The Interaction Between Mutants Of GTFⅡF2 Proteins And CLIC1 Protein

Objective This article aims to demonstrate if there is interaction between GTFⅡF2 mutants and CLIC1, and the effect of general transcription factor GTFⅡF2 expression on the proliferation and migration of esophageal cancer cells. Some experimental techniques were applied to investigate this interaction in vitro(yeast cells and mammalian cells) and in vivo respectively, such as plasmid comstruction, Yeast two-hybrid, western blot, immunofluorescence, co-imumoprecipitation, laying the cytological foundation for exploring the functions of GTFⅡF2 mutants and CLIC1.Methods Design upstream and downstream primers of GTFIIF2 mutants according to the needs of different experimental by PCR. GTFⅡF2-N: GTFⅡF2-(1-165 aa); GTFⅡF2(1-100 aa); GTFⅡF2(1-142 aa)and GTFⅡF2-C:GTFⅡF2(161-249 aa); GTFⅡF2(101-249 aa); GTFⅡF2(143-249 aa), the correct enzyme digestion of recombinant plasmids were delivered to sequenced. In endogenous co-immunoprecipitation experiments, no plasmids were transfected, total cellular protein was extracted to incubate by GTFⅡF2 and CLIC1 antibody respectively and precipitate by Agarose beads, then the beads were collected for Western blot. According to the need of the yeast two hybrid, then constructing the yeast expression plasmids p GΑDT7- GTFⅡF2(1-165aa), p GΑDT7- GTFⅡF2(161-249aa), p GBKT7- GTFⅡF2(1-165aa), p GBKT7- GTFⅡF2(161-249aa) were constructed in turn, and were co-transformed into yeast AH109 cells with yeast expression plasmids p GBKT7-CLIC1, p GΑDT7-CLIC1, respectively, then screening cells on the SD3-(-Leu/-His/-Trp) medium, determining the activities of X-α--gal of cells, todemonstrate if there is interaction between GTFⅡF2 mutants and CLIC1 by observing if observing whether the color of cells turn blue; To construct the eukaryotic expression plasmids pc DNA3.1 and p CDGFP were constructed. pc DNΑ3.1-CLIC1-FLΑG and pc DGFP-GTFⅡF2 mutants or FLΑG-GTFⅡF2 mutants and pc DGFP-CLIC1 were co-transfected into mammalian cells in COS7 to observe whether GTFⅡF2 mutants colocalization with CLIC1 by laser confocal. In exogenous co-immunoprecipitation experiments, pc DNA3.1-CLIC1-FLAG and pc DGFP-GTFⅡF2 mutants were co-transfected into mammalian cells in HEK 293 T, total cellular protein was extracted to incubate by FLAG antibody and precipitate by Agarose beads, then the beads were collected for Western blot. In GST pulldown experiment, plasmids with GFP-tag were transfected into HEK 293 T and mixed with GST-CLIC1 or GST-GTFⅡF2 mutants fusion protein, and then the glutathione beads were collected for immunoblot to prove whether GTFⅡF2 mutants interact with CLIC1. Synthesized si RNA targeted to GTFⅡF2 gene or negative control si RNA and plasmid pc DNA3.1-GTFⅡF2-FLAG were transfected to esophageal cancer cells respectively. Colony formation assay and wound-healing assay were performed to study the effect of knockdown and overexpression of GTFⅡF2 expression on the proliferation and migration of esophageal cancer cells respectively.Results The recombinant plasmids were proved to be constructed successfully by restriction analysis and sequencing. In endogenous co-immunoprecipitation experiments, total cellular protein incubated by GTFⅡF2 and precipitate by Agarose beads could detect the presence of CLIC1 protein. Total cellular protein incubated by CLIC1 and precipitate by Agarose beads could detect the presence of GTFⅡF2. Yeast two-hybrid experiments show the yeast cells that had co-expressed the GTFⅡF2 and GTFⅡF2(1-165) and CLIC1 could grow normaly in SD3- medium, and the color of cells became blue in α-galactosidase activity assay, which proved the existence of interactionbetween them. However, GTFⅡF2(161-249) and CLIC1 had no interaction with each other. Immunofluorescence experiments showed that FLAG-GTFⅡF2 mutants and CLIC1 were mainly distributed in cytoplasm and nucleus, they had the obvious colocalization in the cytoplasm. However, GFP-GTFⅡF2-N were mainly distributed in cytoplasm, they had the less obvious colocalization with CLIC1 and GFP-GTFⅡF2-C were mainly distributed nucleus, they had the obvious colocalization with CLIC1 in the cytoplasm. In exogenous co-immunoprecipitation experiments showed that the GFP-GTFⅡF2-N could be detected in the experimental group after immunoblotting by GFP antibody, and indicated that GFP-GTFⅡF2-N could be co-precipitated when CLIC1-FLAG be precipitated by FLAG antibody, which proved the existence of interaction between them, and the domain is(1-100 aa). In GST pulldown experiments showed that GFP-CLIC1 + GST-GTFⅡF2-N were able to detect the presence of GTFⅡF2-N, indicating that GST-GTFⅡF2-N could drop down GFP-CLIC1, non-GST-GTFⅡF2-C fusion protein, and also showed that the GTFⅡF2-N could be detected in the experimental group after immunoblotting by GFP antibody, then indicated the GST-CLIC1 fusion protein could drop down the GTFⅡF2-N in vitro, that proved that there was interaction between them and the domin was(1-100). Knockdown of GTFⅡF2 inhibited the proliferation and migration in TE-1, ECA-109 cells, but overexpression of GTFⅡF2 had no significant effect on cell proliferation and migration.Conclusion Endogenous co-immunoprecipitation experiments confirmed the interaction of GTFⅡF2 and CLIC1, some experimental techniques were applied to investigate this interaction in vitro and in vivo respectively, such as Yeast two-hybrid, immunofluorescence, co-imumoprecipitation, GST pulldown. Andthe interaction domain is within the first range of the N-terminal 100 amino acids. Knockdown GTFⅡF2 inhibited proliferation and migration of esophageal cancer cells TE-1,ECA-109, laying the cytological foundation for exploring the functions of GTFⅡF2 mutants and CLIC1.