| CCCTC-binding factor (CTCF) is a multivalent transcription factor which is ubiquitously expressed in eukaryotic somatic cells. Its sequence is highly conserved, and its middle 312 amino acid residues consist of 11 successive zinc fingers. These zinc fingers can bind to a variety of DNA sequences in a combinatorial way. Besides, CTCF also can bind to different factors to form protein-protein compounds. CTCF is involved in different aspects of organism regulation including promoter activation or repression, gene silencing, chromatin insulation, gene imprinting, X-chromasome inactivation, regulation of cell growth or differentiation and tumorigenesis. CTCF has to enter the nucleus through nuclear pore complex to perform these functions. Until now, there is no report about how CTCF enter the nucleus.Based on literature research and our preliminary study, we supposed that the intra-cellular distribution of CTCF might be correlated with the CTCF expression level, and the intra-cellular distribution of CTCF might influence the invasive capability of tumor cells. To confirm the presumption, we investigated as follows.1. The intra-cellular distribution of CTCF is correlated with the CTCF expression level.In low invasive SMMC-7721, HepG2 and HeLa cells, CTCF mostly locates in cellular nucleus. We chose 2 RNAi targets of CTCF and RNAi target of luciferase as control to transfect these cells. Western blot was used to detect the CTCF expression level in cell lysate, and immunofluorescence stain was used to detect the CTCF distribution in these cells. We found that siRNA of 2 RNAi targets successfully knocked down the CTCF expression level. When knocking down the CTCF expression level, the distribution ratio of CTCF in cellular nucleus was decreased and the distribution ratio in cytoplasm was increased. We presumed that knocking down the CTCF expression level might down-regulate the expression of one or several proteins which were related with nucleocytoplasmic transport of high molecular proteins, and then inhibited the nuclear import of CTCF.2. CTCF regulates the IPO13 expression. To find the target genes regulated by CTCF, we used the CHIP-on-chip technique, and detected Importin 13 (IPO13) is a target gene. IPO13 is one of members of Karyopherins super-family which mediates high molecular proteins transport between cytoplasm and cellular nucleus. It has been known that IPO13 mediates nuclear import of NF-Y, myopodin, hUBC9, RBM8(Y14)-MGN, GR (glucocorticoid receptor) and nuclear export of eIF-1A, so we presumed that IPO13 might mediate the nuclear import of CTCF.To avoid pseudo-positive data, we validated the chip data through CHIP-PCR and confirmed there is a CTCF target site in ipo13 -774～-573bp promoter region. We further confirmed that CTCF could regulate the expression level of IPO13. When over expression of CTCF in MDA-MB-157 cell, we detected increased ipo13 transcript level by RT-PCR and increased IPO13 protein level by western blot. These results indicated that over expression of CTCF up-regulated the expression of IPO13 in RNA and protein level. When knocking down the expression of CTCF in HepG2, SMMC-7721 and HeLa cells, we detected decreased IPO13 protein level by western blot. These results demonstrated that knocking down the expression of CTCF down-regulated the expression of IPO13.3. IPO13 mediates the nuclear import of CTCF.We presumed that IPO13 might mediate the nuclear import of CTCF. Because the high molecular weight protein needs to bind its transport receptor for importing into cellular nucleus, there might be interaction between CTCF and IPO13. We expressed GST as control and GST-IPO13 fusion protein in E. coli and expressed CTCF in Wheat Germ Extract in vitro system. We detected the direct interaction between CTCF and IPO13 in vitro without introducing any other eucaryotic proteins by GST pull-down. Besides, we detected their physiological condition interaction using HepG2 cell lysate by Co-IP. Furthermore, we found that IPO13 influenced intra-cellular distribution of CTCF with immunofluorescence stain. When over expression of IPO13 in MDA-MB-231 and MDA-MB-157 cells, the distribution ratio of CTCF in cellular nucleus was increased and the distribution ratio in cytoplasm was decreased. These results suggested that over expression of IPO13 promoted the nuclear import of CTCF. When knocking down the IPO13 expression in HepG2, SMMC-7721 and HeLa cells, the distribution ratio of CTCF in cellular nucleus was decreased and the distribution ratio in cytoplasm was increased. These results suggested that knocking down the IPO13 expression inhibited the nuclear import of CTCF. In addition, when knocking down the CTCF expression and simultaneous over expression of IPO13 in HeLa cell, we confirmed the compensation of IPO13 for the change of CTCF intra-cellular distribution influenced by the expression level of CTCF.In the second and the third part, we revealed a mechanism for CTCF nuclear import. That is, CTCF regulats the expression of IPO13, and IPO13 mediats the nuclear import of CTCF. In other words, it is a nuclear import mechanism regulated by CTCF itself.4. CTCF influences tumor cells invasive capability.In this part, we preliminarily investigated whether CTCF influenced tumor cells invasive capability. After changing the expression level of CTCF, or changing the intra-cellular distribution of CTCF by changing the expression level of IPO13, we carried out Transwell assay to analyze the effect on invasive capability of tumor cells. When knocking down the expression of CTCF in low invasive HepG2 and SMMC-7721 cells, we detected enhanced invasive capability, which suggested knocking down the expression of CTCF enhanced invasive capability of tumor cells. Similarly, when knocking down the expression of IPO13 in these two cells, we also detected enhanced invasive capability. When over expression of IPO13 in highly invasive MDA-MB-157 cell, we detected reduced invasive capability and migratory capability. These results demonstrated that the increased distribution ratio of CTCF in cytoplasm enhanced invasive capability of tumor cells and the increased distribution ratio of CTCF in cellular nucleus reduced invasive capability of tumor cells. Conclusion:Our research revealed the association among the expression level of CTCF, the intra-cellular distribution of CTCF and invasive capability of tumor cells. CTCF regulated the IPO13 expression through the change of CTCF expression level, and IPO13 mediated the nuclear import of CTCF, so the CTCF expression level influenced the intra-cellular distribution of CTCF, and then the different intra-cellular distribution of CTCF influenced invasive capability of tumor cells. |
PDF Full Text Request