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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 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.