OBJECTIVETo study the effect of down-regulation of histone deacetylase 2 (HDAC2) expression on cell proliferation and cell cycle in cervical carcinoma cell lines HeLa.

METHODSHDAC2 siRNA and control siRNA were transfected to HeLa cells. CCK-8 and flow cytometry were used to analyze the changes of cell proliferation and cell cycle, respectively. Western blot was employed to detect the changes of cell proliferation and cell cycle-related proteins.

RESULTSHDAC2 siRNA significantly down-regulated the expression of HDAC2 protein in HeLa cells, resulting in marked inhibition of cell proliferation. In addition, the percentage of cells in G(0)/G(1) phase in HDAC2 siRNA group (63.3% ± 2.0%) was significantly higher than that in untreated group (29.3% ± 1.7%) or control siRNA group (29.4% ± 1.7%), F = 354.181, P = 0.000. Furthermore, Western blot demonstrated that down-regulation of HDAC2 expression decreased the expression of cyclin D1, cyclin E and CDK2 proteins but increased the expression of p21 protein.

CONCLUSIONSDown-regulation of HDAC2 expression mediates proliferation inhibition and cell cycle arrest. It is associated with decrease in cyclin D1, cyclin E and CDK2 protein expression and increase in p21 protein expression.

Cell Cycle ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Down-Regulation ; HeLa Cells ; Histone Deacetylase 2 ; genetics ; metabolism ; Humans ; Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

In this study, DNAzymes against cyclin D1 (cyclin D1-DRz) were designed according to the secondary structure of cyclin D1 mRNA which was computed with RNAdraw and Mfold. Cyclin D1-DRz were transfected into tumor cell line u251 and HeLa by oligofectamine. The expression of cyclin D1 was detected by RT-PCR. It was shown that the expression of cyclin D1 gene was suppressed obviously, and the expressions of other cell-cycle related genes such as cyclin E1, cyclin A1 and cyclin B1 were also declined. The cell cycle analysis of tumor cells tansfected with cyclin D1-DRz revealed an arrestment in the G0/G1 phase. In conclusion, the approach is effective and feasible for designing DNAzyme. Cyclin D1-DRz is useful for interfering with the cell cycle procession of tumor cells.
Base Sequence ; Cell Cycle ; drug effects ; Cyclin D1 ; antagonists & inhibitors ; genetics ; metabolism ; DNA, Catalytic ; genetics ; metabolism ; pharmacology ; HeLa Cells ; Humans ; Molecular Sequence Data ; RNA, Messenger ; genetics ; metabolism ; Transfection

OBJECTIVE: To explore the role of miR-744-5p/CCND1 axis in clear-cell renal cell carcinoma (ccRCC). METHODS: We examined the expression levels of miR-744-5p in 65 pairs of ccRCC and adjacent tissue specimens and in 5 ccRCC cell lines and human renal tubular epithelial (HK2) cells using qRT-PCR. The ccRCC cell lines 786-O and OSRC2 were transfected with miR-744-5p mimic, CCND1 mimic, or their negative control mimics, and the changes in cell proliferation, migration, and invasion were evaluated with CCK-8, wound healing, and Transwell assays. The downstream target molecules of miR-744-5p were predicted by bioinformatics analysis, and the expression level of CCND1 in ccRCC cells was verified by qRT-PCR and Western blotting. The relationship between miR-744-5p and CCND1 was further validated by dual luciferase reporter assay, and the role of the miR-744-5p/CCND1 axis in ccRCC was explored by rescue experiments. RESULTS: MiR-744-5p was significantly downregulated in ccRCC tissues and cell lines (all P < 0.05), and its overexpression inhibited the proliferation, migration, and invasion of ccRCC cells (all P < 0.05). Bioinformatics analysis and dual luciferase reporter assay showed that CCND1 was a downstream target of miR-744-5p. The results of rescue experiments showed that upregulation of CCND1 could partially reverse the inhibitory effect of miR-744-5p overexpression on ccRCC cell proliferation, migration, and invasion (all P < 0.05). CONCLUSION MiR-744-5p inhibits the malignant phenotype of ccRCC cells by targeting CCND1, and the miR-744-5p/CCND1 axis may be a novel target for diagnosis and treatment of ccRCC.
Carcinoma, Renal Cell/metabolism* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Cyclin D1/genetics* ; Humans ; Kidney Neoplasms/metabolism* ; MicroRNAs/metabolism*

This study was aimed to quantitatively detect the levels of beta-catenin and cyclin D1 mRNA in various subgroups of acute myeloid leukemia (AML) and to analyze their potential relationship, so as to provide theoretical basis for exploring the role of Wnt/beta-catenin pathway in the pathogenesis of AML. Real time fluorescent quantitative RT-PCR was used to detect the relative expression levels of beta-catenin and cyclin D1 mRNA, to analyze changes of the two gene expressions and their relationship. The results showed that the beta-catenin mRNA expression level in BMMNC of AML patients was significantly higher than that in benign blood disease patients (p < 0.05), but no statistical difference was found among the various subgroups of AML (p > 0.05). In AML there was overexpression of cyclin D1 mRNA, and its expression level was significantly higher than that in benign blood disease group (p < 0.05), but there was no statistical difference among the subtypes of AML. The expression levels of beta-catenin and cyclin D1 were correlated each other in AML-M(1), M(2) and M(4) (r values were 0.822, 0.627, 0.712 respectively; p values were 0.001, 0.020, 0.002 respectively). It is concluded that the over-expressions of beta-catenin and cyclin D1 exit in AML patients, and the significant correlation appears in part of the subgroups, which means that the Wnt/beta-catenin pathway is aberrantly activated in AML, probably activating the downstream target gene cyclin D1 and participating in the regulation of cell cycle disturbance and abnormal proliferation of leukemic cells.
Adult ; Cyclin D1 ; metabolism ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; RNA, Messenger ; metabolism ; Signal Transduction ; Young Adult ; beta Catenin ; genetics ; metabolism

OBJECTIVETo investigate the effect of antisense cDNA of cyclin D1 on the cyclin D1 gene expression and cell proliferation of human hepatocarcinoma HepG2 cells in vitro.

METHODSPlasmids containing cyclin D1 antisense cDNA were constructed and transfected into HepG2 cells. Their effects on cell proliferation were examined by MTT method, RT-PCR, immunohistochemical means, and flow cytometry.

RESULTSCyclin D1 antisense cDNA significantly inhibited the growth of HepG2 cells. The inhibition peaked at 48 hour after transfection by MTT method. RT-PCR analysis showed that cyclin D1 antisense cDNA down-regulated cyclin D1 at the mRNA levels. Expression level of cyclin D1 protein was also decreased as shown by immunohistochemical studies. Cell-cycle analysis by flow cytometry showed that transfected HepG2 cells were arrested at the G1 phase of the cell cycle.

CONCLUSIONSOur data suggest that cyclin D1 antisense cDNA could specifically inhibit the expression of cyclin D1 mRNA and protein and regulate cell cycle and cell proliferation of HepG2 cells. Cyclin D1 antisense cDNA may serve as a potential antitumor strategy in regulating cell-cyclin treating advanced HCCs.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cyclin D1 ; genetics ; Genes, bcl-1 ; genetics ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Oligodeoxyribonucleotides, Antisense ; genetics

OBJECTIVETo study the inhibition effect of CyclinD1 specific small interfering RNA(siRNA) on CyclinD1 gene expression in human keloid fibroblast, investigating the effect of CyclinD1 specific siRNA (siRNA-CyclinD1) on the cell cycle, multiplication and apoptosis.

METHODSAccording to the principle of siRNA design, siRNA-CyclinD1 was designed and the keloid fibroblast were transfected. RT-PCR was used to examine CyclinD1 mRNA expression. Flow cytometry was used to examine cell cycle. The apoptotic rate was analyzed by using Annexin V-FITC/PI kit. The DNA gragmentation were measured by DNA ladder analysis.

RESULTSAfter transfection, the expression of CyclinD1 mRNA decreased remarkably. Twenty-four, forty-eight and seventy-two hours after transfection, the radio of G1 stage cell was (59.80 +/- 3.06)%, (66.01 +/- 4.03)% and (67.43 +/- 5.35)%, all significantly higher than in the control group (54.50 +/- 5.35)%; the radio of S stage cell was (18.40 +/- 1.42)%, (17.21 +/- 1.76)% and (11.07 +/- 1.00)%, significantly lower than in the control group (22.33 +/- 1.49)%; the proportion of the cells in G1 stage increased and those in the S stage decreased in the keloid fibroblast transfected with siRNA-CyclinD1. The apoptotic rate of the siRNA-CyclinD1 group was (7.82 +/- 0.45)%, (15.71 +/- 1.06)%, (18.32 +/- 1.08)%, all significantly higher than in the control group (0.68 +/- 0.12)%, and the DNA gragmentation can be seen remarkably.

CONCLUSIONSChemically synthesized siRNA- CyclinD1 effectively inhibits. The expression of CyclinD1 in keloid fibroblast thus arresting the cell cycle at G1 stage and enhancing cell apoptosis. Our study provided a preliminary results in seaching of a RNAi therapy of keloid.

Apoptosis ; genetics ; Cells, Cultured ; Cyclin D1 ; genetics ; Fibroblasts ; cytology ; metabolism ; Flow Cytometry ; Gene Silencing ; Humans ; Keloid ; genetics ; metabolism ; pathology ; RNA, Small Interfering ; Transfection

OBJECTIVETo investigate the role that E-cadherin (E-cad) plays on cell adhesion and proliferation of human breast carcinoma.

METHODSE-cad expression vector was transfected into an E-cad-negative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad, β-catenin (β-cat) and cyclin D1 expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/β-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of β-cat.

RESULTE-cad(+) cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When co-cultured with HCT116 cells, the average adhesion rates at 30 min are 39.0%, 60.0% and 59.5% for MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4.2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous β-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2.0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture. β-cat increased in the cytoplasma.

CONCLUSIONSTwo monoclonal tumor cell strains (Ecad-231-7 and Ecad-231-9) stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving β-cat and cyclin D1.

Apoptosis ; Breast Neoplasms ; metabolism ; pathology ; Cadherins ; genetics ; metabolism ; physiology ; Cell Adhesion ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Female ; Genetic Vectors ; Humans ; Plasmids ; Transfection ; beta Catenin ; metabolism

OBJECTIVETo explore the molecular events and mechanism in the carcinogenesis of esophageal epithelium in the high incidence area of esophageal carcinoma.

METHODSEpithelial cells collected from the high incidence area of esophageal carcinoma were used to detect DNA content and ploidy by propidium iodide(PI) stain. The expressions of p53, p16 and cyclin D1 were stained by indirect immunofluorescence of fluorescein isothiocyanate(FTTC), which were detected by flow cytometry (FCM).

RESULTSDuring the process of carcinogenesis, DNA content increased significantly. The diploid cells decreased while heteroploid cells increased sharply, with a heteroploidy rate of 84.2%. At the same time, the p53 protein accumulated and p16 was deleted. The positive rates of p53 and oncogene cyclin D1 were both 100%(5/5, 6/6) in the cancer group.

CONCLUSIONIn the early carcinogenesis of esophageal epithelium, DNA content and heteroploidy rates increase with tumor suppressor gene p16 deletion and p53 protein accumulation while oncogene cyclin D1 is overexpressed. Multiple molecular events have already occurred when esophageal carcinoma develops.

Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; DNA, Neoplasm ; metabolism ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Flow Cytometry ; Gene Expression ; Humans ; Precancerous Conditions ; genetics ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth.

METHODSsiRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup. Cell growth was assessed by MTT assay. AgNORs staining was applied to determine cell proliferation. Plate cell clone test was conducted to examine the capacity of cell clone formation. FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins. In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection.

RESULTSThe inhibition rate of RhoC was 82.3%. From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ± 0.10 vs. 0.73 ± 0.11 and 0.71 ± 0.07 respectively, P < 0.05). AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ± 0.35 vs. 3.47 ± 0.93 and 3.17 ± 0.78, P < 0.01). Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42)% vs. (70.58 ± 10.10)% and (69.83 ± 14.77)%, respectively, P < 0.01]. Cell cycle analysis by FACS showed that G(0)/G(1) cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ± 5.93)% vs. (57.05 ± 5.97)% and (52.99 ± 4.80)%, P < 0.05]. Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1(0.45 ± 0.21 vs. 1.25 ± 0.24 and 1.12 ± 0.15, respectively, P < 0.05)and CDK4 (0.55 ± 0.08 vs. 1.18 ± 0.32 and 1.10 ± 0.29, respectively, P < 0.05); the following genes were notably increased: p16(1.07 ± 0.23 vs. 0.36 ± 0.12 and 0.35 ± 0.13, respectively, P < 0.01)and p21(0.42 ± 0.12 vs. 0.17 ± 0.06 and 0.19 ± 0.08, respectively, P < 0.05). RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line. From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups (0.83 ± 0.10 vs. 0.54 ± 0.11 and 0.58 ± 0.55, respectively, P < 0.05).

CONCLUSIONSRhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.

Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; rho GTP-Binding Proteins ; genetics ; metabolism ; rhoC GTP-Binding Protein

To evaluate the regulating effect of Buyang Huanwu decoction and its simple prescription (Naojian tablet) on CDK4/Cyclin D1 expression in hippocampus tissues of rats with cerebral ischemia, SD rats were divided into the sham-operation group, the model group, the Buyang Huanwu decoction group (ig, 3.15 g · kg⁻¹) and the simple prescription group (ig, 2.41 g · kg⁻¹). Each group was further divided into five subgroups based on time points after the administration, i. e. 1 d, 3 d, 7 d, 14 d and 28 d, respectively. CDK4/Cyclin D1 expressions of the group at different time points were examined by using immunohistochemistry and real-time qPCR. According to the results, the cerebral ischemia model group showed higher CDK4/Cyclin D1 expression than the sham-operation groups (P < 0.05), suggesting that the cell cycle signal pathway would be activated by the cerebral ischemic injury. Both Buyang Huanwu decoction and simple prescription groups showed significantly lower cyclin expression than the model group at 3 d, 7 d, 14 d, 28 d (P < 0.05), indicating both Buyang Huanwu decoction and its simple prescription could play the neuroprotective effect through the cell cycle signal pathway.
Animals ; Brain Ischemia ; drug therapy ; genetics ; metabolism ; physiopathology ; Cell Cycle ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Rats ; Rats, Sprague-Dawley