Depletion of mmu_circ_0001751 (circular RNA Carm1) protects against acute cerebral infarction injuries by binding with microRNA-3098-3p to regulate acyl-CoA synthetase long-chain family member 4

Figures & data

Table 1. General characteristics of the ACI patients

Parameter	Circ-carm1			miR-3098-3p			ACSL4
	Low (n = 13)	High (n = 19)	p-value	Low (n = 22)	High (n = 10)	p-value	Low (n = 15)	High (n = 17)	p-value
Age,years	47.92 ± 5.27	46.89 ± 4.31	0.5490	48.14±4.85	45.50±3.84	0.1410	46.20 ± 3.88	48.29 ± 5.18	0.2104
BMI, kg/m^2	23.00 ± 2.06	23.65 ± 1.41	0.2938	23.08±1.73	24.06±1.51	0.1369	23.89 ± 2.11	22.94 ± 1.14	0.1172
SBP, mmHg	142.20 ± 4.88	138.89 ± 10.12	0.2839	142.51 ± 7.68	137.44 ± 9.78	0.2117	139.46 ± 8.33	140.92 ± 8.75	0.6326
DBP, mmHg	85.63 ± 6.10	86.70 ± 10.44	0.7440	86.67 ± 7.87	85.37 ± 11.08	0.7063	83.87 ± 9.63	88.37 ± 7.74	0.1533
Creatinine (Cr), μmol/L	84.59 ± 11.27	85.28 ± 8.50	0.8452	82.92 ± 7.58	89.58 ± 12.10	0.0666	84.48 ± 8.84	85.46 ± 10.38	0.7763
HDL-C, mmol/L	1.46 ± 0.36	1.31 ± 0.46	0.3317	1.38 ± 0.39	1.35 ± 0.52	0.8708	1.51 ± 0.28	1.24 ± 0.50	0.0808
LDL-C, mmol/L	2.62 ± 0.38	2.86 ± 0.43	0.1167	2.67 ± 0.37	2.97 ± 0.48	0.0623	2.82 ± 0.45	2.71 ± 0.40	0.4717
Triglyceride (TG), mmol/L	1.82 ± 0.48	2.04 ± 0.23	0.0881	1.91 ± 0.4	2.05 ± 0.27	0.3265	1.97 ± 0.45	1.94 ± 0.28	0.8389
Total cholesterol (TC), mmol/L	5.27 ± 0.88	5.05 ± 0.50	0.3849	4.99 ± 0.59	5.47 ± 0.78	0.0653	5.22 ± 0.71	5.07 ± 0.66	0.5329

Figure 1. Circ-Carm1 is highly expressed in the ACI. (a) Volcano map of dysregulated circRNAs identified in ACI. (b) circ-Carm1 expression in the serum of patients with ACI and healthy volunteers was measured by RT-qPCR. (c) circ-Carm1 expression in HT22 cells treated with OGD/R performance, 4.6 μM Erastin, 5 μM ferrostatin-1 (Fer-1), 22 μM liproxstatin-1 (Lip-1), and 0.5 µM RSL3 separately was detected by RT-qPCR.

***P < 0.001, vs. normal and control group. ^##P < 0.01, vs. OGD/R group.

Figure 2. Depletion of circ-Carm1 restores cell viability and inhibits ferroptosis of HT22 cells. (a) circ-Carm1 expression levels were determined by RT-qPCR after circ-Carm1 knockdown. (b) Cell viability of HT22 cells was detected using the CCK-8 assay before and after circ-Carm1 knockdown. (c-d) TUNEL staining was used to detect cell death before and after circ-Carm1 knockdown. (e-f) Levels of Fe²⁺ and malondialdehyde (MDA) were determined using an enzyme-linked immunosorbent assay. (g-j) Protein levels of transferrin receptor 1 (TFR1), glutathione peroxidase 4 (GPX4), and glutathione (GSH) were detected by Western blotting.

**P < 0.01, vs. si-NC and control group. ^#P < 0.05, ^##P < 0.01, vs. OGD/R group.

Figure 3. Carm1 sponges miR-3098-3p. (a) The binding sites between miR-3098-3p and circ-Carm1 were predicted by bioinformatics analysis. (b) Relative luciferase activity of HT22 cells co-transfected with wt circ-Cram1 and miR-3098-3p mimics. (c) RT-qPCR analysis of circ-Carm1 expression enriched in biotin containing miR-3098-3p. (d) The FISH assay was used to detect the location of circ-Carm1 and miR-3098-3p. (e) miR-3098-3p expression in the serum of patients with ACI and healthy volunteers was measured by RT-qPCR. (f) RT-qPCR analysis of miR-3098-3p expression in OGD/R-induced HT22 cells. (g) RT-qPCR analyses of miR-3098-3p expression in HT22 cells transfected with si-circ-Carm1.

**P < 0.01, ***P < 0.001, vs. mimic NC, Biotin-NC, normal, control, and si-NC group.

Figure 4. Downregulation of miR-3098-3p reverses the effects of circ-Carm1 deficiency on cell viability and ferroptosis of HT22 cells. (a) miR-3098-3p expression levels were detected using RT-qPCR after transfection. (b) Cell viability of HT22 cells co-transfected with si-circ-Carm1 and miR-3098-3p inhibitor and was detected using the CCK-8 assay. (c-d) TUNEL staining was conducted to detect cell death in cells co-transfected with si-circ-Carm1 and miR-3098-3p inhibitor. (e-f) Levels of Fe²⁺ and malondialdehyde (MDA) were determined using an enzyme-linked immunosorbent assay. (g-j) Protein levels of transferrin receptor 1 (TFR1), glutathione peroxidase 4 (GPX4), and glutathione (GSH) were detected by Western blotting.

**P < 0.01, ***P < 0.001,vs. NC mimic and control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. NC inhibitor and OGD/R group. ^&P < 0.05, ^&&P < 0.01, vs. OGD/R + si-circ-Carm1 + NC inhibitor group.

Figure 5. ACSL4 is a target gene of miR-3098-3p. (a) Binding sites between miR-3098-3p and ACSL4 were predicted by bioinformatics analysis. (b) Relative luciferase activity of HT22 cells co-transfected with wt ACSL4 and the miR-3098-3p mimic. (c) RT-qPCR analyses of ACSL4 expression enriched in biotin containing miR-3098-3p. (d) RT-qPCR analysis of ACSL4 expression in OGD/R-induced HT22 cells. (e) ACSL4 protein and mRNA expression in HT22 cells transfected with the miR-3098-3p mimic.

**P < 0.01, vs. mimic NC, Biotin-NC, control, and mimic NC group.

Figure 6. Overexpression of ACSL4 inhibits the functions of miR-3098-3p. (a) miR-3098-3p expression levels were detected using RT-qPCR after transfection. (b) Cell viability of HT22 cells co-transfected with miR-3098-3p mimic and overexpressing ACSL4 plasmids were detected using the CCK-8 assay. (c-d) TUNEL staining was used to detect cell death in cells co-transfected with the miR-3098-3p mimic and overexpressed ACSL4 plasmids. (e-f) Levels of Fe²⁺ and malondialdehyde (MDA) were determined using an enzyme-linked immunosorbent assay. (g-j) Protein levels of transferrin receptor 1 (TFR1), glutathione peroxidase 4 (GPX4), and glutathione (GSH) were detected by Western blotting.

**P < 0.01, ***P < 0.001,vs. vector and control group. ^#P < 0.05, ^##P < 0.01, vs. OGD/R group. ^&P < 0.05, ^&&P < 0.01, vs. OGD/R + mimic + vector group.

Data Availability Statement

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.