| Tumor is one of the most lethal diseases of humans and animals. A lot of factors induce tumor, such as immune surveillance, virus infectin, ultraviolet exposure Cancer research is not only beneficial for therapies, but also is helpful to answer huge number of biology questions.CCCTC-binding factor (CTCF) is an evolutionarily conserved nuclear protein which is widely expressed in eukaryotic organisms. The 11-zinc finger protein CTCF uses different conbinations of zinc fingers to bind varying CTCF-target sequences (CTSs), that mediates the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). CTCF is widely expressed in various of tumor cells. There is a significant correlation between CTCF levels and tumor histological grades. Docquier etal found that up-regulation of CTCF was linked to apoptosis resistance of breast cancer cells.In this study, we wanted to know if CTCF was associated with apoptosis-resistance of HeLa cells. For this purpose, apoptotic cell ratio was monitored following knockdown of CTCF in HeLa cells. We have obtained a cell line, HeLa-CTCF, which was of about 70% reduced CTCF level by stably expression of CTCF shRNA. Both HeLa cells and HeLa-CTCF cells were treated with ultraviolet irradiation, sodium butyrate or cisplatin, respectively. The percentage of apoptotic cells was calculated 24 hours after stimulation. Apoptotic cell ratio was assessed by Hoechst33342/PI staining method. Statistical analysis was done using Student's t test. In UV treatment experiments, the apoptotic rate of HeLa-CTCF cells was (29.59±3.50)%, which was higher than (11.00±3.06)% of HeLa cells. In Ara-C treatment experiments, the apoptotic rate of HeLa-CTCF cells and of HeLa cells were (4.98±0.25)% and (2.01±0.24)%, respectively. In cisplatin treatment experiments, the apoptotic rate of HeLa-CTCF cells was (18.02±3.96)%, and the apoptotic rate of HeLa cells was (10.01±1.55)%. For all above experiments, the increased apoptotic ratio of HeLa-CTCF cells was statistical significant (p<0.05), comparing to HeLa cells'.Taken together, these results suggested that CTCF show antiapoptotic function in HeLa cells. To explore the molecular mechanism by which CTCF regulate the cell anti-apoptosis capability, we did whole genome expression analysis by using gene chips. The result revealed thatX- linked inhibitor of apoptosis protein (XIAP) was a potential target of CTCF.XIAP is a member of the inhibitor of apoptosis protein family (IAPs). XIAP is related with cancer and neuronal apoptosis. XIAP is expressed in the majority of human malignancies at elevated levels and play an active role in promoting tumor maintenance through the inhibition of cellular death and participation in signaling pathways associated with malignancies.We observed XIAP level in HeLa and HeLa-CTCF cells using PCR and Western-blot. Western blot analysis revealed that XIAP levels in HeLa-CTCF cells were decreased 48% compared with HeLa cells. CTCF positively regulates the expression of XIAP.HeLa cells were transiently transfected with pEGFP-Cl by different reagents and various conditions. The best transfection effect was achieved by using 1.2\ig of pEGFP-Cl together with 2.4|il of Entranster-D as transfeciont reagent. To further validate XIAP as a potential CTCF target, a Western blot analysis of HeLa cells transiently transfected with pCMV6-XL5-CTCF was carried out. The pCMV6-XL5-CTCF vector for transient transfection was made by insertion of the full-length CTCF cDNA into the pCMV6-XL5 vector. HeLa cells were transiently transfected with 1.2|ig of pCMV6-XL5-CTCF or empty vector. CTCF levels among the transfected cells were detected 48 hours after transfection. There was at least 2.14-fold elevation of CTCF protein in HeLa cells transfected with pCMV6-XL5-CTCF compared with HeLa cells transfected with pCMV6-XL5. Following CTCF up-regulation in HeLa cells transiently transfected with pCMV6-XL5-CTCF, the expression level of XIAP was 1.9-fold increased. The results showed that CTCF regulated the expression of XIAP positively. CTCF overexpression resulted in elevated expression of XIAP.The observations reported in this study showed the over-expression of CTCF may have evolved as a compensatory mechanism to protect HeLa cells from apoptosis. The expression of CTCF protected cells from apoptosis at least partially byregulating the expression of XIA P. Reduced level of CTCF results in XIAP repression, leading to apoptosis. The observations reported here may be helpful to understand more about the CTCF-related molecular mechanisms of cell apoptosis. |
