蛋白4.1R对肝细胞HL-7702增殖、凋亡以及糖酵解的影响

doi:10.12122/j.issn.1673-4254.2024.07.15

摘要/Abstract

摘要：

目的探究蛋白4.1R对肝细胞增殖、凋亡以及糖酵解的影响，并初步阐明其分子机制。方法以肝细胞株HL-7702为材料，利用CRISPR/Cas9技术构建4.1R^-/- HL-7702细胞株。设置对照组为正常培养的4.1R^+/+ HL-7702细胞，实验组为4.1R^-/- HL-7702细胞。细胞在培养24、48以及72 h时，分别用CCK-8及EdU-488染色，然后利用酶标仪以及流式细胞术检测细胞的增殖能力。细胞经Annexin V-FITC/PI染色后，流式细胞术检测HL-7702细胞在培养24、48以及72 h时的凋亡水平。利用生化试剂盒分别检测HL-7702细胞葡萄糖摄取量、乳酸分泌量以及ATP生成的变化。pH计检测培养48 h HL-7702细胞上清培养基的pH值。qRT-PCR检测HL-7702细胞糖酵解过程关键调节酶HK2、PFKL、PKM2以及LDHA的mRNA表达量。Western blotting检测AMPK、p-AMPK、Raptor以及p-Raptor的蛋白表达量。结果 Western blotting以及基因测序结果表明4.1R^-/- HL-7702细胞株构建成功。与对照组相比，4.1R^-/-组的CCK-8和EdU-488实验结果均显示HL-7702细胞的增殖能力降低；细胞凋亡实验结果表明，4.1R^-/- HL-7702细胞凋亡水平升高。蛋白4.1R的缺失导致HL-7702细胞葡萄糖摄取量（P<0.05）、乳酸分泌量（P<0.001）、ATP生成（P<0.001）下降，细胞上清培养基pH值（P<0.01）升高。qRT-PCR实验结果表明糖酵解过程关键调节酶的mRNA表达量均下降（P<0.001）。相较于HL-7702细胞，4.1R^-/- HL-7702细胞的AMPK和Raptor蛋白表达量下降，p-AMPK和p-Raptor蛋白表达量升高。结论蛋白4.1R的缺失导致HL-7702细胞增殖能力降低、凋亡水平增加，糖酵解过程被抑制，其调控机制与下游AMPK-mTORC1信号通路的激活密切相关。

关键词: 蛋白4.1R, 肝细胞, 增殖与凋亡, 糖酵解

Abstract:

Objective To explore the effects of deletion of protein 4.1R on hepatocyte proliferation, apoptosis, and glycolysis and the molecular mechanisms. Methods 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. Results 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. Conclusion 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.

Key words: protein 4.1R, hepatocytes, proliferation and apoptosis, glycolysis

郑孟冬, 刘妍, 刘娇娇, 康巧珍, 王婷. 蛋白4.1R对肝细胞HL-7702增殖、凋亡以及糖酵解的影响[J]. 南方医科大学学报, 2024, 44(7): 1355-1360.

Mengdong ZHENG, Yan LIU, Jiaojiao LIU, Qiaozhen KANG, Ting WANG. Effect of deletion of protein 4.1R on proliferation, apoptosis and glycolysis of hepatocyte HL-7702 cells[J]. Journal of Southern Medical University, 2024, 44(7): 1355-1360.

图/表 5

表1 靶向4.1R基因的sgRNA序列

Tab.1 sgRNA sequences targeting the 4.1R gene

5'- TTTATGTCTTCAGCTTCGGC -3'

3'- AAATACAGAAGTCGAAGCCG -5'

NGG

sgRNA2

5'- ACTGGCCCCGAATCAGACCA -3'

3'- TGACCGGGGCTTAGTCTGGT -5'

sgRNA3

5'- CGAGCGTACAGCAAGTAAAC -3'

3'- GCTCGCATGTCGTTCATTTG -5'

表2 糖酵解过程关键调节酶的引物序列

Tab.2 Primer sequences for RT-qPCR of the key regulatory enzymes in glycolysis

Gene	Primer sequence 5'-3'
HK2	Forward: GACCAACTTCCGTGTGCTTT
	Reverse: TCCATGAAGTTAGCCAGGCA
PFKL	Forward: ATCTACGAGGGCTATGAGGGC
	Reverse: GAGCGCTGCCAATGATAGTG
PKM2	Forward: GGAAGCCTGTCATCTGTGCT
	Reverse: TCCCCTTTGGCTGTTTCTCC
LDHA	Forward: ATGGCAACTCTAAAGGATCAGC
	Reverse: CCAACCCCAACAACTGTAATCT
β-actin	Forward: CGTGCGTGACATTAAGGAGAAG
	Reverse: GGAAGGAAGGCTGGAAGAGTG

图1 构建4.1R-/- HL-7702细胞株

Fig.1 Construction of 4.1R-/- HL-7702 cell line. A: Identification of protein 4.1R expression in HL-7702 cells. B: Schematic representation of the locus of sgRNA targeting the 4.1R gene. C: Sequencing results of recombinant vector px330-mCherry-sgRNA. D, E: Identification of the knockout efficiency. F: Identification of protein 4.1R expression. G: 4.1R-/- HL-7702 cell line sequencing results. H: Comparison of 4.1R gene sequence. *P<0.05, ***P<0.001 vs HL-7702_WT.

图2 敲除蛋白4.1R对HL-7702细胞增殖与凋亡的影响

Fig.2 Effect of knockout protein 4.1R on proliferation and apoptosis of HL-7702 cells. A: Results of CCK-8 experiments. B, C: Detection of cell proliferation by EdU method and flow cytometry. D, E: Detection of apoptosis by Annexin V-FITC/PI staining and flow cytometry. *P<0.05, **P<0.01, ***P<0.001 vs 4.1R+/+.

图3 敲除蛋白4.1R对HL-7702细胞糖酵解的影响

Fig.3 Effect of deletion of protein 4.1R on glycolysis in HL-7702 cells. A: Changes in cellular glucose uptake. B: Changes in extracellular lactate secretion. C: Changes in intracellular ATP levels. D: Changes in pH value of cell culture medium supernatants. E: Levels of mRNA expression of the key regulatory enzymes of glycolysis. F: Western blotting for detecting the downstream signaling pathway AMPK-mTORC1 (Raptor). *P<0.05, **P<0.01, ***P<0.001 vs 4.1R+/+.

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