| At present,schistosomiasis is still a serious impact on China's economic and social development and people's health a major parasitic disease.The main lesion of schistosomiasis japonica is granuloma and fibrosis caused by eggs.Although the pathogenesis of schistosomiasis has not yet been fully elucidated,some studies have suggested some of the effectic cells and effectors that may be involved in the process.Schistosomiasis is an immunopathogenic disease.During the initial stage of schistosomiasis,the Th1 immune response?marked with an increase in IFN-??is moderately activated to protect the migratory parasite in the host and in the mesenteric vein Mature cells;with the eggs deposited in the organization,the host immune response quickly transformed into Th2 immune response?IL-4 and IL-13 increased as a sign?,these cytokines on the occurrence and development of schistosomiasis decisive effect.In the human and mouse schistosomiasis-related hepatic fibrosis model,it has been confirmed that hepatic stellate cells?HSC?are the main effectic cells of schistosomiasis-related liver fibrosis.In view of the central role of HSC in the development and progression of hepatic fibrosis,HSC has become the main target cell for the prevention and treatment of various hepatic fibrosis diseases including schistosomiasis-related liver fibrosis,and inhibition of HSC activation is the main idea of the development of prevention and treatment of hepatic fibrosis.Although studies have initially elucidated the effectic cells and effector factors involved in schistosomiasis,it is not clear how schistosomiasis?egg?act on effectic cells and how to regulate their signaling and corresponding molecular mechanisms at the source.MicroRNA?miRNA?is an endogenous small molecule RNA that has a wide range of functions.In recent years,miRNA has made important progress in the study of the role and mechanism of miRNAs in a variety of human diseases,including infectious diseases.The pathogenesis of schistosomiasis and its mechanism have been carried out in the early stage of this laboratory.TGF-?1 and IL-13 can promote the expression of miR-21 in HSC by activating SMAD signaling pathway,and miR-21 can be further targeted by targeting SMAD7 by using in vivo and in vitro experiments with schistosomiasis-related liver fibrosis mice as an animal model Amplify the SMAD signaling pathway,thereby promoting the formation of hepatic fibrosis.In addition,the laboratory first reported it existd miRNA in schistosomiasis pathogens.A large number of studies have confirmed that parasite-derived miRNA widely involved in the growth and development of parasites and other control.In recent years,studies have shown that schistosomiasis miRNAs can enter the host cells through exosomes,but whether these miRNAs can participate in pathogenic effects on the host has not been reported so far.In this study,we used the HSC cell line model to synthesize and transform mimics of miRNAs,and screened and identified a large number of insect miRNAs that could activate HSC cells and up-regulate the expression of fibrosis-related markers.At the same time,we isolated the original HSCs infected with schistosomiasis mice and identified the insect miRNAs present in HSC by high throughput sequencing.Based on the in vitro activation of HSC-T6 screening and high-throughput sequencing of host HSC to identify insect miRNAs and their specificity,We selected the miRNA?Sja-miR-2162?,which was involved in both hepatic fibrosis and host HSC,as the subject of this study to study its role in the regulation of host liver fibrosis and its molecular mechanisms,including:in vitro HSC to determine its activation of HSC and promote the role of fibrosis;through the normal mice Expressing miRNAs in the infected mice or interfering with insecticidal miRNAs in infected mice to detect the degree of hepatic fibrosis and the activation status of HSC in mice to determine the function of insect miRNAs in schistosomiasis-related liver fibrosis;Source miRNAs and target genes of miRNAs were identified by in vivo and in vitro experiments to elucidate the molecular mechanism of parasite-derived miRNAs in the regulation of schistosomiasis-related liver fibrosis.This study has achieved the following results:1.Screening and identification of S.japonicum miRNAsIn this study,51 miRNAs of S.japonicum were identified in this laboratory and other miRNAs were synthesized and identified.The miRNA mimics were synthesized and identified in this study.HSC-T6 was used as an in vitro cell model.The expression of HSC activation marker gene?Col1?1,Col3?1 and?-SMA?was detected by qPCR technique after 48 hours.The results showed that there were 19 parasite-derived miRNAs with significant increased,and 4 parasite-derived miRNAs were a significant decrease expression.Of the 23 parasite-derived miRNAs,15 were miRNA-specific miRNAs and 8were conserved miRNAs.These results indicate that parasite-derived miRNAs can regulate the activation of host HSC.2.Isolation of HSC from infected mice and detection of parasite-derived miRNAs?1?separation and detection of primary HSCThe primary HSCs were isolated from the first 42,49,and 56 days after infection by density gradient centrifugation.The HSC of the normal mice at the 42th and 56th day of the control group was also isolated.In order to clarify that these isolated mouse primary HSCs were not contaminated with S.japonicum eggs or their debris,we selected two genes?AY223149 and AY812797?specific for ovulation of Schistosoma japonicum,and the two genes were detected by qPCR Expression in two sets of HSC RNA samples.The results showed that neither of these two genes were detected in RNA samples of normal and infected mice with HSC,indicating that the isolated primary HSC was not contaminated by S.japonicum eggs.?2?Second generation sequencing analysis of infected mice miRNAs in HSCThe miRNAs were constructed using the two sets of HSC RNA samples,and the second generation sequencing was performed.The miRNA sequences obtained from the sequencing were compared with the existing host or S.japonicum miRNA data to determine whether miRNAs of S.japonicum were present in the host HSC cells infected with S.japonicum.The criteria for determining the source of S.japonicum miRNAs are that there are three or more bases different from all known miRNA sequences in mice and are fully paired with known S.japonicum miRNA sequences.According to the above criteria,8 S.japonicum miRNAs were identified in the total RNA samples of HSC after infection.Sja-bantam,Sja-miR-190-5p,Sja-miR-125b,Sja-miR-2162-3p,Sja-miR-2a-3p,Sja-let-7,Sja-MiR-10-5p and Sja-miR-1.In addition,we analyzed the conservativeness of miRNAs and host miRNAs in these 8 miRNAs.The results showed that five of them were conserved insect miRNAs,which were Sja-miR-190-5p,Sja-miR-125b,Sja-let-7,Sja-miR-10-5p,Sja-miR-1;the remaining 3 were specific S.japonicum miRNAs,Sja-bantam,Sja-miR-2162-3p and Sja-miR-2a-3p,respectively.We further used qPCR technology to verify the above sequencing results.The results showed that 8 miRNAs were not detected in HSC samples of uninfected mice,and 8 insect miRNAs could be stably detected in HSC samples.The abundance is similar to the abundance of mmu-miR-203 Or mmu-miR-96.These results indicated that some S.japonicum miRNAs can indeed enter the host cell during S.japonicum infection.3.Sja-miR-2162 promotes the activation of HSC and the occurrence of hepatic fibrosisThrough the comprehensive analysis of the above experimental results,this study selected Sja-miR-2162 as the object of study,its role in the host liver fiber and its molecular mechanism.?1?Construction and expression of over-expression plasmid of Sja-pri-miR-2162In order to study the function of promoting the fibrosis of Sja-miR-2162,we constructed a recombinant plasmid of Sja-miR-2162 eukaryotic expression.The primers were designed on the Sja-pri-miR-2162 gene and the genomic DNA of S.japonicum was used as template.The gene fragment was amplified by PCR and ligated to the skeleton of recombinant adeno-associated virus?rAAV?On the plasmid.The recombinant plasmid was transfected into HSC-T6 cell line.After 48 hours,the expression of Sja-pri-miR-2162,Sja-pre-miR-2162 and Sja-miR-2162 were detected by qPCR technique,Schistosome eggs RNA as a positive control..The results of qPCR showed that the expression of Sja-pri-miR-2162,Sja-pre-miR-2162 and Sja-miR-2162 could be stably detected in the RNA samples of S.japonicum,and the expression abundance was Sja-pri-miR-2162,Sja-miR-2162,Sja-pre-miR-2162;primary HSC RNA samples transfected with Sja-pri-miR-2162 recombinant plasmid can also be used to detect the expression of the three Abundance from high to low were Sja-pri-miR-2162,Sja-pre-miR-2162 and Sja-miR-2162.These results indicate that Sja-pri-miRNA gene can be stably expressed in host cells and can be cleaved into functional mature miRNAs.?2?Verification of the effect of Sja-miR-2162 on HSC activation in vitroIn order to further validate the function of Sja-miR-2162 in vitro,we isolated normal mouse primary HSCs and cultured in vitro.After transfection of Sja-miR-2162 mimics or pAV-sja-pri-miR-2162 plasmid.The expression levels of Col1?1,Col3?1 and?-SMA were detected by qPCR after 48 hours.Sja-pri-miR-2162,Sja-pre-miR-2162 and Sja-miR-2162were stably detected in HSC transfected with the rAAV8-Sja-pri-miR-2162 plasmid group compared to the control group.More importantly,the expression of Col1?1,Col3?1 and?-SMA was significantly increased?p<0.001?,and the expression of Col1?1,Col3?1 and?-SMA in primary HSC was transfected with Sja-miR-2162 mimics?p<0.001?,which indicated that the rAAV8-Sja-pri-miR-2162 plasmid could be successfully expressed in primary HSC,which was processed into mature miRNA and up-regulated the expression of fibrosis-related marker gene.In addition,the constructed rAAV8-Sja-pri-miR-2162plasmid was transfected into HSC-T6 cell line in vitro.The expression of Col1?1,Col3?1and?-SMA was also significantly up-regulated by qPCR?p<0.01?.?3?Sja-miR-2162 induced liver fibrosis in miceTo observe whether Sja-miR-2162 induces BALB/c mouse liver fibrosis in vivo,we constructed a rAAV8 vector that expresses Sja-miR-2162.The viral vector is highly hepatophilic and can express foreign genes continuously in hepatocytes.In order to optimize the dose and time of rAAV8 vector injection,we first designed experiments at different dosages and time points.Dose gradient:2×109 v.g.,2×10100 v.g.and 2×1011v.g.;Infection time:20,30 and 40 days after infection,rxAV8 virus group and PBS group with 2×10100 v.g.dose as control group.?Col 1?1,Col 3?1 and?-SMA?were detected in the liver samples of mice in different doses of different days.The results showed that mice were treated with liver fibrosis at a dose of 2×10111 v.g./on the 40th day after infection with Sja-miR-2162 virus.At the same time,we used qPCR to detect the expression of Sja-pri-miR-2162,Sja-pre-miR-2162 and Sja-miR-2162 in the liver tissues of mice in each dose group at 40 days after infection,Schistosome eggs RNA as a positive control.The results showed that Sja-pri-miR-2162,Sja-pre-miR-2162 and Sja-miR-2162 could be stably detected in the RNA samples of the control group,and the expression abundance was high Sja-pri-miR-2162,Sja-miR-2162 and Sja-pre-miR-2162;and in mice infected with rAAV8-Sja-pri-miR-2162 virus,And the expression abundance of the three increased with the increase of the infection dose.Sja-pri-miR-2162,Sja-pre-miR-2162 and Sja-miR-2162 were the highest in the 2×10111 v.g.group.These results indicate that the insect-derived Sja-pri-miRNA sequence can be stably expressed in the host and can be cleaved into functional mature miRNAs.Seven weeks old of BALB/c male mice were injected with 2×10111 v.g.virus through the tail vein according to the optimized dose and time of the rAAV8 vector.The mice were sacrificed on the 40th day after infection.The expression of Sja-pri-miR-2162,Sja-pre-miR-2162 and Sja-miR-2162 was detected by qPCR technique,indicating that Sja-pri-miR-2162 carried by the virus vector could be expressed in the host liver Cells are expressed and can be processed to cut into functional Sja-miR-2162.In addition,the qPCR results showed a significant increase in hydroxyproline levels?p<0.001?in liver samples infected with rAAV8-Sja-pri-miR-2162 virus compared with the control group,and liver fibrosis The expression levels of related genes such as Col1?1,Col3?1,TIMP and Col4?1were significantly increased?p<0.001?,Masson trichrome staining showed that there was significant collagen deposition in mouse liver tissue on 100th day after infection with rAAV8-Sja-pri-miR-2162.The levels of Col1?1,Col3?1 and Col4?1 were significantly increased in HSCs expressing rAAV8-Sja-pri-miR-2162 virus vector infection group?p<0.01?.4.Neutralization of Sja-miR-2162 inhibits liver fibrosis of schistosomiasisThe above experiments have shown that Sja-pri-miR-2162 produced by S.japonicum can secrete to the host cells,including the liver HSC.Sja-pri-miR-2162 into HSC was able to upregulate the expression of hepatic fibrosis-associated marker genes,leading to fibrosis.The main pathological changes of S.japonicum infection are the occurrence of hepatic fibrosis.Previous work has shown that S.japonicum infection induced some of the factors produced by the host,including host miRNAs and IL-13,which are the major contributors to host liver fibrosis.Based on the above results,we propose that the S.japonicum miRNA?Sja-miR-2162?plays a role in promoting hepatic fibrosis in the pathogenesis of S.japonicum and host liver fibrosis.In this regard,the subject design of the following experiments to be verified.?1?The validation in cell model in vitro:Sja-miR-2162 was secreted by the eggs into HSC and regulated fibrosis-related gene expressionIn order to verify that Sja-miR-2162 secreted by S.japonicum can enter the host HSC cells and play a role,we designed an simulated environmental model of eggs and HSCs in vivo:the primary HSC was cultured in the lower layer of the Transwell chamber and the freshly isolated live eggs were cultured in the upper layer.After 48 hours of culture,Sja-miR-2162 in HSC was detected by qPCR and its regulation on Col1?1,Col3?1 and?-SMA expression.The results showed that the presence of Sja-miR-2162 was stably detected in co-cultured HSC,and the expression of Col1?1 and?-SMA was significantly up-regulated?p<0.01?.To confirm the effect of this activated HSC on the contribution of Sja-miR-2162,we previously transfected the Sja-miR-2162 inhibitor in HSC and co-cultured with live eggs.The results showed that the expression of Col3?1 and?-SMA after co-culture was significantly lower than that of the control group?p<0.01?.?2?Down-regulation of Sja-miR-2162 inhibited the activation of HSC in vitroSja-miR-2162 inhibitor was transfected into the primary HSC on day 56 after infection.The Sja-miR-2162 level was detected by qPCR and the activation of HSC was detected 24 hours later.The results showed that Sja-miR-2162 inhibitor significantly reduced the level of Sja-miR-2162 in HSC after infection?p<0.05?.At the same time,down-regulation of Sja-miR-2162 significantly reduced the expression levels of Col1?1and Col3?1 in HSC.This result indicated that Sja-miR-2162 was present in in HSC infected by S.japonicum,and the use of inhibitors to interfere with S.Japonicum miRNA could reduce the production of collagen.?3?The validation of Sja-miR-2162 in vivo could promote the role of liver fibrosis in miceTo investigate the effect of Sja-miR-2162 on the hepatic fibrosis of schistosomiasis in vivo,we constructed a rAAV8-anti-Sja-miR-2162-sponge vector that specifically down-regulates Sja-miR-2162 in the liver.The rAAV8-anti-Sja-miR-2162-sponge virus vector was injected into the 7-week-old BALB/c male mice infected with S.japonicum.The scramble group,PBS group and normal mice group were the control group.After infection The mice were sacrificed on 56th day to take the liver test.The results showed that the hydroxyproline content in the rAAV8-anti-Sja-miR-2162-sponge group was significantly lower than that in the scramble group?p<0.05?.The genes related to hepatic fibrosis,such as Col1?1,Col3?1,?-SMA,TIMP and Col4?1 were significantly decreased?p<0.001?.Masson trichrome staining showed that collagen deposition in liver tissue was significantly decreased?p<0.05?.HE staining showed that the area of granuloma was significantly decreased?p<0.001?.The levels of Col1?1,Col3?1,?-SMA and TIMP in HSC of rAAV8-anti-Sja-miR-2162-sponge virus vector infection group were significantly decreased in the primary HSCs of each group and the expression of related genes was detected?p<0.001?.The above two-part results validate our hypothesis that Sja-miR-2162crosses species to regulate host liver fibrosis.5.The target gene and its pathway of Sja-miR-2162?1?prediction and validation of target genes:We used the miRDB computer software?http://www.mirdb.org/miRDB/custom.html?to predict the target gene of Sja-miR-2162 in host cells.Based on the comprehensive analysis of the predicted results and fibrosis related pathways,transforming growth factor,beta receptor III?TGFR3?was identified as a candidate target gene and verified by experiments.First,the double luciferase reporter gene system was used to verify the target site of TGFR3 mRNA 3'UTR by DNA synthesis and ligated to the double luciferase reporter gene plasmid.Sja-miR-2162 mimics and the recombinant plasmid were co-transfected into 293T cells and the expression of fluorescent protein was detected.The results showed that Sja-miR-2162 mimics significantly reduced the expression of plasmid protein?p<0.01?in the wild plasmid group compared with the NC control group.?2?Up-regulation of Sja-miR-2162 inhibited the expression of host TGFR3With the prolongation of infection time,Sja-miR-2162 gradually accumulated in the liver cells,and the expression of TGFR3 also decreased.After transfection the overexpression plasmid of Sja-miR-2162 in HSC-T6 cell line,the expression level of TGFR3 was significantly decreased?p<0.05?.The expression level of TGFR3 was also decreased after transfection the overexpression plasmid of Sja-miR-2162 in primary HSC?p<0.001?.After expression of Sja-miR-2162,the rAAV8 vector was used in normal liver,the expression levels of TGFR3 in liver tissue and HSC were significantly decreased?p<0.001?.?3?down-regulation of Sja-miR-2162 promoted the expression of TGFR3The expression level of TGFR3 was significantly increased in primary HSC transfected Sja-miR-2162 inhibitor and isolated from the 56th day after infection in mice?p<0.01?.Using the rAAV8 vector in mice infected with S.japonicum,the expression levels of TGFR3 in both liver tissue and HSC were all significantly increased?p<0.01?after Sja-miR-2162 was neutralized.Summary:In this study,Sja-miR-2162 was found in a large number of screening of parasite-derived miRNA in vitro and in the deep sequencing of HSCs in mice infected with S.japonicum.In the process of S.japonicum infection,The cell model shows the effect of promoting fibrosis of Sja-miR-2162.On the basis of this,we further study the promoting mechanism of Sja-miR-2162 and its molecular mechanism.Through the study in primary HSC and in HSC-T6 cell lines,it was found that transfection of Sja-miR-2162 mimics and overexpression vector of Sja-miR-2162 could promote the activation of HSC.After the expression vector of Sja-miR-2162 was transfected into normal mice,It was confirmed that the expression of Sja-pre-miR-2162?precursor?could be processed into mature miRNAs in the host cells.In the process of S.japonicum infection and host liver fibrosis,whether Sja-miR-2162 can control the hepatic fibrosis of the host across the species is an important scientific problem in this subject.The subject has been elucidated from the different perspectives:First,in vitro validation:the transwell chamber was co-cultured with primary HSC and found that Sja-miR-2162 entered HSC and activated HSC,but this activation effect could be blocked by Sja-miR-2162 inhibitor;second,the validation in vitro and in vivo:isolation and culture of infected mice primary HSC,and transfected Sja-miR-2162inhibitor,found that transfection inhibitors in the primary HSC of the liver fibrosis-related gene expression was down;Third,the validation in vivo:the specific down-regulation of Sja-miR-2162 in rAAV8 virus vector transfected mice,the liver fibrosis-related gene expression decreased.The above three aspects of the experimental results showed that Sja-miR-2162 could control the liver fibrosis in host across the species.The last part of this topic showed that TGFR3 is a Sja-miR-2162 target gene,that is,Sja-miR-2162 enhances the expression of hepatic fibrosis-related genes by targeting TGFR3 protein and up-regulating TGF-?1/SMAD signaling pathway.For the first time,this study confirmed that parasite-derived can enter the host cells and play an important role in the pathogenesis of the host,which enriches our understanding of the pathogenesis of S.japonicum and provides new intervention targets and ideas for the prevention and treatment of schistosomiasis. |
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