Demethylation of TIMP2 and TIMP3 inhibit cell proliferation, migration and invasion in pituitary adenoma

Background: Pituitary adenoma (PA) is one of the most common intracranial neoplasms. Tissue inhibitors of metalloproteinases (TIMPs) are prognostic biological markers, but their biological roles remains largely unclear in invasive PA. Methods: The promoter methylation status of TIMP2 and TIMP3 genes in invasive PA tissues and cells was measured by methylation-specific polymerase chain reaction (MSP). The expression of TIMP1-3 was validated by quantitative real time PCR and western blot analysis. Overexpression and knockdown of TIMP2 and TIMP3 in GH3 cells were created by transfection of pcDNA3.0 and siRNA against TIMP2 and TIMP3, respectively. Functional experiments in GH3 cells were performed with CCK-8 assay, wound healing assay and transwell assay. Effects of 5-Azacytidene (5-AzaC) on the methylation of TIMP2 and TIMP3 gene, and DNA methyltransferase 1 (DNMT1), DNMT3a and DNMT3b were determined by western blot analysis. Results: We found the expression of TIMP1, TIMP2 and TIMP3 was down-regulated in invasive PA tissues and cells. Moreover, decreased TIMP2 and TIMP3 expression was closely associated with their promoter methylation. The in vitro experiments showed that overexpression of TIMP2 and TIMP3 exerted suppressive effects, while knockdown of TIMP2 and TIMP3 presented enhanced effects on cell proliferation, migration and invasion in PA cells. Furthermore, 5-AzaC treatment concomitantly up-regulated the protein levels of TIMP2, TIMP3, DNMT1, DNMT3a and DNMT3b. Conclusions: In conclusion, our results support that DNA methylation at least partly accounts for TIMP2 or TIMP3 silencing in invasive PA, which will provide new insights into the mechanisms underlying the function of TIMPs in PA. Key words: Invasive pituitary adenoma, TIMPs, DNA methylation, 5-AzaC, GH3