Exploring The Role Of MiR-200 In Vascular Injury And Remodeling Based On MiRNA/mRNA Co-Profiling

Background: miRNA has shown its versatility in regulating smooth muscle cell fate and behavior in vascular physiological and pathological events such as vascular development and atherosclerotic diseases.While targeting investigations have established the functional certainty of specific miRNAs in modulating the process of SMC differentiation or phenotypic modulation,SMC miRNAome and its contribution in regulating transcriptomic profile changes during phenotypic modulation and vascular remodeling have not been fully characterized.Vascular smooth muscle cell is a major cell type in vascular system.It not only functions by contraction to promote structural integrity of the vascular wall,but also participates nearly all of the vascular events by switching phenotypes.Multiple stimulation factors can induce phenotypic switching of smooth muscle cell,which leads to a disease-promoting pathological cell state characterized as less contraction,more migration and proliferation.However,current functional miRNA studies have not fully deciphered the questions regarding the maintenance of smooth muscle cell quiescence and phenotypic switching.The versatile functions of differentially expressed abundant miRNAs,such as the miR-200 family,have not been extensively investigated.Aim: Three parts of aims are as follows.Part Ⅰ: To establish the miRNA/mRNA co-profiling on healthy control,PDGF-BB induced and H3K4me2 edited smooth muscle cell dedifferentiation models.To predict the overall contribution of differentially expressed miRNA in regulating transcriptome and cell functions.To predict the functional basis of miR-200 family in regulating smooth muscle cell functions during vascular remodeling induced by vascular injury.Part Ⅱ: To validate the differential expression of miR-200 family in multiple treatment conditions from different experiments angles.To validate the function of miR-200 in regulating smooth muscle cell functions and behaviors and to explore and demonstrate the underlying mechanism.Part Ⅲ: To validate the functions of miR-200 b in carotid ligation induced neointima model of smooth muscle cell lineage tracing mice.Method: Three parts of method are as follows.Part Ⅰ: This part is genomic profiling and bioinformatic study.Small RNAseq and mRNAseq were used to establish the profiling of miRNA/mRNA in healthy control and two de-differentiated smooth muscle cell model.Bioinformatic prediction methods and public databases were utilized to generate the network modulation of miRNA and mRNA.The gene ontology of above network were predicted and the contribution and modulation of differentially expressed miRNA,including miR-200,in transcriptome and cellular functions were analyzed.Part Ⅱ: This part is a molecular biological study.The function of miR-200 were validated in human,mouse and rat smooth muscle cells.By using in situ hybridization,miRNA qPCR,Western Blot,migration assays,proliferation assays and immunofluorescent staining,the function and underlying mechanism of miR-200 in regulating smooth muscle cell function were demonstrated.Part Ⅲ: This part is an animal in vivo study.The lineage tracing carotid ligation induced neointima model was established.By transducing miR-200 b overexpressing lentivirus,in situ hybridization and immunofluorescent staining,the functions of miR-200 b were validated on mouse level regarding the efficacy against vascular remodeling.Result: Three parts of results are as follows.Part Ⅰ: The proportion of isomiR/total miRNA was decreased in PDGF-BB and H3K4me2 edited smooth muscle cells(p<0.05)and there were 50 and 148 differentially expressed miRNAs,respectively.Results showed that PDGF-BB and H3K4me2 groups exhibited 13 common decreased and 17 common increased differentially expressed miRNAs and there were 37.8% and 66% of differentially expressed genes predicted to be targets of differentially expressed miRNA,respectively.These predicted gene sets were found to modulate smooth muscle cell differentiation and quiescence.In the miRNA profiling of healthy differentiated smooth muscle cells,top 20 expressing miRNAs were found to take up over 80% of total miRNAs and they were predicted to govern cellular homeostasis(p<0.05).miR-200 family genetic cluster I,including miR-200 a,miR-200 b and miR-429,were predicted to modulate proliferation and migration ability of smooth muscle cell and the expression of miR-200 s were found to be decreased in de-differentiation state.Part Ⅱ: The expression level of miR-200 family genetic cluster I was significantly decreased in human,mouse and rat de-differentiated smooth muscle cell models(p<0.05).Overexpressing miR-200 b was found to inhibit smooth muscle cell migration and proliferation,while inhibiting miR-200 b increased migration and proliferation(p<0.01).The combined inhibition of miR-200 cluster I showed that inhibition of miR-200 b and miR-429 both promoted smooth muscle cell migration(p<0.01)and presented functional redundancy.Overexpressing miR-200 b was validated to modulate a functional gene set,including QKI(p<0.01).miR-200 b directly targeted QKI,leading to decreased QKI mRNA level(p<0.01)and protein level(p<0.001).QKI negatively regulated the proliferation of smooth muscle cell(p<0.05).Part Ⅲ: Carotid ligation induced vascular injury effectively led to neointimal formation and decreased miR-200 b expression in media layer of carotid.Overexpressing miR-200 b significantly reduced carotid injury induced neointima and vascular remodeling.Conclusion: Three parts of conclusions are as follows.(1)In healthy differentiated aortic smooth muscle cells,top 20 expressing miRNAs functions predictively to govern smooth muscle cell homeostasis.During the de-differentiation of smooth muscle cell,miRNA and mRNA transcriptomes undergoes significant changes and differentially expressed miRNAs target transcriptome to modulate smooth muscle cell functions such as migration,proliferation and differentiation.(2)miR-200 family genetic cluster I,a novel functionally-relevant miRNA in aortic smooth muscle cell,is significantly decreased during dedifferentiation.The miR-200 cluster functions to modulate cell migration and proliferation through mechanisms of targeting a series of functional targetome including QKI.(3)miR-200 b inhibits neointimal formation and vascular remodeling induced by vascular injury.