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Objective: To investigate the inhibitory effect of leonurine on cardiomyocyte hypertrophy induced by angiotensin Ⅱ(Ang Ⅱ) and its effect on p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway and miRNA-1.Method: Cardiomyocyte hypertrophy was induced by Ang Ⅱ (0.1 μmol·L-1) in primary neonatal cardiomyocytes. Experiments were designed in 6 groups as following:normal group, model group, p38 MAPK inhibitor group (SB203580, 10 μmol·L-1), low-dose(5 μmol·L-1), middle-dose(10 μmol·L-1) and high-dose(20 μmol·L-1) group. The cardiomyocyte surface area was measured by image software, and the protein contents were detected by Lowry. The concentrations of ANP in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The expression level of miRNA-1 was detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR), and the protein expression levels of endothelin-1 (ET-1), p38 MAPK, p-p38 MAPK, myocyte enhancer factor 2 (MEF2), β-myosin heavy chain (β-MHC), α-myosin heavy chain (α-MHC) were detected by Western blot.Result: Compared with normal group, the surface area of cardiomyocyte, the protein contents, the concentrations of ANP, and the protein expression levels of ET-1, p38 MAPK, p-p38 MAPK, MEF2, β-MHC in model group were higher (Pα-MHC and miRNA-1 were lower than those in normal group (Pβ-MHC in high-dose group were lower (Pα-MHC and miRNA-1 were higher than those in model group (PConclusion: Leonurine (20 μmol·L-1) could inhibit cardiomyocyte hypertrophy induced by AngⅡ, and the mechanism is related to the inhibition of activation of p38 MAPK signaling pathway and up-regulation the expression of miRNA-1.
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Objective@#To investigate the expression of miRNA-1 (miR-1) in colorectal cancer (CRC) tissues and its effect on proliferation and invasion of CRC cells in vitro as well as its mechanism.@*Methods@#A total of 180 CRC tissues from the hospitalized patients who underwent excision surgery and 114 adjacent cancer tissues in the Affiliated Cancer Hospital of Shanxi Medical University between June 2015 and December 2015 were collected. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-1 in 58 cases of CRC tissues and adjacent cancer tissues. Immunohistochemistry was used to detect the expression of stromal cell-derived factor-1 (SDF-1) protein in 122 CRC tissues and effect of overexpression or knockdown of miR-1 on the proliferation, colony formation and invasiveness of DLD1 cells, respectively. Western blot was used to determine the effect of overexpression or knockdown of miR-1 on the expression of SDF-1 in DLD1 cells.@*Results@#RT-qPCR results showed that the expression of miR-1 in CRC tissues was decreased compared with the corresponding adjacent cancer tissues (the relative expression level ratio < 0.5), and the low expression rate of miR-1 in CRC was 82.8% (48/58). Immunohistochemistry results showed that the positive expression rate of SDF-1 in CRC tissues was higher than that in adjacent cancer tissues [81.1% (99/122) vs. 8.9% (5/56), χ2 = 82.415, P < 0.01]. Cell function experiment showed that, compared with the control group, the proliferative activity of DLD1 cells that transfected miR-1 mimics was decreased, meanwhile, the colony formation, invasion and SDF-1 expression were reduced (P < 0.05). The proliferative activity, colony formation, invasion and SDF-1 expression in DLD1 cells that transfected miR-1 inhibitor were improved compared with those in the control group (P < 0.05).@*Conclusions@#The expression of miR-1 in CRC tissues is low and it may act as a tumor suppressor gene through affecting proliferation and invasive potential of CRC cells by regulating the expression of SDF-1.
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Objective To investigate the expression of miRNA-1 (miR-1) in colorectal cancer (CRC) tissues and its effect on proliferation and invasion of CRC cells in vitro as well as its mechanism. Methods A total of 180 CRC tissues from the hospitalized patients who underwent excision surgery and 114 adjacent cancer tissues in the Affiliated Cancer Hospital of Shanxi Medical University between June 2015 and December 2015 were collected. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-1 in 58 cases of CRC tissues and adjacent cancer tissues. Immunohistochemistry was used to detect the expression of stromal cell-derived factor-1 (SDF-1) protein in 122 CRC tissues and effect of overexpression or knockdown of miR-1 on the proliferation, colony formation and invasiveness of DLD1 cells, respectively. Western blot was used to determine the effect of overexpression or knockdown of miR-1 on the expression of SDF-1 in DLD1 cells. Results RT-qPCR results showed that the expression of miR-1 in CRC tissues was decreased compared with the corresponding adjacent cancer tissues (the relative expression level ratio < 0.5), and the low expression rate of miR-1 in CRC was 82.8％ (48/58). Immunohistochemistry results showed that the positive expression rate of SDF-1 in CRC tissues was higher than that in adjacent cancer tissues [81.1％ (99/122) vs. 8.9％ (5/56), χ2= 82.415, P< 0.01]. Cell function experiment showed that, compared with the control group, the proliferative activity of DLD1 cells that transfected miR-1 mimics was decreased, meanwhile, the colony formation, invasion and SDF-1 expression were reduced (P< 0.05). The proliferative activity, colony formation, invasion and SDF-1 expression in DLD1 cells that transfected miR-1 inhibitor were improved compared with those in the control group (P< 0.05). Conclusions The expression of miR-1 in CRC tissues is low and it may act as a tumor suppressor gene through affecting proliferation and invasive potential of CRC cells by regulating the expression of SDF-1.
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OBJECTIVE@#To observe the expression differences of the plasma miRNA-1, miRNA-21 between patients with coronary heart disease (CHD) and without coronary artery lesions, between patients with in-stent restenosis (ISR) and none in-stent restenosis (NISR), and to study their predictive value for ISR occurred after percutaneous coronary intervention (PCI) in patients with CHD and diabetes mellitus (DM).@*METHODS@#The selected subjects were divided into CHD group in which patients were implemented stenting (=187), and control group in which patients were without coronary artery lesions (=195). According to the guidelines, the control group was divided into normal group (=150), simple-DM group (=45); the CHD group was divided into simple-CHD group (=119) and CHD-DM group (=68), the CHD group was also divided into ISR group (=48), NISR group (=139), and the ISR group was divided into simple-ISR group (=26) and ISR-DM group (=22) again. Plasma was collected from each group, and total RNA was extracted, the level of blood miRNA-1, miRNA-21 of each group was detected, and their level differences were analyzed.@*RESULTS@#Compared with control group, the level of miRNA-1 and miRNA-21 of CHD group was increased (<0.05); compared with NISR group, the level of miRNA-1 and miRNA-21 of ISR group was increased (<0.05). The incidence of ISR of CHD-DM group was obviously higher than that of simple-CHD group, ISR-DM group's level of miRNA-21 was higher than that of simple-ISR group (<0.05), and there was no difference of miRNA-1 level between ISR and ISR-DM group (<0.05). In Logistics, for CHD patents, the OR of DM, miRNA-1, miRNA-21 were 2.132, 3.066, 1.924 respectively (<0.05); for CHD patents with ISR, the OR of DM, miRNA-21 were 2.123, 3.066 respectively (<0.05); especially for CHD and DM patents with ISR, the OR of miRNA-21 was 9.148 (<0.05). In ROC curve, for CHD patients with ISR, the AUC of miRNA-1, miRNA-21 were 0.854, 0.857 respectively; for CHD-DM patients with ISR, the AUC of miRNA-21 was 0.783.@*CONCLUSIONS@#To predict the occurrence of ISR for CHD patients, the plasma miRNA-1 and miRNA-21 have a relatively high specificity and sensitivity, for CHD patients with DM, miRNA-21 may have a higher clinical value.
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Humans , Coronary Angiography , Coronary Restenosis , General Surgery , Diabetes Complications , Diabetes Mellitus , MicroRNAs , Blood , Nerve Tissue Proteins , Blood , Percutaneous Coronary Intervention , StentsABSTRACT
OBJECTIVE: To investigate the expression of miRNA-1 in denervated skeletal muscle at different periods, and to explore effects of passive movement on the expression of miRNA-1 and differentiation of myoblasts in denervation-induced skeletal muscle atrophy in rats. METHODS: Twenty-seven Sprague Dawley rats, weighing (200±10) g, were randomly divided into sham-operated group (group A, n=3), denervated group (group B, n=12), and passive movement group (group C, n=12). After the right sciatic nerve was exposed and dissociated, the sciatic nerve of 1 cm in length was removed in groups B and C; resection was not performed in group A. At 1 day after operation, passive flexion and extension movement was performed on the right hind limb in group C. At 6 hours in group A and at 3, 7, 14, and 28 days in groups B and C, 3 rats were sacrificed to measure the wet weight ratio of gastrocnemius muscle, to observe the diameter of the gastrocnemius muscle cell and evaluate the muscle atrophy by HE staining; RT-PCR was used to detect the mRNA expression of miRNA-1 and myocyte differentiation factor (MyoD), and immunohistochemistry to determine the protein expression of MyoD. RESULTS: Atrophy in various degrees was observed in denervated gastrocnemius muscle of groups B and C. The muscle fiber arranged in disorder and the diameter of the muscle cells decreased gradually with the time, without normal structure and morphology. The wet weight ratio and the cell diameter of the gastrocnemius in groups B and C were significantly less than those in group A (P0.05), and had positive correlation at 14 and 28 days (P<0.05); positive correlation was found between the relative expression of MyoD and miRNA-1 mRNA (P<0.05). CONCLUSIONS: Passive movement can prevent amyotrophy by increasing the expression of miRNA-1 and promoting the differentiation of myoblasts.
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Objective To investigate the effect of miRNA -1 00 on the proliferation of human leukemia cells HL -60,and to explore the mechanism of this action.Methods The bioinformatics software and database were applied to predict and analyze target genes of miRNA -1 00.The vector contained the target gene 3′UTR portion cloned into a luciferase reporter construct.A luciferase reporter assay was performed following co -transfection of small molecular miRNA -1 00 mimics and target gene wild -type or mutant plasmid into HEK -293T cells.HL -60 cells were trans-fected with miRNA -1 00 mimics or anti -miRNA -1 00.After transfection,Western blot was applied to validate the expression of carboxy -terminal domain small phosphatase -like protein (CTDSPL),and the viability of HL -60 was measured by using cell counting kit (CCK -8)assay at 24 h,48 h,72 h,96 h.Results Online software predicted that CTDSPL was likely to be the target gene of miRNA -1 00.Dual luciferase reporter gene assay system showed that miRNA -1 00 could significantly suppress the activity of reporter gene containing CTDSPL 3′-UTR which decreased by about 57.1 %(P =0.000 7).Western blot showed that the expression of CTDSPL was increased after being trans-fected with miRNA -1 00 antisense oligonucleotides and decreased after being transfected with miRNA -1 00 mimics.At the same time,the growth rate of cells treated with miRNA -1 00 mimics or CTDSPL miRNA -1 00 was increased com-pared with that in control by CCK -8 test (P <0.05 ).Conclusions CTDSPL is a downstream target gene of miRNA -1 00.miRNA -1 00 can promote leukemia cell proliferation by inhibiting the expression of CTDSPL.
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Objective To investigate the effect of miRNA-1 on the cardiomyocyte apoptosis in rats. Methods MicroRNA-1 mimics was transfected into the cultured H9c2 cell line (miRNA-1 group). Cells transfected with random miR-NA fragment was used as negative control group. The cell apoptosis was evaluated by FCM assay. MTT assay was used to de-tect the cell viability. The expression level of miRNA-1 was detected by real-time PCR. The expression levels of Bcl-2 mRNA and protein were detected by real-time PCR and Western blot assay. Results Compared with normal and negative control groups, the expression level of miRNA-1 was significantly higher in H9c2 cardiomyocytes, the apoptosis rate was in-creased, the cell vitality and Bcl-2 expression level were significantly decreased after transfection of miRNA-1 mimics. Conclusion miRNA-1 mimics can up-regulate miRNA-1 level, inhibit proliferation and induce cardiomyocyte apoptosis.