs-746T cells, a gastric cancer cell line overexpressing CDK5RAP3 (CDK5RAP3 group) was constructed, and the pcDNA3.1 plasmid was transfected into Hs-746T cells as a control group. The changes of CDK5RAP3 expression in the two groups of cells were detected by real-time quantitative PCR (qRT-PCR). The effects of overexpression of CDK5RAP3 on the proliferation and invasion of Hs-746T cells were detected by CCK-8 assay and Transwell assay, respectively. The binding sites of CDK5RAP3 and miR-223-3p were predicted by the starBase v2.0 database. The direct binding of CDK5RAP3 and miR-223-3p was verified by dual-luciferase reporter gene experiment. The expression levels of miR-223-3p in Hs-746T cells in each group were detected by qRT-PCR. Western blot was used to detect the expression levels of proliferation proteins and invasion proteins in Hs-746T cells in each group. The experimental data were analyzed by SPSS 17.0 software, and the measurement data conforming to the normal distribution were expressed as Mean±SD. The t-test was used to compare between two groups, and the one-way analysis of variance was used to compare the means of multiple groups. Results:Compared with adjacent tissues, the expression level of CDK5RAP3 in gastric cancer tissues was significantly lower ( P<0.01). The expressions of CDK5RAP3 in Hs-746T cells in the control group and CDK5RAP3 group were (1.08±0.77) and (10.63±2.14), respectively, and the difference was statistically significant ( P<0.01). Up-regulation of CDK5RAP3 significantly decreased the proliferation activity of Hs-746T cells ( P<0.05). The number of invasive cells in the control group and CDK5RAP3 group were (137.80±28.72) and (57.76±24.95), respectively, and the difference was statistically significant ( P<0.01). CDK5RAP3 could directly bind miR-223-3p ( P<0.01). The expression of miR-223-3p in Hs-746T cells in control group and CDK5RAP3 group were (6.22±1.20) and (1.01±0.98), respectively, and the difference was statistically significant ( P<0.01). Compared with the control group, up-regulation of CDK5RAP3 significantly reduced the expression levels of proliferation and invasive proteins. Conclusion:The expression of CDK5RAP3 is low in gastric cancer tissue, and CDK5RAP3 inhibits the proliferation and invasion of gastric cancer Hs-746T cells by targeting miR-223-3p.
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@#AIM: To investigate the regulatory effect of miR-223-3p on the expression of transcription factor Rbpj and on the differentiation of Th1 and Th17 cells in experimental autoimmune uveitis(EAU)rats.<p>METHODS: The regulatory role of miR-223-3p in Rbpj gene expression was investigated by a dual luciferase expression reporter system. In the present study, 24 female Lewis rats were randomly divided into EAU model group, normal control(NC)group and blank control(BC)group, and each group included 8 rats. The EAU model group was injected with interphotoreceptor retinoid-binding protein(IRBP)emulsion containing Mycobacterium tuberculin H37RA and complete Freund's adjuvant to induce uveitis, while the NC group was injected with an equal volume of emulsion without IRBP peptide. The rats in the BC group received the same volume of sterile saline solution. At 12d after immunization, the spleen, lymph node and eye tissues in both groups were aseptically isolated, and the expression levels of miR-223-3p and Rbpj RNAs were detected by real-time quantitative PCR(Q-PCR); Meanwhile, the expression levels of Rbpj, IFN-γ and IL-17 proteins were detected by ELISA, and the levels of Th1 and Th17 cell lineages in each tissue from each groups were detected by flow cytometry. <p>RESULTS: The results of dual fluorescein assay indicated that Rbpj was the target gene which regulated by miR-223-3p. At 12d after immunization, compared with NC group, the relative expression levels of miR-223-3p in spleen, lymph node and eye tissues from EAU model rats were 0.33±0.29, 0.11±0.12 and 0.18±0.11, respectively, accompanied by the down-regulated expression, and the differences were statistically significant(all <i>P</i><0.05); Rbpj mRNA levels were 3.00±0.06, 1.52±0.12 and 3.01±0.34, respectively, and were all up-regulated, while the differences were statistically significant(all <i>P</i><0.05). Moreover, the differences in miR-223-3p and Rbpj mRNA levels in spleen, lymph node and eye tissues of rats in the blank control group were not statistically significant compared with those in the NC group(<i>P</i>>0.05); ELISA results revealed that the expression levels of RBPJ, IFN-γ and IL-17 proteins in all tissues from EAU rats at 12d after immunization were significantly higher than those in the NC group( all <i>P</i><0.05), and there was no statistically significant difference in the expression levels of Rbpj, IFN-γ and IL-17 protein in all tissues of rats in the blank control group compared with the NC group(<i>P</i>>0.05); Meanwhile, flow cytometry results showed that the proportions of Th1 and Th17 cell lineages in all tissues from EAU model group were significantly higher than those from the NC group at 12d after immunization, and the differences were statistically significant(all <i>P</i><0.05). Furthermore,there was no significant change in the proportion of Th1 and Th17 cells in each tissue in the BC and NC groups(all <i>P</i> >0.05). <p>CONCLUSION: The miR-223-3p can negatively regulate the expression of the transcription factor Rbpj of Notch signaling pathway. The down-regulated miR-223-3p expression in EAU rats can increase the expression levels of Rbpj gene and protein, and aggravate the differentiation of Th1 and Th17 cells and the expression levels of related molecules IFN-γ and IL-17, which in turn affect the development of uveitis.
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【Objective】 To investigate the effect of microRNA （miRNA， miR）-223 on the proliferation and invasion of siha cells in cervical cancer and its mechanism. 【Methods】 Fluorescent quantitative polymerase chain reaction （Real-time PCR） was used to detect the expression levels of miR-223 and arm duplication protein 1 （ARMCX1） in cervical cancer tissues. Siha cell lines overexpressed by miR-223 were established， and the effects of overexpression of miR-223 on biological functions of siha cells were tested by CCK8 and Transwell test. Wild-type and mutant ARMCX1 double luciferase reporter vectors were constructed and luciferase activity was detected. The mRNA and protein expression levels of ARMCX1 were detected by Real-time PCR and Western blotting after overexpression of miR-223 in siha cells. 【Results】 Compared with that of adjacent tissues， the expression level of miR-223 in cervical cancer tissues was significantly decreased （P<0.05）， and the mRNA expression lof ARMCX1 was significantly increased （P<0.05）. Compared with the NC mimic group， the expression of miR-223 in miR-223 mimic group was significantly increased （P<0.05）， and the cell proliferation and invasion ability were significantly reduced （P<005）. However， after transfection with GV230-ARMCX1， cell proliferation and invasion abilities were enhanced in miR-223+GV230-ARMCX group compared with miR-223+GV230-NC group （P<0.05）. The double luciferase reporting experiment confirmed that miR-223 could directly bind with ARMCX1’s 3’UTR region sequence， and Real-time PCR and Western blotting results showed that the expressions of ARMCX1 mRNA and protein were decreased （P<0.05）. 【Conclusion】 miR-223 is underexpressed in cervical cancer and can inhibit the proliferation and invasion of cervical cancer cells by targeting ARMCX1 gene.
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Aim To explore the effect of resveratrol (RSV) on necrotizing enterocolitis (NEC) mouse pyroptosis and its regulatory mechanism. Methods Five-day-old C57BL/6 newborn mice were selected and divided into control group (Control), NEC model group, and RSV treatment group (RSV). The mice in the control group were given normal breastfeeding, while those in the NEC group were given hypoxia stimulation and artificial feeding accompanied by lipopolysaccharide intervention, and the mice in the RSV treatment group were given RSV gavage (1 mg · kg
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@#[Abstract] Objective: To investigate the effects of miR-223-3p on the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells by regulating Ras-related C3 botulinum toxin substrate 1 (RAC1) and its possible mechanism. Methods: Thirty pairs of HCC and corresponding para-cancer tissues resected in Jilin Central Hospital from August 2016 to August 2018 were collected for this study; in addition, human HCC cell lines SMMC-7721, BEL-7402, HepG2 and human normal hepatocyte QSG-7701 were also collected. The expression level of miR-223-3p in HCC tissue and cell lines was detected by qPCR. miR-223-3p mimics, miR-223-3p inhibitor and siRAC1 were transfected into SMMC-7221 cells, respectively. CCK-8 assay, Colony formation assay and Annexin V-FITC/PI staining Flow cytometry were used to detect the proliferation, clone formation and apoptosis of SMMC-7721 cells, respectively. The relationship between miR-223-3p and RAC1 was confirmed by Dual luciferase reporter gene assay. The protein level of RAC1 in SMMC-7721 cells was detected by Western blotting. Results: The expression of miR-223-3p in HCC tissues was significantly lower than that in paracaner tissues (P<0.01), and had significant correlation with pathological characteristics, such as tumor size, TNM stage, EdmondsonSteiner grade (all P<0.05 or P<0.01). miR-223-3p expression in HCC cell lines was significantly lower than that in QSG-7701 cells with the lowest expression in SMMC-7721 cells. Dual luciferase reporter gene assay confirmed that RAC1 was a target gene of miR-223-3p, and miR-223-3p negatively regulated RAC1 expression. Over-expression of miR-223-3p significantly inhibited the proliferation and colony formation (P<0.05 or P<0.01) of SMMC-7721 cells and promoted cell apoptosis (P<0.01). Contrarily, knockdown of miR-223-3p reversed the inhibitory effect of miR-223-3p mimics on cells. Conclusion: miR-223-3p over-expression inhibits proliferation and colony formation and promotes apoptosis of HCC cells, the mechanism of which may be related with its targeted down-regulation of RAC1.
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BACKGROUND: Mast cells (MCs) have been found to play a critical role during development of inflammatory bowel disease (IBD) that characterized by dysregulation of inflammation and impaired intestinal barrier function. However, the function of MCs in IBD remains to be fully elucidated. RESULTS: In our study, we used exosomes isolated from human mast cells-1 (HMCs-1) to culture with NCM460, HT-29 or CaCO2 of intestinal epithelial cells (lECs) to investigate the communication between MCs and lECs. We found that MCs-derived exosomes significantly increased intestinal epithelial permeability and destroyed intestinal barrier function, which is attributed to exosome-mediated functional miRNAs were transferred from HMCs-1 into lECs, leading to inhibit tight junction-related proteins expression, including tight junction proteins 1 (TJP1, ZO-1), Occludin (OCLN), Claudin 8 (CLDN8). Microarray and bioinformatic analysis have further revealed that a panel of miRNAs target different tight junction-related proteins. Interestingly, miR-223 is enriched in mast cell-derived exosome, which inhibit CLDN8 expression in IECs, while treatment with miR-223 inhibitor in HT-29 cells significantly reversed the inhibitory effect of HMCs-1-derived exosomes on CLDN 8 expression. Most importantly, enrichment of MCs accumulation in intestinal mucosa of patients with IBD compared with those healthy control. CONCLUSIONS: These results indicated that enrichment of exosomal miR-223 from HMCs-1 inhibited CLDN8 expression, leading to destroy intestinal barrier function. These finding provided a novel insight of MCs as a new target for therapeutic treatment of IBD.
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Humains , Animaux , Bovins , microARN/métabolisme , Cellules épithéliales/métabolisme , Muqueuse intestinale/métabolisme , Mastocytes/métabolisme , Perméabilité , Maladies inflammatoires intestinales/métabolisme , Cellules cultivées , Cellules Caco-2/cytologie , Biologie informatique , Analyse sur puce à tissus , Exosomes/métabolisme , Claudines/métabolisme , Occludine/métabolisme , Protéine-1 de la zonula occludens/métabolismeRÉSUMÉ
Objective To investigate the radioprotective function and its mechanism of miR-223 in acute radiation-induced lung injury in mice.Methods Forty female C57BL/6 J mice were randomly divided into healthy control group,irradiation group,irradiation plus miR-223 group and irradiation plus NC group.Radiation groups were exposed with a single dose of 15 Gy of 6 MV X-rays delivered by a linear accelerator.The mice in drug group were administered by tail vein injection with miR-223 agomir or agomirNC every other day from 1 d before irradiation to 14 d after irradiation.The lung tissue samples of mice were taken at 14 d post-irradiation.The pathological changes were observed by HE staining.The localization and expressions of IL-1β and IL-18 were observed by immunohistochemistry (IHC).Real-time PCR was used to detect miR-223,but NLRP3 mRNA expression in lung tissue.Western blot was used to detect the protein expressions of NLRP3 and Caspase-1,and ELISA assay was used to detect the expressions of IL-1β and IL-18 in lung homogenate.Results Radiation decreased the expression of miR-223,but increased the expression of NLRP3 in lung tissue.Administration of miR-223 agomir inhibited the expression of NLRP3 and attenuated lung inflammation.HE and IHC staining showed that miR-223 reduced the acute inflammatory response and the expressions of IL-1β and IL-18 in lung tissue compared with irradiation group (t=10.16,6.00,P＜0.05).The expressions of NLRP3 and Caspase-1 protein in lung tissue of irradiated plus miR-223 group was lower than that in the irradiation alone group (t =12.47,4.95,P＜ 0.05).ELISA assay also showed a decrease of inflammatory factors IL-1β and IL-18 in lung tissue homogenate of the irradiation plus miR-223 group (t =8.22,8.47,P＜0.05).Conclusions MiR-223 effectively reduces the secretion of radiation-induced inflammatory factors IL-1β and IL-18 by inhibiting the expression of NLRP3 in lung tissue of mice,and thus has protective effect on radiation-induced lung injury.
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OBJECTIVE To investigate the anti-pyroptotic effects of melatonin in atherosclerotic endothelium and to elucidate the potential mechanisms.METHODS ApoE-/-mice were randomly divid-ed into four groups (n=8): the normal-diet group (ND), the normal-diet group treated with melatonin (10 mg·kg-1)(ND+MLT),the high-fat-diet group(HFD),and the high-fat-diet group treated with melatonin (HFD+MLT).After 12 weeks,the expression levels of pyroptosis related genes including NLRP3,ASC, cleaved-caspase 1,GSDMD-N,IL-1β and IL-18 were examined in aortic endothelium by Western blotting, qRT-PCR and immunofluorescent staining.Besides,levels of MEG3 and miR-223 were also tested by qRT-PCR.The interaction between MEG3 and miR-223 was detected by luciferase assay.For in vitro study,human aortic endothelial cells(HAECs)were transiently transfected with miR-223 mimic,miR-223 inhibitor (AMO-223), MEG3-overexpressing plasmid or negative controls. After 6 h of transfection, the medium was replaced by fresh medium with or without ox-LDL(25 μg·mL-1)for 24 h and then treated with or without melatonin (10 μmol·L-1) for 48 h. Cell pyroptosis was evaluated by Hoechst 33342/PI staining and differentially expressed pyroptosis related genes. RESULTS Melatonin markedly reduced the atherosclerotic plaque in aorta of ApoE-/- mice. Meanwhile, melatonin also attenuated the expression NLRP3, ASC, cleaved-caspase1, NF-κB/GSDMD, GSDMD-N termini, IL-1β, and IL-18 in aortic endo-thelium.Consistent anti-pyroptotic effects were also observed in ox-LDL-treated HAECs.We found that lncRNAMEG3 enhanced pyroptosis in HAECs. Moreover, MEG3 acted as an endogenous sponge by sequence complementarity to suppress the function of miR-223 and to increase NLRP3expression and enhance endothelial cell pyroptosis. Furthermore, knockdown of miR-223 blocked the anti-pyroptotic actions of melatonin in ox-LDL-treated HAECs. CONCLUSION Melatonin prevents endothelial cell pyroptosis via MEG3/miR-223/NLRP3 axis in atherosclerosis and therefore melatonin replacement might be considered a new strategy for protecting endothelium against pyroptosis thereby for the treat-ment of atherosclerosis associated with pyroptosis.
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Objective To explore the effect of lncRNA of growth arrest-specific 5 (lncRNA GAS5) on the radiosensitivity of colon cancer cells by targeting miR-223.Methods The expressions of lncRNA GAS5 in a few of colon cancer cell lines were detected by real-time quantitative PCR (qPCR).The cell lines with low expression level of lncRNA GAS5 were selected for subsequent study.The effect of overexpression of lncRNA GAS5 on the radiosensitivity of colon cancer SW480 cells was detected by cell cloning experiments.The target gene miR-223 of lncRNA GAS5 was predicted and validated by the bioinformatics database starBase and dual luciferase reporter assays.qPCR was used to detect the expression of miR-223 in various colon cancer cell lines and the influence of lncRNA GAS5 overexpression on the expression of miR-223 in SW480 cells.Results Compared with normal human colonic epithelial cells (NCM460),the expressions of lncRNA GAS5 in the colon cancer SW480,LOVO,HT-29 and SW620 cell lines were significantly lower(t =15.25,8.69,14.42,11.62,P ＜ 0.05),with the lowest level in SW480 cells.Both overexpression of lncRNA GAS5 and down-regulation of miR-223 significantly increased the radiosensitivity of colon cancer cells by decreasing cell survival fraction (at 8 Gy,lncRNA GAS5,t =13.51,P ＜ 0.05;anti-miR-223,t =14.93,P ＜ 0.05)and promoting apoptosis (lncRNA GAS5,t =8.30,P ＜ 0.05;anti-miR-223,t =7.32,P ＜ 0.05).Bioinformatics analysis showed that the 3'sequence of lncRNA GAS5 contained the binding sites with miR-223.After overexpression or downregulation of lncRNA GAS5,the expression of miR-223 was enhanced or reduced.Conclusions The lncRNA GAS5 promotes the apoptosis of colon cancer cells and inhibits its survival by targeting miR-223 expression,thereby increases the radiosensitivity of colon cancer cells.
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Objective:To investigate the potential correlation between miR-223 level in leukocytes and platelet responses to clopidogrel in patients with coronary artery disease.Methods:A cohort of 188 outpatients,who conducted percutaneous coronary intervention (PCI) and received dual antiplatelet therapy,were recruited.The patient's electronic health data were collected,and their blood samples were obtained for measurement of adenosine diphosphate (ADP)-induced whole-blood platelet aggregation.Extreme cases ofplatelet responses to clopidogrel (ultra-vs.non-responder) were measured with miR-223-3p levels in leukocytes.Results:Both groups had similar miR-223-3p levels in leukocytes.There were no significant differences in other demographic and clinical data except for metrics of ADP-induced whole-blood platelet aggregation between the 2 group.Conclusion:MiR-223-3p in peripheral leukocytes is not associated with the altered platelet responses to clopidogrel in PCI outpatients.
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Objective To explore the expression of miR-223-3p in the plasma of patients with diabetic kidney disease and its clinical significance. Methods The expression levels of plasma miR-223-3p in normoalbuminuric group (DM) , microoalbuminuria group (Micro-DKD) , macroalbuminuria group (Macro-DKD) and healthy controls were measured by quantitative real-time polymerase chain reaction (qRT-PCR) . To analysis the relationship with clinical pathological parameters,target genes of miR-223-3p were predicted with bioinformatics software. Results The levels of miR-223-3p in the plasma of the remaining three groups were significantly lower than those in the healthy controls (P＜0.001), and the decrease was positively correlated with the severity of the disease. The potential target genes of miR-223-3p identified by bioinformatics softwares include IL6ST and PRKCE. Conclusion The expressionof miR-223-3pin diabetic kidney disease (DKD) patients' plasma decreased and was positively correlated with the severity of the disease. It may play an important role in the development and progression of diabetic kidney disease through its target genes.
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Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.
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Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.
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AIM To explore the effects of Shexiang Wulong Pills (Moschus Artifactus,Aconiti Radix Cocta,Pheretima,etc.) drug serum on the expression levels of miR-146a,miR-130 and miR-223 in peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis (RA).METHODS PBMCs were extracted from blood of 30 cases of RA patients,cells were divided into three groups,blank control group,serum-free groupand Shexiang Wulong Pills drug serum group.After 48 hours,total RNA was extracted,then the expression levels of three miRs were detected by RT-PCR.RESULTS Compared with the serum-free group,the expression levels of miR-146a,miR-130 and miR-223 in the Shexiang Wulong Pills drug serum group were significantly decreased (P ＜ 0.01).CONCLUSION Shexiang Wulong Pills can inhibit inflammation in RA patients by down-regulating the expression levels of miR-223,miR-130 and miR-146a in PBMCs.
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Objective To investigate the expression and effect of miR-22-3p in non-small cell lung cancer (NSCLC). Methods The miR-22-3p expression level in seventy-six NSCLC tissues and para-cancer tissues was detected by qRT-PCR. The relationship between the expression of miR-22-3p and gender,age,tumor size,histolo-gy grade,pathological type and lymph node metastasis was analyzed. The function of miR-22-3p on the prolifera-tion of NSCLC cells was tested by growth curve assay. Target genes of miR-22-3p were predicted by online software Targetscan. Luciferase reporter assay and qRT-PCR was used to certificate the prediction. Results The expression of miR-22-3p was increased in NSCLC tissues than the para-cancer tissues and was correlated to lymph node metas-tasis. Overexpression of miR-22-3p could suppress the proliferation of A549 cells. Astrocyte-Elevated Gene-1(AEG-1) was predicted to be a target of miR-22-3p. MiR-22-3p was revealed to bind to AEG-13′UTR by luciferase report-er assay. Overexpression of miR-22-3p could inhibit the expression of AEG-1 in A549 cells. Suppression of miR-22-3p could increase AEG-1 expression. Conclusion MiR-22-3p could inhibit the proliferation of NSCLC by tar-geting AEG-1.
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Objective To investigate the role of miR-223 in the pathogenesis of acute gouty inflammation.Methods The subjects were divided into 3 groups:65 acute gout patients (AG),50 inter-critical gout patients (IG),and 45 healthy controls (HC).The peripheral blood mononuclear cells (PBMCs) and the clinical laboratory parameters were all collected.The expression of miR-223 in the PBMCs was detected using realtime fluorescent quantitative polymerase chain reaction (RT-qPCR) (TaqMan probe).The PBMCs of 5 healthy people were stimulated with monosodium urate (MSU) (100 μg/ml) for 12 h,and then,miR-223,NLRP3 mRNA and IL-1β production were all measured using RT-qPCR and ELISA respectively.All data were analyzed by SPSS 17.0 statistical software,Wilcoxon rank sum test,t test and Spearman's correlations analysis were used for statistical analysis.Results ① The expression of miR-223 in AG and IG groups was both significantly decreased than that in the HC group (8±17 vs 26±76,P＜0.05;9±17 vs 26±76,P＜0.05;respectively),AG group was significantly decreased than that in the IG group [8(17) vs 9(17),Z=11.387,P＜0.01].② After stimulated with MSU in healthy controls,IL-1β production and NLRP3 mRNA were both significantly increased [(86±5) pg/ml vs (13±6) pg/ml,t=21.042,P＜0.01;5.2±0.4 vs 1.2±0.4,t=14.640,P＜0.01;respectively],while the expression of miR-223 was significantly decreased (0.34±0.20 vs 1.05±0.24,t=-5.164,P＜0.01).Conclusion The data suggests that miR-223 might be involved in the patho-genesis of spontaneous regulation,but further study is needed to discover the exact mechanism.