| Alveolar echinococcosis is a zoonotic disease.WHO shows that the 10-year mortality rate of untreated AE patients is as high as 94%.Xinjiang is one of the areas with high incidence of AE(Echinococcus multilocularis)causes persistent irreversible damage to the liver after portal vein entry into the liver to form lesions.In recent years,miRNAs have been shown not only to regulate the formation,development,and physiology of normal tissues,but also to play a role in the occurrence,development,and diagnosis of diseases.They are involved in the process of injury and repair of diseases and become hot spots of medical research.More and more studies have shown that miRNAs play an important role in viral hepatitis,alcoholic liver injury,hepatocellular carcinoma,and parasite-induced liver damage.In our previous study,we used the Illumina sequencing method to analyze miRNA expression profiles in mouse liver tissues at different infection stages(1montn,3montnh,6montn).The results showed that miRNA-133 a expression was inhibited at all three infection time points at different stages of infected with Echinococcus multilocularis.miR-133 is a myocardial-specific miRNAs,which is also expressed in the liver.However,its function and role in pathological liver injury have been rarely reported.In this study,miR-133 a expression was up-regulated by recombinant adeno-associated virus type 8(rAAV8).The overexpression of miR-133 a hepatocyte injury induced by Echinococcus multilocularis was observed.To investigate the role and mechanism of miR-133 a in hepatocellular injury induced by Echinococcus multilocularis.A mouse model of Echinococcus multilocularis infection was established in vivo.The recombinant adeno-associated virus type 8(rAAV8)was used as a vector specifically expressing miR-133 a.Overexpression of miRNA-133 a was induced by tail vein injection.The hepatic pathological changes of the mice at different time points were observed by HE.qRT-PCR Verifies the Change of miR-133 a at different time,qRT-PCR was used to detect the expression of Caspase3,Caspase9,Col1a1,Col3a1,α-sma at the mRNA level.Immunohistoche-mical technique was used to detect the expression of Caspase3 and Caspase9 proteins related to liver injury.At the cellular level,HL-7702 cells were stimulated by TGFβ1 at24 h and 48 h,MTT assay detected the toxic effects of Me on HL-7702 cells,qRT-PCR was used to detect the expression of miRNA-133 a and apoptosis-related genes Caspase3 and Caspase9 at the mRNA level.The specific results are as follows:(1)Upregulation of miR-133 a on hepatic injury induced by Echinococcus multilocularis.Compared with Sham group,the expression level of miR-133 a in Em group was down-regulated in 1,3,6month,which was basically consistent with the results of microarray.The expression of miR-133 a in the Em+miR-133 a mimic group was significantly increased(P<0.001).After specific up-regulation of miR-133 a,compared with Em+Scramble group,the expression levels of Caspase3 and Caspase9 in Em+miR-133 amimic group were significantly decreased at mRNA and protein levels(P<0.01).Compared with Em+Scramble group,the expression level of Col1a1 in Em+miR-133 amimic group was significantly decreased at 3 months after infection(P<0.001).The expression of Col3a1 and α-sma was significantly decreased at 1month and 3 months after infection(P<0.001).No significant change at other time points.Serum ALT levels in the Em+miR-133 amimic group decreased significantly at6 months of infection(P<0.05).(2)In vitro,co-cultured with HL-7702 cells for 24 h and 48 h,compared with the control group,Em protein induced no toxicity to HL-7702 cells.The expression level of miR-133 a in HL-7702 cells treated with 600 μg/mL Em reduced 1.33 fold(P>0.05)and 2.85 fold(P<0.01).rAAV8-miR-133 a mimic transfection of HL-7702 cells showed that after Em stimulation at 60μg/mL for 24 h and 48 h,the expression of Caspase3 in Em+miR-133 a mimic group was decreased by 1.56-fold(P<0.05)and1.84(P<0.05)times,the expression level of Caspase9 decreased by 2.5(P<0.05)fold and 4.6 fold(P<0.001).After 24 h and 48 h of Em stimulation at 600 μg/mL,the expression of Caspase3 decreased by 1.55 times(P<0.05).The results of this study show that:(1)Successful establishment of a mouse model of balloon Echinococcus multilocularis infection and injection of r VV8-miR-133 a mimic via tail vein can increase the expression of miR-133 a in the liver.(2)specifically up-regulate the expression of miR-133 a in the liver,reduce the expression levels of liver injury and liver fibrosis related indicators,reduce collagen deposition,inhibit liver fibrosis,and reduce liver damage.(3)HL-7702 cells were stimulated with different concentrations of Em protein,and miR-133 a was down-regulated in HL-7702 cells,suggesting that miR-133 a is involved in the regulation of apoptosis in hepatocytes in Echinococcus multilocularis.The results of this study suggest that miR-133 a plays an important regulatory role in the liver injury caused by Echinococcus multilocularis infection.provide a new perspective and direction for exploring the treatment of liver damage induced by miRNA-mediated Alveolar echinococcosis. |
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