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. 2022 Jun 15;17(1):1092-1099.
doi: 10.1515/med-2022-0484. eCollection 2022.

ALKBH5 inhibits TNF-α-induced apoptosis of HUVECs through Bcl-2 pathway

Xiaoshan Zhang  1 , ShiBing Deng  1 , Yang Peng  1 , Han Wei  1 , Zhiming Tian  2
Affiliations

ALKBH5 inhibits TNF-α-induced apoptosis of HUVECs through Bcl-2 pathway

Xiaoshan Zhang et al. Open Med (Wars). .

Abstract

The dysfunction and apoptosis of vascular endothelial cells are the initiating links in the formation of atherosclerosis. N6-methyladenosine (m6A) is an extremely extensive RNA methylation modification and its abnormality leads to the occurrence of various human diseases. In this study, we explored the effects of demethylase α-ketoglutarate-dependent dioxygenase ALKB homolog 5 (ALKBH5) on TNF-α-induced apoptosis of human umbilical vein endothelial cells (HUVECs). In TNF-α-treated HUVECs, the expression of ALKBH5 was significantly decreased. ALKBH5 overexpression promoted the proliferation and inhibited the apoptosis in TNF-α-treated HUVECs, suggesting that ALKBH5 had a protective effect on cell damage induced by TNF-α. Importantly, ALKBH5 promoted the expression of Bcl-2 in HUVECs. Bcl2 overexpression reduced the expression of Gadd45, Bax, and p21, which are transcriptionally activated by p53. But the expression of p53 has not been significantly affected, indicating that Bcl2 might regulate the apoptosis by inhibiting p53 downstream targets. In addition, ALKBH5 overexpression significantly increased the level of pri-miR-7 and decreased the level of miR-7. In conclusion, ALKBH5 attenuated the TNF-α-induced cell injury via promoting Bcl2 expression. Our research expands the understanding of the progression mechanism of atherosclerosis and provides a potential strategy for the protection of vascular endothelial injury.

Keywords: ALKBH5; Bcl-2; N6-methyladenosine; apoptosis; atherosclerosis.

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Conflict of interest statement

Conflict of interest: The authors declare that they have no competing interests exist.

Figures

Figure 1
Figure 1
The model of vascular endothelial cell injury was constructed using TNF-α. (a) HUVECs were treated with 10 ng/mL TNF-α for 24 h, and cell viability was detected using CCK8 assay. PBS buffer was used as the control. The expression of ALKBH5 in TNF-α and control groups after the treatment for 24 h was analyzed using western blot. (b) Cell viability was detected using CCK8 assay. **P < 0.01; ***P < 0.001.
Figure 2
Figure 2
ALKBH5 attenuated the cell injury induced by TNF-α in HUVECs. (a) The overexpression plasmid of ALKBH5 was transfected into TNF-α-treated HUVECs with the empty plasmid as a negative control. Western blot was performed to detect the ALKBH5 protein level. (b) Relative protein level was analyzed using Image J software. (c) Cell viability was detected using CCK8 assay. (d) Flow cytometry analysis was preformed to detect the apoptosis of HUVECs. (e) The percentage of apoptotic cell number was analyzed using flowJo software. **P < 0.01; ***P < 0.001.
Figure 3
Figure 3
Bcl2 blocked the apoptosis induced by TNF-α through inhibiting transcriptional activity of p53. (a) The expression of Bcl2 was determined by western bolt in TNF-α treated HUVECs. (b) The relative protein level was analyzed using Image J software. (c) The mRNA level of Bcl2 was detected using qPCR. (d) The overexpression plasmid of Bcl2 was transfected into TNF-α-treated HUVECs with the empty plasmid as a negative control. Cell viability was detected using CCK8 assay. (e) The expression levels of apoptosis relative protein were detected using western blot. The protein levels of Bcl2 (f), p53 (g), Gadd45 (h), Bax (i), p21 (j) were analyzed using Image J software. **P < 0.01; ***P < 0.001.
Figure 4
Figure 4
ALKBH5 inhibits TNF-α-induced apoptosis of HUVEs through regulating the expression of Bcl2. (a) The overexpression plasmids of ALKBH5 and Bcl2-siRNA were transfected into TNF-α treated HUVECs. Cell viability was detected using CCK8 assay. (b) Flow cytometry analysis was preformed to detect the apoptosis of HUVECs. (c) The percentage of apoptotic cell number was analyzed using flowJo software. (d) The expression levels of apoptosis relative protein were detected using western blot. The protein levels of Bcl2 (e), Gadd45 (f), Bax (g), and p21 (h) were analyzed using Image J software. The levels of miR-7 (i) and pri-miR-7 (j) were detected using qPCR. (k) negative or miR-7 mimic was transfected into HUVECs, and Bcl2 expression was detected using western blot. *P < 0.05 vs TNF-α + ALKBH5 group; **P < 0.01 vs TNF-α + ALKBH5 group; ***P < 0.001 vs TNF-α + ALKBH5 group; ## P < 0.01 vs TNF-α + ALKBH5 + Bcl2 group; ### P < 0.001 vs TNF-α + ALKBH5 + Bcl2 group.
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