Hypermethylation in the promoter region inhibits AJAP1 expression and activates the JAK/STAT pathway to promote prostate cancer cell migration and stem cell sphere formation

Abstract

AJAP1 is down-regulated in multiple cancer types and plays a suppressive role in cancer progression. However, its molecular regulatory mechanism in prostate cancer has not been reported. Bioinformatics methods were employed to analyze AJAP1 expression in prostate cancer tissues and its association with TNM staging. MSP and qRT-PCR were used to quantify promoter methylation and AJAP1 expression after 5-aza-20-deoxycytidine (5-AzaC) treatment. Scratch healing assay and Transwell method were adopted to analyze the effects of aberrant AJAP1 expression, 5-AzaC and AG490 on cell migration and invasion. The levels of AJAP1 protein, EMT-related and JAK/STAT pathway-related proteins were determined by Western blot. The effects of AJAP1 aberrant expression and AG490 treatment on the sphere forming ability of prostate cancer cells were analyzed by sphere formation assay. This study confirmed the significant down-regulation of AJAP1 expression in prostate cancer tissues and cells, and its negative correlation with TNM staging. 5-AzaC treatment led to a significant reduction of AJAP1 methylation level and a significant upregulation of AJAP1 expression, indicating that the methylation level of AJAP1 promoter may affect the expression of AJAP1. Cell function experiments found that overexpression or decreased methylation of AJAP1 inhibited epithelial mesenchymal transition (EMT), migration, and invasion, while silencing or increased methylation of AJAP1 had the opposite functions. JAK2/STAT3 pathway inhibiting assay found that inhibition of JAK2/STAT3 pathway significantly reduced EMT, cell migration, and stem cell sphere formation in prostate cancer. Therefore, investigating the influence of aberrant AJAP1 expression on functions of prostate cancer cells is conducive to our in-depth understanding of the mechanism of prostate cancer genesis and development.