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MiR32-5p promoted vascular smooth muscle cell calcification by upregulating TNF? in the microenvironment.

ABSTRACT: BACKGROUND:Vascular calcification is often associated with chronic inflammation and is a risk factor for brain arterial stiffness. Our previous results showed that miR32-5p was positively correlated with vascular smooth muscle cells (VSMC) calcification, but it is unclear whether miR32-5p promoted VSMC calcification by regulating inflammatory factor production. RESULTS:In this study, bioinformatics analysis was used to select tumour necrosis factor ? (TNF?) as a candidate inflammatory factor associated with calcification. Moreover, alizarin red staining and qRT-PCR analysis revealed that TNF? produced by BV2 cells was the key promoting factor of VSMC calcification. Interestingly, the expression of TNF? was significantly increased at the mRNA and protein levels after miR32-5p mimic treatment but significantly decreased after miR32-5p antagomir treatment. To explore the mechanism of the regulation of TNF? expression by miR32-5p, bioinformatics analysis indicated that PIKfyve was a candidate target gene of miR32-5p, and luciferase assays verified that the expression of PIKfyve was significantly repressed by miR32-5p mimics. Importantly, rescue experiments showed that the expression of TNF? in BV2 cells treated with miR32-5p antagomir and the PIKfyve inhibitor YM201636 was significantly increased. CONCLUSIONS:The production of TNF? in microglia could be affected by miR32-5p targeting PIKfyve, and these results will be beneficial to reveal the mechanism of brain arterial calcification.

SUBMITTER: Cao J

PROVIDER: S-EPMC6967090 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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miR32-5p promoted vascular smooth muscle cell calcification by upregulating TNFα in the microenvironment.

Cao Jingsong J Chen Ling L Zhong Xiaoling X Shen Yingying Y Gao Yan Y Chen Qian Q Zu Xuyu X Liu Jianghua J

BMC immunology 20200117 1

<h4>Background</h4>Vascular calcification is often associated with chronic inflammation and is a risk factor for brain arterial stiffness. Our previous results showed that miR32-5p was positively correlated with vascular smooth muscle cells (VSMC) calcification, but it is unclear whether miR32-5p promoted VSMC calcification by regulating inflammatory factor production.<h4>Results</h4>In this study, bioinformatics analysis was used to select tumour necrosis factor α (TNFα) as a candidate inflamma ...[more]

PMID: 31952480

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Project description:BackgroundVascular calcification (VC) is primarily studied using cultures of vascular smooth muscle cells. However, the use of very different protocols and extreme conditions can provide findings unrelated to VC. In this work we aimed to determine the critical experimental parameters that affect calcification in vitro and to determine the relevance to calcification in vivo.Experimental procedures and resultsRat VSMC calcification in vitro was studied using different concentrations of fetal calf serum, calcium, and phosphate, in different types of culture media, and using various volumes and rates of change. The bicarbonate content of the media critically affected pH and resulted in supersaturation, depending on the concentration of Ca2+ and Pi. Such supersaturation is a consequence of the high dependence of bicarbonate buffers on CO2 vapor pressure and bicarbonate concentration at pHs above 7.40. Such buffer systems cause considerable pH variations as a result of minor experimental changes. The variations are more critical for DMEM and are negligible when the bicarbonate concentration is reduced to ¼. Particle nucleation and growth were observed by dynamic light scattering and electron microscopy. Using 2mM Pi, particles of ~200nm were observed at 24 hours in MEM and at 1 hour in DMEM. These nuclei grew over time, were deposited in the cells, and caused osteogene expression or cell death, depending on the precipitation rate. TEM observations showed that the initial precipitate was amorphous calcium phosphate (ACP), which converts into hydroxyapatite over time. In blood, the scenario is different, because supersaturation is avoided by a tightly controlled pH of 7.4, which prevents the formation of PO43--containing ACP.ConclusionsThe precipitation of ACP in vitro is unrelated to VC in vivo. The model needs to be refined through controlled pH and the use of additional procalcifying agents other than Pi in order to reproduce calcium phosphate deposition in vivo.

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MiR32-5p promoted vascular smooth muscle cell calcification by upregulating TNF? in the microenvironment.

Publications

miR32-5p promoted vascular smooth muscle cell calcification by upregulating TNFα in the microenvironment.

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