Positive feedback of SuFu negating protein 1 on Hedgehog signaling promotes colorectal tumor growth

摘要

A, B Screening for novel downstream target genes of Hh signaling. Venn diagram (A) and heatmap (B) of differentially expressed genes (DEGs) (fold change ≥2 or ≤ 0.05, adjusted p < 0.05) in HT-29 cells treated with GANT61 or expressing Gli2 and cluster analysis of these genes with Gene Ontology (GO) annotation. C Gli2 expression affects SNEP1 mRNA levels. HT-29 cells were transfected with Myc-vector, Myc-Gli2, or Myc-Gli2A for 48 h and harvested for qPCR analysis of SNEP1 mRNA. Bcl2, Ptch1, Sox2 and Gli1, well-known Hh signaling target genes, were used as positive controls. D Gli2 expression affects SNEP1 protein levels. HT-29 cells were transfected with Gli2 constructs for 48 h and harvested for IB with the indicated antibodies. E Gli2 knockdown reduces SNEP1 protein levels. HT-29 cells were transfected with shRNAi-Gli2 plasmids for 72 h and harvested for IB analysis with the indicated antibodies. F Pharmacological repression of Gli2 reduces SNEP1 mRNA levels. HT-29 cells were treated with GANT61 for 48 h and harvested for RNA extraction and qPCR. G Chromatin immunoprecipitation (ChIP) assays for Gli2 at the SNEP1 promoter. Upper: schematic representation of the SNEP1 promoter region showing the putative transcription factor-binding sites. Lower: HT-29 cells were harvested for ChIP assays as described in the Materials and Methods with IgG or anti-Gli2 antibodies for IP followed by qPCR with specific primers for each putative-binding element as indicated. H Luciferase assays for Gli2 transcriptional activity at the SNEP1 promoter. A series of SNEP1-luciferase constructs (left) were transfected into HEK-293T cells, and relative Gli2 transcript levels were measured 48 h after transfection (right). Each experiment was performed in triplicate. *p < 0.05, **p < 0.01.