Y-box Protein-1 Regulates the Expression of Collagen I in Hepatic Progenitor Cells via PDGFR-β/ERK/p90RSK Signalling

Figure 4

YB-1 directly binds to the PDGFR-β promoter and negatively regulates its expression. (a) ChIP-sequencing assays detected that YB-1 directly bound to a region of the PDGFR-β promoter spanning from the site 61044456 to 61044670 on chromatin 18. (b) ChIP-PCR confirmed that PDGFR-β is one of the YB-1 target genes. Chromatin immunoprecipitations were performed using HPC chromatin with normal rabbit IgG, anti-RNA polymerase II, and anti-YB-1 antibodies. Purified DNA was then analysed with PCR using primers specific for YB-1 binding sites on the PDGFR-β promoter or primers for the GAPDH promoter as the positive control. The size of the PCR product for the PDGFR-β promoter and the GAPDH promoter was 82 and 166 bp, respectively. The PCR product was observed in the input, positive, and anti-YB-1 ChIPs (lanes 2, 4, and 5) but not in the no DNA and IgG ChIPs (lanes 1 and 3). (c) YB-1 negatively modulates PDGFR-β promoter activity in HPCs. HPCs were transfected with a scramble lentivirus or YB-1 knockdown lentivirus; PDGFR-β promoter-luciferase constructs and Renilla luciferase constructs were cotransfected into HPCs. Cells were harvested 48 h after the second transfection, and luciferase activity was measured. The results are an average of three independent transfections. (d) YB-1 knockdown enhanced PDGFR-β mRNA expression. In addition, PDGF-β expression was elevated in HPCs with YB-1 silenced. (e) Western blotting revealed that YB-1 knockdown negatively regulated PDGFR-β and PDGF-β expression. Compared with the culture supernatant from HPCs transfected with the scrambled lentivirus, the YB-1 knockdown groups showed higher levels of PDGF-β protein. , compared with the scramble control.

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