Therapeutic Strategy For Hepatic Fibrosis Based On Interrupting The Auto-regulatory Feedback Loop Of MiR-21/PDCD4/AP-1

Sustained activation of hepatic stellate cells(HSCs)leads to hepatic fibrosis which is characterized by the excessive extracellular martrix deposition,especially the over-production of type ? collagen.The HSC activation consists of two stages:initiation and perpetuation.Despite of tremendous progresses in understanding the cellular activities in the initiation phase and underlying signaling cascades,the cellular events underlying the perpetuation stage are only partially understood.Previous studies cannot fully explain the typical phenotype of HSCs in the perpetuation stage,and no drug target has been succesfully developed in pharmaceutical industry,thus unfortunately,there is no durg available in clinic.Thus,more insightful researches are urgently needed to clarify the drinving forces beneth the HSC activation.microRNAs have been reported to be involved in the pathogenesis and progression of multiple deseases,whereas in most of researches so far on miRNA fuction,the model has always been 'one single miRNA versus one single protein target in the pathological context'.Little attention has been paid to their regulatory roles in higher grade and more sophisticated networks.In the present study,the miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide(TAA)or carbon tetrachloride(CC14).A dramatic miR-21 increase was noted in fibrotic liver,and moreover,predominantly in activated HSCs.We further found that miR-21 maintained itself at constant high levels by employing a microRNA-21/Programmed cell death protein 4/Activation Protein-1(miR-21/PDCD4/AP-1)feedback loop.MiR-21 could decrease PDCD4 expression,whereas PDCD4 could down-regulate AP-1 activity.Thus this auto-regulatory feedback loop enabled the maximal AP-1 activity in HSCs,resulting in continuous expression of Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-beta(TGF-?)signaling pathway by degrading inhibitory Smad7 and promoting p-Smad2/Smad2 activities.Besides,miR-21 mediated PDCD4 down-regulation protected activated HSCs from death,leading to more collagen-producing cells in the fibrotic context.Disrupting this loop with miR-21 antagomiR or AP-1 inhibitors significantly suppressed HSCs activation and fibrogenic activities in HSCs;and in vivo studies demonstrated that treating with antagomiR-21 or AP-1 inhibitors could ameliorate liver fibrosis in mice.In contrast,reinforcing this loop with small interfering RNA(siRNA)against PDCD4 promoted fibrogenesis in HSCs.In summary,we suggest that by regulating multiple targets at a higher network scope,this miR-21/PDCD4/AP-1 auto-regulatory loop is one of the main driving forces for hepatic fibrosis progression.Targeting this aberrantly activated feedback loop may provide new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.Considerring the important role of miR-21 mediated feebback loop in the pathogenesis and progression of hepatic fibrosis,here in this section,we are trying to screen a chemical candidate that could target this aberrantly activated feedback loop.3'3-diindolylmethane(DIM)is a natural autolytic product in plants and can down-regulate miR-21 expression.The aim of this study was to examine the therapeutic effects of DIM against hepatic fibrosis and to investigate the underlying mechanisms.Firstly,the AP-1 responsive luciferase plasmid was constructed and transfected into cells,and DIM treatment could significantly inhibit the luciferase activity in cells.Further analysis indicated that DIM treatment could down-regulate c-Jun and Fra-1 expression of AP-1 protein family,followed by miR-21 down-regulation in HSCs.The impact of DIM on HSC activation was tested by analyzing smooth muscle alpha actin(a-SMA)and collagen I expression in both HSC-T6 cell line and primary HSCs;DIM blunted the activation phenotype of both HSC-T6 cell line and primary HSCs.DIM suppressed the central transforming growth factor beta(TGF-?)signaling pathway underlying HSC activation through down-regulating the miR-21 expression.Moreover,the therapeutic effect of DIM was further studied in the thioacetamide(TAA)-induced murine hepatic fibrosis model.Administration of DIM was demonstrated to attenuate experimental liver fibrosis,as assessed by the collagen deposition and profiles of profibrogenic markers.In addition,DIM cannot down-regulate serum ALT/AST levels,indicating DIM could directly target on the pro-fibrogenic activies,instead of by protecting hepatocytes from necrosis.In summary,we recommend DIM be further trialed as a therapeutic agent for the treatment of hepatic fibrosis.Another promising therapeutic strategy is to desigen a drug carrier that could specifically target HSCs in the liver.One of the most specific characteristics of HSCs is to absorb and store retinol from blood circulation,in which retinol binding protein(RBP)facilitate this active process.In this section,we designed a vehicle for ASO delivery by conjugating small molecular weight polyetherimine(PEI)to retinol via an N,N'-carbonyldiimidazole(CDI)-activation method.The retinol module in this delivery system could specifically associate with the RBP in circulation.Following the specific interaction of retinol/RBP with the receptors on HSCs,this delivery vehicle could achieve active targeting at the cellular level.In carbon tetrachloride(CCl4)-induced murine liver fibrosis model,the Retinol-c-PEI/ASO complex was preferentially accumulated in HSCs during liver fibrosis.Moreover,the type I collagen expression was much more sufficiently suppressed in comparison with the naked ASO.All these findings suggest that this delivery system could provide a tool for antisense-based therapeutics against hepatic fibrosis.