miR-182-5p attenuates Schistosoma japonicum-induced hepatic fibrosis by targeting tristetraprolin

Abstract

Egg granuloma formation in the liver is the main pathological lesion caused by Schistosoma japonicum infection, which generally results in liver fibrosis and may lead to death in advanced patients. MicroRNAs (miRNAs) regulate the process of liver fibrosis, but the putative function of miRNAs in liver fibrosis induced by S. japonicum infection is largely unclear. Here, we detect a new miRNA, miR-182-5p, which shows significantly decreased expression in mouse livers after stimulation by soluble egg antigen (SEA) of S. japonicum or S. japonicum infection. Knockdown or overexpression of miR-182-5p in vitro causes the increased or decreased expression of tristetraprolin (TTP), an important immunosuppressive protein in the process of liver fibrosis. Furthermore, knockdown of miR-182-5p in vivo upregulates TTP expression and significantly alleviates S. japonicum-induced hepatic fibrosis. Our data demonstrate that downregulation of miR-182-5p increases the expression of TTP in mouse livers following schistosome infection, which leads to destabilization of inflammatory factor mRNAs and attenuates liver fibrosis. Our results uncover fine-tuning of liver inflammatory reactions related to liver fibrosis caused by S. japonicum infection and provide new insights into the regulation of schistosomiasis-induced hepatic fibrosis.

Keywords: liver fibrosis; miR-182-5p; microRNA; tristetraprolin.

Figure 1

The expression of miR-182-5p following SEA, IFNγ stimulation and S. japonicum infection in vitro and in vivo (A) The expression of miR-182-5p in AML12 and primary hepatocytes exposed to SEA (AML12, 30 μg/mL, mouse primary hepatocytes, 60 μg/mL) for 12 and 24 h. (B) The expression of pre-miR-182 in HL7702 and U937 cells after exposure to SEA (30 μg/mL) for 12 and 24 h. (C) The expression of pre-miR-182 in mouse livers 4 and 8 weeks after S. japonicum infection. (D) The expression of miR-182-5p in mouse livers (GSE134866) and splenic B cells (GSE115443) 9 and 13 weeks after S. japonicum infection. (E) pri-miR-182 and pre-miR-182 expression in AML12 cells exposed to IFNγ for up to 8 h. (F) pri-miR-182 and pre-miR-182 expression in HL7702 cells exposed to IFNγ for up to 8 h. * P<0.05.

Figure 2

TTP expression is inhibited in cells transfected with siDicer and siDrosha in vitro (A) The efficiency of siRNA transection. (B) The mRNA level of TTP in cells transfected with siDicer and siDrosha. * P<0.05.

Figure 3

miR-182-5p regulates TTP expression in hepatocytes (A) The putative binding sites for miR-182-5p in TTP. (B) TTP mRNA and protein levels in cells transfected with miR-182-5p mimic. (C) TTP mRNA and protein levels in cells transfected with miR-182-5p inhibitor. * P<0.05.

Figure 4

IFNγ increases TTP expression in vitro (A) AML12 and HL7702 cells were exposed to IFNγ for up to 12 h, followed by real-time PCR. (B) AML12 and HL7702 cells were exposed to IFNγ for up to 48 h, followed by western blot analysis. * P<0.05.

Figure 5

miR-182-5p regulates TTP mRNA in a 3′UTR-dependent manner (A) HL7702 cells were cotransfected with pMIR-TTP-3′UTR, pMIR-TTP-∆3′UTR, pMIR-TTP-FL-3′UTR or pMIR-TTP-FL-∆3′UTR together with miR-182-5p mimics. (B) HL7702 cells were cotransfected with pMIR-TTP-3′UTR, pMIR-TTP-∆3′UTR, pMIR-TTP-FL-3′UTR or pMIR-TTP-FL-∆3′UTR together with miR-182-5p inhibitor. (C) The stability of TTP mRNAs was calculated in HL7702 cells transiently transfected with miR-182-5p mimics. Actinomycin D (Act D) was then added, and the cells were collected for real-time PCR. The stability of mRNAs is presented as the relative amount of mRNA to the control. * P<0.05. ns, no significance.

Figure 6

miR-182-5p influences the expression of fibrosis-related cytokines (A) The expressions of selected fibrosis-related cytokines in HL7702 cells transfected with miR-182-5p mimics or miR-182-5p inhibitor. (B) Real-time PCR for fibrosis-related cytokine mRNA in HL7702 cells transiently transfected with PHAGE-TTP and in stable TTP-knockdown HL7702 cells. (C) The protein levels of IL-6, IL-8, and TNFα were detected in HL7702 cells transiently transfected with miR-182-5p inhibitor and in stable TTP-knockdown HL7702 cells. (D) The expression level of TTP in U937 cells after exposure to SEA (30 μg/mL) for 24 h. * P<0.05.

Figure 7

miR-182-5p knockdown attenuates schistosomiasis-induced hepatic fibrosis (A) Schematic overview of parasite infection and administration of plasmid vectors and samples collected. (B) Real-time PCR analysis of the levels of mature miR-182-5p and western blot analysis of TTP expression in liver tissues. (C) Real-time PCR analysis of the levels of Col1α1, Col3α1, and α-SMA mRNA in liver tissues. (D) Masson’s trichrome staining and H&E staining of liver sections. Liver sections were immunohistochemically stained for α-SMA and COL1A1. Scale bar: 50 μm. (E) Granuloma size was measured by H&E staining and ECM deposits were detected by Masson’s trichrome staining in liver sections. * P<0.05.