The Effect Of MiR-21-5p In Hepatocyte Exosome On Liver Fibrosis And Its Molecular Mechanism

Hepatic fibrosis is a process of chronic liver injury caused by various pathogenic factors,resulting in excessive deposition of extracellular matrix(ECM).Hepatic stellate cells(HSCs)account for 15% of the total number of intrinsic cells in the liver,and play a key role in the process of liver fibrosis.HSCs transforme into activated state under the action of various pathogenic factors,secrete excessive ECM,participate in the process of liver fibrosis.Exosomes are extracellular vesicles with a size of 30-150 nm,which can participate in information exchange between cells and play an important role in disease diagnosis and treatment.Hepatocyte exosomes have been the focus of research,and the role of micro RNAs(miRNAs)in cell-to-cell communication in liver disease is just beginning to be explored.Under the action of different stimulus factors,such as palmitic acid and hepatitis virus,hepatocytes can release exosomes containing different miRNAs to regulate the activation of HSCs,while normal hepatocyte exosomes can inhibit the activation of HSCs and alleviate liver fibrosis.However,it is still unclear how the contents of hepatocyte exosomes change in the process of liver fibrosis,and the specific mechanism of hepatocyte exosomes regulating HSCs.In order to accurately understand the changes of exosomes in the process of liver fibrosis,we established a mouse model of liver fibrosis,isolated normal and fibrotic mouse hepatocytes,extracted exosomes from normal and fibrotic mouse hepatocytes,screened the changes of exosomes differential miRNA,and further explored the mechanism of the differential miRNA in the process of liver fibrosis,provide a possible biomarker and target for the diagnosis and treatment of liver fibrosis.Based on the above research purposes,this topic carries on the related research from the following five parts.PART Ⅰ The effect of hepatocyte exosomes on the activation of hepatic stellate cellsObjective: To investigate the effect of normal and fibrotic hepatocyte exosomes on the activation of HSCs.Methods:(1)C57BL/6 mice were injected intraperitoneally with 20%CCl4 to establish liver fibrosis model.(2)H&E staining was used to clarify the pathological changes of liver tissues.Primary hepatocytes and HSCs were isolated from normal and fibrotic mice by collagenase in situ perfusion and density gradient centrifugation respectively.Cell morphology was observed under light microscope,hepatocytes were identitied by glycogen staining and cytokeratin(CK18)immunocytechemical staining,and HSCs were identified byα-SMA cellular immunofluorescence.(3)The supernatants of human hepatocyte line(L02)、normal and fibrotic primary mouse hepatocytes were collected.The exosomes were extracted by ultra-high-speed centrifugation.The uptake of exosomes by human hepatic stellate cell line(LX-2)was observed by confocal microscope.The exosomes of L02 、 normal and fibrotic primary mouse hepatocytes were co-cultured with HSCs respectively.After 24 hours,m RNA expressions of α-SMA and COL1A1 in HSCs were detected by q RT-PCR.Results: Results:(1)After C57BL/6 mice were intraperitoneally injected with20% CCl4 for 4-6 weeks,hepatic fibrosis was observed by H&E staining,and pseudolobular structure was observed at 8 weeks.(2)Collagenase in situ perfusion method could smoothly extract hepatocytes from normal and liver fibrosis groups.The morphology of hepatocytes,glycogen of hepatocytes and anti-CK-18 immunocytochemical staining confirmed that the purity of primary hepatocytes was more than 95%.The primary mouse HSCs showed obvious proliferation about 1 week.The result of immunofluorescence staining showed that the positive rate of α-SMA reached more than 90%.(3)Hepatocyte exosomes were successfully extracted by ultra-high-speed centrifugation.The uptake of hepatocyte exsomes by LX-2 was observed by confocal microscope.The results of q RT-PCR detection of related gene expressions of HSCs showed that exosomes of normal L02 decreased the expressions of α-SMA and COL1A1 m RNA in LX-2.Compared with HSCs co-cultured with normal hepatocyte exosomes,the expressions of α-SMA and COL1A1 m RNA were significantly increased in HSCs co-cultured with fibrosis hepatocyte exosomes(P < 0.05).Conclusion: Hepatocyte exosomes can be taken up by LX-2.Compared with normal hepatocyte exosomes,fibrosis hepatocyte exosomes can promote HSCs activation and collagen expression.Part Ⅱ Screening and validation of differential miRNAs in hepatocyte exosomesObjective:To extract exosomes from normal hepatocytes and fibrosis hepatocytes,screen differential miRNAs of hepatocyte exosomes by high-throughput sequencing.q RT-PCR was used to detect the expressions of differential miRNAs in hepatocyte exosomes of normal and fibrosis groups.Methods:(1)The exoRNeasy Maxi Kit was used to extract hepatocyte exosomes from normal and fibrosis groups.The gene expression profiles of the two groups were detected by high-throughput sequencing technology,and the differentially expressed genes(DEGs)of the two groups were screened.Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed on DEGS target genes of hepatocyte exosomes in normal group and fibrosis group.(2)q RT-PCR was used to verify the expressions of DEGs in the exosomes of normal hepatocytes and fibrosis hepatocytes.Results: Hepatocyte exosomes from the normal group and the fibrosis group could be successfully extracted by exo RNeasy Maxi Kit.High-throughput sequencing results showed that there were 38 differential miRNAs between two groups,compared with the normal group,there were 32 up-regulated miRNAs and 6 down-regulated miRNAs in hepatocyte exosomes of fibrosis group.The GO and KEGG enrichment analysis of DEGs target genes suggested that these miRNAs were associated with liver fibrosis.Five miRNAs with high expression abundance and significant difference between two groups were selected for q RT-PCR verification.The results showed that the expressions of miR-21a-5p, miR-30a-3p,miR-20a-3p,miR-143-3p and miR-199a-5p in fibrosis hepatocyte exosomes were significantly higher than those in normal hepatocyte exosomes(P< 0.05),and the expression of miR-21a-5p was the highest,which was consistent with the results of high-throughput sequencing.Conclusion: The gene expressions of miR-21a-5p,miR-30a-3p,miR-20a-3p,miR-143-3p and miR-199a-5p in fibrosis hepatocyte exosomes are significantly higher than those in normal hepatocyte exosomes(P < 0.05),and miR-21a-5p has the highest expression abundance.In the process of liver fibrosis,hepatocyte exosomes may affect liver fibrosis by delivering miR-21a-5p to target cells.Part Ⅲ Study on the correlation between exosome derived miR-21-5p and liver fibrosisObjective: To compare the expression of miR-21-5p in serum exosomes of cirrhotic patients and normal adults.and investigate the correlation between miR-21-5p expressions of serum exosomes and liver histopathological changes,HSCs activation and collagen deposition through animal models.Methods:(1)human serum exosomes were extracted by ultrahigh speed centrifugation,and the expressions of miR-21-5p was detected by q RT-PCR.(2)C57BL/6 mice were intraperitoneally injected with 20%CCl4 to establish liver fibrosis model.The degree of liver fibrosis was detected by H&E and Masson staining,and α-SMA immunohistochemical staining was used to detect the activation of HSCs;(2)exo RNeasy serum/plasma MIDI kit was used to extract serum exosomes of mice,and q RT-PCR was used to detect the expression of miR-21-5p in serum exosomes and liver tissue.Results:(1)The results of q RT-PCR showed that compared with normal group,the expressions of miR-21-5p in serum exosomes of cirrhotic patients increased significantly(P < 0.001).(2)H&E staining of liver tissue confirmed the successful construction of liver fibrosis.Masson staining showed that the positive area of collagen deposition in liver of model group at 4 and 8 weeks was significantly higher than that of normal group and control group(P < 0.05);immunohistochemical staining showed that compared with normal group and control group,the positive area of α-SMA expression in liver of model group increased obviously at 4 and 8 weeks(P < 0.05).(3)q RT-PCR detection showed that compared with normal group and control group,the expressions of miR-21-5p in serum exosomes and liver tissue of model group increaesed significantly at 4 and 8 weeks(P < 0.05).In addition,the expressions of miR-21-5p in serum exosomes were positively correlated with the expressions of miR-21-5p,α-SMA and collagen deposition in liver tissue(P < 0.01).Conclusion: The expression of miR-21-5p in serum exosomes is positively correlated with liver tissue collagen deposition and HSCs activation.The expresson of miR-21-5p in serum exosomes may be used as a biological marker for the diagnosis of liver fibrosis.Part Ⅳ Mi R-21-5p targets Smad7 to regulate the activation of hepatic stellate cells and promote the development of hepatic fibrosisObjective: Double luciferase report experiment was used to verify whether miR-21-5p can bind to Smad7 site.To explore the molecular mechanism of miR-21-5p affecting the activation of HSCs and promoting liver fibrosis.Methods:(1)The Target Scan website was used to predict the miR-21-5p potential target gene Smad7 and its binding site.293 T cells were transfected with Smad7 WT and Smad7 MUT vectors,the cells were divided into WT+NC group、WT+miR-21-5p mimic Group、MUT+NC group and MUT+miR-21-5p mimic group,dual luciferase report experiment was used to verify whether miR-21-5p could bind to Smad7 site.(2)LX-2 cells were transfected with miR-21-5p mimic and miR-21-5p inhibitor by Lipofectamine TM RNAi MAX Transfection Reagent respectively,the cells were divided into mimic NC group,miR-21-5p mimic group,inhibitor NC group and miR-21-5p inhibitor group,The expression levels of Smad7,α-SMA and COL1A1 m RNA and protein were detected by q RT-PCR and Western Blot.cell proliferation of LX-2 were detected by CCK8 and Ed U.Results:(1)The binding sites of miR-21-5p and Smad7 were found through the miRNA target gene prediction website.(2)The dual luciferase report experiment found that compared with WT+NC group,the relative luciferase reading of the WT+miR-21-5p mimics group decreased significantly(P < 0.0001);there were no difference between MUT+NC group and MUT+ miR-21-5p mimics group.(3)QRT-PCR and Western Blot showed that compared with the mimic NC group,the expression levels of α-SMA and COL1A1 m RNA and protein in the miR-21-5p mimic group significantly increased(P < 0.05),and the expression levels of Smad7 m RNA and protein significantly decreased(P<0.05);compared with inhibitor NC group,the expression levels of α-SMA and COL1A1 m RNA and protein in the miR-21-5p inhibitor group significantly decreased(P < 0.05),and the expression levels of Smad7 m RNA and protein significantly increased(P < 0.05).(4)CCK8 and Ed U results confirmed that miR-21-5p mimic promoted the proliferation of LX-2,while miR-21-5p inhibitor inhibited the proliferation of LX-2.Conclusion: miR-21-5p may regulate the TGF-β/Smad pathway by inhibiting Smad7,promote HSCs activation,proliferation and collagen synthesis,and regulate the development of liver fibrosis...