敲低Cav1基因可抑制小鼠肝细胞线粒体功能及关键基因mRNA的m6A修饰和表达

doi:10.12122/j.issn.1673-4254.2025.12.08

摘要/Abstract

摘要：

目的探讨Cav1对小鼠肝细胞线粒体功能和mRNA的m⁶A甲基化修饰及表达水平的影响。方法采用慢病毒转染敲低小鼠肝细胞AML12中Cav1基因的表达（shCav1组），转染阴性对照病毒作为对照组。采用TMRE荧光探针检测线粒体膜电位变化，结合Seahorse能量代谢分析系统检测线粒体呼吸功能。采用甲基化RNA免疫共沉淀技术（MeRIP）联合m⁶A表观转录组芯片检测mRNA的m⁶A甲基化修饰和表达水平变化，通过富集分析筛选线粒体功能相关基因，并用qPCR验证目标基因mRNA的m⁶A 修饰和表达水平。结果与对照组相比，shCav1组线粒体膜电位（P<0.05）和线粒体呼吸能力降低（P<0.01）。m⁶A表观转录组芯片检测结果显示，shCav1组有7814个mRNA的m⁶A甲基化修饰水平发生变化（变化倍数>1.5，P<0.05），其中152个上调、7662个下调。联合表达差异分析显示，有2497个mRNA的m⁶A甲基化修饰和表达水平存在协同变化。协同变化的mRNA富集到氧化磷酸化通路，其中Usp15、Suclg2、Ppa2 mRNA的m⁶A甲基化修饰变化百分比最高。芯片和qPCR结果均显示shCav1组中Usp15、Suclg2、Ppa2 mRNA的m⁶A修饰和表达水平均较对照组降低（P<0.05）。结论 Cav1敲低的小鼠肝细胞mRNA的m⁶A甲基化修饰变化显著，该变化与mRNA的表达有关，Cav1对线粒体功能的调节作用可能依赖于其对Usp15、Suclg2、Ppa2 mRNA的m⁶A修饰调控。

关键词: Cav1基因, m⁶A RNA甲基化, mRNA表达, 线粒体膜电位, 线粒体呼吸

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

丁珊珊, 廖颖, 白雪, 黄娇阳, 浅川哲也. 敲低Cav1基因可抑制小鼠肝细胞线粒体功能及关键基因mRNA的m⁶A修饰和表达[J]. 南方医科大学学报, 2025, 45(12): 2607-2615.

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.

图/表 8

表1 MeRIP-qPCR引物序列

Tab.1 Primer sequences for MeRIP-qPCR

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

图1 shCav1慢病毒转染AML12细胞下调Cav1的表达

Fig.1 Knockdown of Cav1 by lentivirus-mediated transfection with shCav1 in AML12 cells. A: Phase-contrast and fluorescence microscopy of the cells under bright-field conditions (GFP, Original magnification: ×100). B: Real-time PCR analysis of Cav1 mRNA expression levels. C: Western blotting of Cav1 protein expression. Control : Untreated control; NC: Control shRNA; shCav1: Cav1 shRNA. **P<0.01 vs NC group.

图2 Cav1敲低损伤AML12细胞线粒体膜电位和线粒体呼吸

Fig.2 Cav1 knockdown impairs mitochondrial membrane potential and respiratory function in AML12 cells. A: TMRE staining for analysis of mitochondrial membrane potential (ΔΨm) (×100). B: Seahorse XF metabolic analysis of basal respiration, maximal respiration and ATP production. CCCP: A mitochondrial uncoupler, used as a positive control to induce ΔΨm collapse (dim red fluorescence); OCR: Oxygen consumption rate. *P<0.05, **P<0.01 vs NC group.

图3 MeRIP质量控制良好, 芯片数据可靠

Fig.3 Quality control of MeRIP and reliability of microarray. A: Agarose gel electrophoresis of the total RNA to confirm RNA integrity. B: MeRIP quality control demonstrating efficient m6A enrichment (%MeRIP confirming assay specificity). C: Representative RNA microarray hybridization image (Cy5 and Cy3). Positive controls: m6A-modified RNAs; Negative controls: Non-methylated RNAs. **P<0.01 vs negative control.

图4 Cav1敲低改变AML12细胞mRNA的m6A修饰和表达

Fig.4 Cav1 knockdown alters mRNA m6A modification and expression profiles in AML12 cells. A: Heatmap of differentially methylated mRNAs. B: Heatmap of differentially expressed mRNAs. C: Nine-quadrant analysis of coordinated m6A methylation and mRNA expression changes. D: Gene Ontology and KEGG pathway enrichment analysis of genes with concurrent m6A and expression changes. |log2 fold change|>0.58, P<0.05.

表2 Cav1敲低导致AML12细胞m6A修饰上调前20位和下调前20位的mRNA

Tab.2 Top 20 upregulated and downregulated m6A-modified mRNAs in AML12 cells with Cav1 knockdown

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%

表3 Cav1敲低导致AML12细胞表达上调前20位和下调前20位的mRNA

Tab.3 Top 20 upregulated and downregulated mRNAs in AML12 cells with Cav1 knockdown

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

图5 Cav1敲低调节AML12细胞线粒体功能相关mRNA的m6A修饰和表达

Fig.5 m6A modification and relative expression levels of mitochondrial function-associated mRNAs in AML12 cells. A: Microarray and qPCR analysis of relative m6A modification of Usp15, Suclg2 and Ppa2 mRNA. B: Microarray and qPCR analysis of relative mRNA expression of Usp15, Suclg2 and Ppa2. *P<0.05, **P<0.01 vs NC group.

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敲低Cav1基因可抑制小鼠肝细胞线粒体功能及关键基因mRNA的m⁶A修饰和表达

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

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