Effects of RNA interference targeting CDC25a gene on proliferation of human liver cancer HepG2 cells / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effect of silencing cell division cycle 25a ( CDC25a) gene on the prolifera-tion of human hepatoma HepG2 cells.METHODS:CDC25a gene in human hepatoma HepG2 cells was silenced by RNA interference.Real-time PCR was applied to detect the expression of CDC25a, cyclin E and CDK2 at mRNA levels in the HepG2 cells.Western blotting was applied to detect the expression of CDC25a at protein level.In addition, MTT assay, Giemsa staining and flow cytometry were used to measure the proliferation of human hepatoma HepG2 cells.RESULTS:The expression of CDC25a at mRNA and protein levels in RNA silence group was lower than those in negative control group and normal control group (P<0.05).The mRNA expression of cyclin E and CDK2 in silence group was lower than that in negative control group and normal control group (P<0.05).The cell proliferation in silence group was lower than that in negative control group and normal control group ( P<0.05) .The results of flow cytometry revealed that the cells in silence group were blocked in G1 phase.CONCLUSION:Infection of LV-CDC25a-RNAi recombinant to the HepG2 cells effec-tively inhibits the CDC25a gene expression and the proliferation of human hepatoma cells, and arrests the cells in G1 phase, suggesting that CDC25a gene may be a key target for the treatment of liver cancer.

Construction and ldentification of Cell Division Cycle 2 Promoter Reporter Gene Vector / 天津医药

Objective To construct the luciferase reporter gene vector of cell division cycle 2 (Cdc2) gene promoter and determine its transcriptional activity. Methods Primers were designed based on human Cdc2 promoter sequence from UCSC software. Then Cdc2 promoter from human genome DNA was replicated. After pGL3-Basic vector and Cdc2 promoter were digested with restriction enzymes SacⅠand XhoⅠseparately, Cdc2 promoter was inserted into pGL3-Basic vector. The recombinant plasmid named pGL3-Cdc2-promoter was transiently co-transfected into U2OS cells with control vector pRL-SV40, and then the activity of dual luciferase was detected. Results pGL3-Cdc2-promoter was constructed successfully. The restriction analysis and sequencing proved the entirely correct sequencing results. The luciferase activity was higher in pGL3-Cdc2-promoter/pRL-SV40 group than that of pGL3-Basic/pRL-SV40 group (1.591 5±0.199 8 vs 0.049 9±0.010 4). Conclusion pGL3-Cdc2-promoter can be transcribed and activated in U2OS cells. This study provided an important basis for screening and evaluation of anticancer drugs.

The influence of hepatic ischemia-reperfusion injury to the hepatic cell cycle G2/M-related gene Gadd45a and Egr-1 expression / 中华器官移植杂志

ObjectiveTo explore the possible mechanism that Gadd45a and Egr-1together participate in the DNA damage repair,by analyzing the influence of hepatic ischemia-reperfusion injury (IRI) on the rnRNA and protein expression of hepatic cell cycle-related genes.Method Methods Forty-two SD rats were divided into normal control group,ischemia 30 min group and reperfusion 30 min,1h,2 h,3 h and 24 h groups.The hepatic specimens of rats at the corresponding time points after success of modeling were obtained. Chip experiment was used to screen out the key genes Gadd45a,Egr-1and GAPDH for target genes.We used Northern blot and RT-PCR to confirm the chip's results. The expression of related proteins was detected by using immunofluorescence and immunohistochemistry.Results Northern blot detection results showed that the RT-PCR products and the target genes for testing were concordant.The expression spectral chip signal and RT-PCR detection revealed that the expression of target genes had a 4-fold increase at early reperfusion period (1h) (up-regulation of Egr-1,Gadd45a and HSP70-1a by 9.39,8.28 and10.8 respectively.At the later stage of reperfusion (24 h),the expression of Egr-1,Gadd45a and Hspala was reduced to the normal levels, and the chip screening results were consistent with the RT-PCR test results.Fluorescence immunohistochemistry demonstrated that in the reperfusion1h and 2 h groups,the expression of Egr-1in nuclei of liver cells was positive. Immunohistochemistry indicated that the Gadd45 expression in the nucleus and cytoplasm was gradually increased with increasing reperfusion time.ConclusionGadd45a and Egr-1are the key genes of the nuclear factor (NF)-kB regulatory pathways,and involved in the regulation of cell cycle arrest at G2/M phase and repair of damaged DNA in rat hepatic IRI.

Effects of lentiviral-delivered CDC25B2 siRNA on biological behaviors of human pancreatic cancer cell line CFPAC-1 / 国际肿瘤学杂志

Objective To establish CFPAC-1 cell lines deficient in CDC25B2 by recombinant lentivirus, and to investigate the role of this gene. Methods After CFPAC-1 cells were transduced with recombinant lentivirus producing CDC25B2 siRNA, stably transduced cells with green fluorescent protein were selected by flow cytometer. The mRNA and protein expression of CDC25B2 was examined by RT-PCR and Western blot analysis. The effect of the lentivirus on the cell proliferation, cell cycle, clone-forming, migration and invasion ability was analyzed by MTr method, flow cytometer, plate clone-forming assay and Transwell chamber method respectively. Results CDC25B2 siRNA knocked down CDC25B2 expression in CFPAC-1 cells significantly. The silencing efficiency of siRNA transduction by recombinant lentivirns was very high. Proliferation, cloneforming, migration and invasion ability of human pancreatic cancer cell line CFPAC-I were significantly in-creased, while cell cycle was not affected. Conclusion CDC25 B2 plays an important role in cell proliferation, clone-forming, migration and invasion of pancreatic cancer. This research provides experimental evidences for targeting CDC25B2 in gene therapy against pancreatic cancer.

THE EXPRESSION OF CDC25B IN COLORECTAL CANCER AND ADENOMA AND ITS CLINICAL SIGNIFICANCE / 解放军医学杂志

To explore the role of CDC25B in pathogenesis and development of colorectal cancer and to investigate the relation between the expression of CDC25B and the clinical pathological character of colorectal cancer and the prognosis of patients. The expression of CDC25B in 168 patients with colorectal cancer and 25 patients with adenoma, and normal colorectal mucosa from 20 individuals was assayed by immunohistochemistry (S-P method). The expression of CDC25B was not detected in adenoma and normal colorectal mucosa. The expression rate of CDC25B in colorectal cancer was 71.4%(120/168). The expression of CDC25B had a positive correlation with distant metastasis and the increase of CEA,while it had no relationship with the tumor stage, grade, lymph node metastases, gender, age or the size of tumor . The patients in whom expression of CDC25B was detected had a markedly low 5-year survival rate. The results suggested that expression of CDC25B in colorectal cancer might accelerate the transformation of cell cycle,which promoted metastasis to distant organ, indicating that the expression of CDC25B might play a role in the development of colorectal cancer. The expression of CDC25B was a risk factor of poor prognosis.

Primary analysis on interaction between hCDC14A and BRAP2 / 中国病理生理杂志

AIM: To search for new candidate partners of human cell division cycle gene 14A (hCDC 14A) and explore their functional relationships.METHODS: Yeast two-hybrid screening was employed to find hCDC 14A new partners. Physical interaction between two proteins was verified using Pulldown and co-immunoprecipitation assays. Subcellular localizations were revealed by immunofluorescence. In vivo ubiquitination test implied their potential functional relationship.RESULTS: BRAP2 (BRCA1 associated protein 2) was found to be a new candidate partner of hCDC 14A. hCDC 14A was modified by ubiquitination, and BRAP2 increased this modification in vivo. As expected, hCDC 14A and BRAP2 co-localized on mitotic spindles in HeLa cells.CONCLUSION: BRAP2 may be an ubiquitin E3 ligase of hCDC 14A.