The Effects Of MicroRNA Let-7b On Schistosoma Liver Fibrosis And Its Preliminary Mechanism

[Objective]In this research,we upregulated the expression of microRNA let-7b in vivo and in vitro,then explore the effect of let-7b on regulation of T?RI and its anti-fibrosis effect and possible mechanism in Schistosoma japonicum-infected mice.[Methods]To identify the roles of let-7b on the T?RI in vitro,let-7b mimics,let-7b mimic negative control,let-7b inhibitor mimics,let-7b inhibitor negative control or PBS were transiently transfected into THP-1 cells,respectively.QRT-PCR and western blot determined the mRNA and protein expression of T?RI.We conducted the recombinant Lentivirus?Lenti-let-7b?and the negative control Lentivirus?Lenti-NC?by lentivurus recombination technique.Mice were divided into 4 groups,named as uninfected group,PBS group,Lenti-let-7b group and Lenti-NC group.To construct the models of Schistosoma liver fibrosis?SLF?,the mice of PBS,Lenti-NC and Lenti-let-7b were percutaneously infected with 15±2 cercariae of S.japonicum.The mouse was percutaneously infected with 15±2 cercariae of S.japonicum.Mice were injected PBS or Lenti-let-7b?1×10⁹ TU/mL,150?L?or Lenti-NC(1×10⁹ TU/mL,150?L)via the tail vein after S.japonicum infection for 10 days,and sacrificed to harvest the livers and serum samples on week 4,6,8 and 10 after infection.The expression of hepatic let-7b,profibrogenic markers and inflammatory cytokines including transforming growth factor-?1?TGF-?1?,TGF-?receptor I?T?RI?,?-SMA,colla I,IL-4 and IFN-?were detected by qRT-PCR.IFN-?and IL-4 level of serum among different groups and its dynamic change were detected by ELISA.Western blot analyzed the protein level of T?RI.The deposition of S.japonicum egg granulomas and collagenous fiber was demonstrated by H&E and Masson staining.Quantitative analysis revealed that the contents of hepatic hydroxyproline and alanine aminotransferase?ALT?of serum.[Results]The results in cells experiment showed that over-expression of let-7b down-regulated the expression of T?RI in THP-1 cells transfected with let-7b mimics,while T?RI was up-regulated after treated with let-7b inhibitor?P<0.05?.The results in animal experimen showed that the expression level of let-7b in the S.japonicum-infected liver samples decreased on week 2,4,6,8 and10,and the value was 0.91,0.59,0.48,0.13,0.08 respectively.After treated with Lenti-let-7b by tail injection on day 10 post-infection,the expression of let-7b was significantly increased on week 4,6,8 and 10post-infection,and the value was 11.30,5.39,3.93,2.55 respectively?P<0.05?.Pro-fibrotic genes test showed that the fibrosis gene including TGF-?1,T?RI,colla I and?-SMA were all increased in S.japonicum-infected liver in the group of PBS and Lenti-NC,compared with uninfected?P<0.05?,while the expression level of fibrosis gene of Lenti-let-7b group were significantly reduced,compared with the group of PBS and Lenti-NC?P<0.05?.H&E and Masson staining showed that the deposition of japonicum egg granulomas and collagenous fiber in the livers was decreased after 6w infection.While Lenti-let-7b-mediated over-expression of let-7b could reduce the morphology changes of livers,compared to the groups of PBS and Lenti-NC.The results of quantitative analysis showed that the level of hydroxyproline was increased in S.japonicum-infected liver in the group of PBS and Lenti-NC,compared with uninfected?P<0.01?,and the level was increased with the progress of infection.Upregulation of let-7b significantly decreased hydroxyproline content in liver on week 6,8 and 10 after infection.The level of ALT in serum was increased on week 4 and 6 post-infection,while the level was decreased with the progress of infection.The level of ALT of Lenti-let-7b group was significantly reduced on week 4,6 after infection,compared with PBS and Lenti-NC group?P<0.05?.Cytokines test results showed that the level of IFN-?in the liver and serum was significantly higher on week 4 and 6 post-infection in PBS and Lenti-NC group?P<0.05?,the IFN-?reached peak value at 4w.The level of IL-4 in the liver and serum was significantly higher on week 4⁸ post-infection in PBS and Lenti-NC group,compared with the group of uninfected,the IL-4 reached peak value at 8w.The level of IL-4 of Lenti-let-7b group was reduced on week 4 and 8 after infection,compared with PBS and Lenti-NC group?P<0.05?.[Conclusion]Lentivirus-mediated elevates the expression of let-7b in liver can suppress liver fibrosis induced by S.japonicum.The mechanism may be related to the down-regulation of T?RI and the reduction of the level of pro-fibrotic factors by let-7b.