Knockdown of Cav1 inhibits mitochondrial function and mRNA m6A modification and expression of key genes in mouse hepatocytes

doi:10.12122/j.issn.1673-4254.2025.12.08

Abstract

Abstract:

Objective To investigate the role of Cav1 gene in regulating mitochondrial function and mRNA m⁶A modification and expressions of the key genes in mouse hepatocytes. Methods In mouse hepatocyte AML12 cells, the effects of lentivirus-mediated Cav1 knockdown and transfection with a negative control virus on mitochondrial membrane potential and mitochondrial respiratory function were analyzed using the TMRE fluorescent probe and the Seahorse extracellular flux analysis system. Methylated RNA immunoprecipitation (MeRIP) combined with m⁶A microarray was utilized to evaluate the changes in mRNA m⁶A modification and expression levels, followed by enrichment analysis to identify the functionally relevant genes. The m⁶A modification and expression levels of the mRNAs were validated by qPCR. Results Compared with the negative control group, the cells with Cav1 knockdown exhibited significantly reduced mitochondrial membrane potential and respiratory capacity. m⁶A microarray analysis revealed significant changes in m⁶A modification levels (fold change>1.5) of 7814 mRNAs, including 152 upregulated and 7662 downregulated mRNAs. Integrated expression analysis identified 2497 mRNAs showing coordinated changes in m⁶A modification and expression levels. These mRNAs were enriched in the oxidative phosphorylation pathway, with Usp15, Suclg2, and Ppa2 exhibiting the highest percent changes in m⁶A modification. Both microarray and qPCR results showed that the m⁶A modification and expression levels of Usp15, Suclg2 and Ppa2 mRNAs were significantly reduced in cells with Cav1 knockdown compared to the NC group. Conclusion Cav1 knockdown induces significant alterations in mRNA m⁶A modification as well as their expression levels. The regulatory effects of Cav1 on mitochondrial function may be mediated by modulation of m⁶A modification of Usp15, Suclg2, and Ppa2 mRNAs.

Key words: Cav1 gene, m⁶A RNA methylation, mRNA expression, mitochondrial membrane potential, mitochondrial respiratory

Shanshan DING, Ying LIAO, Xue BAI, Jiaoyang HUANG, Tetsuya ASAKAWA. Knockdown of Cav1 inhibits mitochondrial function and mRNA m⁶A modification and expression of key genes in mouse hepatocytes[J]. Journal of Southern Medical University, 2025, 45(12): 2607-2615.

Figures/Tables 8

References 33

[1]	范建高, 徐小元, 南月敏, 等. 代谢相关(非酒精性)脂肪性肝病防治指南(2024年版)[J]. 实用肝脏病杂志, 2024, 27(4): 494-510.
[2]	Eslam M, Sarin SK, Wong VW, et al. The Asian Pacific Association for the Study of the Liver clinical practice guidelines for the diagnosis and management of metabolic associated fatty liver disease[J]. Hepatol Int, 2020, 14(6): 889-919. doi：10.1007/s12072-020-10094-2
[3]	Radosavljevic T, Brankovic M, Samardzic J, et al. Altered mitochondrial function in MASLD: key features and promising therapeutic approaches[J]. Antioxidants (Basel), 2024, 13(8): 906. doi：10.3390/antiox13080906
[4]	Shin S, Kim J, Lee JY, et al. Mitochondrial quality control: its role in metabolic dysfunction-associated steatotic liver disease (MASLD)[J]. J Obes Metab Syndr, 2023, 32(4): 289-302. doi：10.7570/jomes23054
[5]	Bhowmick S, Biswas T, Ahmed M, et al. Caveolin-1 and lipids: association and their dualism in oncogenic regulation[J]. Biochim Biophys Acta Rev Cancer, 2023, 1878(6): 189002. doi：10.1016/j.bbcan.2023.189002
[6]	Jiang Y, Krantz S, Qin X, et al. Caveolin-1 controls mitochondrial damage and ROS production by regulating fission - fusion dynamics and mitophagy[J]. Redox Biol, 2022, 52: 102304. doi：10.1016/j.redox.2022.102304
[7]	Fu DD, Wu S, Jiang XF, et al. Caveolin-1 alleviates acetaminophen-induced vascular oxidative stress and inflammation in non-alcoholic fatty liver disease[J]. Free Radic Biol Med, 2023, 195: 245-57. doi：10.1016/j.freeradbiomed.2022.12.095
[8]	Tang WX, Li YS, Li Y, et al. Caveolin-1, a novel player in cognitive decline[J]. Neurosci Biobehav Rev, 2021, 129: 95-106. doi：10.1016/j.neubiorev.2021.06.044
[9]	Timmins LR, Ortiz-Silva M, Joshi B, et al. Caveolin-1 promotes mitochondrial health and limits mitochondrial ROS through ROCK/AMPK regulation of basal mitophagic flux[J]. FASEB J, 2024, 38: e23343. doi：10.1096/fj.202201872rr
[10]	You TY, Li Y, Li BW, et al. Caveolin-1 protects against liver damage exacerbated by acetaminophen in non-alcoholic fatty liver disease by inhibiting the ERK/HIF-1α pathway[J]. Mol Immunol, 2023, 163: 104-15. doi：10.1016/j.molimm.2023.09.003
[11]	Deng GH, Wu CF, Li YJ, et al. Caveolin-1 is critical for hepatic iron storage capacity in the development of nonalcoholic fatty liver disease[J]. Mil Med Res, 2023, 10(1): 53. doi：10.1186/s40779-023-00487-3
[12]	Ćorović M, Hoch-Kraft P, Zhou Y, et al. m⁶A in the coding sequence: linking deposition, translation, and decay[J]. Trends Genet, 2025: S0168-9525(25)00132-5. doi：10.1016/j.tig.2025.06.002
[13]	Xie XZ, Fang Z, Zhang HY, et al. The role of N(6)-methyladenosine (m6a) modification in cancer: recent advances and future directions[J]. EXCLI J, 2025, 24: 113-50.
[14]	Ming XY, Chen SR, Li HJ, et al. m⁶A RNA methylation and implications for hepatic lipid metabolism[J]. DNA Cell Biol, 2024, 43(6): 271-8. doi：10.1089/dna.2023.0410
[15]	Yan W, Saqirile, Li K, et al. The role of N6-methyladenosine in mitochondrial dysfunction and pathology[J]. Int J Mol Sci, 2025, 26(8): 3624. doi：10.3390/ijms26083624
[16]	Fan JH, You JB, Liu ZY, et al. Novel landscapes of N6-methyladenosine modification of mitochondrial oxidative stress in organ fibrosis[J]. Eur J Pharmacol, 2025, 1003: 177888. doi：10.1016/j.ejphar.2025.177888
[17]	Fromenty B, Roden M. Mitochondrial alterations in fatty liver diseases[J]. J Hepatol, 2023, 78(2): 415-29. doi：10.1016/j.jhep.2022.09.020
[18]	丁珊珊, 庄妍, 廖颖, 等. 二陈汤通过抑制mTORC1/SREBP1/CAV1通路改善高脂饮食小鼠肝脏线粒体功能的作用研究[J]. 中国中药杂志, 2024, 49(3): 763-9.
[19]	Xu HL, Li Y, Guo N, et al. Caveolin-1 mitigates the advancement of metabolic dysfunction-associated steatotic liver disease by reducing endoplasmic reticulum stress and pyroptosis through the restoration of cholesterol homeostasis[J]. Int J Biol Sci, 2025, 21(2): 490-506. doi：10.7150/ijbs.100794
[20]	Razani B, Combs TP, Wang XB, et al. Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyc-eridemia with adipocyte abnormalities[J]. J Biol Chem, 2002, 277(10): 8635-47. doi：10.1074/jbc.m110970200
[21]	Xu YQ, Chen BW, Yi J, et al. Buyang Huanwu Decoction alleviates cerebral ischemic injury through modulating caveolin-1-mediated mitochondrial quality control[J]. Front Pharmacol, 2023, 14: 1137609. doi：10.3389/fphar.2023.1137609
[22]	Wu S, Guo N, Xu HL, et al. Caveolin-1 ameliorates hepatic injury in non-alcoholic fatty liver disease by inhibiting ferroptosis via the NOX4/ROS/GPX4 pathway[J]. Biochem Pharmacol, 2024, 230(Pt 2): 116594. doi：10.1016/j.bcp.2024.116594
[23]	Kahl M, Xu ZF, Arumugam S, et al. m6A RNA methylation regulates mitochondrial function[J]. Hum Mol Genet, 2024, 33(11): 969-80. doi：10.1093/hmg/ddae029
[24]	Wang SW, Zhang WY, Wang ZJ, et al. Mettl3-m6A-YTHDF1 axis promotion of mitochondrial dysfunction in metabolic dysfunction-associated steatotic liver disease[J]. Cell Signal, 2024, 121: 111303. doi：10.1016/j.cellsig.2024.111303
[25]	Huangfu LT, Zhu HB, Wang GJ, et al. The deubiquitinase USP15 drives malignant progression of gastric cancer through glucose metabolism remodeling[J]. J Exp Clin Cancer Res, 2024, 43(1): 235. doi：10.1186/s13046-024-03152-2
[26]	Fang RH, Jia ZG, Xin YH, et al. N6-methyladenosine-modification of USP15 regulates chemotherapy resistance by inhibiting LGALS3 ubiquitin-mediated degradation via AKT/mTOR signaling activation pathway in hepatocellular carcinoma[J]. Cell Death Discov, 2025, 11(1): 3. doi：10.1038/s41420-024-02282-y
[27]	Wu BW, Qiu JT, Zhao TV, et al. Succinyl-CoA ligase deficiency in pro-inflammatory and tissue-invasive T cells[J]. Cell Metab, 2020, 32(6): 967-80.e5. doi：10.1016/j.cmet.2020.10.025
[28]	Zhang XM, Liu J, Cheng YJ, et al. Metabolic enzyme Suclg2 maintains tolerogenicity of regulatory dendritic cells diffDCs by suppressing Lactb succinylation[J]. J Autoimmun, 2023, 138: 103048. doi：10.1016/j.jaut.2023.103048
[29]	Hu QF, Xu J, Wang L, et al. SUCLG2 regulates mitochondrial dysfunction through succinylation in lung adenocarcinoma[J]. Adv Sci (Weinh), 2023, 10(35): e2303535. doi：10.1002/advs.202303535
[30]	Yu HF, Zeng QR, Xiao PY, et al. Hippo-YAP signaling alleviates copper-induced mitochondrial dysfunction and oxidative damage via the ATOX1-PPA2 pathway[J]. Int J Biol Macromol, 2025, 290: 138908. doi：10.1016/j.ijbiomac.2024.138908
[31]	Zhang X, Di YQ, Wang YP, et al. SIRT5-mediated desuccinylation of PPA2 enhances HIF-1alpha-dependent adaptation to hypoxic stress and colorectal cancer metastasis[J]. EMBO J, 2025, 44(9): 2514-40. doi：10.1038/s44318-025-00416-1
[32]	Zhou Y, Wang Q, Deng HF, et al. N6-methyladenosine demethylase FTO promotes growth and metastasis of gastric cancer via m⁶A modification of caveolin-1 and metabolic regulation of mitochondrial dynamics[J]. Cell Death Dis, 2022, 13(1): 72. doi：10.1038/s41419-022-04503-7
[33]	Golubeva VA, Das AS, Rabolli CP, et al. YTHDF1 is pivotal for maintenance of cardiac homeostasis[J]. J Mol Cell Cardiol, 2024, 193: 25-35. doi：10.1016/j.yjmcc.2024.05.008

Gene	Forward primers sequences	Reverse primers sequences
Usp15	CCGTGGATGAAAACCTGAGTAG	TTCTCTTAGGCAGACAGGGATAA
Suclg2	CCAGCGAACTTCTTGGACCT	CACAGTTGACGATCCCACCA
Ppa2	CGGTGGACAAATGCCAAGATG	TTCGGTGTGTAGCGTAGCTT
Gapdh	CACTGAGCAAGAGAGGCCCTAT	GCAGCGAACTTTATTGATGGTATT

Gene	Forward primers sequences	Reverse primers sequences
Usp15	CCGTGGATGAAAACCTGAGTAG	TTCTCTTAGGCAGACAGGGATAA
Suclg2	CCAGCGAACTTCTTGGACCT	CACAGTTGACGATCCCACCA
Ppa2	CGGTGGACAAATGCCAAGATG	TTCGGTGTGTAGCGTAGCTT
Gapdh	CACTGAGCAAGAGAGGCCCTAT	GCAGCGAACTTTATTGATGGTATT

Gene	FC	P	Reg	%m⁶A	Gene	FC	P	Reg	%m⁶A
Ifrd1	0.07	0.002	Down	-55%	Olfr175-ps1	19.55	0.001	Up	54%
Zfp934	0.08	0.000	Down	-54%	Crhr2	13.70	0.000	Up	52%
Kif18a	0.06	0.001	Down	-54%	Glis1	13.21	0.008	Up	49%
Kif20b	0.07	0.000	Down	-51%	Olfr175-ps1	21.62	0.000	Up	47%
Tcaf1	0.12	0.001	Down	-51%	Fam19a2	2.14	0.045	Up	40%
Zufsp	0.08	0.004	Down	-50%	Gss	11.18	0.001	Up	37%
Zfp932	0.09	0.000	Down	-50%	Slc13a4	7.35	0.000	Up	36%
Klhl32	0.13	0.000	Down	-49%	Elavl2	4.16	0.003	Up	33%
Slc35d1	0.15	0.003	Down	-49%	Crlf1	4.78	0.032	Up	33%
Mis18bp1	0.12	0.028	Down	-47%	Mdc1	2.62	0.042	Up	32%
Samd9l	0.14	0.002	Down	-46%	Grm2	7.02	0.004	Up	32%
Smek1	0.13	0.001	Down	-45%	Tulp2	4.39	0.001	Up	31%
Fastkd2	0.19	0.000	Down	-45%	Dynap	2.69	0.020	Up	28%
Zfp280d	0.13	0.001	Down	-45%	Adamtsl1	3.05	0.008	Up	26%
Sema6d	0.10	0.002	Down	-44%	Txnip	2.68	0.005	Up	26%
Pole4	0.15	0.006	Down	-44%	Dpp4	13.55	0.000	Up	26%
Kif20b	0.10	0.000	Down	-43%	Il5ra	7.25	0.000	Up	25%
Pcdhb18	0.14	0.000	Down	-42%	Klrc1	3.28	0.001	Up	24%
Wac	0.14	0.005	Down	-42%	Txnip	3.17	0.004	Up	24%
Cdadc1	0.15	0.015	Down	-42%	Kcne3	2.70	0.048	Up	23%

Gene	FC	P	Reg	%m⁶A	Gene	FC	P	Reg	%m⁶A
Ifrd1	0.07	0.002	Down	-55%	Olfr175-ps1	19.55	0.001	Up	54%
Zfp934	0.08	0.000	Down	-54%	Crhr2	13.70	0.000	Up	52%
Kif18a	0.06	0.001	Down	-54%	Glis1	13.21	0.008	Up	49%
Kif20b	0.07	0.000	Down	-51%	Olfr175-ps1	21.62	0.000	Up	47%
Tcaf1	0.12	0.001	Down	-51%	Fam19a2	2.14	0.045	Up	40%
Zufsp	0.08	0.004	Down	-50%	Gss	11.18	0.001	Up	37%
Zfp932	0.09	0.000	Down	-50%	Slc13a4	7.35	0.000	Up	36%
Klhl32	0.13	0.000	Down	-49%	Elavl2	4.16	0.003	Up	33%
Slc35d1	0.15	0.003	Down	-49%	Crlf1	4.78	0.032	Up	33%
Mis18bp1	0.12	0.028	Down	-47%	Mdc1	2.62	0.042	Up	32%
Samd9l	0.14	0.002	Down	-46%	Grm2	7.02	0.004	Up	32%
Smek1	0.13	0.001	Down	-45%	Tulp2	4.39	0.001	Up	31%
Fastkd2	0.19	0.000	Down	-45%	Dynap	2.69	0.020	Up	28%
Zfp280d	0.13	0.001	Down	-45%	Adamtsl1	3.05	0.008	Up	26%
Sema6d	0.10	0.002	Down	-44%	Txnip	2.68	0.005	Up	26%
Pole4	0.15	0.006	Down	-44%	Dpp4	13.55	0.000	Up	26%
Kif20b	0.10	0.000	Down	-43%	Il5ra	7.25	0.000	Up	25%
Pcdhb18	0.14	0.000	Down	-42%	Klrc1	3.28	0.001	Up	24%
Wac	0.14	0.005	Down	-42%	Txnip	3.17	0.004	Up	24%
Cdadc1	0.15	0.015	Down	-42%	Kcne3	2.70	0.048	Up	23%

Gene	FC	P	Reg	Gene	FC	P	Reg
Cav1	0.05	0.000	Down	Olfr175-ps1	12.25	0.000	Up
Dek	0.14	0.000	Down	Olfr175-ps1	8.42	0.000	Up
Dek	0.15	0.000	Down	Dpp4	8.14	0.000	Up
Upf3b	0.16	0.000	Down	Glis1	6.03	0.000	Up
Tpr	0.18	0.000	Down	Gss	5.82	0.000	Up
Arid4b	0.19	0.000	Down	Crhr2	5.64	0.000	Up
Dtnbp1	0.19	0.000	Down	Slc13a4	4.73	0.000	Up
Hells	0.20	0.010	Down	Il5ra	4.49	0.000	Up
Zfml	0.20	0.000	Down	Slc45a4	4.44	0.000	Up
Kif18a	0.20	0.000	Down	Pdzd2	4.14	0.000	Up
Ccl2	0.21	0.000	Down	Grm2	3.99	0.000	Up
Dek	0.21	0.000	Down	Cdh26	3.73	0.000	Up
Kif20b	0.21	0.000	Down	Ube2i	3.13	0.040	Up
Thoc2	0.21	0.000	Down	Tulp2	2.96	0.000	Up
Ccar1	0.22	0.000	Down	Olfr70	2.81	0.000	Up
Zfml	0.22	0.000	Down	Chpf2	2.73	0.000	Up
mt-Nd1	0.22	0.000	Down	Slc6a20b	2.66	0.040	Up
Zfp950	0.22	0.000	Down	Msantd3	2.61	0.020	Up
Zfp960	0.22	0.000	Down	Atp2c1	2.59	0.010	Up
Ccar1	0.23	0.010	Down	Fam32a	2.58	0.050	Up

Knockdown of Cav1 inhibits mitochondrial function and mRNA m⁶A modification and expression of key genes in mouse hepatocytes

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