CircLZIC regulates ox-LDL-induced HUVEC cell proliferation and apoptosis via Micro-330-5p/NOTCH2 axis in atherosclerosis

Abstract

Atherosclerosis (AS) is a major chronic non-communicable disease and a primary cause of cardiovascular disease. Recent studies have shown that circRNAs are potential epigenetic factors that regulate vascular endothelial inflammatory responses and AS progression. Therefore, identification of the circRNAs that regulate ox-LDL levels is a critical step to understanding the pathology of AS. Our study is aim to investigate how circLZIC regulates atherosclerosis (AS) via the Micro-330-5p/NOTCH2 regulatory axis. The results showed that CircLZIC and NOTCH2 are highly expressed in human AS clinical samples, while Micro-330-5p is expressed locally. The CCK-8 experiment results showed that circLZIC promotes the proliferation of HUVECS cells. Flow cytometry analysis showed that circLZIC act as an inhibitor of HUVEC cell apoptosis. The expression level of Micro-330-5p can be up-regulated by transfection of small interfering RNA against circLZIC. Further, Starbase predicted that Micro-330-5p could target and regulate NOTCH2. Next, we confirmed that overexpression of Micro-330-5p could significantly reduce the expression of fluorescein using the double Luciferase reporter assay. RIP-qRT-PCR experiment showed that Micro-330-5p and NOTCH2 mRNAs are effectively enriched by ago2 protein. Further, we found that knocking down circLZIC increases the expression of Micro-330-5p and promotes cell apoptosis, while inhibiting the expression of NOTCH2 and cell activity. On the other hand, co-transfection of Micro-330-5p inhibitor decreases Micro-330-5p expression and inhibit cell apoptosis, while increasing NOTCH2 expression and cell activity. In conclusion, CircLZIC regulates HUVEC cell activity by the Micro-330-5p/NOTCH2 signaling pathway, suggesting that circLZIC plays a key role in atherosclerosis development.

Keywords: CircLZIC; Micro-330-5p/NOTCH2; apoptosis; atherosclerosis; proliferation.

Fig. 1

CircLZIC is upregulated in atherosclarsis serum and ox-LD-treated vascular endothelial cells. (A) Heat map showing circRNA expression levels in GEO database. (B) Volcano plot showing circRNA expression pattern. (C) The diagram of CircLZIC formation. (D) qRT-PCR was performed to detect circLZIC expression in serum samples of AS patients and normal volunteers. (E) QRT-PCR was implemented to detect circLZIC expression in each group of cells. (F) qRT-PCR was used to quantify linear LZIC and circLZIC. (G) qRT-PCR was used to quantify circLZIC in the cytoplasm and nucleus of HUVECs.

Fig. 2

CircLZIC regulates ox-LDL-induced vascular endothelial cell proliferation and apoptosis. (A) qRT-PCR was used to detect the knockdown/overexpression efficiency of circLZIC in HUVECs. (B) The CCK-8 experiment detected the changes in cell activity of HUVECs treated with ox-LDL after circLZIC knockdown or overexpression. (C) PCNA and ki67 expression in HUVECs treated with ox-LDL after circLZIC knockdown or overexpression was detected by Western blot. (D) Cell apoptosis in HUVECs treated with ox-LDL after knockdown/overexpression of circLZIC was assessed by flow cytometry analysis. (E) The expression of apoptosis-related proteins in HUVECs treated with ox-LDL after knocking down/overexpressing circLZIC was determined by Western blot.

Fig. 3

CircLZIC directly targets Micro-330-5p in HUVECs. (A) Circular interaction prediction revealed Micro-330-5p as a putative target ofcircLZIC.(B) The interaction between circLZIC and Micro-330-5p was investigated by the luciferase fluorescent reporter assay. (C) qRT-PCR was used to detect the expression of Micro-330-5p in the serum of 20 AS patients and 25 normal volunteers. (D) qRT-PCR was used to detect the expression of Micro-330-5p in each group of cells. (E) qRT-PCR was used to detect the expression of Micro-330-5p in HUVECs after knocking down or overexpressing circLZIC.

Fig. 4

NOTCH2 is a target gene of Micro-330-5p. (A) Starbase predicted NOTCH2 as a candidate gene targeted by Micro-330-5p. (B) Luciferase fluorescent reporter assay was performed to validate the binding of Micro-330-5p tothe 3’-UTR region of NOTCH2. (C) RIP-qRT-PCR experiments were conducted using Ago2 antibodies in HUVECS cells. (D) qRT-PCR was used to detect NOTCH2 expression in the serum of AS and healthy controls. (E) qRT-PCR was used to detect NOTCH2 expression in cells of each group. (F) Western blot was performed to assess NOTCH2 expression in each group of cells.

Fig. 5

CircLZIC regulates ox-LDL-induced vascular endothelial cell proliferation and apoptosis via Micro-330-5p/NOTCH2 axis. (A) qRT PCR was used to detect the expression of Micro-330-5p in different groups. (B) qRT PCR was used to detect the expression of NOTCH2 in different groups. (C) CCK-8 detected the changes in the activity of HUVECS cells in different groups. (D) Flow cytometry apoptosis assay was used to detect the changes in the proportion of HUVECs undergoing apoptosis in different groups. (E) Western blot was used to detect the expression of proliferation- and apoptosis-related proteins in different groups of HUVECs.