MicroRNA-29b-3p prevents Schistosoma japonicum-induced liver fibrosis by targeting COL1A1 and COL3A1.

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, causing granulomatous inflammation and cumulative fibrosis. This study explored in vivo and vitro effects of miR-29b-3p in granulomatous liver fibrosis by targeting COL1A1 and COL3A1 in Schistosoma japonicum infection. Thirty male Balb/c mice were assigned to normal control and model (percutaneous infection of cercariae of S. japonicum) groups. NIH-3T3 mouse embryonic fibroblasts were designated into blank, NC, miR-29b-3p mimic, TGF-ß1, TGF-ß1 + NC, and TGF-ß1 + miR-29b-3p mimic groups. HE and Masson staining were employed to observe the pathological changes and collagenous fibrosis. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was determined by immunohistochemistry. The RT-qPCR, Western blotting and immunofluorescence staining were conducted to determine expression of miR-29b-3p, COL1A1, and COL3A1. CCK-8 assay and flow cytometry were performed to evaluate viability and apoptosis. The relative expression of miR-29b-3p decreased in the model group. The model group showed marked fibrosis in liver tissues. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was higher in the model group than that in the normal control group. Dual luciferase reporter gene assay revealed that miR-29b-3p directly targeted COL1A1 and COL3A1. Compared with the blank, NC, TGF-ß1 and TGF-ß1 + NC groups, the miR-29b-3p mimic group exhibited up-regulated expression of miR-29b-3p and MMP-9 but down-regulated expression of TIMP-1, HSP47, α-SMA, COL1A1, and COL3A1; while lower cell viability but higher apoptosis rate showed. It indicated that miR-29b-3p prevents S. japonicum-induced liver fibrosis by inhibiting COL1A1 and COL3A1.

[Construction of HSP47-shRNA and its impact on HSP47 at cellular level].

OBJECTIVE: To construct a short hairpin RNA (shRNA) against HSP47 gene, assess the expression level of HSP47 gene in NIH/3T3 cells, and observe the influence on cell function. METHODS: The HSP47-shRNA sequence presented at the downstream of the U6 promoter. The shRNA expression constructs were created using PCR- based methods. The PCR product was digested with Nhe I/Hind Ill and ligated into pGCsi/U6/Neo vector to produce HSP47-pGCsi-U6-shRNA (HSP47-1-pGCsi-U6-shRNA, HSP47-2-pGCsi-U6-shRNA and HSP47-3-pGCsi-U6-shRNA). The non-interference vector and non-related interference vector served as control. The vectors were transfected into NIH/3T3 fibroblast cells by liposome mediated gene transfection method. Transfection efficiency and fluorescence intensity were determined by fluorescence microscopy at 12, 24, 48, and 72 hours after transfection, respectively. Cells were collected before transfection, and at 24, and 48 hours post-transfection, respectively, HSP47 mRNA and protein expression levels were assessed by real-time PCR and Western blotting. The mRNA expression of TGF-pl, collagen types I and Ill in NIH/3T3 cells, and TGF-beta1 levels in cell culture supernatant were determined. RESULTS: HSP47-shRNA vector was transfected into NIH/3T3 cells by liposome-mediated transfection. The transfection efficiency in each HSP47-shRNA plasmid interference group was about 60.0%, and there is no statistical difference among the interference groups (P> 0.05). A small amount of green fluorescent cells were found at 12 h post-transfection. The number of green fluorescent cells increased with the transfection time, and reached strongest at 72 h after transfection. shRNA interference significantly inhabited HSP47 expression in NIHI/3T3 cells. At 24 h after transfection with HSP47-1-shRNA, the inhibition effect was the strongest, and the relative silence efficiency of HSP47 mRNA was (25.83?1.79)%, lower than that of control group and non-related group (P<0.05). Collagen synthesis and secretion by NIH/3T3 cells reduced significantly at 24 and 48 hours post-transfection with HSP47-1-shRNA; and there was a significant difference between HSP47-1-shRNA intervention group and non-related controls. After transfection for 24 and 48 h, mRNA expression of collagen types I and IlI decreased to (56.52 +/- 1.64)% and (53.48 +/- 2.54)%, (54.17 +/- 2.63)% and (50.21 +/- 2.34)%, respectively, significantly lower than that of the control group and non-related group (P<0.05); however, no significant difference was found among the interference groups (P>0.05). In each HSP47-shRNA plasmid interference group, TGF-p1 mRNA expression was the lowest at 24 h post-transfection. The relative mRNA expression level was (63.23?2.18)%, (64.5+3.17)%, and (75.19 +/- 4.20)% in HSP47-1-shRNA, HSP47-2-shRNA, HSP47-3-shRNA groups (P>0.05), respectively, lower than that of the control group and non-related group (P<0.01). At 24 and 48 h post-transfection, TGF-P131 expression was the lowest at 24 h post-transfection, and the relative expression level in HSP47-1-shRNA, HSP47-2-shRNA, HSP47-3-shRNA groups was (51.79 +/- 3.12)%, (66.67 +/- 2.13)%, and (69.61 +/- 3.65)%, respectively. Compared with control group, the expression of TGF-beta1 in HSP47-1-shRNA and HSP47-2-shRNA2 groups was significant inhibited, but there was no significantly difference between control group and HSP47-3-shRNA group (P>0.05). CONCLUSION: HSP47-shRNA. interference plasmid is constructed. HSP47-shRNA effectively inhibits protein expression of HSP47, and results in changes of cell function.

Involvement of heat shock protein 47 in Schistosoma japonicum-induced hepatic fibrosis in mice.

Chronic infection with the blood fluke Schistosoma japonicum is associated with both liver cirrhosis and liver cancer. Previously, heat shock protein 47, a collagen-specific molecular chaperone, was shown to play a critical role in the maturation of procollagen. However, less is known about the role of heat shock protein 47 in S. japonicum-induced hepatic fibrosis. We therefore investigated the expression of heat shock protein 47 in S. japonicum-induced liver fibrosis and attempted to determine whether inhibition of heat shock protein 47 could have beneficial effects on fibrosis in vitro and in vivo. In this study, we found that the expression of heat shock protein 47 was significantly increased in patients with Schistosoma-induced fibrosis, as well as in rodent models. Immunohistochemistry revealed heat shock protein 47-positive cells were found in the periphery of egg granulomas. Administration of heat shock protein 47-targeted short hairpin (sh)RNA remarkably reduced heat shock protein 47 expression and collagen deposition in NIH3T3 cells and liver tissue of S. japonicum-infected mice. Life-table analysis revealed a dose-dependent prolongation of survival rates with the treatment of heat shock protein 47-shRNA in murine fibrosis models. Moreover, serum alanine aminotransferase and aspartate transaminase activity, splenomegaly, spleen weight index and portal hypertension were also measured, which showed improvement with the anti-fibrosis treatment. The fibrosis-related parameters assessed were expressions of Col1a1, Col3a1, TGF-ß1, CTGF, IL-13, IL-17, MMP-9, TIMP-1 and PAI-1 in the liver. This study demonstrated that heat shock protein 47-targeted shRNA directly reduced collagen production of mouse liver fibrosis associated with S. japonicum. We conclude that heat shock protein 47 plays an essential role in S. japonicum-induced hepatic fibrosis in mice and may be a potential target for ameliorating the hepatic fibrosis caused by this parasite.

[Expression of transforming growth factor-beta1 and connective tissue growing factor in schistosomal hepatic fibrosis of mice].

OBJECTIVE: To establish the model of hepatic fibrosis in mice infected with Schistosoma japonicum and observe the expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growing factor (CTGF) in mice model. METHODS: Thirty BALB/c mice were randomly assigned to control group and model group. Each mouse of model group was infected with (30 +/- 1) S. japonicum cercariae through the abdominal skin. Serum samples were collected at 4, 6, 8, 10, and 12 weeks after infection, and were analyzed for the levels of ALT and AST. Pathological changes and proliferation of hepatic collagen fibers in liver tissue were observed after HE staining and Masson staining. Immunohistochemistry and real-time PCR were used to detect the protein and mRNA expression of CTGF and TGF-pl. RESULTS: 6-12 weeks after infection, there was significant difference in ALT and AST between model group and control group (P43.05). At 12th week, ALT [(173.53 +/- 31.12) U/I] and AST [(301.00 +/- 34.87) U/LI in model group were higher than those in control group [(42.00 +/- 3.53) and 96.58 +/- 11.26) U/L] . In model group, egg granulomas formed in the liver, and the formation of hepatic fibrosis was significant in portal areas, and there was tree-like hepatic fibrosis around the portal vein branch. 8 weeks after infection, hepatic fibrosis area in mice of model group increased considerably, and there was significant difference in percentage of positive area of collagen between 12th week [(23.83 +/- 1.68) %] and control group [(1.23 +/- 0.14) %] (P < 0.05). 10 and 12 weeks after infection, the percentage of positive area of TGF-beta1 [(22.34 +/- 2.58)% and (25.82 +/- 3.01) %] and CTGF [(1 l.32 +/- 2.44)% and (14.51 +/- 2.05) %] was higher respectively than that of the control [(2.56 +/- 0.87)%, and (1.09 +/- 0.73)% (P < 0.05). 6, 8, 10, and 12 weeks after infection, both TGF-beta1 and CTGF mRNA increased gradually, higher than that in control group (P < 0.05). 10 weeks after infection, TGF-beta1 mRNA relative transcription level was the highest (0.0721 +/- 0.0187) and it was 0.0089 +/- 0.0037 in control group. CTGF mRNA relative transcription level reached the highest value (0.1136 +/- 0.0365) in 12 weeks after infection, while it was 0.0293 +/- 0.0184 in control group. CTGF mRNA expression was positively correlated with the duration of infection (r = 0.927, NO.05). CONCLUSION: The area and cell types of TGFb1 positive expression is the same as that of CTGF in liver tissue of schistosome infected mice (BALB/c). CTGF mRNA expression is significantly related to the duration of infection, but it is not the case for TGFbl.