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

doi:10.12122/j.issn.1673-4254.2024.07.15

Abstract

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

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.

Figures/Tables 5

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'

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

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.

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+/+.

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+/+.

References 31

1	Trefts E, Gannon M, Wasserman DH. The liver[J]. Curr Biol, 2017, 27(21): R1147-51.
2	Zhou Z, Xu MJ, Gao B. Hepatocytes: a key cell type for innate immunity[J]. Cell Mol Immunol, 2016, 13(3): 301-15.
3	Rui LY. Energy metabolism in the liver[J]. Compr Physiol, 2014, 4(1): 177-97.
4	Ding HR, Wang JL, Ren HZ, et al. Lipometabolism and glycometabolism in liver diseases[J]. Biomed Res Int, 2018, 2018: 1287127.
5	Conboy J, Kan YW, Shohet SB, et al. Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton[J]. Proc Natl Acad Sci U S A, 1986, 83(24): 9512-6.
6	Baines AJ, Lu HC, Bennett PM. The Protein 4.1 family: hub proteins in animals for organizing membrane proteins[J]. Biochim Biophys Acta, 2014, 1838(2): 605-19.
7	Sun CX, Robb VA, Gutmann DH. Protein 4.1 tumor suppressors: getting a FERM grip on growth regulation[J]. J Cell Sci, 2002, 115(Pt 21): 3991-4000.
8	李博文. 蛋白4.1R对白血病K562细胞功能的影响及机制研究[D]. 郑州: 郑州大学, 2021.
9	桑思瑶. 蛋白4.1R对LPS诱导的小鼠脓毒症肝损伤的作用研究[D]. 郑州: 郑州大学, 2020.
10	Kubes P, Jenne C. Immune responses in the liver[J]. Annu Rev Immunol, 2018, 36: 247-77.
11	Adeva-Andany MM, Pérez-Felpete N, Fernández-Fernández C, et al. Liver glucose metabolism in humans[J]. Biosci Rep, 2016, 36(6): e00416.
12	An XL, Mohandas N. Disorders of red cell membrane[J]. Br J Haematol, 2008, 141(3): 367-75.
13	唐治航. 蛋白4.1R对红系祖细胞增殖的影响及其在AML中的表达[D]. 郑州: 郑州大学, 2021.
14	Yuan JP, Xing HX, Li YK, et al. EPB41 suppresses the Wnt/β-catenin signaling in non-small cell lung cancer by sponging ALDOC[J]. Cancer Lett, 2021, 499: 255-64.
15	Yang XY, Yu DK, Ren YL, et al. Integrative functional genomics implicates EPB41 dysregulation in hepatocellular carcinoma risk[J]. Am J Hum Genet, 2016, 99(2): 275-86.
16	Kang QZ, Yu Y, Pei XH, et al. Cytoskeletal protein 4.1R negatively regulates T-cell activation by inhibiting the phosphorylation of LAT[J]. Blood, 2009, 113(24): 6128-37.
17	Liang TT, Guo YY, Li MJ, et al. Cytoskeleton protein 4.1R regulates B-cell fate by modulating the canonical NF-κB pathway[J]. Immunology, 2020, 161(4): 314-24.
18	梁桃桃. 蛋白4.1R对B细胞活化调控机制研究[D]. 郑州: 郑州大学, 2021.
19	Hardie DG, Carling D, Carlson M. The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell?[J]. Annu Rev Biochem, 1998, 67: 821-55.
20	Kemp BE, Mitchelhill KI, Stapleton D, et al. Dealing with energy demand: the AMP-activated protein kinase[J]. Trends Biochem Sci, 1999, 24(1): 22-5.
21	Mulukutla BC, Yongky A, Le T, et al. Regulation of glucose metabolism-A perspective from cell bioprocessing[J]. Trends Biotechnol, 2016, 34(8): 638-51.
22	Zhang BB, Zhou GC, Li C. AMPK: an emerging drug target for diabetes and the metabolic syndrome[J]. Cell Metab, 2009, 9(5): 407-16.
23	Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease[J]. Cell, 2017, 169(2): 361-71.
24	Liu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease[J]. Nat Rev Mol Cell Biol, 2020, 21(4): 183-203.
25	Draberova L, Draberova H, Potuckova L, et al. Cytoskeletal protein 4.1R is a positive regulator of the FcεRI signaling and chemotaxis in mast cells[J]. Front Immunol, 2020, 10: 3068.
26	Park JS, Burckhardt CJ, Lazcano R, et al. Mechanical regulation of glycolysis via cytoskeleton architecture[J]. Nature, 2020, 578(7796): 621-6.
27	Zanotelli MR, Zhang J, Reinhart-King CA. Mechanoresponsive metabolism in cancer cell migration and metastasis[J]. Cell Metab, 2021, 33(7): 1307-21.
28	Nunomura W, Takakuwa Y, Parra M, et al. Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins[J]. J Biol Chem, 2000, 275(9): 6360-7.
29	Nunomura W, Gascard P, Takakuwa Y. Insights into the function of the unstructured N-terminal domain of proteins 4.1R and 4.1G in erythropoiesis[J]. Int J Cell Biol, 2011, 2011: 943272.
30	Khan AA, Hanada T, Mohseni M, et al. Dematin and adducin provide a novel link between the spectrin cytoskeleton and human erythrocyte membrane by directly interacting with glucose transporter-1[J]. J Biol Chem, 2008, 283(21): 14600-9.
31	丁滪, 苏旸, 姜维华, 等. 以带3蛋白为核心的红细胞膜蛋白复合体研究[C]. 第十次中国生物物理学术大会论文摘要集. 青岛, 2006: 108.

[1]	Xiaohui WEN, Shiya HUANG, Xuehong LIU, Kunyin LI, Yongge GUAN. Role of Notch 1 signaling and glycolysis in the pathogenic mechanism of adenomyosis [J]. Journal of Southern Medical University, 2024, 44(8): 1599-1604.
[2]	MAN Hao, WANG Jianwei, WU Mao, SHAO Yang, YANG Junfeng, LI Shaoshuo, LÜ Jinye, ZHOU Yue. Jisuikang formula promotes spinal cord injury repair in rats by activating the YAP/PKM2 signaling axis in astrocytes [J]. Journal of Southern Medical University, 2024, 44(4): 636-643.
[3]	WANG Zining, YANG Ming, LI Shuanglei, CHI Haitao, WANG Junhui, XIAO Cangsong. A transcriptomic analysis of correlation between mitochondrial function and energy metabolism remodeling in mice with myocardial fibrosis following myocardial infarction [J]. Journal of Southern Medical University, 2024, 44(4): 666-674.
[4]	YAN Qiuxia, ZENG Peng, HUANG Shuqiang, TAN Cuiyu, ZHOU Xiuqin, QIAO Jing, ZHAO Xiaoying, FENG Ling, ZHU Zhenjie, ZHANG Guozhi, HU Hong, CHEN Cairong. RBMX overexpression inhibits proliferation, migration, invasion and glycolysis of human bladder cancer cells by downregulating PKM2 [J]. Journal of Southern Medical University, 2024, 44(1): 9-16.
[5]	FENG Wen, LAI Yuexing, WANG Jing, XU Ping. Long non-coding RNA ABHD11-AS1 promotes glycolysis in gastric cancer cells to accelerate tumor progression [J]. Journal of Southern Medical University, 2023, 43(9): 1485-1492.
[6]	WANG Qiusheng, ZHANG Zhen, WANG Lian, WANG Yu, YAO Xinyu, WANG Yueyue, ZHANG Xiaofeng, GE Sitang, ZUO Lugen. High expression of death-associated protein 5 promotes glucose metabolism in gastric cancer cells and correlates with poor survival outcomes [J]. Journal of Southern Medical University, 2023, 43(7): 1063-1070.
[7]	WANG Huijie, SUN Zhengui, ZHAO Wenying, GENG Biao. S100A10 promotes proliferation and invasion of lung adenocarcinoma cells by activating the Akt-mTOR signaling pathway [J]. Journal of Southern Medical University, 2023, 43(5): 733-740.
[8]	. Long non-coding RNA UPK1A-AS1 promotes glycolysis in hepatocellular carcinoma cells via stabilization of HIF-1α [J]. Journal of Southern Medical University, 2021, 41(2): 193-199.
[9]	. Shikonin induces cell death by inhibiting glycolysis in human testicular cancer I-10 and seminoma TCAM-2 cells [J]. Journal of Southern Medical University, 2020, 40(09): 1288-1294.
[10]	. Gefitinib inhibits glycolysis and induces programmed cell death in non-small cell lung cancer cells [J]. Journal of Southern Medical University, 2020, 40(06): 884-892.
[11]	. Recombinant methioninase regulates PI3K/Akt/Glut-1 pathway and inhibits aerobic glycolysis to promote apoptosis of gastric cancer cells [J]. Journal of Southern Medical University, 2020, 40(01): 27-33.
[12]	. Effects of sera of rats fed with Huganqingzhi tablets on endoplasmic reticulum stress in a HepG2 cell model of nonalcoholic fatty liver disease [J]. Journal of Southern Medical University, 2018, 38(11): 1277-.
[13]	. 2-deoxyglucose inhibits angiogenesis of rheumatoid arthritis via activating AMPK pathway [J]. Journal of Southern Medical University, 2018, 38(08): 962-.
[14]	. Macrophage migration inhibitory factor promotes lung fibrosis via reactive oxygen species-mediated up-regulation of aerobic glycolysis [J]. Journal of Southern Medical University, 2018, 38(07): 873-.
[15]	. Monocarboxylate transporter 1 enhances the sensitivity of breast cancer cells to 3-bromopyruvate in vitro [J]. Journal of Southern Medical University, 2017, 37(05): 588-.