Analysis of hsa_circ_0136256 as a biomarker for fibrosis in systemic sclerosis

Abstract

Background: Exploration of whether circRNAs in the skin of systemic sclerosis (SSc) model mice interact with 4E-BP1 protein to mediate the mTOR signaling pathway to regulate SSc fibrosis is crucial to identify homologous human circRNAs as markers to guide the diagnosis and treatment of SSc.

Methods: C57BL/6 mice aged 6-8 weeks and weighing approximately 20 g were subcutaneously injected with bleomycin (BLM) to establish an SSc model. High-throughput sequencing was used to screen the differentially expressed circRNA in the skin of SSc model mice and control mice. RNA immunoprecipitation and RNA pulldown confirmed the interaction between circRNA and 4E-BP1 protein. SSc model mice were treated with empty plasmid (OE-NC), overexpression plasmid of mmu_circ_0005372 (OE-circ_0005372), interference plasmid of mmu_circ_0005372 (sh-circ5372), mutant plasmid of mmu_circ_0005372 (circ5372-MT), mTOR activator (MHY1485), mTOR inhibitor (omipalisib), or JAK1/2 inhibitor (ruxolitinib). Sections of mouse skin tissue were stained with Hematoxylin and eosin and Masson's stain. The collagen volume fraction (CVF) was calculated as CVF = area of blue collagen/total area with ImageJ. The correlation between homologous human circRNAs and clinical data was analyzed.

Results: Compared to the control group, 21,839 circRNAs were upregulated and 27, 946 circRNAs were downregulated in the skin tissue of mice in the SSc model group. Among them was mmu_circ_0005372, which is derived from the FZD3 gene, is closely related to fibrosis, and is involved in the mTOR signaling pathway. Hsa_circ_0136256 was identified as the homologous human circRNA of mmu_circ_0005372. RT-qPCR confirmed that the expression of mmu_circ_0005372 was significantly reduced in the skin tissue of SSc mice, and the expression of hsa_circ_0136256 was significantly reduced in the peripheral blood mononuclear cells of patients with SSc. The interaction between mmu_circ_0005372 and 4E-BP1 protein was inhibited in the skin tissue of SSc model mice. The results showed that the CVF of OE-circ_0005372 group was significantly lower than that of the sh-circ5372, circ5372-MT, and MHY1485 groups, indicating that OE-circ5372 significantly improved skin fibrosis in the SSc mice. ROC curve analysis was performed on hsa_circ_0136256 (AUC = 0.719, P = 0.035). The expression of hsa_circ_0136256 was negatively correlated with COL IV, RDW-SD, and RDW-CV, and positively correlated with VC, PLT, and PCT. The results suggested that hsa_circ_0136256 may have important roles in the clinical diagnosis of SSc.

Conclusion: Mmu_circ_0005372 and homologous human hsa_circ_0136256 may be biomarkers and therapeutic targets for SSc fibrosis.

Keywords: Fibrosis; MTOR/4E-BP1; Systemic sclerosis; circRNA.

Fig. 1

The study design

Fig. 2

Analysis of differentially expressed circRNAs in the skin tissues of SSc model mice and control mice. A The number of circRNAs in the circBase database in the skin tissues of SSc model mice and control mice was classified by high-throughput screening (n = 3). B The circRNAs were divided into intragenic circRNAs (introns and exons) and intergenic circRNAs. C The differentially expressed circRNAs in the skin tissue of mice were analyzed by DEGseq (n = 3). Red represents upregulation, blue represents downregulation, and gray represents no difference. D Heatmap showing hierarchical cluster analysis of differentially expressed circRNAs in the skin tissue of SSc model mice and healthy control mice. The X-axis represents 16 samples from the cluster analysis, and the Y-axis represents differential circRNAs. Color represents expression normalized by the z-score. Red indicates upregulation and blue indicates downregulation (n = 3). E Mmu_circ_0005372 was highly homologous to hsa_circ_0136256 on the BLAST website. Range: the length of the alignment sequence. Score: alignment score, in which the longer the matching fragment, the higher the similarity, the higher the score. Expect: likelihood of a random match, with smaller expect values indicating more similar sequences. Identities: sequence similarity, number of matched bases as a percentage of total sequence length. Gaps: allows for gaps (base deletions or insertions) in the aligned sequence. F Genome structure map of mmu_circ_0005372. G Genome structure map of hsa_circ_0136256

Fig. 3

The mmu_circ_0005372 and 4E-BP1 protein interaction is inhibited by reduced or absent expression of mmu_circ_0005372. A The interaction matrix of 4E-BP1 and mmu_circ_0005372 predicted by catRNPID, in which the red shaded part of the figure is the binding site. B The interaction matrix of 4E-BP1 and hsa_circ_0136256 was predicted by catRNPID online software, and the red shaded part in the figure is the binding site. C The expression of mmu_circ_0005372 in 3T3-L1 cells transfected with sh-circ5372, OE-circ5372, and MT-circ5372 was detected by RT-qPCR. D, E WB was used to detect the expression of 4E-BP1 and GAPDH in OE-circ5372, sh-circ5372, and WT-circ5372 groups before RIP. F, G The enrichment of mmu_circ_0005372 in transfected 3T3-L1 cells after RIP was detected by 19 RT-qPCR. H The expression of 4E-BP1 in 3T3-L1 cells transfected with sh-circ5372 and OE-circ5372 was detected by WB after RIP. I, J The enrichment of mmu_circ_0005372 in transfected 3T3-L1 cells after RNA pulldown by RT-qPCR. K The expression of 4E-BP1 in 3T3-L1 cells transfected with sh-circ5372 and OE-circ5372 after RNA pulldown was detected by WB. *P < 0.05; *P < 0.01. All the blots were from different gels without cropping. IgG means the enrichment of 4E-BP1 protein in immunoprecipitation with IgG antibody; IP means the enrichment of 4E-BP1 protein in immunoprecipitation with 4E-BP1 antibody; Input means cell lysates without immunoprecipitation

Fig. 4

Histopathological analysis and Masson’s staining of the skin tissue of SSc mice after treatment. A HE staining of skin tissue from treated mice (scale bar: 100 μm). B Masson’s staining of skin tissue of treated mice. Muscle fibers are shown in red and collagen fibers in blue (scale bar: 100 μm). C The collagen volume fraction (CVF) in the OE-circ5372 group was significantly lower than that in the sh-circ5372 group, circ5372-MT group, and MHY1485 group *P < 0.05

Fig. 5

Hsa_circ_0136256 may be a biomarker for the clinical diagnosis and treatment of SSc. A The expression of hsa_circ_0136256 was significantly decreased in the PBMCs of patients 24 with SSc (n = 16). HC: Healthy control, SSc: Patients with systemic sclerosis (SSc). *P < 0.05. B ROC curve analysis of hsa_circ_0136256 suggested that hsa_circ_0136256 plays key roles in the clinical diagnosis of SSc (n = 16). (C–H) hsa_circ_0136256 expression was negatively correlated with COL IV, RDW-SD, and RDW-CV, and positively correlated with VC, PLT, and PCT