Role And Mechanism Of HucMSC-ex Derived MiR-373 And Let-7b In Liver Fibrosis

Objective: Liver fibrosis is a pathological process in the early stage of liver cirrhosis and liver cancer,characterized by activated hepatic stellate cell(HSC),which leads to excessive deposition of extracellular matrix(ECM)in liver.Human umbilical cord mesenchymal stem cell(huc MSC)derived exosomes(huc MSC-ex)were found to alleviate liver fibrosis in mice,but the specific mechanism was not clear.Micro RNA(mi RNA)is a non-coding single strand RNA,which is involved in the post transcriptional regulation of genes and plays a key role in liver fibrosis.Here this study aims to explore the role and mechanism of mi R-373 and Let-7b delivered by huc MSC-ex in the development of liver fibrosis,which will provide new targets for the diagnosis and treatment of liver fibrosis.Methods: 1)The mi RNeasy serum/plasma kit was used to extract the mi RNAs from the serum exosome of healthy people and patients with liver fibrosis.The mi RNAs were extracted from the serum exosome and liver tissue of healthy and liver fiber mice.QPCR was applied to detect the expression of mi R-373 in each group.2)QPCR,Western blot and immunofluorescence were used to detect the expressions of Col1,Col3,Col4 and FAP in hepatic stellate cells(LX-2)over-expressing mi R-373.Target Scan was used to predict the target genes of mi R-373,and the gene was verified by the dual luciferase reporter gene.After over-expression or knockdown of mi R-373,the expression of TGF-?R2 in LX-2 cells was estimated by q PCR,Western blot and immunofluorescence.3)Huc MSC was isolated and cultured,and the huc MSC-ex was extracted by Exo Quick-TCTM.The huc MSC-ex observed and identified by transmission electron microscope and Nanosight particle tracer analyzer.Western blot detected the expression of CD9 and CD63 in huc MSC-ex.The liver fibrosis model of mice was established by intraperitoneal injection of CCl4 and followed by administration of huc MSC-ex and PBS.Maestro in Vivo Imaging System and confocal microscopy were applied to observe the distribution of the dye marker huc MSC-ex in vivo and in vitro.HE,Masson and Sirius Red staining observed the tissue structure and distribution of collagen in liver tissue.Tissue immunocytochemistry,q PCR and Western blot were used to compare the expression of Col1,Col3,Col4 and FAP in three groups.LX-2 cells induced by TGF-?1 co-culture with huc MSC-ex.The expressions of Col1,Col3,FAP and TGF-? associated genes in LX-2 cells were detected by q PCR and Western blot.The huc MSC-ex loaded with mi R-373(huc MSC-ex-mi R-373)by ultrasonic was performed by q PCR and flow imaging comparing with room temperature incubation.After LX-2 cells treated with huc MSC-ex-mi R-373,the expressions of Col1,Col3 and FAP were estimated by q PCR,Western blot and immunofluorescence.The q PCR,Western blot and immunofluorescence were applied to analyse the expressions of TGF-?R2 and p-smad2/3.PBS,huc MSC-ex and huc MSC-ex-mi R-373 were injected to liver fibrosis mice by tail vein.HE,Masson and Sirius Red staining observed liver tissue structure and the distribution of the collagen in liver tissue.Tissue immunochemical,liver soluble collagen and Western blot were used to compare the changes of the liver fibrosis related protein in three groups.The expressions of TGF-?R2 and p-samd2/3 were detected by immunochemistry,q PCR and Western blot in liver tissue.4)Tissue immunohistochemistry,q PCR and Western blot were applied to analyse the effect of huc MSC-ex on the expression of TGF-?R1.Target Scan was used to predict the associated mi RNAs of TGF-?R1.q PCR,Western blot and immunofluorescence were used to detect the expression of TGF-?R1 in LX-2 cells overexpressing Let-7b.Results: 1)QPCR showed that the expression of mi R-373 in the serum exosome and liver tissue of fibrotic mouse was significantly lower than that in the healthy group.The expression level of mi R-373 in serum exosomes of liver fibrosis patients was also lower than that in the healthy group.2)The collagen synthesis in LX-2 cells over-expressing mi R-373 was significantly weakened,while the expressions of collagen related proteins in the cells increased significantly due to the knockdown of mi R-373.Target Scan and dual Luciferase reporter revealed that mi R-373 target to TGF-?R2.QPCR,Western blot and cell immunofluorescence determined that mi R-373 could down-regulate the expression of TGF-?R2 in LX-2 cells,while the expression of TGF-?R2 increased significantly after the LX-2 cells knockdown of miR-373.3)In vivo imaging,huc MSC-ex with dye markers was expressed in liver tissue of fibrotic mice.Confocal microscope observed that huc MSC-ex with dye markers presented in LX-2 cells by endocytosis.HE,Masson and Sirius Red staining proved that huc MSC-ex reduced the expression of collagen fibers in liver tissues.Compared with incubation at room temperature,ultrasonication promoted the loading of huc MSC-ex on mi R-373.Huc MSC-ex-mi R-373 significantly reduced the expressions of collagen,TGF-?R2 and p-smad2/3 in LX-2 cells,and improved liver fibrosis.In vivo,compared with huc MSC-ex,huc MSC-ex-mi R-373 decreased the expression of collagen,TGF-?R2 and p-smad2/3 in the liver tissues and significantly improved liver fibrosis.4)huc MSC-ex could down regulate the expression of TGF-?R1 in vivo.Target Scan showed that TGF-?R1 has a similar sequence with Let-7b,and the expression of TGF-?R1 in LX-2 cells reduced significantly after the over-expression of Let-7b.Conclusion: The expression level of mi R-373 in serum exosome could be a biomaker for the patients with liver fibrosis.Huc MSC-ex may alleviate liver fibrosis through delivering mi R-373 and Let-7b to hepatic stellate cell.Huc MSC-ex modified with mi R-373 and Let-7b offers a new approach and proof for the treatment of liver fibrosis.