Detection of dynamic changes of mitochondrial ROS in HepG2 cells based on mitochondria-targeted mt-roGFP2 biosensor

LIU Xiaoning; BO Hui; LIU Cuie

doi:10.3969/j.issn.1002-266X.2018.08.004

Abstract：Objective To detect the dynamic changes of mitochondrial ROS in human hepatoblastoma HepG2 cell line(hereafter called HepG2 cells) by mitochondria-targeted mt-roGFP2 biosensor, and to lay the foundation for the chemotherapy of liver cancer targeting mitochondrial ROS. Methods The lentivirus vector pLenti-CMV-mt-roGFP2-PGK-puro was constructed by seamless cloning technology, and was identified by DNA sequencing. The lentivirus mt-roGFP2 was prepared by co-transfecting the human embryonic kideny 293T cells with the lentivirus vector pLenti-CMV-mt-roGFP2-PGKpuro and packaging plasmids pVSVg, pRev, and pGag-Pol, and then its virus titer was tested. The expression of roGFP in the HepG2 cells infected by the prepared mt-roGFP2 lentivirus particles was analyzed through fluorescence microscope and Western blotting. The HepG2 cells expressing mt-roGFP2 (HepG2-mt-roGFP2 cells) in logarithmic growth phase were incubated with MitoTracker probe, and then the subcellular localization of mt-roGFP2 biosensor was investigated by confocal laser scanning microscopy. The HepG2-mt-roGFP2 cells in the logarithmic growth phase treated with 0. 5 mmol/L H2O2 and 1 mmol/L DTT were imaged by using Nikon A1R confocal laser scanning system, and the fluorescence images were exported to Image J software. Results The lentivirus vector pLenti-CMV-mt-roGFP2-PGK-puro was successfully constructed. The mean titer of mt-roGFP2 was 4. 04 × 108 IU/mL. roGFP biosencor was stably expressed in the HepG2 cells and correctly targeted to mitochondria, indicating the successful construction of HepG2-mt-roGFP2 cells. The addition of exogenous oxidant H2O2 led to a significant increase of mitochondrial ROS in the HepG2-mt-roGFP2 cells, with the 405 nm/488 nm fluorescence ratios ranging from 1. 19 to 3. 98, and the injection of reductant DTT declined the fluorescence ratio to 1. 25. Conclusion The mitochondria-targeted mt-roGFP2 biosensor can real-time and reversibly detect the changes of mitochondrial ROS in the HepG2 cells and display the real-time imaging.