Effect of deletion of protein 4.1R on proliferation, apoptosis and glycolysis of hepatocyte HL-7702 cells

Abstract

To explore the effects of deletion of protein 4.1R on hepatocyte proliferation, apoptosis, and glycolysis and the molecular mechanisms. A 4.1R-/- HL-7702 cell line was constructed using CRISPR/Cas9 technique, and with 4.1R+/+HL-7702 cells as the control, its proliferative capacity and cell apoptosis were assessed using CCK-8 assay, EdU-488 staining, flow cytometry and Annexin V-FITC/PI staining at 24, 48, 72 h of cell culture. The changes in glucose uptake, lactate secretion, ATP production and pH value of the culture supernatant of 4.1R-/- HL-7702 cells were determined. The mRNA expressions of the key regulatory enzymes HK2, PFKL, PKM2 and LDHA in glycolysis were detected with qRT-PCR, and the protein expressions of AMPK, p-AMPK, Raptor and p-Raptor were determined using Western blotting. Western blotting and sequencing analysis both confirmed the successful construction of 4.1R-/- HL-7702 cell line. Compared with the wild-type cells, 4.1R-/- HL-7702 cells exhibited a lowered proliferative activity with increased cell apoptosis. The deletion of protein 4.1R also resulted in significantly decreased glucose uptake, lactate secretion and ATP production of the cells and increased pH value of the cell culture supernatant. qRT-PCR showed significantly decreased mRNA expressions of the key regulatory enzymes in glycolysis in 4.1R-/- HL-7702 cells. Compared with those in HL-7702 cells, the expression levels of AMPK and Raptor proteins were decreased while the expression levels of p-AMPK and p-Raptor proteins increased significantly in 4.1R-/- HL-7702 cells. Deletion of protein 4.1R in HL-7702 cells results in reduced proliferative capacity, increased apoptosis and suppression of glycolysis, and this regulatory mechanism is closely related with the activation of the downstream AMPK-mTORC1 signaling pathway.