TRIM62通过mTOR途径调控主动脉平滑肌细胞自噬的作用

蔡凤娇; 伍锦鸿; 王丽丽; 马逸超; 鲁燕

doi:10.16766/j.cnki.issn.1674-4152.004404

TRIM62通过mTOR途径调控主动脉平滑肌细胞自噬的作用

doi: 10.16766/j.cnki.issn.1674-4152.004404

1.
山西医科大学第一临床医学院，山西太原 030001
2.
山西医科大学第一医院心血管内科，山西太原 030001

基金项目:

山西省科技厅自然科学研究面上项目 202103021224393

详细信息

通讯作者:
鲁燕，E-mail: luyansxmu@163.com

中图分类号: R329.28 R332 R543
计量
- 文章访问数: 12
- HTML全文浏览量: 5
- PDF下载量: 2
- 被引次数: 0
出版历程
- 收稿日期: 2025-09-15
- 网络出版日期: 2026-06-02

The role of TRIM62 in regulating autophagy in aortic smooth muscle cells through the mTOR pathway

1.
First Hospital of Shanxi Medical University, Taiyuan, Shanxi 030001, China

摘要

摘要: 目的探索TRIM62调控主动脉平滑肌细胞(ASMCs)自噬及其可能机制，评估其对血管钙化的影响。方法分离培养SD大鼠ASMCs，构建过表达TRIM62组(OE-TRIM62)、空载体组(Vector)、敲除TRIM62组(KO-TRIM62)及阴性对照组(NC)稳转细胞株，每组样本量均为12。采用β-甘油磷酸钠诱导血管钙化模型。通过单丹磺酰尸胺(MDC)荧光流式检测、透射电子显微镜观察自噬泡、LC3荧光成像、腺嘌呤核苷三磷酸(ATP)活性测定评估细胞自噬水平，采用蛋白免疫印迹法(WB)检测自噬相关蛋白(P62、ATG3、LC3)及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路蛋白(mTOR、p-mTOR)的表达。结果与Vector组相比，OE-TRIM62组MDC荧光强度中位数显著升高(156.7±12.4 vs. 108.6±9.1，P＜0.05)，透射电镜观察显示自噬体数量增加，LC3阳性细胞率显著升高(24.3%±2.7% vs. 13.1%±1.8%，P＜0.05)，ATP活性显著降低(P＜0.05)，p-mTOR/mTOR比值显著降低(P＜0.05)，P62、ATG3、LC3-Ⅱ蛋白相对表达量均显著增高(P＜0.05)。与NC组相比，KO-TRIM62组MDC荧光强度中位数显著降低(78.4±6.2 vs. 105.3±8.7，P＜0.05)，透射电镜显示自噬体数量减少，LC3阳性细胞率显著降低(6.8%±0.9% vs. 12.5%±1.6%，P＜0.05)，p-mTOR/mTOR比值显著升高(P＜0.05)，P62、ATG3、LC3-Ⅱ蛋白相对表达量均显著降低(P＜0.05)。结论 TRIM62可能通过抑制p-mTOR依赖的自噬通路，解除mTOR通路对自噬的抑制作用，从而促进ASMCs的自噬过程，进而可能参与血管钙化进程的调控。
- 血管钙化 /
- TRIM62 /
- 细胞自噬
Abstract: Objective To explore the role of TRIM62 in regulating autophagy in aortic smooth muscle cells (ASMCs) and its potential mechanisms, and to evaluate its impact on vascular calcification. Methods Aortic smooth muscle cells were isolated and cultured from Sprague-Dawley rats. Stable cell lines were constructed for overexpression of TRIM62 (OE-TRIM62), empty vector control (Vector), knockout of TRIM62 (KO-TRIM62), and negative control (NC) groups with 12 samples in each group. Vascular calcification was induced using sodium β-glycerophosphate. Autophagy levels were assessed by monodansylcadaverine (MDC) fluorescent flow cytometry, observation of autophagosomes by transmission electron microscopy, LC3 fluorescent imaging, and adenosine triphosphate (ATP) activity assay. The expression of autophagy-related proteins (P62, ATG3, LC3) and mammalian target of rapamycin(mTOR) signaling pathway proteins (mTOR, p-mTOR) was detected by Western blot (WB). Results Compared with the Vector group, the median fluorescence intensity of MDC in OE-TRIM62 group was significantly increased (156.7±12.4 vs. 108.6±9.1, P < 0.05), and transmission electron microscopy showed that the number of autophagosomes increased. The ratio of p-mTOR/mTOR significantly increased (1.12±0.09 vs. 0.86±0.07, P < 0.05), and the relative expression levels of P62, ATG3, and LC3-Ⅱ proteins significantly decreased (P < 0.05). The rate of LC3 positive cells was significantly increased (24.3%±2.7% vs. 13.1%±1.8%, P < 0.05), and ATP activity was significantly decreased (P < 0.05). The p-mTOR/mTOR ratio was significantly decreased (P < 0.05), and the relative expressions of P62, ATG3 and LC3-Ⅱ proteins were significantly increased (P < 0.05). Compared with NC group, the median fluorescence intensity of MDC in KO-TRIM62 group was significantly decreased (78.4±6.2 vs. 105.3±8.7, P < 0.05). Transmission electron microscopy showed that the number of autophagosomes and the rate of LC3 positive cells were significantly decreased (6.8%±0.9% vs. 12.5%±1.6%, P < 0.05), the p-mTOR/mTOR ratio was significantly increased (P < 0.05), and the relative expressions of P62, ATG3 and LC3-Ⅱ proteins were significantly decreased (P < 0.05). Conclusion TRIM62 may promote the autophagy process in ASMCs by inhibiting the p-mTOR-dependent autophagy pathway, thereby relieving the suppression of autophagy by the mTOR pathway. This, in turn, may be involved in regulating the process of vascular calcification.
- Vascular calcification /
- TRIM62 /
- Cellular autophagy

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图 1 透射电子显微镜观察自噬体(白色箭头)的情况

Figure 1. Transmission electron micrographs of aortic smooth muscle cells

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图 2 各组ASMcs自噬相关蛋白及mTOR通路蛋白表达

Figure 2. Expression of autophagy-related proteins and mTOR pathway proteins in ASMCs from different groups

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表 1 β-甘油磷酸钠诱导的ASMCs钙化模型验证指标(x±s)

Table 1. Validation indicators of β-glycerophosphate-induced ASMCs calcification model (x±s)

组别	n	碱性磷酸酶活性 (U/g蛋白)	钙含量 (μg/g蛋白)
对照组	6	15.8±2.1	0.28±0.04
钙化模型组	6	68.4±7.9	0.45±0.05
t值		15.732	6.513
P值		< 0.001	< 0.001

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表 2 不同条件下ASMCs自噬活性相关指标比较(x±s)

Table 2. Comparison of autophagy activity-related indicators in ASMCs under different groups (x±s)

组别	n	MDC荧光强度中位数	ATP水平(μmol/mg)
NC	12	105.3±8.7	0.89±0.07
KO-TRIM62	12	78.4±6.2^a	0.92±0.08
Vector	12	108.6±9.1	0.87±0.06
OE-TRIM62	12	156.7±12.4^b	0.62±0.05^b
F值		139.686	52.574
P值		＜0.001	< 0.001
注：与NC组比较，^aP < 0.05;与Vector组比较，^bP < 0.05。

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表 3 不同条件下ASMCs的LC3阳性细胞率比较(x±s，%)

Table 3. Comparison of LC3-positive cell rate in ASMCs under different groups (x±s, %)

组别	n	LC3阳性细胞率
NC	12	12.5±1.6
KO-TRIM62	12	6.8±0.9^a
Vector	12	13.1±1.8
OE-TRIM62	12	24.3±2.7^b
F值		35.442
P值		< 0.001
注：与NC组比较，^aP < 0.05;与Vector组比较，^bP < 0.05。

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表 4 不同条件下ASMCs自噬相关蛋白表达及p-mTOR/mTOR比值比较(x±s)

Table 4. Comparison of autophagy-related protein expression and pmTOR/mTOR ratio in ASMCs under different condition (x±s)

组别	n	P62/GAPDH	ATG3/GAPDH	LC3-Ⅱ/GAPDH	p-mTOR/ mTOR比值
NC	12	0.38±0.04	0.45±0.05	0.52±0.06	0.86±0.07
KO-TRIM62	12	0.22±0.03^a	0.28±0.04^a	0.31±0.04^a	1.12±0.09^a
Vector	12	0.41±0.05	0.47±0.06	0.55±0.07	0.83±0.06
OE-TRIM62	12	0.74±0.08^b	0.82±0.09^b	0.91±0.10^b	0.42±0.05^b
F值		26.883	29.447	31.225	24.116
P值		< 0.001	< 0.001	< 0.001	< 0.001
注：与NC组比较，^aP < 0.05;与Vector组比较，^bP < 0.05。

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参考文献(21)

[1]	VILLA-BELLOSTA R. Vascular calcification: key roles of phosphate and pyrophosphate[J]. Int J of Mol Sci, 2021, 22(24): 13536. DOI: 10.3390/ijms222413536.
[2]	LI M, WANG Z W, FANG L J, et al. Programmed cell death in atherosclerosis and vascular calcification[J]. Cell Death Dis, 2022, 13(5): 467. DOI: 10.1038/s41419-022-04923-5.
[3]	YIN Z, ZHANG J S, SHEN Z C, et al. Regulated vascular smooth muscle cell death in vascular diseases[J]. Cell Prolif, 2024, 57(11): e13688. DOI: 10.1111/cpr.13688.
[4]	LIU N, YANG Y, LI X, et al. PARylation of POLG mediated by PARP1 accelerates ferroptosis-induced vascular calcification via activating Adora2a/Rap1 signaling[J]. Arterioscler Thromb Vasc Biol, 2025, 45(6): 1207-1221.
[5]	ZHANG L, LI D Z, AIERKEN Y, et al. KPV and RAPA self-assembled into carrier-free nanodrugs for vascular calcification therapy[J]. Adv Healthc Mater, 2024, 13(32): e2402320. DOI: 10.1002/adhm.202402320.
[6]	王琳茹, 张晶, 赵冬婵, 等. 沉默FOXO1基因对人主动脉血管平滑肌细胞自噬和凋亡的影响[J]. 吉林大学学报(医学版), 2024, 50(2): 431-441. WANG L R, ZHANG J, ZHAO D C, et al. Effect of silencing FOXO1 gene on autophagy and apoptosis in human aortic vascular smooth muscle cells[J]. Journal of Jilin University (Medicine Edition), 2024, 50(2): 431-441.
[7]	LI P, DONG X R, ZHANG B, et al. Molecular mechanism and therapeutic targeting of necrosis, apoptosis, pyroptosis, and autophagy in cardiovascular disease[J]. Chin Med J (Engl), 2021, 134(22): 2647-2655. doi: 10.1097/CM9.0000000000001772
[8]	KLIONSKY D J, PETRONI G, AMARAVADI R K, et al. Autophagy in major human diseases[J]. EMBO J, 2021, 40(19): e108863. DOI: 10.15252/embj.2021108863.
[9]	LI X, WANG X, LIU Y, et al. Downregulation of miR-3568 protects against ischemia/reperfusion-induced cardiac dysfunction in rats and apoptosis in H9C2 cardiomyocytes through targeting TRIM62[J]. Front Pharmacol, 2020, 11: 17. DOI: 10.3389/fphar.2020.00017.
[10]	ONNIS C, VIRMANI R, KAWAI K, et al. Coronary artery calcification: current concepts and clinical implications[J]. Circulation, 2024, 149(3): 251-266. doi: 10.1161/CIRCULATIONAHA.123.065657
[11]	MOSQUREA J V, AUGUSTE G, WONG D, et al. Integrative single-cell meta-analysis reveals disease-relevant vascular cell states and markers in human atherosclerosis[J]. Cell Rep, 2023, 42(11): 113380. DOI: 10.1016/j.celrep.2023.113380.
[12]	王艺凯, 郭利伟. 成纤维细胞因子23-Klotho轴在血管钙化中的作用机制研究进展[J]. 新乡医学院学报, 2023, 40(11): 1083-1086. WANG Y K, GUO L W. Research progress on the mechanism of fibroblast growth factor 23-Klotho axis in vascular calcification[J]. Journal of Xinxiang Medical University, 2023, 40(11): 1083-1086.
[13]	VARGAS J N S, HAMASAKI M, KAWABATA T, et al. The mechanisms and roles of selective autophagy in mammals[J]. Nat Rev Mol Cell Biol, 2023, 24(3): 167-185.
[14]	AHSAN N, SHARIQ M, SUROLIA A, et al. Multipronged regulation of autophagy and apoptosis: emerging role of TRIM proteins[J]. Cell Mol Biol Let, 2024, 29(1): 13. DOI: 10.1186/s11658-023-00528-8.
[15]	TANG J, CHEN Q, XIANG L, et al. TRIM28 Fosters microglia ferroptosis via autophagy modulation to enhance neuropathic pain and neuroinflammation[J]. Mol Neurobiol, 2024, 61(11): 9459-9477. doi: 10.1007/s12035-024-04133-4
[16]	CHEN J, CAO W, HUANG X, et al. TRIM21 enhances bortezomib sensitivity in multiple myeloma by halting prosurvival autophagy[J]. Blood Adv, 2023, 7(19): 5752-5770. doi: 10.1182/bloodadvances.2022008241
[17]	张永琪, 邹震海, 吴梦琦, 等. 敲低EPHA2通过mTOR磷酸化调控膀胱癌细胞自噬和生物学行为[J]. 中华全科医学, 2023, 21(7): 1117-1120, 1129. doi: 10.16766/j.cnki.issn.1674-4152.003063 ZHANG Y Q, ZOU Z H, WU M Q, et al. Knockdown of EPHA2 regulates autophagy and biological behavior of bladder cancer cells through mTOR phosphorylation[J]. Chinese Journal of General Practice, 2023, 21(7): 1117-1120, 1129. doi: 10.16766/j.cnki.issn.1674-4152.003063
[18]	XU Y F, WANG Q, WANG J, et al. The cGAS-STING pathway activates transcription factor TFEB to stimulate lysosome biogenesis and pathogen clearance[J]. Immunity, 2025, 58(2): 309-325.
[19]	ZHOU X, XU S N, YUAN S T, et al. Multiple functions of autophagy in vascular calcification[J]. Cell Biosci, 2021, 11(1): 159. DOI: 10.1186/s13578-021-00639-9.
[20]	LI F X Z, LIU J J, XU F, et al. Cold exposure protects against medial arterial calcification development via autophagy[J]. J Nanobiotechnol, 2023, 21(1): 226. DOI: 10.1186/s12951-023-01985-1.
[21]	ZHOU Z K, LI Y, JIANG W, et al. Molecular mechanism of calycosin inhibited vascular calcification[J]. Nutrients, 2023, 16(1): 99. DOI: 10.3390/nu16010099.