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BACKGROUND:microRNA-26b(miR-26b)plays an important regulatory role in a variety of stem cell functions,but its effects on the biological properties of stem cells from human exfoliated deciduous teeth and human umbilical cord mesenchymal stem cells are unknown. OBJECTIVE:To investigate the effects of miR-26b on the proliferation,migration and osteogenic differentiation of stem cells from human exfoliated deciduous teeth and human umbilical cord mesenchymal stem cells. METHODS:Stem cells from human exfoliated deciduous teeth and human umbilical cord mesenchymal stem cells were cultured and identified.miR-26 mimics(experimental group)and miRNAs mimics control(control group)were used to transfect above mentioned two kinds of cells and construct overexpressed models for subsequent experiments.CCK-8 assay was applied to detect the proliferation ability of overexpressed miR-26b cells.Transwell and scratch assay were employed to analyze the migration ability of overexpressed miR-26b cells.RT-qPCR was utilized to examine the expression of osteogenic markers after osteogenic induction of overexpressed miR-26b cells. RESULTS AND CONCLUSION:(1)Transfection of miR-26b mimics increased miR-26b expression in the two kinds of cells and promoted the proliferation of stem cells from human exfoliated deciduous teeth,with no significant effect on the amplification of human umbilical cord mesenchymal stem cells.(2)Compared with the control group,the migration ability was enhanced after two types of cells overexpressing miR-26b.(3)miR-26b expression decreased during osteogenic differentiation of the two kinds of cells.(4)Compared with the control group,the levels of osteogenesis-related genes osteocalcin,osteopontin,alkaline phosphatase,and human type I collagen mRNA were downregulated after overexpression of miR-26b in the two kinds of cells.The results showed that overexpression of miR-26b promoted the proliferation and migration of stem cells from human exfoliated deciduous teeth and inhibited their osteogenic differentiation;it promoted the migration of human umbilical cord mesenchymal stem cells and inhibited their osteogenic differentiation,but had no significant effects on their proliferation.
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Objective:To investigate the diagnostic value of ultrasound elastography combined with serum microRNA-26b-5p(miR-26b-5p)and cyclooxygenase-2(COX-2)detection for early hysteromyoma.Methods:A total of 228 patients with suspected early hysteromyoma who were diagnosed in the 90th Hospital of the Joint Service Support Center from October 2020 to September 2022 were selected as the observation objects.Based on the results of pathological section examination as the"gold standard",all subjects were divided into a positive group(124 cases)and a negative group(104 cases),and ultrasound elasticity imaging examination was performed in both groups.The expression level of miR-26b-5p in serum was detected by real-time fluorescent quantitative polymerase chain reaction(Rt-PCR),and the level of serum COX-2 was detected by enzyme-linked immunosorbent assay(ELISA),and receiver operating characteristic(ROC)curve was applied to analyze the diagnostic values of serum miR-26b-5p and COX-2 for hysteromyoma.The four tables were applied to analyze the diagnostic values of ultrasound elastography and the combination of ultrasound elastography,serum miR-26b-5p and COX-2 for hysteromyoma.Results:The results of pathological examination indicated that 124 cases of 228 patients were positive result of hysteromyoma and 104 cases were negative result.The results of ultrasound elastography showed that 117 cases were positive,and 111 cases were negative,and the diagnostic sensitivity,specificity and accuracy of ultrasound elastography detection were respectively 74.19%,75.96%and 75.00%.Serum miR-26b-5p level of positive group was significantly lower than that of negative group,while the COX-2 level of positive group was significantly higher than that of negative group,and the differences of them between the two groups were statistically significant(t=4.519,5.601,P<0.05),respectively.The area under curve(AUC)value of ROC curve,sensitivity,specificity and the best cut-off value of serum miR-26b-5p were respectively 0.749,95.97%,46.15%and 1.10 in diagnosing hysteromyoma.The above indicators of serum COX-2 were respectively 0.835,66.13%,84.62%and 40.58 mg/L in diagnosing hysteromyoma.The sensitivity,specificity and accuracy of ultrasound elastography combined with serum miR-26b-5p and COX-2 were respectively 93.55%,86.54%and 90.35%,the differences were statistically significant(x2=23.158,17.169,P<0.05),respectively.Conclusion:Ultrasound elastography combined with serum miR-26b-5p and COX-2 has higher effectiveness in diagnosing the early hysteromyoma.
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Objective To explore the regulatory effect of miR-26b-5p on PI3K/PIP3 signaling path-way and its effect on L-type calcium channel of atrial cardiomyocytes in rats with ischemic ar-rhythmia.Methods A total of 72 SD rats were randomly divided into sham operation group,mod-el group,LY294002 group,negative control group,overexpression group and combination group,with 12 rats in each group.In 24 h after corresponding intervention,rat model of ischemic arrhyth-mia was established in the other groups except the sham operation group(only thoracotomy but without ligation).During the modeling process,arrhythmia indexes were detected in each group,and Curtis-Walker scoring was used for arrhythmia score.Fluorescence quantitative PCR was em-ployed to detect the expression of miR-26b-5p in atrial myocardiocytes,whole-cell patch-clamp technique was used to record the ICa-L of atrial myocardiocytes,and Western blotting was applied to measure the expression of CACNA1C,calmodulin and PI3K/PIP3 pathway related proteins.Re-sults Compared with the model group,the number of left ventricular preventricular contrac-tions,duration of ventricular tachycardia,duration of ventricular fibrillation,arrhythmia score,ab-solute value of ICa-L peak density of atrial myocardiocytes,and expression levels of CACNA1C and calmodulin were significantly increased,and the levels of miR-26b-5p,p-PI3K,PIP3 and p-Akt were obviously decreased in the LY294002 group(P＜0.05).While the overexpression group had decreased number of left ventricular preventricular contractions,shorter duration of ventricular tachycardia,shorter duration of ventricular fibrillation,lower arrhythmia score,reduced absolute value of ICa-L peak density,and lower expression levels of CACNA1C and calmodulin,and in-creased levels of miR-26b-5p,p-PI3K,PIP3 and p-Akt than the model group(P＜0.05).The number of left ventricular preventricular contractions,duration of ventricular tachycardia,dura-tion of ventricular fibrillation,arrhythmia score,absolute value of ICa-L peak density and expres-sion levels of CACNA1C and calmodulin were significantly increased,and the expression of p-PI3K,PIP3 and p-Akt were obviously decreased(0.82±0.08 vs 1.09±0.11,0.91±0.09 vs 1.17± 0.11,0.94±0.09 vs 1.20±0.12,P＜0.05)in the combination group than the overexpression group.Conclusion Overexpression of miR-26b-5p may block the L-type calcium channel in atrial myocardiocytes in ischemic arrhythmia rats by activating PI3K/PIP3 related signaling pathway,and thus improve the performance of arrhythmia in rats.
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【Objective】 To investigate the effect of ethanol extracts of Siwei Dihuang on the apoptosis of cardiomyocytes and its mechanism. 【Methods】 The experiment set the control group， model group， model group + ethanol extracts of Siwei Dihuang-L group， model group + ethanol extracts of Siwei Dihuang-M group， model group + ethanol extracts of Siwei Dihuang-H group， model group + miR-NC group， model group + miR-26b group model group + ethanol extracts of Siwei Dihuang-M + anti-miR-NC group， and model group + ethanol extracts of Siwei Dihuang-M + anti-miR-26b group. Flow cytometry was used to detect apoptosis； lactate dehydrogenase （LDH） kit， superoxide dismutase （SOD） kit， and malondialdehyde （MDA） kit were used to detect LDH， SOD activity and MDA content， respectively. Real-time quantitative PCR （RT-qPCR） was used to detect miR-26b and MAPK mRNA levels； luciferase report experiment was conducted to detect the targeting relationship between miR-26b and MAPK. 【Results】 Compared with those in control group， the apoptosis rate of the cardiomyocytes of the model group was significantly increased， MDA content and LDH activity were significantly increased， the activity of SOD was significantly decreased， miR-26b expression was significantly decreased， but MAPK mRNA expression was significantly increased （P<0.05）. Treatment with low， medium and high concentrations of ethanol extracts of Siwei Dihuang could reduce apoptosis rate， MDA content and LDH activity， increase SOD activity and increase miR-26b expression， and decrease MAPK mRNA expression （P<0.05）. Overexpression of miR-26b inhibited high glucose-induced cardiomyocyte apoptosis and oxidative stress. Inhibition of miR-26b reversed the inhibitory effect of ethanol extracts of Siwei Dihuang on H9C2 apoptosis and oxidative stress in high glucose-induced cells. miR-26b targeted and regulated MAPK. 【Conclusion】 Ethanol extracts of Siwei Dihuang can inhibit the apoptosis and oxidative stress of H9C2 cells in high glucose， and its mechanism may be related to the upregulation of miR-26b and MAPK expressions.
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Objective Diabetic cardiomyopathy (DCM) is one of the complications of diabetes, which is closely related to the change of miRNA. In this study, we observed the characteristic expression of miR-26b in the tissues of the C57BL/6J mouse and in the heart, adipose tissue and liver of the ob/ob mouse, and investigated the effect of high glucose (Glu) on the expression of miR-26b in H9C2 cardiomyocytes. Methods Using RT-PCR, we measured the levels of miR-26b in the heart, adipose tissue, liver and other tissues of C57BL/6J and ob/ob mice. H9C2 cardiomyocytes were treated with Glu at 5.5, 15, 25 and 35 mmol/L for 0, 24, 48, 72, 96 and 120 hours, followed by detection of the proliferation of cardiomyocytes by CCK-8 and determination of thelevels miR-26b. Results The expression of miR-26b was the highest in the heart of the C57BL/6J mice, significantly higher than in the cardiac and white adipose tissues of the ob/ob mice (P < 0.05). The proliferation of cardiomyocytes was markedly increased in the 15, 25 and 35 mmol/L Glu groups at 24, 48, 72, 96 and 120 hours as compared with that in the 5.5 mmol/L Glu group (P < 0.05), higher in the 25 than in the 15 mmol/L Glu group at 24 hours (0.74±0.02 vs 0.72±0.01, P<0.05), but lower in the 35 than in the 15 mmol/L Glu group at 48 hours (0.92±0.01 vs 0.94±0.01, P<0.05), in the 25 and 35 mmol/L Glu groups at 96 hours (P < 0.05), in the 35 mmol/L Glu group at 120 hours (1.12±0.02 vs 1.19±0.05, P<0.05), in the 35 than in the 25 mmol/L Glu group at 24 and 48 hours (P<0.05). The expression of miR-26b in the H9C2 cardiomyocytes was significantly down-regulated in the 25 and 35 mmol/L Glu groups in comparison with that in the 5.5 mmol/L Glu group (P<0.05), remarkably lower in the 25 mmol/L Glu group at 96 and 120 hours than at 0 hour (P<0.05). Conclusion High glucose can down-regulate the expression of miR-26b in H9C2 cardiomyocytes, which suggests that miR-26b may participate in the pathogenesis of DCM.
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Objective:To investigate the effects of 7-difluoromethy-5,4:dimethoxygenistein (DFMG) on stress urinary incontinence (SUI) model in Sprague Dawley (SD) rats and its possible mechanisms.Methods:SD rat model of SUI was established through simulating pregnancy,birth trauma and ovarian castration.The rats were divided into a normal control group,a SUI group,and a DFMG group at 10 or 20 mg/kg.They were treated with 10 mg/kg normal saline (NS),10 mg/kg NS,10 mg/kg DFMG and 20 mg/kg DFMG,respectively,via gastric gavage every other day.Maximal bladder capacity (MBC),leak point pressure (LPP),abdominal leak point pressure (ALPP),hematoxylin-eosin (HE) staining,and Masson staining were performed to detect the index for the model.MiR-26b and its down-stream gene phosphatase and tensin homolog deleted on chromosome 10 (PENT) mRNA in urethral sphincter muscles cells (USMCs) were analyzed by RT-PCR.The protein levels of PENT,phosphatidylinositol 3-kinase (PI3K),protein kinaseB (AKT),B-cell lymphoma 2 (Bcl-2),Bcl-2 associated X protein (Bax),cytochrome C(Cyt-c) and caspase-3 were examined by Western blot.The apoptotic rate of USMCs was determined by flow cytometry (FCM),and the proliferative rate of USMCs was examined by MTT assay.Results:The SD rat model of SUI was successfully established.HE staining and Masson staining showed that the pathological features of urethral sphincter were improved in the DFMG-treated groups compared with the SUI group.The urine dynamics indexes of model rats,such as MBC,LPP and ALPP,were improved (all P＜0.05).The results of RT-PCR showed that the miR-26b mRNA was up-regulated (P＜0.05) and PENT mRNA was down-regulated (P＜0.05) in the DFMG-treated groups compared with the SUI group.Simultaneously,compared with the SUI group,the protein levels of PENT,Bax,Cyt-c and caspase-3 were down-regulated (all P＜0.05) and the protein levels of PI3K,AKT and Bcl-2 protein were up-regulated (all P＜0.05),accompanied by the decreased apoptotic rate of USMCs (P＜0.05) and the increased proliferative rate of USMCs (P＜0.05) in the DFMG-treated groups.Conclusion:The DFMG can significantly improve the symptoms of urinary dynamics,which might be related to the up-regulation of miR-26b expression and the regulation of PI3/AKT-Bcl-2/ Bax signaling pathways.
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Objective To study the cognitive impairment in SD rats after intermittent hypoxia (IH),and explore the relation of miR-26b up-regulated expression and neuron apoptosis in the hippocampus of SD rats after IH.Methods Eight-week-old male SD rats (n=20,each weighing approximately 300±10 g) were randomly divided into normal oxygen control group,IH 1-week group,IH 2-weeks group and IH 4-weeks group (n=5).Rats in the later three groups were given IH for different times,and rats in the normal oxygen control group were given normal oxygen.The spatial learning and memory abilities were detected by Morris Water Maze (MWM) in the normal oxygen control group and IH 4-weeks group.The levels of apoptosis proteins Caspase3 and Bax and anti-apoptosis protein Bcl-2 in the hippocampus of 4 groups were detected by Western blotting.The miR-26b expression level in the 4 groups was detected by real time-PCR.Results (1) The results of MWM revealed that the mean escape latency in the IH 4-weeks group was significantly prolonged as compared with that in the normal oxygen control group (P＜0.05);the time entering into the target quadrant in the IH 4-weeks group ([22.0±6.7] s) was significantly shorter than that in the normal oxygen control group ([39.8±8.8] s,P＜0.05).(2) Western blotting indicated that up-regulated expressions of apoptosis proteins Bax and Casepase3 and down-regulated expression of anti-apoptosis protein Bcl-2 in the IH 1-week group,IH 2-weeks group and IH 4-weeks group were noted as compared with those in the control group,with significant differences (P＜0.05);significantly higher apoptosis protein Bax and Casepase3 expressions in the IH 1-week group were noted as compared with those in the IH 2-weeks group and IH 4-weeks group (P＜0.05),while significantly decreased Bcl-2 expression in the IH 1-week group was noted as compared with that in the IH 2-weeks group and IH 4-weeks group (P＜0.05).(3) The results of real time-PCR revealed that the miR-26b expression level in the hippocampus was up-regulated in the IH 1-week group,IH 2-weeks group and IH 4-weeks group as compared with that in the control group,with significant differences (P＜0.05);miR-26b expression level in the IH 1-week group was significantly higher as compared with that in the IH 2-weeks group and IH 4-weeks group (P＜0.05).Conclusion The miR-26b up-regulated expression in the hippocampus might refer to Bax /Bcl-2-related mitochondrial apoptotic signaling pathway after IH brain injury;miR-26b could be a potential mean ofgene therapy after IH brain injury.
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Objective:To investigate the effect of miR-26b on the invasion and migration of lung cancer cell and to explore its mechanism.Methods:qPCR was used to detect the expression of miR-26b in lung cancer.Luciferase reporter gene was used to detect interaction between miR-26b and hENT1.Transwell assay was used to detect invasion ability after treatment of miR-26b mimics.Scratch assay was used to detect migration ability after treatment of miR-26b mimics.The expressions of hENT1,ROCK-I and RhoA were detected by Western blot.The changes of cytoskeleton after miR-26b mimics treatment with phalloidin were observed.The effect of miR-26b mimics on the tumor size and volume of lung cancer was determined by subcutaneous tumor formation in nude mice.Results:MiR-26b expression was significantly reduced in lung cancer.With the progress of lung cancer,the expression of miR-26b was reduced.With the progress in differentiation of lung cancer,the expression of miR-26b was decreased.Decrease of miR-26b was associated with lung cancer lymph node metastasis.HENT1 was the direct target of miR-26b;miR-26b regulated the invasion and migration ability of human lung carcinoma A549 cells.MiR-26b regulated the expression of hENT1,ROCK-1 and RhoA.After the treatment with miR-26b mimics,the F-actin staining was significantly reduced,whereas the formation of wrinkles and the formation of pseudopodia were significantly reduced.Subcutaneous tumor formation in nude mice showed that miR-26b mimics treatment significantly reduced the tumor size and mass.Conclusion:MiR-26b plays a role in tumor suppression in lung cancer.miR-26b can regulate the invasion and migration ability of lung carcinoma A549 cells by targeting hENT1 depending on the RhoA/ROCK-1 pathway.
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Objective To reveal the pancreatic cancer cell drug resistance mechanism to provide a basis for clinical treatment by studying miR-26b targeting p53 for promoting the drug resistance of pancreatic cancer cell line PANC-1 .Methods (1) The over-expression and knockdown plasmid of p53 and the fluorescent reporter vector containing 3′Untranslated region(3′UTR) were constructed respectively .(2) The effect of p53 and miR-26b on the growth and proliferation of PANC-1 was investigated by methyl thiazolyl tetrazolium(MTT) in the presence of gemcitabine .(3) The target relationship between miR-26b and p53 was determined by bioinformatics ,real-time polymerase chain reaction(PCR) ,fluorescent reporter vector and Western blot experiment .(4) The effect of p53 on the growth of miR-26b was investigated by salvage experiments .Results (1) The MTT experiment confirmed that ,in the presence of gemcitabine ,over-expression of p53 could inhibit the proliferation of pancreatic cancer cell line PANC-1 ,and knock-down of p53 could promote the growth and proliferation of PANC-1;(2) the bioinformatics prediction showed that miR-26b targeted p53 ,real-time PCR ,Western blot and fluorescent reporter vector experiment confirmed that p 53 is the target gene of miR-26b , and miR-26b inhibits transcription and translation of p53 by targeting the 3′UTR of p53 gene;(3) the MTT experiment confirmed that in the presence of gemcitabine ,over-expression of miR-26b could promote the growth and proliferation of PANC-1 cells ,and enhanced its resistance to gemcitabine ;(4) the rescue experiment confirmed that the simultaneous over-expression of p53 rescued the drug resistance promoting effect of miR-26b on PANC-1 .Conclusion miR-26b inhibits the expression of p53 by targeting 3′UTR of the p53 gene and enhances the drug resistance of pancreatic cancer cell line PANC-1 to gemcitabine .
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Objective To investigate the inlfuence of overexpression of miR-26b on the secretion of adipokines dur-ing human adipocyte differentiation. Methods Human preadipocytes were infected with the hsa-miR-26b over-expressing lentivirus and were induced to differentiate, and then the levels of adipokines (IL-6, leptin, resistin, TNF-α) at different time points during differentiation were measured by ELISA. Results Compared with control group, decreased secretions of both IL-6 and leptin, and increased secretion of resistin were found during the differentiation of human adipocytes in miR-26b overexpressed group. However, the secretion of TNF-αwas not measured in both groups. Conclusion The miR-26b can improve the inlfammation and insulin resistance of human adipocytes, which will provide potential targets for obesity treat-ment.