Profiling of EMT-associated mitochondrial RNAs in hepatocellular carcinoma cells

Mitochondria, the powerhouse of the cell, has been recognized as the key players in cancer cell biology, including cancer metabolism, metastasis, and drug resistance. Recent studies have demonstrated a functional interplay between mitochondria and epithelial-mesenchymal transition (EMT) in cancer. To delineate the role of mitochondrial components in this interplay, we induced the EMT in hepatoma HepG2 cells. Mitochondria was isolated from EMT-HepG2 cells, untreated HepG2 cells, and normal hepatic HL7702 cells. Mitochondrial RNAs were isolated for Illumina sequencing. Identification of mitochondrial RNAs that regulate EMT or mitochondria function may serve as potential therapeutic targets for developing new strategies to treat cancers. Overall design: HepG2 cells were seeded into 6-well plates, and EMT was induced by 10 ng/ml TGF-ß1 in the medium was used to replace the common medium for 24-72 h. Cells were characterized by examining the EMT markers using Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (#9782, Cell Signaling Technology). After confirming the EMT, cells were collected and mitochondria was isolated using Qproteome Mitochondria Isolation Kit (Cat.37612, Qiagen). Mitochondrial RNAs were extracted using the miRNeasy Mini Kit (Cat# 217004, Qiagen). Qualified RNAs were further purified by RNAClean XP Kit (Cat.A63987, Beckman Coulter, CA) and were treated with RNase-Free DNase Set (Cat.79254, QIAGEN, CA). Ribosomal RNA was removed by TruSeq Stranded Total RNA LT-(Ribo-Zero Gold rRNA Removal Kit)-Set A/B (#RS-122-2301/2302, Illumina, CA). RNA transcriptome sequencing (RNA-Seq) was performed to identify mitochondrial RNAs that are associated with EMT.