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Our previous research studies have demonstrated the role of microRNA133b (miR133b) in healing the contused spinal cord when administered either intranasally or intravenously 24 h following an injury. While our data showed beneficial effects of exogenous miR133b delivered within hours of a spinal cord injury (SCI), the kinetics of endogenous miR133b levels in the contused spinal cord and rostral/caudal segments of the injury were not fully investigated. In this study, we examined the miR133b dysregulation in a mouse model of moderate unilateral contusion injury at the fifth cervical (C5) level. Between 30 min and 7 days post-injury, mice were euthanized and tissues were collected from different areas of the spinal cord, ipsilateral and contralateral prefrontal motor cortices, and off-targets such as lung and spleen. The endogenous level of miR133b was determined by RT-qPCR. We found that after SCI, (a) most changes in miR133b level were restricted to the injured area with very limited alterations in the rostral and caudal parts relative to the injury site, (b) acute changes in the endogenous levels were predominantly specific to the lesion site with delayed miR133b changes in the motor cortex, and (c) ipsilateral and contralateral hemispheres responded differently to unilateral SCI. Our results suggest that the therapeutic window for exogenous miR133b therapy begins earlier than 24 h post-injury and potentially lasts longer than 7 days.
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Médula Cervical , Contusiones , MicroARNs , Traumatismos de la Médula Espinal , Animales , Ratones , Contusiones/metabolismo , Modelos Animales de Enfermedad , MicroARNs/metabolismo , MicroARNs/uso terapéutico , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/terapia , Médula Cervical/lesionesRESUMEN
Objective: To investigate the mechanism of moxibustion in the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D), by observing the effects of moxibustion at Tianshu (ST25) and Shangjuxu (ST37) on microRNA-133b (miRNA-133b), pituitary homeobox family factor 3 (Pitx3)/tyrosine hydroxylase (TH), and neurotransmitters in the brain tissue of IBS-D rats. Methods: Healthy Sprague-Dawley rats were randomly divided into a normal group, a model group, a moxibustion group, and a Western medicine group, with 12 rats in each group. Except for the normal group, the IBS-D rat model was established by mother-offspring separation and acetic acid enema combined with restraint stress stimulation in all the other groups. No intervention was performed in the normal and model groups. Mild moxibustion was applied to both Tianshu (ST25) and Shangjuxu (ST37) in the moxibustion group. Rifaximin was given by gavage in the Western medicine group. The physical status of rats in each group was observed at different periods. After the intervention, hematoxylin- eosin staining was performed to observe the histopathological morphology of rat colon; enzyme-linked immunosorbent assay was used to measure the levels of dopamine (DA), noradrenaline (NE), and 5-hydroxytryptamine (5-HT) in plasma, colon, and midbrain tissue of rats; the relative expression levels of miRNA-133b, Pitx3 mRNA, and TH mRNA in the midbrain tissue were measured by real-time fluorescence quantitative polymerase chain reaction, and the relative expression levels of Pitx3 and TH proteins in the midbrain tissue were measured by Western blotting and immunofluorescence. Results: The body weights of rats among groups and at different time points were statistically different (P<0.01). The body weight of the normal group was higher than that of the other groups over time (P<0.01). After modeling, the minimum volume threshold of abdominal withdrawal reflex (AWR) was significantly lower (P<0.01) and the loose stool rate was significantly higher (P<0.01) in the model, moxibustion, and Western medicine groups compared with the normal group; the miRNA-133b expression in the midbrain tissue was significantly lower (P<0.01), the expression levels of Pitx3 and TH in the midbrain tissue were significantly higher (P<0.01), and the levels of DA, NE, and 5-HT in plasma, colon and midbrain tissue were significantly higher (P<0.01). After the intervention, the minimum volume threshold of AWR was significantly higher (P<0.01), the loose stool rate was significantly lower (P<0.01), the miRNA-133b expression was significantly increased (P<0.01 or P<0.05) and the expression levels of Pitx3 and TH were significantly decreased (P<0.01) in the midbrain tissue, the levels of DA, NE, and 5-HT in plasma, colon, and midbrain tissue were significantly reduced (P<0.01) in the moxibustion and Western medicine groups compared with the model group; the levels of 5-HT in the colon and midbrain tissue of the moxibustion group were significantly lower than those in the Western medicine group (P<0.05), and there was no statistical difference compared with the remaining groups (P>0.05). Linear correlation analysis showed that miRNA-133b was negatively correlated with Pitx3 (r<0, P<0.01); Pitx3 with TH, TH with DA, and NE with 5-HT were positively correlated (r>0, P<0.01).Conclusion: Moxibustion at Tianshu (ST25) and Shangjuxu (ST37) improves diarrhea symptoms and visceral hypersensitivity in IBS-D rats. The mechanism may be related to up-regulating miRNA-133b, inhibiting Pitx3/TH, and reducing neurotransmitter expression levels in the midbrain tissue.
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Objective To investigate the effect of microRNA-133b(miR-133b)on cardiac fibrosis and its mechanism.Methods Human cardiac fibroblasts(CFs)were harvested.The proliferation of CFs was detected by CCK8 during the overexpression and knock-down of miR-133b.The expressions of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),collagen Ⅰ,and collagen Ⅲ were detected with qRT-PCR and Western blot analysis after miR-133b overexpression or downexpression.Target genes of miR-133b were predicted by bioinformatics software.Dual-luciferase activity assay were used to verify a target gene of miR-133b.Results qRT-PCR showed that the expression level of miR-133b in the miR-133b mimic group was significantly higher than that in the negative control group(=26.219,=0.000).The expression level of miR-133b in the miR-133b inhibitor group was significantly lower than that in the negative control group(=6.738,=0.003).After 21,45,69,93,and 117 hours of transfection,the proliferation ability of CFs significantly decreased in the miR-133b mimic group but significantly increased in the miR-133b group(all <0.05,compared with the negative control group).After overexpression of miR-133b,the mRNA and protein levels of CTGF(=9.213,=0.001;=8.195,=0.001),α-SMA(=6.511, =0.003;=4.434,=0.011),collagenⅠ(=3.172,=0.034;=4.053,=0.015)and collagen Ⅲ(=6.404,=0.003;=5.319,=0.006)were significantly down-regulated.After the expression of miR-133b was knocked down,the mRNA and protein levels of CTGF(=9.439,=0.001;=14.100,=0.000),α-SMA(=4.519,=0.011;=4.377,=0.012),collagen Ⅰ(=5.966,=0.004;=5.514,=0.005)and collagen Ⅲ(=4.622,=0.010;=4.996,=0.008)were significantly increased.The relative luciferase activity of the cells co-transfected with miR-133b mimic and WT 3'UTR expression vector was significantly lower than that of the cells co-transfected with mimic control and WT 3'UTR expression vectors(=5.654,=0.005);however,there was no significant difference in relative luciferase activity between cells co-transfected with miR-133b mimic and MUT 3'UTR expression vectors and cells co-transfected with mimic control and MUT 3'UTR expression vectors(=0.380,=0.724).Conclusion miR-133b may affect the activation and proliferation of CFs by targeting CTGF and thus improve cardiac fibrosis.
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Humanos , Actinas , Metabolismo , Proliferación Celular , Células Cultivadas , Colágeno , Metabolismo , Factor de Crecimiento del Tejido Conjuntivo , Metabolismo , Fibroblastos , Biología Celular , Fibrosis , MicroARNs , Genética , Miocardio , PatologíaRESUMEN
Objective To investigate the expression level of miR-133b in cancer tissues of patients with prostate cancer and its effect on the proliferation of prostate cancer cells.Methods The total RNAs in resected prostate cancer tissues and adjacent tissues from 30 patients with prostate cancer were extracted and reversely transcripted into cDNA,and then the expression levels of miR-133b were detected by real-time quantitative PCR.The correlations between the expression levels of miR-133b and the patients' clinicopathological features were analyzed.The expression of positive regulatory domain I-binding factor 16 (PRDM16) and proliferation of PC-3 cells transfected with miR-133b mimics by LipofectamineTM 3000 were determined by real-time quantitative PCR and the CCK8 method,respectively.Results The expression levels of miR-133b in prostate cancer tissues [(16.85 ± 0.94) × 10-4] was significantly lower than that in adjacent tissues [(22.95 ± 1.567) × 10-4,t =3.335,P < 0.01].The expression levels of PRDM16 in PC-3 cells transfected with miR-133b mimics were significantly lower than that in the control group (0.371 ±0.031 vs 1.000 ±0.022,t =12.53,P < 0.01).The proliferation ability of PC3 cells transfected with miR-133b mimics for 72 hours was significantly lower than that in the control group (t =6.811,P < 0.01).Similarly,the proliferation ability of PC-3 cells transfected with PRDM16 inhibitor for 72 hours was also significantly lower than that in the control group (t =9.048,P <0.01).Conclusion The expression levels of miR-133b in prostate cancer tissues are significantly down-regulated,which regulate the proliferation of prostate cancer cells possibly through PRDM16.
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Background Ultraviolet B (UVB) is one of the main causes of cataract formation,and its mechanism is associated with the apoptosis of lens epithelial cells (LECs).MiroRNA-133b (miR-133b) can regulate oxidative stress-induced LECs apoptosis.However,whether miR-133b is associated with UVB-induced cataract is not elucidated.Objective This study was to observe the inhibitory effects of miR-133b on UVB-induced cataract and its regulating mechanism.Methods Twenty 8-week-old C57BL/6 mice were randomized into cataract model group and normal control group.The mouse eyes in the cataract model group exposed to 302 nm UVB for 5 minutes once per day for consecutive 1 week,with the irradiation intensity of 300 W/cm2,and the mice in the normal control group did not receive any intervention.Five mice in each group were sacrificed and 10 eyeball sections were prepared.Human LECs (SRA01/04) were exposed to UVB for 25 minutes and served as UVB-induced group,the cells in the normal control group did not receive any intervention.The UVB-induced cells were inoculated to 96-well plate and divided into 4 groups,and 50 nmol/L miR-133b mimic,miR-133b mimic control agent,miR-133b inhibitor and miR-133b inhibitor control agent were transfected into the cells with lipofectamine2000.The expression of miR-133b mRNA and a target gene BCL2L2,which was identified by online miRNA database (www.mirab.org) in the cells were detected by real-time quantitative PCR to evaluate the transfected efficacy,and the apoptosis of the cells in mouse lens tissue and different transfected groups were assayed by TUNEL.The use and care of the mice followed ARVO Statement.Results The arrangement of LECs was regular and no apoptotic cell was seen in the normal control group,and the apoptotic cells showed the red fluorescence in the cataract model group.The apoptotic rate of human LECs was (43.90±9.30) % in the UVB-induced group,and that in the normal control group was (1.08±0.49)%,showing a significant difference between the two groups (t =-7.963,P =0.015).The relative expression levels of miR-133b mRNA in the model mouse lens and UVB-induced human LECs were evidently lower and relative expression levels of BCL2L2 mRNA were higher than those in normal mice and normal LECs (miR-133b mRNA:t =-2.958,P =0.042;t =-6.195,P =0.003;BCL2L2 mRNA:t =3.761,P =0.020;t =12.437,P =0.000).The relative expression level of miR-133b mRNA was significantly increased and the relative expression level of BCL2L2 mRNA was reduced in the miR-133b mimic group in comparation with the miR-133b mimic control group (t=10.883,-5 927.617;both at P< 0.01);compared with the miR-133b inhibitor control group,the relative expression level of miR-133b mRNA was significantly decreased and that of BCL2L2 mRNA was evidently increased in the miR-133b inhibitor group (t =-1 606.622,17.556;both at P < 0.01).The apoptotic rate of human LECs was (43.62 ± 9.19) % and (17.55 ± 4.24) % in the miR-133b mimic control group and miR-133b mimic group,with a significant difference between them (t =-4.462,P =0.011),and the apoptotic rate in the miR-133b inhibitor group was (78.23 ± 12.42) %,which was significantly higher than (48.01 ±9.68) % in the miR-133b inhibitor control group (t =3.324,P =0.029).Conclusions miR-133b can prevent UVB-induced cataract probably by negatively targeting the BCL2L2 expression to regulate the apoptosis of LECs.
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Objective To investigate the role of microRNAs (miRs) in the proliferation of vascular smooth muscle cells (VSMCs)induced by endothelial insulin-like growth factor-1 (IGF-1) under low shear stress (LowSS). Methods Endothelial cells (ECs) and VSMCs were co-cultured and exposed to normal shear stress (NSS, 1.5 Pa) and LowSS (0.5 Pa) for 12 h with parallel plate flow chamber system, respectively. Real-time PCR was used to examine the expression levels of miRs. The target genes of miR-133b were predicted by multiple algorithms. The expression of polypyrimidine tract binding protein 1 (Ptbp1) and N-myc downstream regulated 1 (Ndrg1) in VSMCs was detected by Western blotting. The VSMC proliferation was detected by EdU flow cytometry assay. Results After treated with recombinant IGF-1, the expression of both miR-133b and miR-378a in VSMCs was increased. Compared with NSS, LowSS significantly induced the expression of miR-133b in the co-cultured VSMCs, but had no obvious effect on miR-378a. In VSMCs, the protein and mRNA levels of Ptbp1 and Ndrg1 were down-regulated by miR-133b mimics. miR-133b inhibitor up-regulated the mRNA levels of Ptbp1 and Ndrg1. miR-133b overexpression promoted the proliferation of VSMCs significantly. Conclusions IGF-1 secreted by ECs in response to LowSS can upregulate the expression of miR-133b in the co-cultured VSMCs, which subsequently depresses the expression of Ptbp1 and Ndrg1, and induces the proliferation of VSMCs eventually. The research findings provide a potential new target for cardiovascular disease therapy.