ABSTRACT
ObjectiveTo investigate the mechanism of Biejiajian Wan in the intervention of primary liver cancer based on long non-coding RNA SNHG5 （lncRNA SNHG5）/micro RNA-26a-5p （miRNA-26a-5p）/glycogen synthase kinase-3β （GSK-3β） signal axis. MethodDouble luciferase reporting assay was used to verify the targeted interaction between lncRNA SNHG5 and miRNA-26a-5p， miRNA-26a-5p， and GSK-3β in HepG2 cells. Nude-mouse transplanted tumor model of human HepG2 were established and randomly divided into model group， Biejiajian Wan low-dose group （0.5 g·kg-1）， medium-dose group （1.0 g·kg-1）， and high-dose group （2.0 g·kg-1）， and sorafenib group （100 mg·kg-1）， with 10 mice in each group. The mice were given intragastric administration of normal saline or drug for 28 days， and the tumor volume was measured at different time. Hematoxylin-eosin （HE） staining was used to observe the histological changes of tumors. The nucleic acid levels of lncRNA SNHG5， miRNA-26a-5p， GSK-3β， and β-catenin mPNA in tumor tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of GSK-3β and β-catenin in tumor tissue were detected by western blot. ResultCompared with the SNHG5-WT （wild type） + miRNA NC （negative control） group， the relative luciferase activities of the SNHG5-WT + miRNA-26a-5p mimic group were decreased （P<0.05）. Compared with the GSK-3β-WT + miRNA NC group， the relative luciferase activity of the GSK-3β-WT + miRNA-26a-5p mimic group was decreased （P<0.05）. Compared with the model group， the tumor volume of Biejiajian Wan low-dose， medium-dose， and high-dose groups was significantly decreased （P<0.05， P<0.01）. Compared with the model group， the cells in the tumor tissue of nude mice in each dose group of Biejiajian Wan were sparsely arranged with necrocytosis， which showed concentration-dependent changes. Compared with the model group， the expression levels of lncRNA SNHG5， GSK-3β， and β-catenin were decreased （P<0.05， P<0.01）， while the expression of miRNA-26a-5p was increased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. Compared with the model group， the protein expression levels of GSK-3β and β-catenin were decreased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. ConclusionBiejiajian Wan may affect the necrosis of liver cancer cells through lncRNA SNHG5/miRNA-26a-5p/GSK-3β signal axis and thus play an anti-tumor role. This research will provide more theoretical basis for the clinical application of Biejiajian Wan.
ABSTRACT
PURPOSE: Bioactive molecules critical to intracellular signaling are contained in extracellular vesicles (EVs) and have cardioprotective effects in ischemia/reperfusion (IR) injured hearts. This study investigated the mechanism of the cardioprotective effects of EVs derived from hypoxia-preconditioned human mesenchymal stem cells (MSCs). MATERIALS AND METHODS: EV solutions (0.4 µg/µL) derived from normoxia-preconditioned MSCs (EVNM) and hypoxia-preconditioned MSCs (EVHM) were delivered in a rat IR injury model. Successful EV delivery was confirmed by the detection of PKH26 staining in hearts from EV-treated rats. RESULTS: EVHM significantly reduced infarct size (24±2% vs. 8±1%, p < 0.001), and diminished arrhythmias by recovering electrical conduction, INa current, and Cx43 expression. EVHM also reversed reductions in Wnt1 and β-catenin levels and increases in GSK3β induced after IR injury. miRNA-26a was significantly increased in EVHM, compared with EVNM, in real-time PCR. Finally, in in vitro experiments, hypoxia-induced increases in GSK3β expression were significantly reduced by the overexpression of miRNA-26a. CONCLUSION: EVHM reduced IR injury by suppressing GSK3β expression via miRNA-26a and increased Cx43 expression. These findings suggest that the beneficial effect of EVHM is related with Wnt signaling pathway.
Subject(s)
Animals , Humans , Rats , Arrhythmias, Cardiac , Connexin 43 , Extracellular Vesicles , Heart , In Vitro Techniques , Mesenchymal Stem Cells , Real-Time Polymerase Chain Reaction , Reperfusion Injury , Wnt Signaling PathwayABSTRACT
Objective To detect the expression of miR-26a/b in the aorta and serum of mice so as to explore the role of miR-26a/b in vascular remodeling of hypertension.Methods C576L/BJ male mice were randomly divided into AngⅡ group and control group.Mini-osmotic pump was implanted subcutaneously into the back of mice, and the model of blood vessel remodeling in mice was established by continuous infusion of AngⅡ(2.0mg/kg·d).The mice in control group were injected with saline.Blood pressure was taken before the intervention and at 3, 5, 7, 10 and 14 days after the intervention.After 2 weeks, the mice were killed, the serum and aorta tissues were collected, and the expression of miR-26a/b was determined by RT-PCR.HE staining, Masson staining and immunohistochemistry were performed to observe changes in vascular morphology, fibrosis and protein expression.Results After the intervention, systolic blood pressure and diastolic blood pressure were significantly higher in AngⅡ group than in control group (P<0.05).HE staining showed that the vessel wall of AngⅡ group was thicker than that of control group.Masson staining showed more blue collagen deposition in the middle of aorta in AngⅡ group but no obvious collagen deposition in control group.RT-PCR showed that the expression of miRNA-26a/b in the serum and aorta of AngⅡ group was significantly lower than in control group (P<0.05).Immunohistochemistry indicated that the expressions of CTGF, collagen Ⅰ and collagen Ⅲ all increased after AngⅡ infusion (P<0.05).Conclusion MiR-26a/b, CTGF, collagen Ⅰ and collagen Ⅲ may be involved in AngⅡ-induced vascular remodeling in hypertension.MiR-26a/b may be a new therapeutic target of vascular remodeling in hypertension.
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Objective To assess the influence of miRNA (miR)-124 ,miR-26A and miR-126 single nucleotide polymorphism (SNPs) on the prognosis in patients with colorectal cancer (CRC) .Methods A total of 724 cases of patients with CRC treated in this hospital from July 2011 to December 2013 served as the research subjects .The peripheral blood samples were collected ,and genotype analysis of miR-124 rs531564 ,miR-26A rs7372209 and miR-126 rs4636297 SNPs locus were performed by using the PCR-LDR .The patient′s overall survival (OS) and progression-free survival (PFS) were calculated by using Kaplan-Meier method ,and the univariate log-rank method was used to analyse the influence of different genotypes and haplotypes of SNPs on OS and PFS .Re-sults The OS after operation in CRC patient′s carrying miR-124 rs531564 CG genotype was decreased compared with that in pa-tients carrying CC genotype ,and the patient′s death risk was increased[HR2 =1 .450 ,95% CI(1 .043-2 .017) ,P=0 .026];the pa-tient′s death risk in patients carrying GG genotype was also increased compared with that in patients carrying CC genotype [HR2 =2 .339 ,95% CI(1 .171-4 .674)];in the dominant gene model ,the OS in patients carrying CG +GG genotype was significantly de-creased compared with patients carrying CC genotype [HR2 = 1 .532 ,95% CI(1 .119 -2 .096) ,P= 0 .008];in the recessive gene model ,no statistically significant difference was found in OS between patients carrying GG genotype and those carrying CC + CG genotype[HR2 =1 .975 ,95% CI(1 .000-3 .900) ,P=0 .0052];in the co-dominant model ,compared with patients carrying CC+CG genotype ,the OS in patients carrying CG genotype was decreased and their death risk was increased [HR2 =1 .395 ,95% CI(1 .007-1 .931)] .There was no correlation of miR-26A rs7372209 locus and miR-126 rs4636297 locus gene SNPS to OS and PFS in CRC patients (P>0 .05) .Conclusion miR-124 rs531564 gene polymorphism might be an independent influence factor of the prognosis in CRC patients .