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Objective:Trigeminal neuralgia(TN)is a common neuropathic pain.Voltage-gated potassium channel(Kv)has been confirmed to be involved in the occurrence and development of TN,but the specific mechanism is still unclear.MicroRNA may be involved in neuropathic pain by regulating the expression of Kv channels and neuronal excitability in trigeminal ganglion(TG).This study aims to explore the relationship between Kv1.1 and miR-21-5p in TG with a TN model,evaluate whether miR-21-5p has a regulatory effect on Kv1.1,and to provide a new target and experimental basis for the treatment of TN. Methods:A total of 48 SD rats were randomly divided into 6 groups:1)a sham group(n= 12),the rats were only sutured at the surgical incision without nerve ligation;2)a sham+ agomir NC group(n=6),the sham rats were microinjected with agomir NC through stereotactic brain injection in the surgical side of TG;3)a sham+miR-21-5p agomir group(n=6),the sham rats were microinjected with miR-21-5p agomir via stereotactic brain injection in the surgical side of TG;4)a TN group(n=12),a TN rat model was constructed using the chronic constriction injury of the distal infraorbital nerve(dIoN-CCI)method with chromium intestinal thread;5)a TN+antagonist NC group(n=6),TN rats were microinjected with antagonist NC through stereotactic brain injection method in the surgical side of TG;6)a TN+miR-21-5p antagonist group(n=6),TN rats were microinjected with miR-21-5p antagonist through stereotactic brain injection in the surgical side of TG.The change of mechanical pain threshold in rats of each group after surgery was detected.The expressions of Kv1.1 and miR-21-5p in the operative TG of rats were detected by Western blotting and real-time reverse transcription polymerase chain reaction.Dual luciferase reporter genes were used to determine whether there was a target relationship between Kv1.1 and miR-21-5p and whether miR-21-5p directly affected the 3'-UTR terminal of KCNA1.The effect of brain stereotaxic injection was evaluated by immunofluorescence assay,and then the analogue of miR-21-5p(agomir)and agomir NC were injected into the TG of rats in the sham group by brain stereotaxic apparatus to overexpress miR-21-5p.The miR-21-5p inhibitor(antagomir)and antagomir NC were injected into TG of rats in the TN group to inhibit the expression of miR-21-5p.The behavioral changes of rats before and after administration were observed,and the expression changes of miR-21-5p and Kv1.1 in TG of rats after intervention were detected. Results:Compared with the baseline pain threshold,the facial mechanical pain threshold of rats in the TN group was significantly decreased from the 5th to 15th day after the surgery(P<0.05),and the facial mechanical pain threshold of rats in the sham group was stable at the normal level,which proved that the dIoN-CCI model was successfully constructed.Compared with the sham group,the expression of Kv1.1 mRNA and protein in TG of the TN group was down-regulated(both P<0.05),and the expression of miR-21-5p was up-regulated(P<0.05).The results of dual luciferase report showed that the luciferase activity of rno-miR-21-5p mimics and KCNA1 WT transfected with 6 nmol/L or 20 nmol/L were significantly decreased compared with those transfected with mimic NC and wild-type KCNA1 WT,respectively(P<0.001).Compared with low dose rno-miR-21-5p mimics(6 nmol/L)co-transfection group,the relative activity of luciferase in the high dose rno-miR-21-5p mimics(20 nmol/L)cotransfection group was significantly decreased(P<0.001).The results of immunofluorescence showed that drugs were accurately injected into TG through stereotaxic brain.After the expression of miR-21-5p in the TN group,the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were increased.After overexpression of miR-21-5p in the sham group,the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were decreased. Conclusion:Both Kv1.1 and miR-21-5p are involved in TN and miR-21-5p can regulate Kv1.1 expression by binding to the 3'-UTR of KCNA1.
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BACKGROUND:Bone marrow mesenchymal stem cells(BMSCs)can release a large number of exosomes(Exos).The effect of Exos derived from BMSCs on hepatocyte apoptosis and the specific mechanism has not been fully clarified. OBJECTIVE:To explore the effect of miR-21-5p carried by Exos derived from BMSCs on apoptosis of rat liver cells and its mechanism. METHODS:Rat BMSCs were isolated and miR-21-5p NC or miR-21-5p inhibitor was transfected into BMSCs.The Exos were extracted by ultracentrifugation and named(BMSCs+miR-21-5p NC)-Exos and(BMSCs+miR-21-5p inhibitor)-Exos.BMSCs-derived Exos were co-cultured with rat hepatocytes to observe the effect of inhibiting miR-21-5p expression on the apoptosis of rat hepatocytes.The targeting relationship between miR-21-5p and PIK3R1 was verified by double luciferase reporter gene detection.TUNEL was used to detect the effect of miR-21-5p directly targeting PIK3R1 in Exos to activate the PI3K/AKT signaling pathway on hepatocyte apoptosis in BRL rats. RESULTS AND CONCLUSION:(1)The double luciferase reporting system confirmed that when PI3KR1 wild type vector and miR-21-5p mimics co-transfected 293T cells,the luciferase activity decreased significantly compared with the PI3KR1 mutant vector co-transfected group,indicating that miR-21-5p could target PIK3R1.(2)TUNEL test results showed that compared with(BMSCs+miR-21-5p NC)-Exos group,(BMSCs+miR-21-5p inhibitor)-Exos treatment significantly increased the apoptosis rate.Compared with the(BMSCs+miR-21-5p NC)-Exos group,after the addition of AKT inhibitor LY294002,the apoptosis rate was significantly increased.(3)The results indicate that Exos may inhibit the apoptosis of BRL rat hepatocytes through miR-21-5p,in which miR-21-5p directly targets PIK3R1 to activate PI3K/AKT signaling pathway.
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Objective To investigate the action mechanism of cyclic RNA0001287(circ_0001287)and miR-21 in the pathogenesis of diabetic retinopathy(DR).Methods Primary human retinal pigment epithelium(phRPE)cells were iso-lated for circRNA microarray analysis.Arising retinal pigment epithelium(ARPE)-19 cells were cultured in vitro and divid-ed into the blank group,high-glucose group,negative group,si-circ group,circ_0001287 group,circ_0001287+negative group,and circ_0001287+miR-21 group.Small interfering RNA(siRNA)oligonucleotides against circ_0001287,mimics containing miR-21 sequences and miR-21 mimic plasmids were constructed.In the negative group,si-circ group,circ_0001287 group,circ_0001287+negative group and circ_0001287+miR-21 group,the empty plasmid,circ_0001287 siRNA,circ_0001287 mimics,circ_0001287 mimics+miRNA disordered sequence,and circ_0001287 mimics+miR-21 mimic plasmid were transfected into ARPE-19 cells using Lipofectamine 2000 Transfection Reagent.After transfection for 6 h,the Opti-MEM medium was replaced with a fresh normal medium.Cells in the blank group and the high-glucose group were not transfected.Cells in the blank group were cultured with culture solution containing 5.5 mmol·L-1 glucose,and cells in the high-glucose group were cultured with culture solution containing 15.5 mmol·L-1,25.5 mmol·L-1 and 35.5 mmol·L-1 glucose,respectively.Cells in other groups were treated with 35.5 mmol·L-1 glucose for 48 h.The expressions of circ_0001287 and miR-21 were detected by reverse transcription polymerase chain reaction(RT-PCR),cell proliferation activity was detected by Cell Counting Kit-8,and the targeting relationship between circ_0001287 and miR-21 was detected by Dual Luciferase Reporter Assay.RNA immunoprecipitation(RIP)assay and biotin-coupled probe pull-down assay were used to verify the targeting relationship between circ_0001287 and miR-21.Western blot was used to detect protein expression.Re-sults After screening by circRNA,the expression of hsa_circ_0001287 in phRPE cells was significantly reduced.RT-PCR detection showed that compared with the blank group,circ_0001287 expression in ARPE-19 cells in the high-glucose group decreased(P<0.05)in a dose-dependent manner,and miR-21 expression in ARPE-19 cells gradually increased with the in-crease of glucose concentration(P<0.05).After co-transfection of siRNA with circ_0001287 mimics,siRNA also reduced circ_0001287 expression,and the relative expression of circ_0001287 in the circ_0001287+negative group(0.70±0.03)was significantly lower than that in the negative group(0.98±0.04,P<0.05).For cells transfected with the circ_0001287-WT plasmid,compared with the control simulation group(0.98±0.03),the relative luciferase activity of the miR-21 simulation group(0.59±0.02)decreased(P<0.05).However,for cells transfected with circ_0001287-MUT plasmid,the relative ac-tivity of luciferase was almost the same in the control simulation group(0.96±0.05)and the miR-21 simulation group(1.00±0.04,P>0.05).In the anti-Ago RIP experiment,miR-21 was significantly enriched in the circ_0001287 group com-pared with the control group,indicating that miR-21 could be significantly pulled down by the biotinylated circ_0001287 probe.Pull-down analysis demonstrated that compared with the control IgG,circ_0001287 specific probe pull-down sam-ples showed significant enrichment of circ_0001287 and miR-21.In this experiment,the cell proliferation rate of the circ_0001287+miR-21 group(78.25%±3.01%)was lower than that of the circ_0001287+negative group(90.88%±3.51%,P<0.05).Compared with the blank group,the expression of PTEN protein in ARPE-19 cells in the high-glucose group treated with 35.5 mmol·L-1 glucose was significantly down-regulated(P<0.05),the expression of PTEN protein in ARPE-19 cells in the circ_0001287 group was higher than that in the negative group(P<0.05),and the expression of PTEN protein in ARPE-19 cells in the circ_0001287+miR-21 group was higher than that in the circ_0001287 group(P<0.05).Conclusion The expression of circ_0001287 is down-regulated in phRPE cells and high-glucose induced ARPE-19 cells,and up-regulated circ_0001287 can inhibit the injuiy of diabetic RPE cells by adsorption of miR-21 and activation of PTEN expression.
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microRNA-21(miR-21) is an endogenous non-coding RNA and plays a key regulatory role in the process of cell proliferation and differentiation. In recent years, miR-21, as a widely studied miRNA, has attracted much attention for its role in skin- related diseases and wound healing. The study shows that miR-21, as a " broad factor", affects the proliferation, apoptosis, migration and invasion of different cells (keratinocytes, T cells, fibroblasts, etc.) by inhibiting the transcription and translation of different target genes (PTEN, TIMP, PDCD4, etc.) . At the same time, it plays a crucial role in skin tumors, skin immune diseases, skin inflammatory diseases, skin wounds and scar tissue formation by promoting inflammation through different signaling pathways. In this study, we reviewed the regulatory roles of miR-21 in different skin diseases (melanoma, cutaneous squamous cell carcinoma, T-cell lymphoma, psoriasis, scleroderma, etc.) and wound healing, aiming at deepening the understanding of miR-21 molecule in skin-related diseases and wound healing. The potential of miR-21 as a biomarker for skin disease diagnosis and its ability to evaluate the efficacy of drugs were also discussed. miR-21 is expected to become a new target of skin disease and wound healing, which may provide a new direction for clinical research.
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Abstract Recent studies have found that lncRNA-MEG3(MEG3) plays an important role in the development of EMs (Endometriosis), but the specific mechanism needs to be further explored. This study aimed to investigate the effect of MEG3 on the proliferation, invasion of EMs cells. The authors used RT-qPCR to detect the expression of MEG3 and miR-21-5p in EMs tissues and hESCs cells, MTT and Transwell to detect cell proliferation and invasion, western blotting assay to detect the expression of DNMT3B and Twist, MSP to detect the methylation of Twist. The present study's detection results showed that MEG3 was lowly expressed in EMs tissues and hESCs cells, and overexpression of MEG3 could down-regulate miR-21-5p and inhibit endometrial cell proliferation and invasion. In addition, overexpression of MEG3 upregulated the expression of DNMT3B and promoted the methylation of TWIST. In conclusion, the present findings suggest that MEG3 is downregulated in EMs tissues, and overexpression of MEG3 can promote the activity of DNA methyltransferase DNMT3B by downregulating miR-21-5p, thereby promoting the methylation of Twist, downregulating Twist level to inhibits hESCs cells proliferation and invasion.
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BACKGROUND: The main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulatory dysfunction, which are caused by excessive apoptosis of ovarian granulosa cells. Acupuncture has been shown to improve follicular development abnormalities in patients with PCOS, but its mechanism is unknown. This study hypothesized that the mechanism of acupuncture on follicular development abnormalities in PCOS patients is the inhibition of granulosa cell apoptosis through LncMEG3-mediated regulation of miR-21-3p. METHODS: A PCOS-like rat model was established using subcutaneous injection of dehydroepiandrosterone (DHEA). Acupuncture was performed on rats for 15 d (CV-4, RN-3, CV-6, SP-6 and EX-CA 1). Ovarian morphology was observed by HE staining, and sex hormone and AMH levels were detected by ELISA. Primary granulosa cells were isolated from each group of rats to assess the association of acupuncture treatment, LncMEG3, miR-21-3p, and granulosa cell apoptosis in rats with PCOS. RESULTS: LncMEG3 and miR-21-3p were highly expressed in the ovarian granulosa cells of rats with PCOS, and LncMEG3-mediated regulation of miR-21-3p was involved in the development of PCOS in rats. Silencing of MEG3 attenuated sex hormone dysregulation and ovarian histopathological changes in PCOS rats and promoted follicle cell development and maturation. In addition, silencing MEG3 increased the viability and number of granulosa cells. In addition, silencing MEG3 further inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats. Acupuncture improved polycystic ovarian morphology and sex hormone levels in PCOS rats. Acupuncture intervention increased the viability and number of granulosa cells. Acupuncture intervention inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats by targeting miR-21-3p via LncMEG3. CONCLUSION: These results suggest that acupuncture can downregulate LncMEG3, thereby targeting and regulating miR-21-3p to suppress early and late granulosa cell apoptosis and normalize their proliferation. These factors ultimately compensate for abnormal follicular development. These findings shed light on the clinical potential of acupuncture as a safe treatment for follicular developmental abnormalities in PCOS. Highlights LncMEG3-mediated inhibition of miR-21-3p regulates ovarian granulosa cell apoptosis. LncMEG3 and miR-21-3p are involved in the occurrence and development of PCOS-related abnormal follicular development. CuONPs induce co-occurrence of autophagy activation and autophagic flux blockade. Acupuncture can improve the sex hormone levels and follicular development in the context of PCOS. The underlying mechanism of acupuncture in the treatment of PCOS abnormal follicular development was revealed.
Subject(s)
Humans , Animals , Female , Rats , Polycystic Ovary Syndrome/therapy , Acupuncture Therapy , MicroRNAs , RNA, Long Noncoding , Apoptosis , Granulosa CellsABSTRACT
Objective:To explore the effect of miR-21 on cell proliferation, apoptosis, invasion and radiosensitivity of cervical cancer HeLa cells and unravel the underlying mechanism.Methods:RT-qPCR assay was used to detect the expression levels of miR-21 in cervical cancer tissues and adjacent non-tumor tissues, normal cervical epithelial cells (H8) and cervical cancer cell lines (HeLa, SiHa, ME180). HeLa cell line with inhibition of miR-21 or knockdown of RECK were constructed. CCK-8, Caspase3/7 live cell apoptosis detection, wound healing test, Transwell invasion, clone formation assay, Western blot and immunofluorescence were performed to detect cell viability, apoptosis, migration, invasion, radiosensitivity and related proteins. The dual luciferase assay verified whether miR-21 targeted RECK.Results:MiR-21 level in the cervical cancer tissues was significantly higher than that in its corresponding adjacent non-tumor tissues ( P<0.05). The expression levels of miR-21 in cervical cancer cell lines HeLa, SiHa and ME180 were significantly up-regulated compared with those in normal cervical epithelial cells H8(all P<0.05). MiR-21 knockdown significantly inhibited HeLa cell viability, promoted cell apoptosis, reduced radiation tolerance, down-regulated the expression of Cyclin D 1,Bcl-2, MMP-2 and MMP-9, and up-regulated the expression P21 and Bax proteins (all P<0.05). miR-21 targeted the 3’-UTR of RECK mRNA and negatively regulated the expression of RECK. Silencing RECK reversed the effects of miR-21 knockdown on HeLa cell apoptosis, migration, invasion and radiosensitivity. Conclusions:Inhibiting the expression of miR-21 significantly decreases cell viability, induces cell apoptosis, weakens cell migration and invasion capabilities, and enhances the radiosensitivity of HeLa cells. The potential mechanism is closely related to the targeted up-regulation of RECK.
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Objective:To investigate the implication of micro RNA-21(miR-21) in Endostar combined with X-ray irradiation of cardiac fibroblasts (CF).Methods:Rat CFs were used in this experiment and been divided into the blank control group, 10 Gy X-ray irradiation group, Endostar group, 10 Gy X-ray+ Endostar group, 10 Gy X-ray+ Endostar+ NC mimic group (negative control 1), 10 Gy X-ray+ Endostar+ miR-21 mimic group, 10 Gy X-ray+ Endostar+ NC inhibitor group (negative control 2) and 10 Gy X-ray+ Endostar+ miR-21 inhibitor group. The proliferation of CF was determined by Methyl thiazolyl tetrazolium (MTT) assay. The expression level of Collagen Ⅰ protein was analyzed by Western blot. The expression levels of Collagen Ⅰ and miR-21 mRNA were assayed by real-time quantitative polymerase chain reaction (q-PCR).Results:In the 10 Gy X-ray+ Endostar+ miR-21 mimic group, the CF proliferation, Collagen Ⅰ and miR-21 mRNA were increased significantly compared with those in the blank control group, 10 Gy X-ray+ Endostar group, and negative control group 1 (all P<0.05). In the 10 Gy X-ray+ Endostar+ miR-21 inhibitor group, the CF proliferation and expression levels of Collagen Ⅰ mRNA were decreased significantly compared with those in the blank control group, 10 Gy X-ray+ Endostar group and negative control group 2(all P<0.05). Conclusions:The CF proliferation and Collagen Ⅰ expression are increased when the expression level of miR-21 gene is simulated. When inhibiting the expression of miR-21 gene, the CF proliferation and Collagen Ⅰ expression are reduced.
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【Objective】 To investigate the effect and mechanism of arctigenin (ARG) on hypoxia-reoxygenation (H/R) induced pyroptosis of cardiomyocytes. 【Methods】 H9C2 cells were cultured in vitro, and underwent hypoxia for 2 hours and reoxygenation for 4 hours to establish H/R cell injury model. The cells were divided into control group (Control), model group (H/R), ARG group, miR-21 simulation group (miR-21 mimic), and ARG+miR-21 inhibitor group (ARG+miR-21 inhibitor). TUNEL staining was used to detect the pyroptosis index of H9C2 cells; the lactate dehydrogenase (LDH) kit was used to detect the release of LDH in each group of cells; the enzyme-linked immunosorbent assay (ELISA) was used to detect the content of interleukin-1β (IL-1β) and interleukin-18 (IL-18). Western blotting was used to detect the expressions of pyroptosis-related proteins (Caspase-1, GSDMD, IL-1β and IL-18) in each group. 【Results】 Compared with those in the control group, the pyroptosis index, the release of LDH, IL-1β and IL-18, and the protein expressions of Caspase-1p20, GSDMD-N, IL-1β and IL-18 in the H/R group were significantly increased (P<0.01). Compared with H/R group, ARG group and miR-21 mimic group had significantly reduced pyroptosis index, LDH, IL-1β and IL-18 release, and protein expressions of Caspase-1 p20, GSDMD-N, IL-1β and IL-18 (P<0.01), and the above-mentioned index changes could be reversed after treatment with +miR-21 inhibitor. 【Conclusion】 ARG can inhibit H/R-induced pyroptosis of cardiomyocytes, and its mechanism is related to the promotion of miR-21 expression.
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Objective To investigate the effect of PTENP1 on the proliferation and apoptosis of colorectal cancer cells and its molecular mechanism. Methods We selected 107 cases of colorectal cancer and corresponding adjacent tissues as the research objects. The expression level of PTENP1 was analyzed by fluorescence quantitative PCR. Colon cancer HT29 cells with PTENP1 overexpression (PTENP1 group) and empty vector cell line (control group) were established by lentivirus. The cell proliferation and apoptosis were analyzed by CCK8 and flow cytometry. The PTENP1 target gene was analyzed by bioinformatics and double luciferase reporter genes. The expression level of target protein was analyzed by Western blot. Results The expression of PTENP1 in colorectal cancer tissues was significantly lower than that in adjacent tissues (P < 0.05). The expression level of PTENP1 in the control group was significantly lower than that in the PTENP1 group (P < 0.05). Compared with the control group, the cell proliferation ability of the PTENP1 group was significantly decreased (P < 0.05), the apoptosis level was significantly increased (P < 0.05). miR-21 was complementary to PTENP1. Compared with the control group, the expression of miR-21 in the PTENP1 group was significantly down-regulated (P < 0.05), and the expression of PTEN protein was significantly up-regulated (P < 0.05). Conclusion PTENP1 and miR-21 competitively bind to regulate the expression of PTEN, and then affect the proliferation and apoptosis of colorectal cancer cells.
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AIM: To reveal that the targeted regulation of TGF-β1 by miR-21-5P is the key mechanism that mediates the activation of myocardial fibroblasts, and to clarify the intervention ofRadix Angelica Sinensis and Radix Hedysari ultrafiltration on the mechanism of miR-21-5P targeting to regulate TGF-β1 effect. METHODS: (1) The cells were randomly divided into normal group and irradiation group. The irradiation group received 6Gry single irradiation, and then RT-PCR was used to detect miR-21-5P, and Western Blot was used to detect the expression of α-SMA and TGF-β1. (2) The cells were randomly divided into normal group, irradiation group, miR-21-5P
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@#[Abstract] Objective: To explore the effects of miR-21 targeting PDCD4 (programmed cell death factor 4) on proliferation and migration of non-small cell lung cancer (NSCLC) A549 cells and the possible mechanism. Methods: The miR-21 mimics, miR-21 inhibitors and miR-NC plasmids were transfected into A549 cells in logarithmic growth phase by liposome transfection technology. Forty-eight hours after transfection, the transfection efficiency was observed under a fluorescence microscope, and the mRNA expression levels of miR-21 and PDCD4 in A549 cells were detected by qPCR. Dual luciferase reporter gene experiment was used to detect the targeting relationship between miR-21 and PDCD4, MTT method was used to detect cell proliferation, Transwell chamber method was used to detect cell migration ability, and ELISA was used to detect the content of TNF-α in each group of cell culture fluids. WB was used to detect the protein expression levels of PDCD4, NF-κB p65 and p-NF-κB p65 in cells. Results: The A549 cell line with miR-21 over-expression or knockdown was successfully constructed. Dual luciferase reporter gene assay confirmed that miR-21 targetedly inhibited PDCD4 expression. Over-expression of miR-21 could significantly inhibit the mRNA expression of PDCD4 in A549 cells (P<0.01), promote cell proliferation and migration (P<0.05 or P<0.01), increase the secretion level of TNF-α (P<0.01), down-regulate the expression of PDCD4 protein (P<0.01), and up-regulate p-NF-κB p65 protein level (P<0.05). The effect of silencing miR-21 on cells was opposite to the effect of miR-21 over-expression. Conclusion: Over-expression of miR-21 can promote the proliferation and migration ability of A549 cells, which may be related to its targeted inhibition of PDCD4 and activating the NF-κB/TNF-α pathway.
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La diabetes Tipo 1 (DT1) es una compleja enfermedad autoinmune con una etiología aún desconocida. La vitamina D ha sido ampliamente estudiada debido a su potencial terapéutico en los potenciales nuevos casos de DT1. Por otra parte, los microARNs (miRs) han sido propuestos como posibles biomarcadores en diversos procesos biológicos como en la apoptosis e inflamación. El objetivo de este estudio fue evaluar el efecto de la suplementación con vitamina D sobre el perfil de expresión del miR-21 y marcadores de apoptosis tales como: BCL2, STAT3, TIPE2 y DAXX, en células mononucleares periféricas provenientes de pacientes con DT1 y sujetos controles. RESULTADOS: El perfil de expresión de miR-21 se encontró disminuido en los pacientes con DT1 en comparación con los controles. La expresión relativa de BCL2 se encontró aumentada en controles al comparar con pacientes DT1 en todas las condiciones experimentales. La expresión relativa de DAXX mostró un perfil de expresión diferencial al comparar pacientes con DT1 versus controles (p=0.006). CONCLUSIÓN: El estímulo con vitamina D parece tener un posible efecto regulador sobre los genes BCL2 y DAXX.
Type 1 diabetes (T1D) is a complex chronic autoimmune disease. Vitamin D has been one of the most studied therapeutic potential outbreaks related to T1D. Specific miRNAs have been proposed as potential biomarkers in several biological processes as apoptosis and inflammation. The aim of this study was to evaluate the effect of vitamin D on the expression profiles of miR-21 and apoptotic markers BCL2, STAT3, TIPE2 and DAXX, in PBMCs from T1D patients and control subjects. RESULTS: miR-21 expression was increased in controls regarding T1D patients. BCL2 was increased in controls compared to T1D patients in all experimental conditions. DAXX showed different expression patterns between T1D patients and controls (p=0.006). CONCLUSION: Vitamin D showed a possible regulation effect on apoptosis markers mainly through the regulation of BCL2 and DAXX
Subject(s)
Humans , Child , Adolescent , Vitamin D/administration & dosage , Apoptosis , Diabetes Mellitus, Type 1/metabolism , Vitamin D/metabolism , Biomarkers , Molecular Chaperones/drug effects , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Proto-Oncogene Proteins c-bcl-2/drug effects , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , MicroRNAs/drug effects , MicroRNAs/genetics , MicroRNAs/metabolism , STAT3 Transcription Factor/drug effects , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Co-Repressor Proteins/drug effects , Co-Repressor Proteins/genetics , Co-Repressor Proteins/metabolism , Glucose/administration & dosageABSTRACT
The objective of this study was to investigate the effect of human esophageal fibroblast-derived exosomal miR-21 on cisplatin sensitivity against esophageal squamous EC9706 cells. EC9706 cells were co-cultured indirectly with human esophageal fibroblasts (HEF) or miR-21 mimics transfected-HEF in the transwell system. The exosomes in HEF-culture conditioned medium were extracted by differential ultracentrifugation. EC9706 cells were co-cultured with HEF-derived exosomes directly. The cisplatin sensitivity against EC9706 cells was revealed via half maximal inhibitory concentration (IC50) values using MTT assay. The expressions of miR-21, programmed cell death 4 (PDCD4) mRNA, and gene of phosphate and tension homology deleted on chromosome ten (PTEN) mRNA were determined by qRT-PCR. The changes of the protein level were detected using western blot assay. IC50 values of cisplatin against EC9706 cells were increased after EC9706 cells were co-cultured with either HEF or exosomes derived from miR-21 mimics-transfected HEF. Following the increased level of miR-21, the mRNA expression and protein levels of PTEN and PDCD4 were decreased in EC9706 cells. The cisplatin sensitivity to EC9706 cells was reduced by HEF-derived exosomal miR-21 through targeting PTEN and PDCD4. This study suggested that non-tumor cells in the tumor micro-environment increased the tumor anti-chemotherapy effects through their exosomes.
Subject(s)
Humans , Esophageal Neoplasms/genetics , Esophageal Neoplasms/drug therapy , Carcinoma , MicroRNAs/genetics , Cisplatin/pharmacology , RNA-Binding Proteins , Apoptosis , Cell Line, Tumor , Cell Proliferation , Apoptosis Regulatory Proteins/metabolism , Tumor Microenvironment , Fibroblasts/metabolismABSTRACT
Objective: To explore the role of the new regulator pseudogene phosphatase and tensin homolog pseudogene 1 (PTENp1) in the regulation of PTEN mRNA and gene expression in oral squamous cell carcinoma (OSCC) so as to provide a new target for the prevention, treatment and outcome of OSCC. Methods: First, we collected 42 specimens of OSCC and normal tissues, extracted RNA, detected the expressions of PTENp1, PTEN and miR-21 by qRT-PCR, and studied their correlation by Pearson correlation analysis. HEK293 cells were cultured and transfected with luciferase plasmid of 3'UTR of PTEN and mimic or inhibitor of miR-21 or full-length PTENp1 3'UTR plasmid, respectively. The regulatory role of PTENp1 in PTEN-miR-21 axis and its cancer promoting function were verified by luciferase activity test. Results: qRT-PCR showed that the expressions of PTEN (85.7%) and PTENp1 (90.4%) were significantly repressed in the OSCC tissues while miR-21 expression (76.2%) was remarkably increased. Pearson correlation analysis showed that PTEN expression was negatively correlated with miR-21 expression (R=0.123 5, P<0.001) but positively correlated with PTENp1 expression (R=0.051 8, P=0.01). The results of luciferase activity showed that PTEN expression was significantly up-regulated by overexpression of PTENp1, suggesting that PTENp1 could target competitive binding miR-21 to regulate PTEN expression. Results: PTENp1, as the ceRNA of PTEN, competitively binds the miR-21, which provides a new idea for predicting the early marker and targeting therapy of OSCC in the future.
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Subject(s)
Humans , Apoptosis , B-Lymphocytes , Blotting, Western , Carcinogenesis , Caspase 3 , Cell Line , Cell Proliferation , Curcumin , Immunoprecipitation , In Situ Hybridization , Lymphoma , Lymphoma, B-Cell , MicroRNAs , Real-Time Polymerase Chain Reaction , RNA , RNA, MessengerABSTRACT
BACKGROUND: MicroRNA-21 (miR-21) is a regulator of osteoclastogenesis and a promoter of osteoclast differentiation, but its role in periodontitis remains unclear. OBJECTIVE: To investigate whether miR-21 is involved in bone destruction in periodontitis. METHODS: Real-time PCR was used to detect and analyze the differential expression of miR-21 in periodontitis samples. Using liposome transfection method, miR-21 mimics (up-regulating miR-21) or miR-21 inhibitor (down-regulating miR-21) was used to transfect osteoclasts. Expressions of miR-21 and bone destruction markers TRAP and CTSK were detected by real-time PCR. Cell counting kit-8 was used to detect the miR-21 effect on osteoclast proliferation. RESULTS AND CONCLUSION: (1) MiR-21 expression increased in periodontitis samples. (2) When miR-21 mimics was transfected into osteoclasts, miR-21, TRAP and CTSK mRNA expression increased; when miR-21 inhibitor was transfected into osteoclasts, miR-21, TRAP and CTSK mRNA expression decreased. (3) Transfection with miR-21 mimics promoted the proliferation of osteoclasts, while transfection with miR-21 inhibitor inhibited the proliferation of osteoclasts. To conclude, miR-21 can be used as an important target for the treatment of periodontitis.
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AIM: To investigate the inhibitory effect of Yiqi Huoxue decoction on the malignant biological behavior of lung cancer cells and its mechanism. METHODS: Human lung cancer A549 cells were treated with different doses of Yiqi Huoxue Decoction serum (5%, 10%, 15%). CCK-8 assay, transwell chamber experiment, flow cytometry, Western blot and qRT-PCR method were used to study the effect of different doses of Yiqi Huoxue Decoction-containing serum to cell proliferation, cell migration and invasion, cell apoptosis, PTEN protein and miR-21 expression. RESULTS:Compared with the drug-free serum group, survival rate, migration and invasion ability of A549 cells decreased after treatment with different doses of drug-containing serum. The apoptosis rate of A549 cells increased, PTEN mRNA and the expression of its protein increased, the expression of miR-21 decreased, and the medium-dose (10%) drug-containing serum group had the best effect. After the transfection of miR-21 mimics, miR-21 expression was up-regulated, while PTEN protein expression was down-regulated in cells. PTEN protein expression was up-regulated after treatment with medium-dose (10%) drug-containing serum. CONCLUSION: Yiqi Huoxue Decoction can effectively inhibit the malignant cell biological behavior of human lung cancer A549 cells and may be related to the regulation of the miR-21/PTEN signaling pathway.
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Objective To investigate the protective effect of Jiawei Danshen Yin combined with microRNA-21 on IRI myocardial cells,and to study its protective mechanism. Methods H9C2 cardiomyocytes were cultured and divided into four groups: Blank group, IRI model group of cardiomyocytes,miR-21 group,miR-21 combined with Jiawei Danshen Yin group (JWDSY group). Apoptotic rate, apoptosis-related protein and PTEN signaling pathway expression of cardiomyocytes were detected. Results Electron microscopy showed that mitochondria in model group were damaged significantly , and ultrastructural damage in JWDSY group was alleviated; ELISA demonstrated that cTnl was elevated in model group and decreased in JWDSY group with statistical significance (P < 0. 01); flow cytometry re vealed that the apoptotic rate was significantly increased in model group (36. 79 ±2. 12) ,but markedly decreased in JWDSY group (14. 65 ± 0. 94), and the difference was statistically significant ( P < 0. 05 ). Western blot results showed that PTEN expression was uP-regulated and P-Akt expression down-regulated in model group,while PTEN expression decreased and P-Akt expression increased in JWDSY group, and the difference was statistically significant ( P < 0. 05 ). Conclusions miR-21 combined with Jiawei Danshen Yin can inhibit cardiomyocyte apoptosis and protect IRI cardiomyocytes by reducing PTEN expression and activating PI3K/Akt signaling pathway,.
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@#[Abstract] Objective: To investigate the effects of exosome originated from bone marrow mesenchymal stem cell (BMSCs) on proliferation, migration and invasion of prostate cancer PC-3 cell and its mechanism. Methods: qPCR was used to detect the expression level of miR-21-5p in prostate cancer cell lines. The morphology of exosomes isolated from BMSCs was observed with an electron microscope. Western blotting was used to detect the expressions of exosome surface markers and the epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin and Vimentin). Dual luciferase reporter gene experiment was used to detect the targeted regulation relationship between miR-21-5p and PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2). PC-3 cells were co-cultured with 10 μl BMSCs exosomes suspension (Exo group), transfected with sh-PHLPP2 or antagomiR, then CCK-8 and Transwell experiments were used to detect changesinproliferation,migrationandinvasionofPC-3cell.Results: miR-21-5p was highly expressed in prostate cancer PC-3 cell line. The exosomes in the supernatant of BMSCs culture fluid were successfully isolated, and the typical vesicle-like structures of exosomes were observed under transmission electron microscope. Exosomes expressed specific proteins such as CD9, CD63 and CD81. In the Exo group, the proliferation, invasion, migration, as well as the expressions of N-cadherin, Vimentin and miR-21-5p in PC-3 cells were significantly higher than those in the control group (all P<0.05). PHLPP2 is a target gene of miR-21-5p. Compared with the control group, the expression of PHLPP2 in PC-3 cells of Exo group and sh-PHLPP2 group was significantly reduced (0.66±0.09, 0.42±0.05 vs 1.09±0.08, all P<0.01); cell viability, invasion and migration were significantly improved (all P<0.01); and E-cadherin expression level was significantly reduced while N-cadherin and Vimentin expressions were significantly increased (both P<0.05). Conclusion: miR-21-5p is highly expressed in prostate cancer PC-3 cell line. BMSC exosome miR-21-5p can increase the proliferation, migration and invasion ability of PC-3 cells through targeted down-regulation of PHLPP2.