Circular RNA CREBBP Suppresses Hepatic Fibrosis Via Targeting the hsa-miR-1291/LEFTY2 Axis

Abstract

CircRNAs (circRNAs) are commonly dysregulated in a variety of human diseases and are involved in the development and progression of cancer. However, the role of circRNAs in hepatic fibrosis (HF) is still unclear. Our previous high throughput screen revealed changes in many circRNAs in mice with carbon tetrachloride (CCl4)-induced HF. For example, circCREBBP was significantly down-regulated in primary hepatic stellate cells (HSCs) and liver tissue of HF mice induced by CCl4 compared to those in the vehicle group. Overexpression of circCREBBP with AAV8-circCREBBP in vivo prevented CCl4-induced HF worsening by reducing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents, liver hydroxyproline levels, collagen deposition, and levels of pro-fibrosis genes and pro-inflammatory cytokines. Furthermore, in vitro function loss and function gain analysis showed that circCREBBP inhibited HSCs activation and proliferation. Mechanically, circCREBBP acts as a sponge for hsa-miR-1291 and subsequently promotes LEFTY2 expression. In conclusion, our current results reveal a novel mechanism by which circCREBBP alleviates liver fibrosis by targeting the hsa-miR-1291/LEFTY2 axis, and also suggest that circCREBBP may be a potential biomarker for heart failure.

Keywords: HF; LEFTY2; biomarker; circCREBBP; hsa-miR-1291; miR-17-5p.

FIGURE 1

The description of cirRNA and high-throughput sequencing..

FIGURE 2

CircRNAs expression profile in HF in huaman by high-throughput sequencing. Scatter plot of circRNAs expression correlation, volcano map of differentially expressed circRNAs and Heatmap among samples (A–C). the scatter plot of GO enrichment of differentially expressed circRNAs parental gene (D and E). the analysis results of KEGG pathway in differentially expressed circRNAs parental gene (F).

FIGURE 3

Expression level of circCREBBP decreased in HF tissues. The malregulated circRNAs using qRT-PCR, which was consistent with circRNA-seq data. We focused on circCREBBP (hsa_circ_0007637), which significantly downregulated in HF tissues (A–C). The expression level of hsa_circ_0007637 in in human L0-2 and LX-2 cells stimulated by TGF-β1 and we found that hsa_circ_0007637 was decreased (D,E). It was also established a mouse model of HF (F–H).

FIGURE 4

CircCREBBP suppresses activation and proliferation of LX-2 cells in vitro. The up-regulated the expression level of hsa_circ_0007637 in LX-2 cells and the efficiency of overexpression hsa_circ_0007637 (A–C). The up-regulated expression of circCREBBP subsequently decreased the mRNA and protein levels of α-SMA and Col1A1 (D–H). CircCREBBP could arrest cycle and inhibit cell proliferation (I,J).

FIGURE 5

Anti-fibrotic effects of circCREBBP in HF mice in vivo. AAV2/8-mmu_circ_0006288 was injected into the tail vein of mice (A). Liver parenchyma and vascular architecture distortion, collagen deposition were consistently reduced in HF mice following AAV2/8-mmu-circ-0006288 administration (B,C). The expression levels of ALT and AST in serum were reduced in AAV2/8-mmu-circ-0006288-treated HF mice (D,E). The fibrosis factor (α-SMA and COL1A1) were down-regulated after circCREBBP was overexpressed (F–I).

FIGURE 6

Microarray analysis and identification of circCREBBP-hsa-miR-1291 connectivity. To assess the potential miRNAs bind to circCREBBP, and identify promising novel miRNAs relate to HF, miRNA expression profile in HF tissues was analyzed by microarray. Importantly, we found 14 miRNAs downregulated in HF mice, along with 11 miRNAs upregulated in HF mice (A–C). Network based on the correlations between differentially expressed miRNAs and their differentially expressed circRNA targets was showed in a diagram (D). Based on sequence pairing, binding sites of hsa-miR-1291 were identified within the circCREBBP sequences (E,F).

FIGURE 7

CircCREBBP upregulates the expression of LEFTY2 by sponging hsa-miR-1291. The qRT-PCR results showed that the expression level of hsa-miR-1291 were up-regulated in TGF-β1-indcued LX-2 cells, HF patients and mouse tissues (A–C). When circCREBBP expression was increased, hsa-miR-1291 expression level was down-regulated (D,E). α-SMA and Col1A1 protein and mRNA expression levels in LX-2 cells transfected with over-expressed hsa-miRNA-1291 were decreased (F–I). An interaction network of circRNAs-miRNAs-mRNAs was established based on the negative regulatory relationship between differentially expressed miRNAs and their differentially expressed target circRNAs and mRNAs (J).

FIGURE 8

LEFTY2 is one of the target genes of hsa-miR-1291 (A,B). The expression level of LEFTY2 was down-regulated in HF mouse tissues (C–E). The expression level of LEFTY2 was increased in AAV2/8-mmu_circ_0006288-treated HF mice (F–H). When hsa-miRNA-1291 expression was increased or decreased, LEFTY2 expression level was down-regulated or up-regulated (I–N).