PO-218 MicroRNA in mitochondria of multidrug resistant hepatocellular carcinoma

Abstract

IntroductionMitochondria consist of a unique genome which can encode proteins involved in the electron transport chain (ETC) for energy production. Mitochondrial dysfunction can lead to cancer formation.MicroRNAs (miRNAs) are evolutionary conserved, non-coding RNA molecules. MicroRNAs together with Argonaute (Ago) protein are essential for post-transcriptional gene regulation. Growing evidence suggested that nuclear-encoded miRNAs are imported to mitochondria. Despite their existence, their biogenesis and action mechanisms remain unexplored.This project aims at investigating the roles of miRNAs in mitochondria. In particular, we are going to examine whether miRNAs can regulate oxidative phosphorylation (OXPHOS) and their contribution to the development of multidrug resistance (MDR) in hepatocellular carcinoma (HCC).Material and methodsAccumulation of doxorubicin (DOX) in cells after co-treatment with ETC inhibitor for 4 hours was examined by flow cytometer. Mitochondria were purified by affinity column. Total proteins and RNAs isolated from mitochondria were subjected to Western blotting and real time-PCR analysis respectively.Results and discussionsDoxorubicin resistant HepG2 (R-HepG2) cell line has been developed in our lab. P-glycoprotein and Bcl-2, which are vital for MDR are highly upregulated in R-HepG2 cells compared to HepG2 cells. We confirmed that R-HepG2 cells produced more ATP than HepG2 cells. The amount of DOX retained inside R-HepG2 cells increased significantly after compromising the efficiency of OXPHOS by ETC inhibitors treatment. We believe that OXPHOS plays an essential role in maintaining MDR.Subsequently, we identified several miRNAs had differential expression level inside the mitochondria of HepG2 and R-HepG2 cells. Besides, Ago 2 protein, which is the key component in mediating RNA silencing, can also be detected in isolated mitochondria.ConclusionThe current effort has preliminarily demonstrated OXPHOS are responsible for MDR in HCC. Moreover, Ago 2 protein and miRNAs can be found in mitochondria of MDR cancer cells. Further investigation on the relation between miRNAs inside mitochondria and mitochondrial-encoded proteins may shed light on their function in controlling OXPHOS.