Integrated and comparative miRNA analysis of starvation-induced autophagy in mouse embryonic fibroblasts

Abstract

MiRNAs have recently been implicated in the regulation of autophagy. The present study focuses on how miRNA expression profiling is linked to the regulation of starvation-induced autophagy. Atg5 wild-type (WT) and knockout (KO) mouse embryonic fibroblasts (MEFs) were starved in Earle's balanced salt solution (EBSS) for 3h, and miRNA microarray was then performed to compare the miRNA expression profiles. Our results showed that: (1) one hundred miRNAs were significantly altered in both Atg5 WT and KO MEFs during starvation-induced autophagy; (2) among those miRNAs with significant changes upon starvation, 60 of them were upregulated in both Atg5 WT and KO MEFs and only 24 miRNAs were upregulated exclusively in Atg5 KO MEFs; (3) qRT-PCR validation analysis of 8 selected miRNAs showed a high correlation coefficient (r=0.95456) with microarray results; (4) many significantly altered miRNAs were mapped to several key signaling pathways and autophagy-related genes (Atgs) involved in the autophagy process, including (i) the Beclin1-Class III phosphatidylinositol 3-kinase (PI3KC3) complex, (ii) the ULK1 complex, (iii) the RAG/mechanistic target of rapamycin (mTOR) pathway, (iv) the liver kinase B1 (LKB1)–AMP-activated protein kinase (AMPK)–mTOR, and the class I phosphatidylinositol 3-kinase (PI3KC1)–Akt–mTOR pathways. The systematical analysis of the miRNA expression profiling and preliminary network analysis reveal that most of these miRNAs play important roles in autophagy regulation. Our results clearly demonstrate that miRNAs are involved in the autophagy process and understanding the functions of miRNAs provides novel insights into the molecular mechanisms underlying starvation-induced autophagy.