ABSTRACT
Objective: To observe the expression of DNA dependent protein kinase catalytic subunit (DNA-PKcs) in the hepatoma tissues and mouse liver tissues of different developmental stages, so as to understand the role of DNA-PKcs in cell proliferation and tumorigenesis. Methods: Immunohistochemistry and Western blotting assay were used to observe the protein expression of DNA-PKcs in liver tissues and hepatoma tissues. The siRNA technique was used to silence the expression of DNA-PKcs in the HepG2 cells; cell proliferation assay and tumor transplantation test in nude mice were performed to evaluate the changes of the proliferation ability and tumorigenesis. Western blotting assay was also conducted to examine the expression of proliferation related proteins p-GSK3β and c-myc. Results: DNA-PKcs expression decreased in the liver tissues with the decrease of cell proliferation ability during the development of mice, and the expression level of DNA-PKcs was weak in liver tissues of adult mouse; but the DNA-PKcs protein level in the hepatoma tissues was significantly elevated (P<0.01). Cell growth curve showed that the proliferation of HepG2 cells was significantly decreased after suppression of DNA-PKcs with siRNA(P< 0.01), accompanied by a suppressed tumorigenesis ability. The expression of signal pathway related protein p-GSK3β and c-myc was inhibited after DNA-PKcs silencing in HepG2 cells(P<0.01). Conclusion: DNA-PKcs expression level is closely related to the proliferation ability of liver cells. Overexpression of DNA-PKcs may participate in the development and progression of hepatoma through mediating cell proliferation via the Wnt/GSK/c-myc related signal pathway.
ABSTRACT
Objective: To investigate the regulatory effect of HIV-1 Tat on mitotic centromere-associated kinesin (MCAK) expression and the related mechanism. Methods: Tat-expression TE671 cell model and purified prokaryotically expressed Tat protein were used to verify the down-regulated expression of MCAK using Northern blotting and Western blotting analysis. Various DNA fragments in the promoter region of the MCAK gene were amplified with PCR, linked to the luciferase reporter plasmid, and then transferred into TE671 cells. Luciferase activity analysis was performed to measure the promoter activity of various DNA fragments, so as to determine the active promoter region of MCAK gene. Moreover, HIV-1 Tat protein was co-incubated with TE671 cells, and the promoter activity was detected to analyze the modulating effect of Tat on promoter activity in vitro. Results: The results of Northern blotting and Western blotting analysis indicated that the mRNA and protein levels of MCAK were strongly decreased by Tat protein. The core region of MCAK promoter was located in -399∼+1 bp region, and the promoter activity was strikingly inhibited by Tat protein. Conclusion HIV-1 Tat has a marked inhibitory effect on MCAK expression, which might be related to the decreased promoter activity caused by Tat protein.
ABSTRACT
Objective:To investigate the effects of estradiol on ~(60)Co?-ray induced apoptosis of bone marrow hematopoietic cells of mice,and to discuss the related anti-irradiation mechanism.Methods:KM mice were randomly divided into 3 groups(15 mice/each group):control group(without radiation),pure radiation group and estradiol+radiation group(ER group).The pure radiation group was irradiated by 4.0 Gy?-ray at a dose rate of 1.15Gy/min;the ER group was administered with 0.1 mg estradiol(IM)at 10 days before 4.0 Gy?-ray radiation;and the control group received no special treatment.The apoptotic DNA segments of bone marrow hematopoietic cells were analyzed by DNA agarose gel electrophoresis;flow cytometry was used to examine the apoptosis rate of cells and expression of Fas and Bcl-2 at 4 h,8 h,and 12 h after irradiation.Results:Eight hours after radiation,the apoptotic DNA segments were obviously increased and apoptotic DNA ladder appeared,which was not seen in the other 2 groups.The apoptosis rate of bone marrow hematopoietic cells in ER group was significantly lower than that in the pure radiation group at 4,8,and 12 h after irradiation(P