PIPKIγ Regulates CCL2 Expression in Colorectal Cancer by Activating AKT-STAT3 Signaling

Figure 3

STAT3 transcriptionally regulates PIP5Iγ to increase CCL2 expression. (a) Stable PIPKIγ-expressing HCT116 and SW620 cells were transfected with siP65, siSTAT3, siSTAT1, siTwist1, or siETS1 for 48 h, and the CCL2 mRNA level was measured by quantitative PCR with reverse transcription. (b) CCL2 mRNA expression levels were detected in stable PIPKIγ-expressing HCT116 and SW620 cells treated with JSH-23 and Stattic in FBS-free medium. (c) Stable PIPKIγ-expressing HCT116 and SW620 cells were transfected with si p65 and siSTAT3 for 48 h, and the CCL2 protein level was measured by ELISA. (d) CCL2 protein expression levels were detected in stable PIPKIγ-expressing HCT116 and SW620 cells treated with JSH-23 and Stattic in FBS-free medium. (e) The predicted STAT3 binding site on the upstream 0-2000 bp and the STAT3 binding motif. HEK 293 cells were transiently cotransfected with luciferase reporter plasmid (pGL3) containing the CCL2 DNA promoter region [-2000/0]) or different promoter fragment constructs, Renilla, vector, or STAT3 as indicated. After 30 h, luciferase activities were determined by dual luciferase assay. Luciferase activities were normalized to Renilla luciferase activities. The values indicated represent normalized luciferase activities and are shown as the from triplicate assays. (f) Luciferase assay for HEK 293 cells transiently cotransfected with luciferase reporter plasmid (pGL3) containing the CCL2 DNA promoter region [-2000/0]) or motif mutant constructs, Renilla, vector, or STAT3 as indicated. (g) ChIP-PCR analysis of STAT3 binding to the CCL2 promoter in the presence or absence of PIPKIγ silencing in SW480 and LOVO cells.

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