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CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin

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Abstract

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM.

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Data availability

The datasets used in the study are available from the corresponding author upon reasonable request.

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Funding

This study was supported by the National Natural Science Foundation of China (NO. 82271336; NO.81770409; NO.81970694) and Basic Medical and Health Science and Technology Project of Wenzhou Municipal Science and Technology Bureau (Y20210926).

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Author notes

  1. Hao-jie Jin and Zi-heng Wu participated equally to this work and should share “first authorship”.

  2. Wan-tie Wang and Xiang-tao Zheng participated equally to this work and should share “Corresponding authorship”.

Authors and Affiliations

  1. Department of Vascular Surgery, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325015, China

    Hao-jie Jin, Bao-fu Zhang, Jie Deng, Yin-dong Xu & Xiang-tao Zheng

  2. Department of Vascular Surgery, School of Medicine, the First Affiliated Hospital, Zhejiang University, Hangzhou, 310003, China

    Zi-heng Wu

  3. Institute of Ischemia-Reperfusion Injury, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China

    Xin-yu Wang, Zheng-yang Song & Wan-tie Wang

  4. Department of Vascular Surgery, Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, China

    Xin-wu Lu

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Supplementary Figure 1

(a) The interaction between CDKN2B-AS1 and SM22α encoding gene TAGLN. (b-b'') The fluorescent in situ hybridization (FISH) assay was performed to detect the location of CDKN2B-AS1 (scale bar = 10 μm). MOVAS cells were divided into con + shRNA-NC, con + sh-SM22α, insulin + shRNA-NC, and insulin + sh-SM22α groups. N = 3. (c) Relative mRNA levels of SM22α were measured using qRT-PCR. (d-d') Relative protein levels of SM22α were measured using Western blot. GAPDH served as an internal control. (e) Cell viability was measured using an MTT assay. (f) Four PCR primers were designed for the 2 kb upstream sequence of the SM22α promoter and a chromatin immunoprecipitation assay was performed. ***P < 0.001. (g) MSP products for SM22α promoter regions on an agarose gel. GAPDH served as an internal control. One-way ANOVA followed by the LSD post hoc test was used. Data were obtained from three independent replications. (TIF 1993 KB)

Supplementary Figure 2

MOVAS were transfected with CDKN2B-AS1 overexpression vector. N = 3. The expressions of CDKN2B (a) and CDKN2A (b) were detected 48 h later by qRT-PCR. **P < 0.01. Student’s t-test. Data were obtained from three independent replications. (TIF 161 KB)

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Jin, Hj., Wu, Zh., Zhang, Bf. et al. CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin. Cell Tissue Res 394, 455–469 (2023). https://doi.org/10.1007/s00441-023-03836-9

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