| Objective To study the effects of p27 and miR-27 b on recombinant Schistosoma japonicum p40(rSjp40)–induced inhibition of HSC activation and to explore its potential mechanisms.Methods1.The effects of rSjp40 on p27 promoter activity and its mechanisms(1)Dual-lucifera reporter assay:(1)The sequence of the p27 promoter(-1737 bp to +24bp)was cloned into luciferase vector pGL3-Basic vector,labeled as pGL3-p27.Then pGL3-p27 was transfected into human hepatic stellate cell line LX-2 cells which were treated with rSjp40.The p27 promoter activity was then detected by Dual-lucifera reporter assay(experimental groups: PGL3-Basic,p GL3-Basic+rSjp40,pGL3-p27,p GL3-p27+rSjp40).(2)The truncated fragments of pGL3-p27,labeled as p GL3-p27 a,pGL3-p27 b,pGL3-p27 c and pGL3-p27 d,were transfected into LX-2 cells.The effects of rSjp40 on promoter activities of these fragments were measured by Dual-lucifera reporter assay(experimental groups: p GL3-p27,pGL3-p27+rSjp40,p GL3-p27 a,pGL3-p27a+rSjp40,pGL3-p27 b,pGL3-p27b+rSjp40,pGL3-p27 c,pGL3-p27c+rSjp40,pGL3-p27 d,pGL3-p27d+rSjp40).(3)E2F1 interference plasmid(siRNA E2F1)and pGL3-p27 were co-transfected into LX-2 cells,and the effects of E2F1 on p27 promoter activity were detected by the fluorescent reporter assay(groups: Control,siRNA NC,siRNA NC+rSjp40,siRNA E2F1,siRNA E2F1+rSjp40).(4)The E2F1 binding sites in p27 promoter were mutated and labeled as pGL3-p27 mut.pGL3-p27 mut was transfected into LX-2 cells and treated with rSjp40.Then the activity of pGL3-p27 mut was detected(groups: pGL3-p27,pGL3-p27+rSjp40,pGL3-p27 mut,pGL3-p27mut+rSjp40).(2)Western blot:(1)The protein levels of E2F1 and p27 in rSjp40-treated LX-2 cells were measured by Western Blot(groups: Control,rSjp40).(2)The expression levels of p27 and ?-SMA were investigated in si RNA E2F1-treated LX-2 cells(experimental groups: siRNA NC,siRNA NC+rSjp40,siRNA E2F1,siRNA E2F1+rSjp40).(3)CHIP: The primers for E2F1 binding sites on p27 promoter were designed and the presence of the E2F1 binding sites on the p27 promoter was confirmed by CHIP(experimental groups: input,anti-IgG antibody,anti-E2F1 antibody).2.The role of mi R-27 b on rSjp40-induced inhibition of HSC activation and its mechanisms(1)Dual-lucifera reporter assay:PPAR?-3'UTR plasmids,in which 3'UTR of PPAR? was ligating into the psi-CHECK2 vector,were co-transfected into the LX-2 cells with miR-27 b mimic or miR-27 b inhibitor.The activities of PPAR?-3'UTR affected by miR-27 b were examined(experimental groups: NC mimic+ PPAR?-3'UTR,miR-27 b mimic+ PPAR?-3'UTR,NC inhibitor+ PPAR?-3'UTR,miR-27 b inhibitor+ PPAR?-3'UTR).(2)Western blot:(1)LX-2 cells were treated with rSjp40 for 24 h,48h and 72 h,then the protein levels of PPAR? were observed by Western Blot(24h Control,24 h rSjp40,48 h Control,48 h rSjp40).(2)miR-27 b mimic or miR-27 b inhibitor were transfected into LX-2 cells to examin the expression changes of PPAR?(experimental groups: NC mimic,miR-27 b mimic,NC inhibitor,mi R-27 b inhibitor).(3)r Sjp40 and 5'azacytidine(5'AZA)were used to treat LX-2 cells.The expression changes of PPAR? were observed by Western Blot.(3)qRT-PCR:(1)The expression of miR-27 b was investigated by qRT-PCR after treated with rSjp40 in LX-2 cells.(2)LX-2 cells were co-treated with rSjp40 and 5'AZA.The expression changes of miR-27 b were tested by qRT-PCR.(4)Oil red O staining: LX-2 cells were treated with rSjp40 and stained with oil red O.The formation of lipid droplets was observed(groups: Control,rSjp40).Results1.The effects of rSjp40 on p27 promoter activity and its mechanisms(1)The results of Dual-lucifera reporter assay showed that rSjp40 could enhance the activity of p27 promoter in rSjp40-treated cells.(2)The promoter fluorescence activity and protein levels of p27 were significantly inhibited in LX-2 cells co-transfected with E2F1 interference plasmid and pGL3-p27 plasmid.(3)The enhancement of p27 promoter activity and protein level induced by rSjp40 were also suppressed when E2F1 silencing.2.The roles of miR-27 b on rSjp40-induced inhibition of HSCs activation and its mechanisms(1)The expression of PPAR? was increased in rSjp40-treated LX-2 cells,while miR-27 b was decreased,and this change may be due to the increase of miR-27 b methylation levels induced by rSjp40.(2)The protein levels of PPAR? were significantly inhibited by miR-27 b in LX-2 cells.The results of Dual-lucifera reporter assay further demonstrated the inhibitory effects of miR-27 b on PPAR?.(3)miR-27 b mimic was transfected into LX-2 cells and then the cells were treated with rSjp40.The down-regulated effects of miR-27 b on PPAR? were partially reversed by rSjp40.Conclusions1.rSjp40 can increase the expression of transcription factor E2F1,thereby enhancing the activity of p27 promoter.2.miR-27 b could target PPAR? and then involve in rSjp40-induced inhibition of HSC activation. |
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