MiR-130a-3p Alleviates Liver Fibrosis by Suppressing HSCs Activation and Skewing Macrophage to Ly6Clo Phenotype

Abstract

Emerging evidences have highlighted the crucial role of microRNAs (miRNAs) in the liver cirrhosis, but the relationship between miR-130a-3p and liver cirrhosis is not entirely clear. As we all know, schistosomiasis, as one of the zoonoses, can lead to liver cirrhosis when it advances. In this study, we investigated the biological functions of miR-130a-3p on the liver fibrosis of schistosomiasis in vivo and in vitro. The mice infected with Schistosoma japonicum (S. japonicum) were treated with lentivirus vector (LV)-miR-130a-3p by hydrodynamic injection through the tail vein. Our findings showed significantly decreased expression of miR-130a-3p both in the serum of patients with cirrhosis and in the liver of mice infected with S. japonicum. The results showed that LV-miR-130a-3p could effectively enter into the liver and alleviate liver granulomatous inflammation and collagen deposition. Simultaneously, LV-miR-130a-3p-promoted macrophages presented the Ly6C^lo phenotype, concomitant with the decreased expression of the tissue inhibitor of metalloproteinases (TIMP) 1, and increased the expression of matrix metalloproteinase (MMP) 2, which contributed to the dissolution of collagen. Furthermore, overexpression of miR-130a-3p not only inhibited the activation and proliferation of hepatic stellate cells (HSCs) but also induced the apoptosis of HSCs. In addition, we also confirmed that miR-130a-3p enables to bind with mitogen-activated protein kinase (MAPK) 1 and transforming growth factor-beta receptors (TGFBR) 1 and TGFBR2 genes and inhibit the expressions of these genes. Our findings suggested that miR-130a-3p might represent as the potential candidate biomarker and therapeutic target for the prognosis identification and treatment of schistosomiasis liver fibrosis.

Keywords: HSCs; Ly6Clo; liver fibrosis; miR-130a-3p; schistosomiasis.

Figure 1

Validation of the levels of miR-130a-3p in the livers of fibrotic mice and in the serum of fibrotic patients. The levels of miR-130a-3p in the liver of fibrotic mice and in the serum of cirrhosis patients were analyzed by quantitative real-time PCR (qRT-PCR). The expression of miR-130a-3p in S. japonicum-infected mice liver (A) and in serum of cirrhosis patients (B). *p < 0.01 and ***p < 0.001 (n = 4 each group).

Figure 2

LV-miR-130a-3p alleviated the pathological progression of liver fibrosis in S. japonicum infection. The mice were infected with 14 ± 1 cercariae of S. japonicum through abdominal skin in different groups. We injected LV-miR130a-3p, LV-NC, or PBS into the mice through the tail vein. After 72 h, the distributions of green fluorescent protein (GFP) in the heart, liver, spleen, lung, and kidney were detected by In Vivo Imaging System (A). After 8 weeks, the serum and the liver of different groups were collected. The photos of mice livers were captured (B). Eggs in the liver were counted (C). Liver tissue section was stained with H&E and the original magnification of stained liver sections was 100×. The granulomas were showed with quantitative analysis (D, E). Liver tissue section was stained with Masson, and the original magnification of stained liver sections was 100×. The fibrosis was showed with quantitative analysis (F, G). Sera HA was assayed by enzyme-linked immunosorbent assay (H). Liver HYP was checked by the alkaline lysis method (I). Data represent mean ± SD from multigroup experiments. **p < 0.01 and ***p < 0.001 (n = 4 each group). ns, no significance.

Figure 3

LV-miR-130a-3p decreased the levels of Col I by reduced expression of proinflammatory factor. The α-SMA protein level in the liver was evaluated by IHC with quantitative analysis of optical density (A, B). The Col I protein level in the liver was evaluated by IHC with quantitative analysis of optical density (C, D). Liver RNA was extracted and the expressions of α-SMA and Col I were analyzed by qRT-PCR (E, F). Liver RNA was extracted and the expressions of proinflammatory factor, TGF-β, IL-4, IL-13, MMP2, and TIMP1were analyzed by qRT-PCR (G–K). Data represent mean ± SD from multigroup experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (n = 4 each group). ns, no significance.

Figure 4

LV-miR130a-3p drove liver macrophages to Ly6C^lo skewing. Macrophages were isolated from the liver in S. japonicum-infected mice from different groups by density gradient centrifugation and then were purified according to the surface marker of F4/80 and CD11b by FCM. The expressions of Ly6C on the LV-miR130a-3p-M surface were respectively detected by FCM (A, B). The liver macrophage RNA was extracted to analyze inflammatory chemokines (C-F). The liver macrophage RNA was extracted to analyze chemokines, CCL2, CXCL2, CCL3, and CCL4 (G–J). Data represent mean ± SD from multigroup experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (n = 4 each group). ns, no significance.

Figure 5

Overexpression of miR130a-3p reduces the activation of JS1 and the expression of fibrogenic factors. JS1 cells were transfected with Agomir-130a-3p/Agomir NC or Antagomir-130a-3p/Antagomir NC (A). The transfected JS1 cells were collected after 72 h; the expression of miR-130a-3p was validated by qRT-PCR (B). The transfected JS1 were collected after 72 h to assay the protein level of α-SMA and Col I by WB (C), and the mRNA levels of α-SMA, Col I, IL-13, and TGF-β1 in the gene level were analyzed by qRT-PCR (D–K). Data represent mean ± SD from multigroup experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (n = 4 each group). ns, no significance.

Figure 6

Overexpression of miR-130a-3p inhibited the proliferation and growth, and induced apoptosis of JS1. The proliferation of transfected JS1 cells was tested at 0, 24, 48, 72, and 96 h by CCK8 assay (A). The transfected JS1 cells were collected after 72 h; cell apoptosis was assessed by FCM (B–E). Data represent mean ± SD from multigroup experiments. *p < 0.5 (n = 4 each group). **p < 0.01; ns, no significance.

Figure 7

miR-130a-3p target genes prediction. Venn diagram showing the overlap of the miR-130a-3p regulated target genes in the following five algorithms: TargetScan, mirDIP, miRDB, mirTar, and mirDIP (A). JS1 cells were transfected with the Agomir-130a-3p/Agomir NC, Antagomir-130a-3p/Antagomir NC for 72 h; the protein and mRNA expression of ERK1/2, TGFBR1, and TGFBR2 were detected by WB and qRT-PCR, respectively (B, C). The binding sites of miR-130a-3p in 3′ UTR of MAPK1, TGFBR1, and TGFBR2 were predicted (D). The wild-type and mutants 3’ UTR of MAPK1, TGFBR1, and TGFBR2 were cloned into pmirGLO, dual-luciferase reporter assay performed on 293 T cells transfected with MAPK1, TGFBR1, and TGFBR2 UTR reporter plasmid together with Agomir-130a-3p or Agomir N (E). **p < 0.01 and ***p < 0.001 (n = 4 each group).

Figure 8

The graphical abstract of probable cellular and molecular mechanisms by which miR-130a-3p delivery mitigates liver fibrosis of schistosomiasis. Overexpression of miR-130a-3p increases HSC apoptosis and reduces proliferation and activation of HSCs. Moreover, miR-130a-3p could also upregulate the expression of Ly6C^lo macrophages, concomitant with decreased expression of TIMP1, and increased expression of MMP2, contributing to the dissolution of collagen.