Knockout of circRNA single stranded interacting protein 1 (circRBMS1) played a protective role in myocardial ischemia-reperfusion injury though inhibition of miR-2355-3p/Mammalian Sterile20-like kinase 1 (MST1) axis

Abstract

Evidence suggests circRBMS1 regulates mRNA to mediate cell apoptosis, inflammation, and oxidative stress in different diseases. MST1 is reported to be the target and activator of apoptosis-related molecules and signaling pathways. Hence, the present study aims to investigate the role of circ-RBMS1/miR-2355-3p/MST1 in the development of I/R injury. In vitro experiments showed increased circ-RBMS1 and decreased miR-2355-3p in H/R-induced HCMs. CircRBMS1 served as a sponge for miR-2355-3p and miR-2355-3p targeted MST1. Furthermore, knockout of circRBMS1 attenuated cell apoptosis, oxidized stress, and inflammation in H/R-induced HCMs. In vivo experiments indicated circRBMS1 knockdown attenuated cardiac function damage, cell apoptosis, oxidative stress injury and inflammatory response through miR-2355-3p/MST1 axis in mice. In summary, these results demonstrated circRBMS1 played a protective role in myocardial I/R injury though inhibition of miR-2355-3p/MST1 axis. It might provide a new therapeutic target for cardiac I/R injury.

Keywords: MST1; Myocardial I/R injury; circRBMS1; miR-2355-3p.

Graphical abstract

Figure 1.

Knockdown of circRBMS1 attenuated H/R-induced cell injury. (a-b) qRT-PCR analysis of circRBMS1 expression. H/R-induced HCMs were transfected with sh-RBMS1 or sh-NC. (c) CCK-8 assay for cell viability. (d) TUNEL assay for cell apoptosis. Scale bars: 50 μm. (e) Western blot analysis of cleaved-caspase 3, Bax and Bcl-2. (f) ROS generation was detected using immunofluorescence assay. Scale bars: 100 μm. (g-h) Detection of MDA contents and SOD level. (i-k) ELISA data of TNF-α, IL-1βand IL-6. **P < 0.01 compared with control or H/R+ sh-NC.

Figure 2.

CircRBMS1 targeted miR-2355-3p. (a) circRBMS1 was predicted to bind to miR-2355-3p (Starbase). (b) qRT-PCR determined miR-2355-3p expression in HCMs transfected with miR-2355-3p mimics, NC mimics or the control. (c) Detection of luciferase activity. (d) Pull-down assay showed elevated enrichment of miR-2355-3p in HCMs transfected with biotin-labeled circRBMS1. (e) qRT-PCR analysis for miR-2355-3p expression. **P < 0.01 compared with control, miR-NC, bio-NC or H/R+ sh-NC.

Figure 3.

Elevated miR-2355-3p alleviated H/R-induced cell injury. (a) CCK-8 assay results of cell viability in Control, H/R, H/R+ miR-NC, and H/R+ miR-2355-3p. (b) TUNEL assay analyzed cell death. Scale bars: 50 μm. (c) ROS generation was analyzed using immunofluorescence assay. Scale bars: 100 μm. (d) ELISA detection for TNF-α, IL-1β and IL-6. **P < 0.01 compared with miR-NC, bio-circRBMS1 or H/R+ sh-circRBMS1.

Figure 4.

CircRBMS1 mediated H/R-induced cell injury by targeting miR-2355-3p/MST1 axis. I/R induced HCMs were transfected with sh-NC, sh-circRBMS1 or sh-circRBMS1 and miR-2355-3p inhibitor. (a) Predicted binding site for miR-2355-3p and MST1. (b) Detection of luciferase activity. (c) MST1 protein was determined. (d) Detection of miR-2355-3p was conducted using qRT-PCR. (e) Western blot for MST1 quantification. (f) Assessment of cell viability. (g) Calculation of TUNEL positive cells. Scale bars: 50 μm. (h) Western blot for cleaved-caspase 3, Bax and Bcl-2. (i) ROS generation was detected using immunofluorescence assay. Scale bars: 100 μm. (j-l) ELISA results for the contents of TNF-α, IL-1β and IL-6. *P < 0.05, **P < 0.01 compared with control, miR-NC, H/R+ sh-NC or H/R+ sh-circRBMS1+ anti-miR-2355-3p.

Figure 5.

circRBMS1 knockdown mediated myocardial I/R injury by regulating miR-2355-3p/MST1 in mice. The I/R mouse model was established. (a) H&E staining for myocardial tissue in control and sham group. Scale bars: 100 μm. (b) The mice were grouped: Sham, I/R, I/R+ lenti-sh-NC and I/R+ lenti-sh-circRBMS1. Enzyme activity of LDH, CK and CK-MB. (c) Echocardiographic parameters LVEF and LVFS were recorded. (d) H&E staining for myocardial tissue. Scale bars: 100 μm. Swollen and necrotic cardiomyocytes and disordered arrangement of myocardial bundles were observed in myocardial tissue of I/R-induced mice, conversely, sh-circRBMS1 significantly attenuated structural damage of myocardial tissue. (e) qRT-PCR analysis of circRBMS1. (f) qRT-PCR analysis of miR-2355-3p and MST1. (g) TUNEL assay for cell apoptosis. Compared to the sham group, ratio of TUNEL positive cell was increased in myocardial tissue of I/R group, while sh-circRBMS1 reduced the ratio of TUNEL positive cell in I/R group. (h) Detection of serum levels of MDA and SOD. (i) ELISA data for serum TNF-α, IL-1β and IL-6. *P < 0.05, **P < 0.01 compared with sham or H/R+ sh-NC.