Stable knockout of Mcart-1 down-regulates the proliferation and oxidative phosphorylation of RAW264.7 macrophages

Abstract

Objective To construct a RAW264.7 macrophage cell strain with stable knockout of Mcart-1 by using CRISPR/Cas9 gene editing technique, and to detect its biological function. Methods Cas9 and sgRNA lentivirus were used to infect RAW264.7 macrophages in two steps. Positive cells were screened with puromycin and hygromycin, and single cells were plated by flow cytometry to obtain monoclonal cells; Expression of Cas9 and Mcart-1 was detected by qPCR and Western blot, and the mutation site was confirmed by sequence analysis. Cells number counting and carboxyfluorescein diacetate succinimdyl ester (CFSE) staining were used to detect cell proliferation; ATP detection kit was used to measure total ATP content in cells; Seahorse bioenergy analyzer was used to detect cell oxygen consumption rate(OCR) and glycolysis rate. Results Successfully constructed RAW264.7 cell strain with Mcart-1 stably knocked out, denoted as Mcart-1-/--RAW264.7; A frameshift mutation occurred in Mcart-1 gene in this cell strain; Compared with the wild-type RAW264.7 cell line (RAW264.7), the OCR increased and the extracellular acidification rate(ECAR)of Mcart-1-/--RAW264.7 cells decreased; The total ATP content in Mcart-1 knocked out cells decreased(P＜0.05),mitochondrial ATP production rate decreased (P＜0.01), while glycolytic ATP production rate increased (P＜0.01); The proliferation activity of RAW264.7 cells decreased after Mcart-1 was knocked out(P＜0.001). Conclusions The proliferation activity and oxidative phosphorylation level of RAW264.7 cells were both down-regulated after the Mcart-1 was stably knocked out. This cell strain is an important tool for exploring the function of cells in the tumor microenvironment.

Keywords

• crispr/cas9|macrophage|mcart-1|metabolic phenotype