Journal: Molecular Psychiatry

Article Title: A novel circular RNA, circIgfbp2, links neural plasticity and anxiety through targeting mitochondrial dysfunction and oxidative stress-induced synapse dysfunction after traumatic brain injury

doi: 10.1038/s41380-022-01711-7

Figure Lengend Snippet: A , B The network map of the differential circRNAs-miRNAs-mRNAs genes regulatory network screening by DEG algorithm, showing the top 5 up-regulated circRNAs (sham group vs. TBI group) (|fold-change| > 2, Q < 0.01). Red: circRNAs; Green: miRNAs; Black: mRNAs. C Relative circIgfbp2 levels were measured by qRT-PCR in the sham and injured brain tissue 3 days after TBI. n = 5 mice per group. **** p < 0.0001, two-tailed t-test. D Relative circIgfbp2 levels in the H 2 O 2 -treated HT22 cells and control group. n = 3 replications, *** p < 0.001, two-tailed t-test. E Homology analysis between mice circIgfbp2 and human circRNAs. F The relative expression level of hsa_circ_0058195 in the serum of acute TBI patients, which was highly homologous to circIgfbp2, and up-regulated after TBI. n = 50 TBI patients, n = 20 volunteers as control, **** p < 0.0001, two-tailed t-test. G The anxiety-like behaviors were analyzed using the Self-Rating Anxiety Scale in patients 3 months after TBI. The scale score had a positive linear relationship with the expression of hsa_circ_0058195. n = 50 TBI patients, n = 20 volunteers as control, p = 0.0051. H The depression-like behaviors were analyzed using the Self-Rating Depression Scale in patients 3 months after TBI. The scale score had no linear relationship with the expression of hsa_circ_0058195. n = 50 TBI patients, n = 20 volunteers as control, p = 0.7519. I ROC curve in the evaluation of the diagnostic value of serum hsa_circ_0058195 for anxiety after TBI. Area under curve (AUC) = 0.7883; p = 0.0006. J Schematic representation of the circularization of Igfbp2 1-2 exons to form circIgfbp2. The results of Sanger sequencing of the spliced junction resulting from the divergent primers are shown. K The presence of circIgfbp2 was validated in HT22 cells by PCR and agarose gel electrophoresis (AGE). Divergent primers amplified circIgfbp2 from cDNA but not from gDNA. L CircIgfbp2, linear Igfbp, and GAPDH levels were detected by PCR and AGE treated with RNase R for 0 to 120 min. All data were represented as mean ± SEM.

Article Snippet: The isolation of mitochondria from brain specimens or HT22 cells was performed according to a manufacturer’s protocol (Nanjing Jiancheng, Nanjing, China).

Techniques: Quantitative RT-PCR, Two Tailed Test, Control, Expressing, Diagnostic Assay, Sequencing, Agarose Gel Electrophoresis, Amplification

Journal: Molecular Psychiatry

Article Title: A novel circular RNA, circIgfbp2, links neural plasticity and anxiety through targeting mitochondrial dysfunction and oxidative stress-induced synapse dysfunction after traumatic brain injury

doi: 10.1038/s41380-022-01711-7

Figure Lengend Snippet: A RNAhybrid predicted circIgfbp2 and mmu-miR-370-3p binding sites: site1: mfe (minimum free energy) = −31 kcal/mol, site2: mfe = −31.6 kcal/mol, site3 = −34.8 kcal/mol. B The expression of miR-370-3p after biotin-labeled wild-type or mutated circIgfbp2 probe pull-down miR-370-3p in HT22 cells. n = 3 replication, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test. C The expression of circIgfbp2 after the biotin-labeled miR-370-3p pull-down circIgfbp2, n = 3replications, oe-circIgfbp2 + biotin-miR-370-3p(WT) vs. oe-circ-NC + biotin-miR-370-3p(WT), *** p < 0.001; oe-circIgfbp2 + biotin-miR-370-3p(mut) vs.oe-circIgfbp2 + biotin-miR-370-3p(WT), *** p < 0.001. Two-way ANOVA followed by Tukey’s multiple comparisons test. D hsa_circ_0058195 have homologous sequences with circIgfbp2 (the same bases in green and different bases in red). The binding site1 of circIgfbp2 with miR-370-3p and the circIgfbp2 mut1 site are shown. E Relative luciferase activity in 293 T cells transfected with a plasmid containing wild-type/mut circIgfbp2 and miR-370-3p mimics/miR-NC. n = 3 replications, circIgfbp2-WT + miR-370-3p vs. circIgfbp2-WT + miR-NC, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test. F FISH probes indicated that circIgfbp2 and mmu-miR-370-3p were enriched at the cytoplasm in HT22 cells. G The predicted binding sites of miR-370-3p and BACH1 3’UTR (mouse and human). H Relative luciferase activity in 293 T cells transfected with the wild-type/mut BACH1 3’UTR plasmid or miR-370-3p mimics/miR-NC, n = 3 replications. BACH1 3’UTR-WT + mmu-miR-370-3p vs. BACH1 3’UTR-WT + mmu-miR-NC, **** p < 0.0001. One-way ANOVA followed by Tukey’s multiple comparisons test. I The BACH1 level was analyzed in the injured brain cortex tissue with overexpression circIgfbp2 3 days after TBI in mice by Western blot, n = 5 mice per group. TBI vs. sham, * p < 0.05, TBI + oe-circIgfbp2 vs. TBI + oe-circ-NC, ** p < 0.01. One-way ANOVA followed by Tukey’s multiple comparisons test. J The BACH1 level was analyzed in the injured brain cortex tissue with knockdown circIgfbp2 3 days after TBI in mice by Western blot, n = 5 mice per group. TBI vs. sham, **** p < 0.0001, TBI + sh-circIgfbp2 vs. TBI + sh-circ-NC, *** p < 0.001. One-way ANOVA followed by Tukey’s multiple comparisons test. ns: no significance. All data were represented as mean ± SEM.

Article Snippet: The isolation of mitochondria from brain specimens or HT22 cells was performed according to a manufacturer’s protocol (Nanjing Jiancheng, Nanjing, China).

Techniques: Binding Assay, Expressing, Labeling, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Western Blot, Knockdown

Journal: Molecular Psychiatry

Article Title: A novel circular RNA, circIgfbp2, links neural plasticity and anxiety through targeting mitochondrial dysfunction and oxidative stress-induced synapse dysfunction after traumatic brain injury

doi: 10.1038/s41380-022-01711-7

Figure Lengend Snippet: A The double immunofluorescence staining showed the colocalization of PSD95 and HO-1, PSD95 and Syn in control, H 2 O 2 , H 2 O 2 + miR-370-3p mimics, and H 2 O 2 + anti-miR-370-3p group. B The expression of BACH1, HO-1, PSD95 and Syn proteins were analyzed in the HT22 cells transduced with miR-NC or miR-370-3p mimics for 48 h and treated with H 2 O 2 (600 µmol/L) for 6 h by Western blot, n = 3 replication. BACH1: H 2 O 2 vs. control, **** p < 0.0001, H 2 O 2 + miR-370-3p mimics vs. H 2 O 2 + miR-NC, **** p < 0.0001,;HO-1: H 2 O 2 vs. control, **** p < 0.0001, H 2 O 2 + miR-370-3p mimics vs. H 2 O 2 + miR-NC, *** p < 0.001,;PSD95: H 2 O 2 vs. control, *** p < 0.001, H 2 O 2 + miR-370-3p mimics vs. H 2 O 2 + miR-NC, * p < 0.05; Syn: H 2 O 2 vs. control, *** p < 0.001, H 2 O 2 + miR-370-3p mimics vs. H 2 O 2 + miR-NC, * p < 0.05. One-way ANOVA followed by Tukey’s multiple comparisons test. C The expression of BACH1, HO-1, PSD95 and Syn were analyzed in the HT22 cells transduced with anti-miR-NC or anti-miR-370-3p for 48 h and treated with H 2 O 2 (600 µmol/L) for 6 h by Western blot, n = 3 replications.BACH1: H 2 O 2 vs. control, * p < 0.05, H 2 O 2 + anti-miR-370-3p vs. H 2 O 2 + anti-miR-NC, *** p < 0.001;HO-1; H 2 O 2 vs. control, **** p < 0.0001,H 2 O 2 + anti-miR-370-3p vs. H 2 O 2 + anti-miR-NC, ** p < 0.01; PSD95: H 2 O 2 vs. control, **** p < 0.0001, H 2 O 2 + anti-miR-370-3p vs. H 2 O 2 + anti-miR-NC, ** p < 0.01; Syn: H 2 O 2 vs. control, *** p < 0.001, H 2 O 2 + anti-miR-370-3p vs. H 2 O 2 + anti-miR-NC, *** p < 0.001. One-way ANOVA followed by Tukey’s multiple comparisons test. D The content of ATP with miR-370-3p overexpression or knockdown in HT22 cells treated with H 2 O 2 (600 µmol/L) for 6 h, n = 3 replications.H 2 O 2 vs. control, **** p < 0.0001, H 2 O 2 + miR-370-3p mimics vs. H 2 O 2 + miR-NC, *** p < 0.001, H 2 O 2 + anti-miR-370-3p vs. H 2 O 2 + anti-miR-NC, ** p < 0.01. One-way ANOVA followed by Tukey’s multiple comparisons test. E Representative fluorescent staining of Mitosox for mitochondrial ROS with miR-370-3p overexpression or knockdown in HT22 cells treated with H 2 O 2 (600 µmol/L) for 6 h, and quantitative analysis of the mean optical density analysis of Mitosox, n = 3 replications. H 2 O 2 vs. control,*** p < 0.001,H 2 O 2 + miR-370-3p mimics vs. H 2 O 2 , * p < 0.05,H 2 O 2 + anti-miR-370-3p vs. H 2 O 2 , * p < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test. All data were represented as mean ± SEM.

Article Snippet: The isolation of mitochondria from brain specimens or HT22 cells was performed according to a manufacturer’s protocol (Nanjing Jiancheng, Nanjing, China).

Techniques: Double Immunofluorescence Staining, Control, Expressing, Transduction, Western Blot, Over Expression, Knockdown, Staining

Journal: Molecular Psychiatry

Article Title: A novel circular RNA, circIgfbp2, links neural plasticity and anxiety through targeting mitochondrial dysfunction and oxidative stress-induced synapse dysfunction after traumatic brain injury

doi: 10.1038/s41380-022-01711-7

Figure Lengend Snippet: A Western blot analysis of BACH1, HO-1, PSD95 and Syn in HT22 cells transduced with oe-circIgfbp2 lentivirus for 7 days, and then transduced with miR-NC or miR-370-3p mimics for 48 h, finally H 2 O 2 (600 µmol/L) treated HT22 cells for 6 h. The results showed that transfection with oe-circIgfbp2 alone aggravated H 2 O 2 -induced mitochondrial oxidative stress-mediated synapse dysfunction, the expression of BACH1was upregulated, and HO-1, PSD95 and Syn were downregulated. The co-transfection with miR-370-3p mimics reversed these effects. n = 3 replications. BACH1:H 2 O 2 + oe-circIgfbp2+miR-NC vs. H 2 O 2 + miR-NC, **** p < 0.0001; H 2 O 2 + oe-circIgfbp2+miR-370-3p mimics vs. H 2 O 2 + oe-circIgfbp2+miR-NC, ** p < 0.01; HO-1:H 2 O 2 + oe-circIgfbp2+miR-NC vs. H 2 O 2 + miR-NC, **** p < 0.0001;H 2 O 2 + oe-circIgfbp2+miR-370-3p mimics vs. H 2 O 2 + oe-circIgfbp2+miR-NC, **** p < 0.0001; PSD95: H 2 O 2 + oe-circIgfbp2+miR-NC vs. H 2 O 2 + miR-NC, *** p < 0.001, H 2 O 2 + oe-circIgfbp2+miR-370-3p mimics vs. H 2 O 2 + oe-circIgfbp 2 + miR-NC, *** p < 0.001; Syn: H 2 O 2 + oe-circIgfbp2+miR-NC vs. H 2 O 2 + miR-NC, *** p < 0.001, H 2 O 2 + oe-circIgfbp2+miR-370-3p mimics vs. H 2 O 2 + oe-circIgfbp2+miR-NC, *** p < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test. B Western blot analysis of BACH1, HO-1, PSD95, Syn in HT22 cells transduced with circIgfbp2 shRNA lentivirus cells for 7 days, and then transduced with anti-miR-NC or anti-miR-370-3p for 48 h, finally treated with H 2 O 2 (600 µmol/L) for 6 h. The transfection with sh-circIgfbp2 alone attenuated H 2 O 2 -induced mitochondrial oxidative stress-mediated synapse dysfunction, the expression of BACH1was downregulated; HO-1, PSD95 and Syn were upregulated, while co-transfection with anti-miR-370-3p reversed these effects, n = 3 replications. BACH1: H 2 O 2 + sh-circIgfbp2+anti-miR-NC vs. H 2 O 2 + anti-miR-NC, **** p < 0.0001, H 2 O 2 + sh-circIgfbp2+anti-miR-370-3p vs. H 2 O 2 + sh-circIgfbp 2 + anti-miR-NC, *** p < 0.001; HO-1: H 2 O 2 + sh-circIgfbp2+anti-miR-NC vs. H 2 O 2 + anti-miR-NC, **** p < 0.0001, H 2 O 2 + sh-circIgfbp2+anti-miR-370-3p vs. H 2 O 2 + sh-circIgfbp2+anti-miR-NC, *** p < 0.001; PSD95: H 2 O 2 + sh-circIgfbp2+anti-miR-NC vs. H 2 O 2 + anti-miR-NC, ** p < 0.01, H 2 O 2 + sh-circIgfbp2+anti-miR-370-3p vs. H 2 O 2 + sh-circIgfbp2+anti-miR-NC, * p < 0.05; Syn: H 2 O 2 + sh-circIgfbp2+anti-miR-NC vs. H 2 O 2 + anti-miR-NC, **** p < 0.0001, H 2 O 2 + sh-circIgfbp2+anti-miR-370-3p vs. H 2 O 2 + sh-circIgfbp2+anti-miR-NC,**** p < 0.0001 one-way ANOVA followed by Tukey’s multiple comparisons test. C Transduction of HT 22 cells with the miR-370-3p mimics significantly reversed the oe-circIgfbp2 -induced decrease the content of ATP, n = 3 replications. H 2 O 2 + oe-circIgfbp2+miR-NC vs. H 2 O 2 + miR-NC, ** p < 0.01, H 2 O 2 + oe-circIgfbp2+miR-370-3p mimics vs. H 2 O 2 + oe-circIgfbp2+miR-NC, * p < 0.05; one-way ANOVA followed by Tukey’s multiple comparisons test. D Transduction of HT22 cells with the anti-miR-370-3p mimics significantly attenuated the sh-circIgfbp2 -induced increase the content of ATP. n = 3 replications. H 2 O 2 + sh-circIgfbp2+anti-miR-NC vs. H 2 O 2 + anti-miR-NC, *** p < 0.001, H 2 O 2 + sh-circIgfbp2+anti-miR-370-3p vs. H 2 O 2 + sh-circIgfbp2+anti-miR-NC, * p < 0.05.One-way ANOVA followed by Tukey’s multiple comparisons test. E Transduction of HT22 cells with the miR-370-3p mimics significantly reversed the oe-circIgfbp2-induced increase the content of mitochondrial ROS; transduction of HT22 cells with the anti-miR-370-3p significantly attenuated the sh-circIgfbp2-induced the decrease of mitochondrial ROS, n = 3 replications. H 2 O 2 + oe-circIgfbp2+miR-370-3p mimics vs. H 2 O 2 + oe-circIgfbp2+miR-NC, **** p < 0.0001, H 2 O 2 + sh-circIgfbp2+anti-miR-370-3p vs. H 2 O 2 + sh-circIgfbp2+anti-miR-NC, **** p < 0.0001;one-way ANOVA followed by Tukey’s multiple comparisons test. ns: no significance. All data were represented as mean ± SEM.

Article Snippet: The isolation of mitochondria from brain specimens or HT22 cells was performed according to a manufacturer’s protocol (Nanjing Jiancheng, Nanjing, China).

Techniques: Western Blot, Transduction, Transfection, Expressing, Cotransfection, shRNA