BackgroundMicroRNA-24 (miR-24) plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy.

MethodsTwelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randomly divided into two groups: a transverse aortic constriction (TAC) group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay. Immunofluorescence labeling was used to measure the cell surface area, and H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes (NRCMs) with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P < 0.05 was considered as the threshold for significance.

ResultsThe expression of miR-24 was abnormally increased in TAC rat cardiac tissue (t = -2.938, P < 0.05). TargetScans algorithm-based prediction demonstrated that CDKN1B (p27, Kip1), a cell cycle regulator, was a putative target of miR-24, and was confirmed by luciferase assay. The expression of p27 was decreased in TAC rat cardiac tissue (t = 2.896, P < 0.05). The overexpression of miR-24 in NRCMs led to the decreased expression of p27 (t = 4.400, P < 0.01), and decreased G0/G1 arrest in cell cycle and cardiomyocyte hypertrophy.

ConclusionMiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression.

Animals ; Cardiomegaly ; genetics ; pathology ; Cell Cycle ; genetics ; physiology ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Male ; MicroRNAs ; genetics ; Myocardium ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate whether the connection of p27(Kip1) to S-phase kinase-associated protein 2 (Skp2) plays an oncogenic role in intraductal proliferative lesions of the breast.

METHODSHere we investigated the mechanism involved in association of Skp2’s degradation of p27(Kip1) with the breast carcinogenesis by immunohistochemical method through detection of Skp2 and p27(Kip1) protein levels in 120 paraffin-embedded tissues of intraductal proliferative lesions including usual ductal hyperplasia (UDH, n=30), atypical ductal hyperplasia (n=30), flat epithelial atypia (FEA, n=30), and ductal carcinoma in situ (DCIS, n=30). Moreover, the expression status of Skp2 and p27(Kip1) in 30 cases of the normal breast paraffin-embedded tissues were explored.

RESULTSThe DCIS group was with the highest Skp2 level and the lowest p27(Kip1) level, and the UDH group was with the lowest Skp2 level and the highest p27(Kip1) level.Both Skp2 and p27(Kip1) levels in the DCIS group were significantly different from those in the UDH group (all P<0.01).The levels of Skp2 and p27(Kip1) in the FEA group were significantly different from both the DCIS and UDH groups (all P<0.05).p27(Kip1) was negatively correlated with Skp2 in both the UDH group (r=-0.629, P=0.026) and DCIS group (r=-0.893, P=0.000).

CONCLUSIONOverexpression of Skp2 might be the mechanism underlying p27(Kip1) over degradation.

Adult ; Aged ; Breast ; pathology ; Breast Neoplasms ; etiology ; Carcinoma, Intraductal, Noninfiltrating ; etiology ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p27 ; physiology ; Female ; Humans ; Hyperplasia ; Middle Aged ; S-Phase Kinase-Associated Proteins ; physiology

OBJECTIVETo investigate effects of dahuang zhechong pill ( DHZCP) on the cell cycle and the related signal pathways in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF) with the method of serum pharmacology.

METHODSDNA synthesis in VSMCs was examined by detecting 5'-bromo-2'-deoxyuridine incorporation with the immunocytochemical method. The cycle of VSMCs was evaluated with flow cytometry. Expressions of cyclin D1, p27, protein kinase Cα (PKCα), and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) were quantified by Western blot method.

RESULTSDHZCP containing serum significantly inhibited DNA synthesis of PDGF-stimulated VSMCs, arrested the cells in G G(1) phase, modulated the protein expressions of cyclin D D(1) and p27, and suppressed the activation of PKCα and ERK1/2.

CONCLUSIONDHZCP containing serum inhibits VSMCs proliferation via modulating the expressions of cell cycle proteins to arrest the cell in G G(1) phase, which is attributed to, at least in part, suppressing PKCα-ERK1/2 signaling in VSMCs.

Animals ; Aorta, Thoracic ; cytology ; Blood Proteins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; DNA ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; G1 Phase ; drug effects ; physiology ; MAP Kinase Signaling System ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; enzymology ; Platelet-Derived Growth Factor ; pharmacology ; Protein Kinase C-alpha ; metabolism ; Rats ; Rats, Sprague-Dawley

BACKGROUNDRhoA/Rho kinase (ROCK) pathway is involved in pulmonary arterial hypertension (PAH) and pulmonary artery smooth muscle cell (PASMC) proliferation. Inhibition of ROCK has been proposed as a treatment for PAH. But the mechanism of RhoA/ROCK pathway and its downstream signaling in proliferation of human PASMCs is unclear. We investigated the effect of fasudil, a selective ROCK inhibitor, on platelet-derived growth factor (PDGF) induced human PASMC proliferation, and the possible association between RhoA/ROCK and extracellular signal-regulated kinase (ERK), p27(Kip1).

METHODSHuman PASMCs were cultured with the stimulation of 10 ng/ml PDGF, and different concentrations of fasudil were added before the addition of mitogen. Cell viability and cell cycle were determined with MTT and flow cytometry respectively. ROCK activity, ERK activity and protein expression of proliferating cell nuclear angigen (PCNA) and p27(Kip1) were measured by immunoblotting.

RESULTSBy MTT assay, PDGF significantly increased the OD value that represented human PASMC proliferation, and pretreatment with fasudil significantly reversed this effect in a dose-dependent manner. After PDGF stimulation, the percentage of cells in S phase increased dramatically from 15.6% to 24.3%, while the percentage in G(0)/G(1) phase was reduced from 80.6% to 59%. And pretreatment with fasudil reversed the cell cycle effect of PDGF significantly in a dose-dependent manner. PDGF markedly induced ROCK activity and ERK activity with a peak at 15 minutes, which were significantly inhibited by fasudil. In addition, fasudil significantly inhibited PDGF-induced PCNA expression and fasudil also upregulated p27(Kip1) expression in human PASMCs, which decreased after PDGF stimulation.

CONCLUSIONRhoA/ROCK is vital for PDFG-induced human PASMC proliferation, and fasudil effectively inhibited PDGF-induced human PASMC proliferation by up-regulation of p27(Kip1), which may be associated with inhibition of ERK activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Humans ; MAP Kinase Signaling System ; physiology ; Muscle, Smooth, Vascular ; cytology ; Platelet-Derived Growth Factor ; pharmacology ; Protein Kinase Inhibitors ; pharmacology ; Pulmonary Artery ; cytology ; Up-Regulation

OBJECTIVECytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.

METHODSAkt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.

RESULTSCagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.

CONCLUSIONSCagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.

Antigens, Bacterial ; genetics ; physiology ; Bacterial Proteins ; genetics ; physiology ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Gastric Mucosa ; cytology ; enzymology ; microbiology ; Helicobacter pylori ; metabolism ; pathogenicity ; physiology ; Humans ; Interleukin-8 ; secretion ; Intracellular Signaling Peptides and Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transcription, Genetic ; Virulence

OBJECTIVETo observe matrine-induced erythroid differentiation of K562 cells in relation to activation of the apoptotic pathway in vitro.

METHODSK562 cells were cultured in the presence or absence of matrine at different concentrations for 4 days, and the morphological and ultramicrostructural changes of the cells were observed using inverted microscopy and transmission electron microscopy, respectively. The expression of apoptosis-related protein p27kip1 was detected by immunocytochemistry.

RESULTSCompared to untreated K562 cells, the cells treated with matrine at 0.10 g/L exhibited apoptostic characteristics in the cellular morphology and ultramicrostructure, with the expression of p27kip1 protein upregulated in a concentration- and time-dependent manner.

CONCLUSIONMatrine-induced erythroid differentiation of K562 cells is associated with cell apoptosis, and upregulation of p27kip1 protein expression may play a crucial role in the process of apoptosis.

Alkaloids ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; physiology ; Cyclin-Dependent Kinase Inhibitor p27 ; biosynthesis ; Dose-Response Relationship, Drug ; Humans ; Immunohistochemistry ; K562 Cells ; Leukemia, Erythroblastic, Acute ; metabolism ; pathology ; Microscopy, Electron, Transmission ; Quinolizines ; pharmacology ; Signal Transduction ; drug effects ; Time Factors

TGF-beta1-induced glomerular mesangial cell (GMC) injury is a prominent characteristic of renal pathology in several kidney diseases, and a ternary protein complex consisting of PINCH-1, integrin-linked kinase (ILK) and alpha-parvin plays a pivotal role in the regulation of cell behavior such as cell proliferation and hypertrophy. We report here that PINCH-1-ILK-alpha-parvin (PIP) complex regulates the TGF-beta1-induced cell proliferation and hypertrophy in cultured rat GMCs. When GMCs were treated with TGF-beta1 for 1, 2 and 3 days, the PIP complex formation was up-regulated after 1 day, but it was down-regulated on day 2. Cell numbers were significantly elevated on day 2, but dramatically decreased on day 3. In contrast, a significant increase in cellular protein contents was observed 3 days after TGF-beta1-treatment. TGF-beta1 induced early increase of caspase-3 activity. In GMCs incubated with TGF-beta1 for 2 days, cytosolic expression of p27(Kip1) was dramatically reduced, but its nuclear expression was remarkably elevated. A significantly decreased expression of phospho-Akt (Ser 473) was observed in the cells treated with TGF-beta1 for 1 day. TGF-beta1 induced early increase of phospho-p27(Kip1) (Thr 157) expression with subsequent decrease, and similar responses to TGF-beta1 were observed in the p38 phosphorylation (Thr 180/Thr 182). Taken together, TGF-beta1 differently regulates the PIP complex formation of GMCs in an incubation period-dependant fashion. The TGF-beta1-induced up- and down-regulation of the PIP complex formation likely contributes to the pleiotropic effects of TGF-beta1 on mesangial cell proliferation and hypertrophy through cellular localization of p27(Kip1) and alteration of Akt and p38 phosphorylation. TGF-beta1-induced alteration of the PIP complex formation may be importantly implicated in the development and progression of glomerular failure shown in several kidney diseases.
Animals ; *Cell Enlargement ; *Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p27/metabolism ; Cytoskeletal Proteins/*metabolism ; DNA-Binding Proteins/*metabolism ; Male ; Mesangial Cells/drug effects/*physiology ; Microfilament Proteins/*metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases/*metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Transforming Growth Factor beta1/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.

METHODSEctopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.

RESULTSRIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.

CONCLUSIONSRIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.

Cell Cycle ; genetics ; Cell Differentiation ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Plasmids ; genetics ; Transfection ; U937 Cells

BACKGROUNDRenal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor (CTGF) with renal hypertrophy in uninephrectomized diabetic rats.

METHODSSeventy-two Sprague-Dawley (SD) rats were randomly divided into two groups: control group (group C, n = 32) and diabetic nephropathy (group DN, n = 40). Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), 24-h urinary albumin excretion (24hUalb), kidney weight (KW), KW/BW, glomerular tuft area (AG), glomerular tuft volume (VG), proximal tubular area (AT) at each time point, the width of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kip1 were detected by immunohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed.

RESULTSThere was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C (P < 0.05, respectively), diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [(245.7 +/- 103.0) nm vs (121.8 +/- 19.1) nm, P < 0.01] and TBM [(767.7 +/- 331.1) nm vs (293.0 +/- 110.5) nm, P < 0.01] at week 8. There was a weak expression for CTGF and p27kip1 in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kip1 was found in glomeruli and tubuli in diabetic kidney from week 1 onward (P < 0.05, respectively), and the extent of CTGF expression was positively correlated with AG (r = 0.92, P < 0.05), VG (r = 0.86, P < 0.05), AT (r = 0.94, P < 0.01) and positively correlated with the expression of p27kip1 (r = 0.96, P < 0.01).

CONCLUSIONThe expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.

Albuminuria ; etiology ; Animals ; Connective Tissue Growth Factor ; Cyclin-Dependent Kinase Inhibitor p27 ; analysis ; Diabetes Mellitus, Experimental ; pathology ; Hypertrophy ; Immediate-Early Proteins ; analysis ; physiology ; Intercellular Signaling Peptides and Proteins ; analysis ; physiology ; Kidney ; pathology ; Male ; Nephrectomy ; Rats ; Rats, Sprague-Dawley ; Streptozocin

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p27 ; antagonists & inhibitors ; physiology ; Endometrial Neoplasms ; drug therapy ; pathology ; Estradiol ; pharmacology ; Female ; G1 Phase ; drug effects ; Humans ; Intracellular Signaling Peptides and Proteins ; antagonists & inhibitors ; physiology ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; physiology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; physiology ; Signal Transduction ; drug effects ; physiology