OBJECTIVETo assess the effect of small interfering RNA (siRNA)-mediated suppression of CDK6 expression on the proliferation and cell cycles of nasopharyngeal carcinoma (NPC) cells in vitro.

METHODSQRT-PCR was used to examine the differential expression of CDK6 in 30 NPC tissues and 18 normal nasopharyngeal tissues. A siRNA targeting CDK6 was transfected in NPC CNE2 cells, and MTT assay and flow cytometry were used to analyze the changes in cell proliferation and cell cycle distribution. Western blotting was used to examine the expressions of the cell cycle-related factors.

RESULTSCompared with normal nasopharyngeal tissues, NPC tissues showed an increased expression of CDK6 mRNA. Knocking down CDK6 expression obviously inhibited tumor cell growth and cell cycle transition from G1 to S phase and caused reduced expressions of CDK4, CCND1, and E2F1 and enhanced expression of the tumor suppressor p21.

CONCLUSIONNPC tissues overexpress CDK6. Knocking down CDK6 expression inhibits the growth and cell cycle transition of NPC cells in vitro by inhibiting the expressions of CDK4, CCND1, and E2F1 and upregulating tumor suppressor p21 expression.

Carcinoma ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Nasopharyngeal Neoplasms ; pathology ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Up-Regulation

In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene.
Animals ; Batch Cell Culture Techniques ; CHO Cells ; Cell Cycle Proteins ; genetics ; Cell Line ; Cricetinae ; Cyclin-Dependent Kinase 2 ; genetics ; Cyclin-Dependent Kinase 6 ; genetics ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Smad4 Protein ; genetics ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics

OBJECTIVETo evaluate association between DNA methylation of MAL, CDKN2A, and MGMT in stool and development of colorectal cancer, and to evaluate the screening value of these biomarkers in colorectal cancer and pre-malignant lesions.

METHODSMorning stool specimens were collected from 69 patients with colorectal cancer, 24 with colon adenoma, 19 with hyperplastic polyps, and 26 healthy controls. DNA was extracted and treated with bisulfite. Methylation-specific PCR(MSP) was performed for methylation analysis of MAL, CDKN2A and MGMT in DNA samples. Associations between clinicopathological features and gene methylation were analyzed. The sensitivity of diagnosis by combining three methylation markers was compared with fecal occult blood test(FOBT).

RESULTSThe methylation frequencies of MAL, CDKN2A and MGMT were 78.3%, 52.5% and 55.1% in colorectal cancer, 58.3%, 41.7% and 37.5% in colon adenomas, 26.3%, 15.8% and 10.5% in hyperplastic polyps, and 3.8%, 0 and 3.8% in healthy controls, respectively. Significant differences in three genes were found between colorectal cancer and hyperplastic polyp, colorectal cancer and healthy control, colon adenoma and hyperplastic polyp, colon adenoma and healthy control(all P<0.05). The diagnostic sensitivity by combining three methylation markers was 92.8% in colorectal cancer, 70.8% in colon adenomas, significantly higher than FOBT examination (29.0% in colorectal cancer and 25.0% in colon adenomas, all P<0.05). No significant associations existed between three genes methylation of the three genes and clinical characteristic including sex, age, tumor location, lymph node metastases and TNM stage (all P>0.05).

CONCLUSIONDNA methylations levels of MAL, CDKN2A, and MGMT in stools are significantly higher in colorectal cancer and colon adenoma, which may serve as an noninvasive approach for the screening of colorectal cancer and pre-malignant lesions.

Adult ; Aged ; Colorectal Neoplasms ; diagnosis ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Early Detection of Cancer ; Feces ; chemistry ; Female ; Humans ; Male ; Mass Screening ; Middle Aged ; O(6)-Methylguanine-DNA Methyltransferase ; genetics ; Precancerous Conditions ; diagnosis ; genetics ; Promoter Regions, Genetic ; genetics

BACKGROUND: Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells. METHODS: EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991. RESULTS: PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells. CONCLUSIONS PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; metabolism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Mice ; Piperazines ; pharmacology ; Pyridines ; pharmacology

The elucidation of the molecular mechanisms underlying the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSCs) represents a critical step in the development of hADSCs-based cellular therapies. To examine the role of the microRNA-103a-3p (miR-103a-3p) in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. Osteogenic differentiation was induced for 14 days in an osetogenic differentiation medium and assessed by using an Alizarin Red S stain. The regulation of the expression of CDK6 (cyclin-dependent kinase 6), a predicted target of miR-103a-3p, was determined by western blot, real-time PCR and luciferase reporter assays. Overexpression of miR-103a-3p inhibited the proliferation and osteogenic differentiation of hADSCs. In addition, it downregulated protein and mRNA levels of predicted target of miR-103a-3p (CDK6 and DICER1). In contrast, inhibition of miR-103a-3p with 2'O methyl antisense RNA increased the proliferation and osteogenic differentiation of hADSCs. The luciferase reporter activity of the construct containing the miR-103a-3p target site within the CDK6 and DICER1 3'-untranslated regions was lower in miR-103a-3p-transfected hADSCs than in control miRNA-transfected hADSCs. RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation. The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly. These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs.
Adipose Tissue/*cytology ; Cell Differentiation ; *Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase 6/genetics ; DEAD-box RNA Helicases/genetics ; *Gene Expression Regulation ; Humans ; MicroRNAs/genetics/*metabolism ; *Osteogenesis ; Ribonuclease III/genetics ; Stromal Cells/cytology/metabolism

OBJECTIVETo investigate the expressions of the aging gene P16(ink4a) and anti-aging gene HST2 in benign prostatic hyperplasia (BPH).

METHODSTwenty-three BPH and eighteen normal prostate specimens were collected and total RNA was extracted, followed by the reverse transcriptase polymerase chain reaction (RT-PCR). The expressions of P16(ink4a) was detected by semi-quantitative analysis in BPH and normal prostate tissues.

RESULTSP16(ink4a) mRNA, rather than HST2, was expressed in the BPH and normal prostate tissues. Semi-quantitative analysis showed that the P16(ink4a) mRNA expression in the normal prostate tissues (0.4868 +/- 0.545 was significantly higher than in the BPH tissues (0.2783 +/- 0.0268, with a statistical difference in between (P < 0. 05).

CONCLUSIONP16(ink4a) might play an important role in the pathogenesis of BPH and is probably one of the factors of cell aging escape.

Adult ; Aged ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Fibroblast Growth Factor 6 ; genetics ; Gene Expression Profiling ; Humans ; Male ; Pilot Projects ; Prostatic Hyperplasia ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the expression of microRNA-218 (miR-218) and its role in hepatocellular carcinoma (HCC).

METHODSForty-six pairs of fresh surgical specimens of HCC and adjacent tissues were examined for miR-218 expression using qRT-PCR. A miR-218 mimic was transfected into HepG2 cells, and the cell viability and apoptosis were analyzed by MTT assay and flow cytometry, and the potential targets of miR-218 were detected by qRT-PCR and Western blotting.

RESULTSThe expressions of miR-218 in HCC tissues were significantly down-regulated compared to those in the adjacent tissues (P<0.05). Down-regulation of miR-218 was found to correlate significantly with the tumor size (>5 cm) and an advanced TNM stage (III+IV) (P<0.05). Ectopic expression of miR-218 in HepG2 cells resulted in suppressed cell proliferation and enhanced cell apoptosis as well as the down-regulation of Bmi-1 and CDK6 mRNA and protein expressions (P<0.05).

CONCLUSIONThe low-expression of miR-218 is correlated with malignant clinicopathological characteristics of HCC, and miR-218 may inhibit cell proliferation and promote cell apoptosis by down-regulating Bmi-1 and CDK6 in HCC.

Adult ; Aged ; Apoptosis ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Proliferation ; Cyclin-Dependent Kinase 6 ; metabolism ; Female ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Polycomb Repressive Complex 1 ; metabolism

OBJECTIVETo investigate the DNA methylation status of the promoter region within the coding gene hsa-miR-124a in human gastric cancer tissue, and examine its association with the expression of Rb and CDK6 protein and clinicopathological factors.

METHODSMethylation-specific PCR (MS-PCR) was used to detect the DNA methylation status of hsa-miR-124a in gastric cancer tissues and adjacent normal tissues from 30 patients. The expression of Rb and CDK6 protein within these tissues was examined using immunohistochemistry.

RESULTSThe overall methylation rate of gastric cancer tissues was 73.3%, which was significantly higher than that in the adjacent tissues(10.0%, P<0.05). The overall positive rates of Rb and CDK6 protein in gastric cancer tissues were 86.7% and 80.0%, respectively. DNA methylation of hsa-miR-124a was positively correlated with the expression of Rb and CDK6 proteins. Significant associations were found between hypermethylation of hsa-miR-124a and tumor size, differentiation, lymphatic metastasis, and invasion depth(P<0.01).

CONCLUSIONSHypermethylation of hsa-miR-124a is present in gastric cancer, and is associated with increased expression of CDK6 and Rb protein. These may be related to the proliferation and development of malignancy in the stomach.

Adult ; Aged ; Aged, 80 and over ; Cyclin-Dependent Kinase 6 ; metabolism ; DNA Methylation ; Female ; Humans ; Male ; MicroRNAs ; genetics ; Middle Aged ; Promoter Regions, Genetic ; genetics ; Retinoblastoma Protein ; metabolism ; Stomach Neoplasms ; genetics ; metabolism

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.
Animals ; Cell Proliferation ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cytokines ; genetics ; metabolism ; DNA Damage ; Fibroblasts ; drug effects ; Interleukin-6 ; secretion ; Mice ; Mitomycin ; pharmacology ; NIH 3T3 Cells ; Phenotype

OBJECTIVETo study the methylation of O-6-methylguanine-DNA methyltransferase (MGMT) and p16 gene in the sputum cells of radon-exposed population. To provide the experimental base for finding the molecular biomarker of the high risk population of the radon-induced lung cancer.

METHODS91 radon-exposed workers were divided into 4 groups, high dosage group (> 120 WLM), middle dosage group (between 60 and 120 WLM), low dosage group (between 30 and 60 WLB) and lower dosage group (between 2 and 30 WLM) according to the accumulated exposure dosage of the radon daughters. The abnormal methylation of p16 and MGMT gene in the sputum cells of the population in the four groups was detected with the methylation specific PCR (MSP).

RESULTSThere was significantly upward trend for the p16 gene methylation rate (0.00%-20.00%), the MGMT gene methylation rate (0.00%-28.00%) and the total methylation rate (0.00%-40.00%) with the increase of the accumulated exposure dosage of the radon daughters (P < 0.01).

CONCLUSIONThe methylation of p16 and MGMT gene is related to the accumulate exposure dosage of the radon daughters.

Carcinogens, Environmental ; adverse effects ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; Dose-Response Relationship, Radiation ; Humans ; Male ; O(6)-Methylguanine-DNA Methyltransferase ; genetics ; metabolism ; Occupational Exposure ; Radon ; adverse effects ; Radon Daughters ; adverse effects ; Sputum ; metabolism