OBJECTIVETo explore the functions of miR-9 and miR-9(*) in SAMP8 mice during the aging and their possible mechanisms.

METHODSSAMP8 mice(4-,8-,12-month old,respectively)were selected,three age-matched SAMR1 mice were used as the control group with three mice in each group. The brains were collected and then sectioned for in situ hybridization of miR-9 and miR-9(*). Mimics or inhibitors of miR-9 and miR-9(*) were transfected into N2a cells,and the effects of overexpression or knockdown of the microRNAs on the cell cycle were detected by flow cytometry. Target genes were predicted by bioinformatic analysis and confirmed by dual luciferase assay.

RESULTSExpressions of miR-9 and miR-9(*) in hippocampus of SAMP8 mice were lower than those of SAMR1 mice. Knockdown of miR-9 and miR-9(*) induced a prolonged G1 phase and a shortened S phase in N2a cells;in contrast,miR-9 and miR-9(*) overexpression showed opposite effects. The predicted target genes of miR-9 were PSEN1,SCN2B,MAP3K3,and BACE1,and that of miR-9(*) was CDKn1c. Dual luciferase reporter gene assay showed that miR-9 targeted MAP3K3 while miR-9(*) targeted CDKn1c.

CONCLUSIONmiR-9 and miR-9(*) play an important role during aging via the target genes MAP3K3 and CDKn1c in the SAMP8 mice.

Aging ; Animals ; Brain ; Cyclin-Dependent Kinase Inhibitor p57 ; Mice ; MicroRNAs

OBJECTIVETo study the expression of P57(kip2) and Maspin in the pathological scar and their possible role in the pathogenesis of abnormal scars.

METHODSImmunohistochemistry integrated image analysis and reverse transcription polymerase chain reaction (RT-RCR) were performed to detect the expression of P57(kip2) and Maspin in hypertrophic scar, keloid, mature scar and normal skin. Statistics was used to analyze the datas.

RESULTSThe expression of P57(kip2) protein was fixed to fibroblast intranuclear in abnormal scar, and the expression of P57(kip2) protein and P57(kip2) mRNA decreased (P < 0.05). The expression of Maspin protein was fixed to fibroblast cytoplasm and intranuclear in abnormal scar, and the expression of Maspin protein and Maspin mRNA decrease, compared with that in normal group (P < 0.05). There was positive correlation between P57(kip2) protein and Maspin protein expression (P < 0.01).

CONCLUSIONSThe decreased expression of P57(kip2) and Maspin in abnormal scar shows that they are cicatrix-related genes. There is a positive relationship between the two genes. It may be one of the mechanisms of pathogenesis of abnormal scar. It makes effect through fibroblasts.

Cicatrix ; metabolism ; pathology ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p57 ; metabolism ; Fibroblasts ; metabolism ; Humans ; Serpins ; metabolism

OBJECTIVETo investigate the expressions of cyclin E, cyclin dependent kinase 2 (CDK-2) and cyclin-dependent kinase inhibitor p57(KIP2) in human gastric cancer, and to evaluate the relationships between protein levels and clinicopathological parameters.

METHODSWestern blot was used to measure the expressions of cyclin E, CDK-2 and p57(KIP2) proteins in the surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 36 patients.

RESULTSCyclin E and CDK-2 protein levels were higher in gastric cancer tissues in comparison with normal tissues (P < 0.05). Overexpression of cyclin E was correlated with lymph node involvement, poor histological grade and serosa invasion (P < 0.05). Overexpression of CDK-2 was correlated with lymph nodes involvement (P < 0.05). No statistically significant difference between cyclin E and CDK-2 expression was found when samples were stratified according to tumor size (P > 0.05). Expression of cyclin E and CDK-2 showed a positive linear correlation (r = 0.451, P = 0.01). Protein levels of p57(KIP2) were lower in gastric cancer tissues than in the normal mucosa (P < 0.05). Decreased expression of p57(KIP2) was correlated with lymph node involvement (P < 0.05). No statistically significant difference in p57(KIP2) expression was found when sample were stratified according to tumor size, histological grade or serosa invasion (P > 0.05). In metastatic lymph nodes, expression of cyclin E was increased and the expression of p57(KIP2) decreased.

CONCLUSIONOverexpressions of cyclin E, CDK-2 and downregulated expression of p57(KIP2) may play important roles in tumorigenesis and metastatic potential of gastric cancer.

Blotting, Western ; CDC2-CDC28 Kinases ; Cyclin E ; analysis ; physiology ; Cyclin-Dependent Kinase 2 ; Cyclin-Dependent Kinase Inhibitor p57 ; Cyclin-Dependent Kinases ; analysis ; physiology ; Humans ; Lymphatic Metastasis ; Nuclear Proteins ; analysis ; physiology ; Protein-Serine-Threonine Kinases ; analysis ; physiology ; Stomach Neoplasms ; chemistry ; pathology

BACKGROUND/AIMS: The cyclin-dependent kinase inhibitors (CDKI) including p21, p27, and p57 of the kinase inhibitor protein (KIP) family are negative regulators of cell cycle progression and potentially act as tumor suppressor. Tumor behavior and growth are influenced by the extent of tumor cell proliferation. The aim of this study was to evaluate the expression of KIP family CDKI in gastric cancer tissue, and to examine the relationship between these expression and various clinicopathological parameters including tumor cell proliferation. METHODS: We conducted an immunohistochemical analysis of p21, p27, and p57 expression in 109 gastric cancer tissues. Tumor cell proliferation was assessed by immunohistochemistry with antibody against Ki-67. RESULTS: Negative expression of p21, p27, and p57 was demonstrated in 45.9%, 65.1%, and 57.8% of cancer tissues, respectively. Negative expression of p21 correlated with larger tumor size, poor differentiation, depth of invasion, lymph node metastasis and advanced TNM stage (p=0.048, 0.041, 0.001, 0.005, and 0.001 respectively). Negative expression of p21 correlated with poor survival (p=0.037). Tumors with negative p21 expression had higher Ki-67 expression than those with positive p21 expression (p=0.024). No significant correlation could be observed between status of p27 and p57 expression and various clinicopathological parameters including survival and tumor cell proliferation. CONCLUSIONS: These results suggest that negative expression of p21 may play an important role in carcinogenesis by stimulating tumor cell proliferation, and may help in predicting the prognosis of gastric cancer.
Adult ; Aged ; Cell Division ; Cyclin-Dependent Kinase Inhibitor Proteins/*metabolism ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Cyclin-Dependent Kinase Inhibitor p27/metabolism ; Cyclin-Dependent Kinase Inhibitor p57/metabolism ; English Abstract ; Female ; Humans ; Male ; Middle Aged ; Stomach Neoplasms/*metabolism/mortality/pathology ; Survival Rate

OBJECTIVETo investigate the effects of p57(kip2) and cyclinE proteins on the genesis and progression of human pancreatic cancer.

METHODSThe expression of p57(kip2) and cyclinE proteins in tumor tissues and adjacent tissues of pancreatic cancer in 32 patients was detected by SP immunohistochemical technique.

RESULTSThe p57(kip2) protein positive-expression rate in tumor tissues of pancreatic cancer was 46.9%, which was lower than that in adjacent pancreatic tissue (P < 0.05). The p57(kip2) protein positive-expression correlated significantly with tumor cell differentiation (P < 0.05) and did not correlate significantly with lymph node metastasis (P > 0.05). The cyclinE positive-expression rate in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues (P < 0.05). The cyclinE positive-expression also correlated significantly with tumor cell differentiation and lymph node metastasis (P < 0.05). The cyclinE protein positive-expression rate in the tumor tissues of the p57(kip2) protein positive-expression group was lower than that in the p57(kip2) protein negative-expression group, and there were no significant correlation between the two groups (r = -0.112, P > 0.05).

CONCLUSIONDecreased expression of the p57(kip2) protein and/or over-expression of the cyclinE protein may play an important role in the genesis and progression of human pancreatic cancer.

Adult ; Aged ; Cyclin E ; analysis ; Cyclin-Dependent Kinase Inhibitor p57 ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Nuclear Proteins ; analysis ; Pancreatic Neoplasms ; chemistry ; pathology

OBJECTIVETo investigate the regulatory effect of microRNA-221 (MIR221) on CDKN1C/p57 expression in colon carcinoma cells in vitro.

METHODSCaco2 cells were treated with or without anti-p57-siRNA prior to the addition of pre-MIR221 or anti-MIR221. The MIR221 expression pattern was detected by real-time RT-PCR, and the mRNA and protein levels of CDKN1C/p57 expression were detected using semi-quantitative RT-PCR and Western blotting. Caco2 cell proliferation following the treatment was detected with MTT assay. CDKN1C/p57 3'-UTR fragment was amplified by PCR from the genome DNA of human colon and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid construct was then transfected into Caco2 cells along with pre-MIR221 or anti-MIR221, and the luciferase activity in the transfected cells was detected.

RESULTSMIR221-specific inhibitor significantly up-regulated CDKN1C/p57 protein expression in Caco2 cells (P<0.01). Anti-MIR221 could markedly inhibit Caco2 cell proliferation, and the inhibitory effect was obviously abolished by pretreatment with anti-p57-siRNA, suggesting that the inhibition was mediated by CDKN1C/p57 (P<0.01). A significant increase of luciferase activity was detected in Caco2 cells co-transfected with the luciferase reporter plasmid construct and anti-MIR221 (P<0.01).

CONCLUSIONSMIR221 can interact with the target site on the 3'-UTR of CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote colon carcinoma cell proliferation, suggesting the value of MIR221 as a potential target for treatment of colon carcinoma.

3' Untranslated Regions ; Caco-2 Cells ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p57 ; genetics ; metabolism ; Down-Regulation ; Humans ; MicroRNAs ; pharmacology ; RNA, Messenger ; genetics ; metabolism

This study was purposed to explore the expression of p57kip2 in the bone marrow of patients with de novo myelodysplastic syndrome (MDS) and its role in MDS pathogenesis, as well as the relationship between the expression of p57kip2 and SDF-1/CXCR4 signal. The expression of p57kip2 and CXCR4 in 67 de novo MDS patients was measured by real-time quantitative PCR. The percentage of CD34(+) cells in the bone marrow from MDS patients was measured by flow cytometry. 18 healthy volunteers were recruited for control. The effect of SDF-1 on p57kip2 expression in bone marrow mononuclear cell (BMMNC) from MDS or normal controls was investigated in vitro, and difference between them was compared. The results showed that low-risk MDS and high-risk MDS displayed a significant reduction of p57kip2 mRNA expression in BMMNC compared with that in control group (P < 0.001) and there was a negative correlation between p57kip2 expression and percentage of CD34(+) (r = -0.458, P < 0.001); the patients with abnormal karyotype showed lower expression of p57kip2 gene, compared to patients with normal karyotype (P = 0.045). Although the expression of CXCR4 had no difference between MDS patients and normal controls, a positive correlation between p57kip2 and CXCR4 in MDS patients was still found (r = 0.609, P < 0.001). Moreover, SDF-1 increased p57kip2 expression in normal BMMNC in dose-dependent manner, but BMMNC from MDS patients showed no response to SDF-1. SDF-1-induced p57 expression was blocked by AMD3100. It is concluded that the low expression of p57 gene in MDS may play a role in the pathogenesis of MDS. Furthermore, SDF-1-induced p57kip2 expression in BMMNC, and the decreasing response of BMMNC to SDF-1 may contribute to the low expression of p57kip2 in MDS patients.
Case-Control Studies ; Chemokine CXCL12 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p57 ; genetics ; metabolism ; Flow Cytometry ; Humans ; Myelodysplastic Syndromes ; genetics ; metabolism ; Receptors, CXCR4 ; metabolism

In situ hybridization was applied to locate and detect the expression of p57KIP2 in hydatidiform mole (5 cases of partial hydatidiform mole and 18 cases of complete hydatidiform mole) and normal villi (23 cases). The positive signals of p57KIP2 expression were analyzed by HPIAS-1000 Image-Analysis System. p57KIP2 was highly expressed in normal villi but showed distinct low expression in hydatidiform mole (P < 0.01). Furthermore, the locus of low expression of p57KIP2 accorded with the place where lesion of trophoblast occurred. Detection of p57KIP2 made it possible to study the genetics of hydatidiform mole at the transcriptional level. Low expression of p57KIP2 could be a molecular marker in hydatidiform mole and a target for therapy.
Cyclin-Dependent Kinase Inhibitor p57 ; Cyclin-Dependent Kinases ; antagonists & inhibitors ; Enzyme Inhibitors ; metabolism ; Female ; Genomic Imprinting ; genetics ; Humans ; Hydatidiform Mole ; genetics ; metabolism ; Nuclear Proteins ; biosynthesis ; genetics ; Placenta ; metabolism ; Pregnancy ; RNA, Messenger ; biosynthesis ; genetics ; Uterine Neoplasms ; genetics ; metabolism

OBJECTIVE: In this study, we investigated the cell growth inhibition, regulation of cell cycle and induction of apoptosis through recombinant p53 adenoviral vector delivery into cervical cancer cell line SiHa, to explore the possibility of p53 gene therapy. METHODS: We infected SiHa with AdCMVp53 at 50 MOI. After 48 hours, the regulation of cell cycle and apoptosis were investigated with FACS. The gene expression profiling associated with cell cycle was also investigated with cell cycle DNA membrane chip. RESULTS: SiHa cells were arrested in the G1 phase by AdCMVp53 and showed cell growth inhibition via apoptosis. The gene expression profiles involved in cell cycle including cyclin-dependent kinase inhibitor 1C (p57, Kip2), RAD9 (S.pombe) homolog, and MAD2 (mitoticarrest deficient, yeast, homolog)-like 2 were up-regulated by more than three-fold, as compared to control group. In contrast, 6 genes such as retinoblastoma-like 2 (p130), and cyclin H were down-regulated by more than three-fold. Several genes known as being differentially up- or down-regulated compared to control were confirmed by RT-PCR and Western blotting assays. CONCLUSION: The adenoviral p53 gene delivery into cervical cancer cell line, suggesting the possibility of p53 gene therapy in cervical neoplasia make the cell growth inhibition and changes of cell cycle-associated gene expression.
Apoptosis ; Blotting, Western ; Cell Cycle Proteins* ; Cell Cycle* ; Cell Line* ; Cyclin H ; Cyclin-Dependent Kinase Inhibitor p57 ; DNA ; G1 Phase ; Gene Expression ; Gene Expression Profiling ; Genes, p53 ; Membranes ; Transcriptome ; Uterine Cervical Neoplasms* ; Yeasts

Neural stem cells (NSCs) proliferation can be influenced by repetitive transcranial magnetic stimulation (rTMS) in vivo via microRNA-106b-25 cluster, but the underlying mechanisms are poorly understood. This study investigated the involvement of microRNA-106b-25 cluster in the proliferation of NSCs after repetitive magnetic stimulation (rMS) in vitro. NSCs were stimulated by rMS (200/400/600/800/1000 pulses per day, with 10 Hz frequency and 50% maximum machine output) over a 3-day period. NSCs proliferation was detected by using ki-67 and EdU staining. Ki-67, p21, p57, cyclinD1, cyclinE, cyclinA, cdk2, cdk4 proteins and miR-106b, miR-93, miR-25 mRNAs were detected by Western blotting and qRT-PCR, respectively. The results showed that rMS could promote NSCs proliferation in a dose-dependent manner. The proportions of ki-67+ and Edu+ cells in 1000 pulses group were 20.65% and 4.00%, respectively, significantly higher than those in control group (9.25%, 2.05%). The expression levels of miR-106b and miR-93 were significantly upregulated in 600-1000 pulses groups compared with control group (P<0.05 or 0.01 for all). The expression levels of p21 protein were decreased significantly in 800/1000 pulses groups, and those of cyclinD1, cyclinA, cyclinE, cdk2 and cdk4 were obviously increased after rMS as compared with control group (P<0.05 or 0.01 for all). In conclusion, our findings suggested that rMS enhances the NSCs proliferation in vitro in a dose-dependent manner and miR-106b/p21/cdks/cyclins pathway was involved in the process.
Animals ; Animals, Newborn ; Biomarkers ; metabolism ; Cell Proliferation ; genetics ; Cyclin-Dependent Kinase 2 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p57 ; genetics ; metabolism ; Cyclins ; genetics ; metabolism ; Gene Expression Regulation ; Hippocampus ; cytology ; metabolism ; Ki-67 Antigen ; genetics ; metabolism ; Magnetic Fields ; MicroRNAs ; genetics ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Primary Cell Culture ; Rats ; Rats, Sprague-Dawley ; Signal Transduction