Zheng M, Liu Y, Liu J, Kang Q, Wang T
School of Life Sciences, Zhengzhou University, Zhengzhou 450000, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2024 Jul 20;44(7):1355-1360. doi: 10.12122/j.issn.1673-4254.2024.07.15.
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 4.1RHL-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.
探讨蛋白4.1R缺失对肝细胞增殖、凋亡及糖酵解的影响及其分子机制。
利用CRISPR/Cas9技术构建4.1R HL-7702细胞系，以4.1R HL-7702细胞为对照，在细胞培养24、48、72 h时，采用CCK-8法、EdU-488染色、流式细胞术及Annexin V-FITC/PI染色评估其增殖能力和细胞凋亡情况。检测4.1R HL-7702细胞培养上清液中葡萄糖摄取、乳酸分泌、ATP生成及pH值的变化。采用qRT-PCR检测糖酵解关键调节酶HK2、PFKL、PKM2和LDHA的mRNA表达，采用蛋白质免疫印迹法检测AMPK、p-AMPK、Raptor和p-Raptor的蛋白表达。
蛋白质免疫印迹法和测序分析均证实4.1R HL-7702细胞系构建成功。与野生型细胞相比，4.1R HL-7702细胞增殖活性降低，细胞凋亡增加。蛋白4.1R的缺失还导致细胞葡萄糖摄取、乳酸分泌和ATP生成显著减少，细胞培养上清液pH值升高。qRT-PCR显示4.1R HL-7702细胞中糖酵解关键调节酶的mRNA表达显著降低。与HL-7702细胞相比，4.1R HL-7702细胞中AMPK和Raptor蛋白表达水平降低，而p-AMPK和p-Raptor蛋白表达水平显著升高。
HL-7702细胞中蛋白4.1R的缺失导致增殖能力降低、凋亡增加及糖酵解受抑制，且这种调节机制与下游AMPK-mTORC1信号通路的激活密切相关。
