Epigenetic inactivation of ACAT1 promotes epithelial-mesenchymal transition of clear cell renal cell carcinoma

Abstract

Background: Acetyl-CoA acyltransferase 1 (ACAT1) is a key enzyme catalyzing the production of mitochondrial ketone bodies. We have shown that ACAT1 is down-regulated in kidney renal clear cell carcinoma (KIRC) previously.

Objective: To investigate the reasons for downregulation of ACAT1 in KIRC and explore the underlying mechanisms involved in metastatic inhibition regulated by ACAT1.

Methods: The Gene Expression Omnibus (GEO) database was queried for meta-analysis of ACAT1 mRNA expression in KIRC. The UALCAN website was used to compare the methylation levels of the ACAT1 promoter region in KIRC and normal tissues. RT-qPCR was used to quantitate ACAT1 transcription levels. The GCBI and Tarbase V.8 databases were used to predict miRNAs that may target the mRNA of ACAT1. The correlation between mRNA expression of ACAT1, MMP7 (matrix metallopeptidase 7), CDH1 (E-cadherin), EpCAM (epithelial cell adhesion molecule), and VIM (vimentin) was analyzed. Extracellular MMP7 protein was quantitated using an ELISA assay.

Results: The methylation level of the ACAT1 promoter region in KIRC was significantly higher than that in the normal kidney tissues. The ACAT1 mRNA expression in the KIRC cell lines was restored after treatment with 5-aza-dC (p < 0.05). MiR-21-5p is a conserved microRNA targeting ACAT1. It is expressed at a significantly higher level in KIRC than in normal tissues (p < 0.001). MiR-21-5p miRNA expression negatively correlates with ACAT1 mRNA expression. The expression of miR-21-5p is higher at the T3-T4 stages and in the histologic grades G3-G4. Patients with high miR-21-5p expression tended to have lower overall survival, suggesting that miR-21-5p could serve as a potentially valuable diagnostic biomarker for KIRC (AUC = 0.957; p < 0.001). A mimetic of miR-21-5p inhibited the expression of ACAT1 mRNA and protein. In addition, ACAT1 mRNA expression positively correlates with CDH1 and EpCAM but is negatively correlated with VIM. Overexpression of ACAT1 suppresses the secretion of MMP7 in KIRC cells.

Conclusion: Expression of ACAT1 in KIRC is controlled at two levels, firstly by the hypermethylation of the ACAT1 promoter region and secondly by overexpression of miR-21-5p. Downregulation of ACAT1 expression correlates with epithelial-mesenchymal transition (EMT).

Keywords: Acetyl-CoA acyltransferase 1; KIRC; Metastasis; miR-21-5p.