| Objective: Verification of the high expression of miRNA-29 b in peripheral blood exosomes and PDGF in liver tissue of biliary atresia;To explore the mechanism by which highly expressed miRNA-29 b in exosomes activates hepatic stellate cells and participates in hepatic fibrosis with biliary atresia.Methods: The exosomes from the peripheral blood of Biliary atresia(BA)an d Choledochal cysts(CC)patients were collected,and the expression of miRN A-29 b was detected by RT-PCR.The liver tissues of BA and CC patients were collected,and the expression of PDGFA in the liver tissues was detected by RT-PCR and Western Blot.The jurkat cells were infected with lentiviruses with abnormal expression of miRNA-29 b,and the stably transfected strains were ver ified by inverted fluorescence microscope and RT-PCR.The exosomes of each group were collected,and the extracted exosomes were identified by transmissi on electron microscopy,nanoparticle tracking analysis,nano-flow technology,an d RT-PCR.LX-2 were co-cultured with exosomes or exosomes+5Azac respective ly,and the expression of DNMT3a/DNMT3b/PDGFA/COL1A1/α-SMA at the m RNA and protein levels was detected by RT-PCR and Western Blot.The DNA methylation of PDGFA was detected by bisulfite method,and the proliferation ability of cells was detected by CCK-8 assay.Results:(1)The expression of miRNA-29 b in BA peripheral blood exosomes was significantly higher than CC group.(2)The expression of PDGFA m RNA and protein in BA liver tissue was significantly higher than CC group.(3)The abnormally expressed lentivirus of miRNA-29 b successfully infected jurkat cells,and the stable transgenic strains were constructed.The number of cells expressing GFP fluorescence in the stable transgenic strains were greater than 85%.The expression level of miRNA-29 b in jurkat cells infected with lentivirus overexpressing miRNA-29 b was higher than that in blank control and negative control group(76.05±1.24 vs.1.03±0.04 vs.1.05±0.05.P<0.05).and miRNA-29 b was low expressed.The expression of miRNA-29 b in lentivirus-infected jurkat cells was lower than that in blank control and negative control group(0.60±0.02 vs.1.06±0.04 vs.1.03±0.03;P<0.05).(4)Extracted exosomes was confirmed by transmission electron microscopy,nanoparticle tracking analysis,and nano-flow cytometry.The expression level of miRNA-29 b in the exosomes of Jurkat cells overexpressing miRNA-29 b was significantly higher than that in the blank control and negative control group(30.20±0.83 vs.1.05±0.05 vs.0.99±0.05;P<0.05).The expression of miRNA-29 b in the exosomes of jurkat cells with low expression of miRNA-29 b was significantly lower than that in the blank control and negative control group(0.70±0.02 vs.1.07±0.05 vs.1.02±0.06;P<0.05).(5)The m RNA and protein expressions of DNMT3 a and DNMT3 b were down-regulated,the m RNA and protein expressions of PDGFA were up-regulated,and the m RNA and protein expressions of COL1A1 and α-SMA were up-regulated in LX-2 cells co-cultured with miRNA-29 b overexpressed exosomes.(6)In LX-2 cells co-cultured with exosomes with low expression of miRNA-29 b,the m RNA expressions of DNMT3 a and DNMT3 b were up-regulated,the protein expression of DNMT3 a was up-regulated.the m RNA and protein expressions of PDGFA were down-regulated,and the m RNA and protein expressions of COL1A1 and α-SMA were down-regulated.(7)The m RNA and protein expressions of DNMT3 a and DNMT3 b were down-regulated,the m RNA and protein expressions of PDGFA were up-regulated,and the m RNA and protein expressions of COL1A1 and α-SMA were up-regulated in LX-2 cells co-cultured with miRNA-29b-overexpressing exosomes and 5-Azac.(8)The m RNA and protein expressions of DNMT3 a and DNMT3 b were down-regulated,the m RNA and protein expressions of PDGFA were up-regulated,and the m RNA and protein expressions of COL1A1 and α-SMA were up-regulated in LX-2 cells co-cultured with low-expressing miRNA-29 b exosomes and 5-Azac.(9)DNA promoter methylation of PDGFA in LX-2 cells co-cultured with exosomes overexpressing miRNA-29 b was down-regulated,DNA promoter methylation of PDGFA in LX-2 cells co-cultured with exosomes with low expression of miRNA-29 b was up-regulated.(10)The DNA promoter methylation level of PDGFA in LX-2 cells co-cultured with exosomes overexpressing miRNA-29b+5Azac was down-regulated.The DNA promoter methylation level of PDGFA in LX-2 cells co-cultured with exosomes with low expression of miRNA-29b+5Azac was down-regulated.(11)Exosomes overexpressing miRNA-29 b promoted the proliferation of LX-2 cells,and exosomes with low expression of miRNA-29 b inhibited the proliferation of LX-2cells.(12)Exosomes overexpressing miRNA-29b+5Aazc promoted the proliferation of LX-2 cells,and exosomes with low expression of miRNA-29b+5Azac promoted the proliferation of LX-2 cells.Conclusions:(1)miRNA-29 b was highly expressed in BA peripheral blood exosomes,and PDGFA was highly expressed in liver tissue of BA.(2)Exosomes overexpressing miRNA-29 b hypomethylated the DNA promoter region of PDGFA in hepatic stellate cells,promoting high expression of PDGFA,activating hepatic stellate cells,and participating in hepatic fibrosis with biliary atresia. |
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