| Until now,schistosomiasis is still a major infectious disease that seriously affects human health and economic development.Reportedly,in 2006,there were more than 200 million people infected with schistosomiasis.By 2014,the number of people infected had grown to 230 million.In China,only Schistosomiasis japonicum is prevalent.Granuloma and fibrosis caused by the deposition of Schistosomiasis japonicum eggs in the liver are the main pathological changes of schistosomiasis,but its pathogenesis is still complicated and unclear.Hepatic stellate cells(HSC)have been proved to be the main effective cells of various liver fibrosis diseases including schistosomiasis.Under the action of various pathological factors,static HSC can be transformed into muscle fibroblasts,which secrete a large amount of collagen in the liver tissue causing liver fibrosis.Inhibiting the activation of HSC is an important resolution for the prevention and treatment of liver fibrosis.MicroRNA(miRNA)is a class of endogenous non-coding single-stranded RNA molecules containing 21-23 bases.It is widely distributed in animals and plants,regulating gene expression at the transcriptional and post-transcriptional levels.miRNAs play important regulatory roles in the occurrence,development and transformation of various diseases.Our laboratory previously found that schistosoma infection up-regulated the expression of some host miRNAs(such as miR-21,miR-351,miR-203,etc)and regulated the activation of HSC and liver fibrosis of schistosomiasis.Recent studies have shown that miRNAs derived from plants or pathogens can regulate host cell genes expression and phenotypes in a transboundary or cross-species manner.Schistosoma japonicum as eukaryotic pathogen has a large number of miRNAs(called "sja-miRNAs").Studies have shown that during the infection of Schistosoma japonicum,and these sja-miRNAs can be secreted into the hosts' body fluids or enter the host cells through exosomes to regulate the host cell genes or phenotypes across substances.This subject is based on the scientific problem of the laboratory project: Schistosoma japonicum eggs are deposited in the liver,and live mites in the eggs can secrete or transport sja-miRNAs to adjacent host cells through exosomes,including HSC,regulating its activation and liver fibrosis.Previously,some research has been carried out in our laboratory.With sequencing and qPCR identification,some miRNAs from Schistosoma japonicum are found in the host cells,and the functions and mechanisms of Sja-miR-2162 in liver fibrosis have been illuminate.Based on this work,the following studies were mainly carried out in the following two projects:(1)Systematic identification of miRNAs entering HSC during schistosomiasis-infected mice by high-throughput sequencing and real-time PCR(qPCR);(2)Explore the role and molecular mechanism of the parasite-derived Sja-miR-1 in the process of liver fibrosis of schistosomiasis.The results are as follows:1.Identification of Schistosoma-derived miRNAs in primary HSC isolated from mice infected with Schistosoma japonicumIn order to identify the presence of Schistosoma-derived miRNA in Schistosoma-infected mice HSC,we infected BALB/c mice with Schistosoma japonicum cercariae.The mice were killed at 49 days post-infection and primary HSC were isolated to be used to high-throughput sequencing of miRNA.The sequencing results were compared with the miRNAs database of Schistosoma japonicum and mice respectively and miRNAs which sequences were completely matched with Schistosoma japonicum but not completely matched with mice were screened out,namely the Schistosoma-derived miRNA.A total of 27 Schistosoma-derived miRNAs were screened out.In order to verify the accuracy of sequencing results,including sja-bantam,sja-let-7,sja-miR-1,sja-miR-10-5p,sja-miR-125,sja-miR-125 b,sja-miR-190-3p,sja-miR-190-5p,sja-miR-2162-3p,sja-miR-2162-5p,sja-miR-2a-3p,sja-miR-2b,sja-miR-2e-3p,sja-miR-2f,sja-miR-3019,sja-miR-3045,sja-miR-3050,sja-miR-3103,sja-miR-3140,sja-miR-3144,sja-miR-3479-3p,sja-miR-3492,sja-miR-3496,sja-miR-36-3p,sja-miR-7-5p,sja-miR-71 a,sja-miR-71 b.Then we selected 9 Schistosoma-derived miRNAs for qPCR verification.Consistently,we did detect Schistosoma-derived miRNAs in HSC.2.The role and mechanism of sja-miR-1 in schistosomiasis liver fibrosis(1)The activation of HSC in vitro: We ligated the Sja-pri-miR-1 sequence to the adeno-associated virus vector pAV-MIR and constructed a Sja-miR-1 overexpression plasmid named pAV-pri-miR-1.To investigate whether Sja-miR-1 can activate HSC,we transfected pAV-pri-miR-1 plasmid into HSC-T6 cell line and human LX-2 cell line respectively,meanwhile empty vectors were used as control.48 h later after transfection,qPCR were used to detect the expression level of Col1?1,Col3?1 and ?-Sma in HSC.We found that the Col1?1,Col3?1 and ?-Sma levels significantly increased after transfection(p<0.05),indicating that Sja-miR-1 can activate HSC in vitro.(2)Liver fibrosis promoting effect in naive mice: In order to stably express Sja-miR-1 in the liver,we used the type 8 recombinant adeno-associated virus(rAAV8)to infect the liver.The pAV-pri-miR-1 plasmid and the empty vector were packaged and purified to produce recombinant plasmids,named rAAV8-pri-miR-1 and rAAV8-SCR.To observe the effect of liver after overexpressing of Sja-miR-1,we injected rAAV8-pri-miR-1 or equivalent rAAV8-SCR or equivalent PBS into the tail of BALB/c mice.50 days later,liver tissues and primary HSC were isolated and the expression levels of Col1?1,Col3?1 and ?-Sma were detected by qPCR.The hydroxyproline contents in liver tissues were also detected.The results showed that compared with the rAAV8-SCR and PBS groups,the mRNA levels of Col1?1,Col3?1 and ?-Sma in the liver tissues or HSC of the rAAV8-pri-miR-1 group mice were significantly increased(p<0.05).The hydroxyproline contents were also dramatically rising(p<0.05).These results indicate that overexpression of Sja-miR-1 in mouse liver can promote liver fibrosis.(3)Analysis of the roles of Sja-miR-1 in liver fibrosis of schistosomiasis:To investigate whether Sja-miR-1 plays practical roles in schistosomiasis liver fibrosis,we constructed a sponge plasmid that specifically adsorbs Sja-miR-1,namely the anti-miR-1 plasmid,using miRNA sponge principle.We injected rAAV8-anti-miR-1 into the BALB/c mice infected with Schistosoma japonicum to adsorb Sja-miR-1 in the liver cells,and killed the mice 56 days after infection to detect fibrosis in the liver and primary HSC.The results showed that the expression levels of Col1?1,Col3?1 and ?-Sma mRNA in rAAV8-anti-miR-1 group were significantly lower than those in rAAV8-anti-SCR and PBS control group(p<0.05).The hydroxyproline content can reflect the level of collagen in the tissue,and the hydroxyproline content of the rAAV8-anti-miR-1 group was significantly reduced compared with the control group(p<0.05).We analysized of the areas of collagen tissue and the sizes of egg granuloma in Masson staining and HE staining.The results showed that both indexes were significantly reduced after inhibiting Sja-miR-1.In addition,we performed immunohistochemical detection of HSC-activated marker ?-SMA and found that the positive rate of HSC activation in the liver of rAAV8-anti-miR-1 group was significantly declined(p<0.05).These results indicate that the inhibition of Sja-miR-1 in HSC can significantly reduce the HSC activation and severity of fibrosis during schistosomiasis infection,suggesting that the Schistosoma-derived Sja-miR-1 plays roles in promoting liver fibrosis.(4)Schistosoma japonicum egg-derived exosomes can activate HSC in vitro: To investigate the possible mechanisms of how Sja-miR-1 is transferred into HSC,we used PKH67 to label the egg-derived exosomes and added them to HSC in vitro to study its roles in transmitting miRNAs and activating HSC.We found that the egg-derived exosomes can emerge with HSC in vitro and thus deliver Sja-miR-1 to HSC.More importantly,the exosomes can promote the expression of fibrosis-related genes in HSC.This promotion can be removed by Sja-miR-1 inhibitor,indicating that Sja-miR-1 is pertinent to the process of exosomes activating HSC.(5)Target gene of Sja-miR-1: We predicted that the secreted Frizzled-related Protein 1(Sfrp1)was the target gene of Sja-miR-1 in mice through miRDB database and pathway analysis,and the targeting relationship was verified through related experiments.Firstly,we used the dual luciferase reporter gene system to confirm that Sja-miR-1 can target the 3'UTR of Sfrp1.Then,we examined the changing relationship of Sja-miR-1 and Sfrp1 in mice HSC during Schistosoma japonicum infection.The results showed that the expression level of Sja-miR-1 was significantly elevated in HSC of infected mice,while the expression level of Sfrp1 was significantly decreased,showing an significant negative regulatory relationship.In addition,Sfrp1 was significantly reduced in both gene level and protein level after transfection of primary HSC in vitro with Sja-miR-1 mimics.When the Sja-miR-1 of HSC was adsorbed in schistosomiasis-infected mice,the reduced expression level of Sfrp1 caused by infection could be partially reversed,which was significantly higher than that of the control group.These results indicate that Sja-miR-1 can target the Sfrp1 in host cells.(6)Signaling pathway participates in the activation of HSC by Sja-miR-1: Studies have found that the Wnt/?-catenin pathway is closely related to liver fibrosis.Sfrp1 is an inhibitor of Wnt/?-catenin pathway,and inhibition of Sfrp1 expression can activate this pathway.To investigate whether Sja-miR-1 activating of HSC is associated with this pathway,we first transfected Sfrp1 siRNA in primary HSC.Similarly,interfering Sfrp1 expression could also activate HSC.Western blot analysis showed that HSC transfected with Sja-miR-1 mimics or Sfrp1 siRNA could activate Wnt/?-catenin pathway,suggesting that Sja-miR-1 may activate Wnt/?-catenin pathway by inhibiting Sfrp1.We also tested the mice infected with Schistosoma.Compared with the uninfected group,we found that the Wnt/?-catenin pathway was activated in HSC of mice infected with Schistosoma.After inhibiting Sja-miR-1 in HSC,Wnt/?-catenin pathway was also partially inhibited.In summary,based on the previous experimental results,this study identified the Schistosoma-dervied miRNAs in HSC of mice infected with Schistosoma,and explored functions and mechanisms of Sja-miR-1.On the one hand,Sja-miR-1 can enter the host HSC during Schistosoma infection,a process that may require the involvement of exosomes.On the other hand,we confirmed that Sja-miR-1 in the host HSC participates in the development of schistosomiasis-associated liver fibrosis by activating Wnt/?-catenin.Schistosomiasis is still a major parasitic disease that endangers human beings.The results of this study indicate that Schistosoma-dervied Sja-miR-1 could enter the host HSC during the process of schistosome infection,and promote liver fibrosis in mice by targeting Sfrp1 gene in a cross-species manner.At the same time,this study confirms that Sja-miR-1 plays important roles in liver fibrosis in schistosomiasis,which broadens our understanding of the pathogenesis of schistosomiasis and provides new targets and pathways for treating liver fibrosis. |
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