A Study On The Mechanism Of MiR-9-dependent Regulation Of MYOCD In PASMCs Phenotypic Modulation And Proliferation Induced By CBDL Rat Serum

Research background and objective:Hepatopulmonary syndrome(HPS)is a life-threatening complication of liver disease characterised by a triad of advanced liver disease,intrapulmonary vascular dilation(IPVD)and arterial hypoxaemia.The syndrome is a severe pulmonary sequela,which can affect 4-47% of patients with cirrhotic liver disease and has been associated with poorer survival rate 41%.To date,there is no effective treatment for HPS other than liver transplantation.Our previous research demonstrated that HPS rat serum induced phenotypic modulation and excessive proliferation of PASMCs,which have been identified as two major pathophysiological characteristics in HPS-associated pulmonary vascular remodelling(PVR).Myocardin(MYOCD),a robust transcriptional coactivator of serum response factor(SRF),is able to stimulate the expression of vascular smooth muscle cells(VSMCs)marker genes and inhibit the cell cycle.It has been reported that myocardin is expressed in the heart and in most developing and adult SMC compartments.Several lines of evidence suggest that myocardin plays a pivotal role in PVR and various other proliferative vascular diseases.MicroRNAs(miRNAs)are small non-coding RNAs that negatively regulate gene expression via degradation or translational inhibition of protein-encoding mRNAs.Such posttranscriptional regulation affects various biological processes,including cell proliferation and differentiation and cell type-specific function,and is involved in several cardiovascular diseases.Since the first report on the role of miRNA in VSMCs was published in 2007,miRNAs such as miR-143/145,miR-21,and miR-20 a have been shown to regulate various aspects of VSMCs biology.However,it is not yet clear which miRNAs regulate myocardin and whether targeting myocardin by miRNAs can regulate HPS-associated PVR.Therefore,in this study we investigated whether some miRNAs involved in HPS-associated PVR by targeting MYOCD.Methods(four parts):1.The construction of in vivo CBDL animal model and in vitro PASMCs model.Common bile duct ligation(CBDL)rats,an accepted HPS animal model,were established.Serum was separated from the blood samples of CBDL rats.Primary rat PASMCs were isolated from healthy Sprague-Dawley rats and cultured in DMEM with 10% FBS and used for experiments between passages 4 and 9.2.Bioinformatics' analysis and the evaluation of selected miRNAs,myocardin as well as PASMCs phenotypic markers.Related bioinformatics softwares(Targetscan?miRanda and miRDB)were used to search for miRNAs,which can directly target the 3'-UTR of MYOCD mRNA.The selected miRNAs(miR-1?miR-9?miR-128 and miR-186)and myocardin mRNA were evaluated by qRT-PCR in PASMCs treated with normal rat serum or CBDL rat serum for 24h(T1),48h(T2)and 72 h(T3),respectively.In addition,the protein levels of myocardin,SM-?-actin and SM-MHC were assessed by Western blotting.3.Functional analysis of miR-9 in CBDL rat serum-induced phenotypic modulation of PASMCs.Cultured PASMCs were transfected with miR-9 inhibitor,and then treated with CBDL rat serum.The expression levels of myocardin,SM-?-actin and SM-MHC protein were detected by Western blotting.Furthermore,qRT-PCR and double-luciferase reporter assay were used to confirm the effect of miR-9 on myocardin was due to its directly binding to the complementary sites of MYOCD mRNA.4.Functional analysis of miR-9/MYOCD pathway in CBDL rat serum-induced phenotypic modulation and excessive proliferation of PASMCs.Cultured PASMCs were transfected with miR-9 inhibitor or MYOCD expression plasmids respectively,and then treated with CBDL rat serum.The expression levels of SM-?-actin and SM-MHC protein were detected by Western blotting.The 3H-TdR and CCK-8 analysis were performed to further validate the functional role of myocardin in mediating miR-9 effects on CBDL rat serum-induced excessive proliferation of PASMCs.Results:1.CBDL rat serum up-regulates miR-9 expression,down-regulates myocardin mRNA and protein levels and induces phenotypic modulation in rat PASMCs.Among the selected miRNAs,miR-9 showed the highest increase(2.8-,4-and 5.2 fold);miR-1 demonstrated approximately two-fold increase,whereas miR-128 and miR-186 showed no significantly changes under CBDL rat serum stimulation at all time-points.Myocardin mRNA decreased significantly at different time-points.In addition,CBDL rat serum induced a time-dependent decline both in the protein levels of myocardin and PASMCs differentiation markers(SM-?-actin?SM-MHC).2.Knockdown of miR-9 reverses the CBDL rat serum-induced down-regulation of myocardin and differentiation markers in PASMCs.As demonstrated by Western blotting analysis,transfection with miR-9 inhibitor significantly repressed CBDL rat serum-induced down-regulation of myocardin and differentiation markers(SM-?-actin?SM-MHC).3.miR-9 efficiently regulates myocardin expression in cultured PASMCs under the condition of CBDL rat serum.According to qRT-PCR analysis,we observed that the expression levels of myocardin were efficiently down-regulated by miR-9 mimic and up-regulated by miR-9 inhibitor.In addition,luciferase reporter assay demonstrated that miR-9 mimic effectively repressed luciferase activity,but mutations in the putative binding site eliminated miR-9-mediated repression of luciferase activity.4.Myocardin mediates the effects of miR-9 on CBDL rat serum-induced phenotypic modulation and excessive proliferation of PASMCs.Both the inhibition of miR-9 or overexpression of myocardin repressed CBDL rat serum-induced down-regulation of differentiation markers(SM-?-actin?SM-MHC)and excessive proliferation in cultured PASMCs.Conclusions:These findings demonstrate that miR-9 is required in CBDL rat serum-induced phenotypic modulation and excessive proliferation of PASMCs for targeting of myocardin and that miR-9 may serve as a potential target in HPS.