Study On The Mechanism Of MicroRNA Let-7b Regulate Keratinocyte Differentiation In Psoriasis

Psoriasis is a kind of abnormal epidermal proliferation,erythema scales as the clinical features of chronic relapsing inflammatory skin disease,the main pathological changes have formed epidermis cutin cell abnormal proliferation and differentiation,dermal inflammatory cells infiltration and angiogenesis.For many years,the scientific researchers and clinical workers carried out extensive and in-depth research on the etiology and pathogenesis of psoriasis,although a lot of progress has been made,but it has not been fully elucidated.Although the mainstream theory of psoriasis pathogenesis remains a T cell-mediated immune theory,but the epidermis keratinocity is still in the process of the occurrence of psoriasis development plays an important role.Reported psoriasis gene level are mainly concentrated on the protein-coding genes,in recent years microRNAs（miRNAs）plays very important role in regulating gene expression,and participate in the psoriasis skin cutin protein regulation and inflammatory reaction process of proliferation and differentiation,but also not let-7b involved in biological function in patients with psoriasis.Therefore,this thesis we will delve into the let-7b for the pathogenesis of psoriasis,especially the effect and mechanism study of keratinocyte differentiation,and preliminary discussion on the let-7b effects on scleroderma.Skin damage is one of the important factors for psoriasis,and the process of wound healing has a relationship with process of psoriasis.Our previous study revealed that let-7b inhibits re-epithelialization in wound healing by targeting IGF2BP2,so we hypothesis that let-7b have a relationship with psoriasis.We firstly applied imiquimod cream（IMQ）,on the shaved back skin of wild-type mice for 7 consecutive days to induce psoriasis.Real-time PCR showed let-7b expression was significantly decreased in injury of psoriasis skin.Psoriasis is usually characterized by hyperproliferation,apoptosis and abnormal differentiation of keratinocytes.In order to explore the meachine that let-7b regulate keratinocity in level of cell and melocular,we analysis of cell proliferation,apoptosis and differentiation in HaCaT over-expressing and knockdown let-7b by Flow Cytometer and Real-time PCR.We found that let-7b had no effect on cell proliferation and apoptosis,but it promotes keratinocyte differentiation.Previous studies have confirmed the function of let-7b in vitro,but not enough to simulate normal psoriatic model.So we developed transgenic mice with over-expression of let-7b(let-7b^TG)in skin with K5 promoter to achieve keratinocyte-specific let-7b expression by microinjection of fertilized eggs,followed by embryo transplantation,it can explore the meachanism of let-7b in the process of development psoriasis more directly and in-depth.Then,we preliminary analysis of the phenotype of wild-type and transgenic mice,and found the volume of transgenic mice is less than wild type mice,even the reddish skin,hair is more sparse.In order to further study whether let-7b could regulate cell differentiation and affect skin development,skin tissue histology of wild type and transgenic mice were analysied by H&E staining.We could detected mice skin thickness,the difference of number of hair follicle and hair follicle cycle from microscopic observation.Intresting,the results showed that let-7b^TGG mice do not affect the number of hair follicles,but it affects hair follicle cycle.Finally,in order to study the function of let-7b in process of psoriasis,we applied imiquimod cream on the shaved back skin of let-7b^TG and wild-type mice for 7 consecutive days to induce psoriasis.Both let-7b^TG and wild-type mice treated with IMQ developed sharply demarcated erythematous lesions covered with white silvery squama（Fig.1a）.After use the Imiquimod cream application onto the skin of wild-type and let-7b^TG mice,let-7b^TGG mice reduced skin erythema,scaling and thickening.Moreover,compared with IMQ-treated wild-type and to mice treated with an emollient cream as control,let-7b^TGG mice showed a striking decrease of skin inflammation as reflected by modified Psoriasis Area and Severity Index（mPASI）which was adapted to mice pathology.Microscopic examination of haematoxylin and eosin-stained skin sections from IMQ-treated let-7b^TGG mice revealed many histological features of chronic inflammation that are characteristic for human psoriasis including parakeratosis,acanthosis and elongation of the dermal papillae.In addition,our study again illustrated the let-7b had no relationship with proliferation in vivo,only affect cell differentiation by immunofluorescence and Real-time PCR.Collectively,our data indicate that let-7b promotes keratinocyte differentiation and lead to a reduced disease severity in vivo and in vitro.MicroRNA can bind to the 3’untranslated region of its target mRNA and is capable of inducing posttrancriptional gene regulation by degrading the target mRNA or blocking translation.As an important regulatory molecule,it is almost involved in regulating all of hysiological and pathological processes.To gain insights into the molecular events associated with let-7b overexpression,we investigated how this miRNA affects keratinocyte differentiation.Using TargetScan 5.1 prediction software and miRase,a set of mRNAs which encode proteins was identified.There are identified as a potential target gene of let-7b with the predicted binding site at nucleotide positions.We choose IL-6,a proinflammatory cytokine that acts on epithelial barrier function and keratinocyte differentiation.To validate whether IL-6 is a functional target of let-7b,a dual-luciferase reporter system was employed.Data from luciferase assays show that over-expression of let-7b remarkably reduced luciferase activity regulated by the wild-type construct but not by the mutant IL-6 3’UTR construct in HaCaT.We further examined the expression of IL-6 from healthy skin and psoriasis lesions in transgenic mice and found a marked reduction of IL-6 compared with that of control littermates by immunohistochemistry and Real-time PCR.miRNA regulation of target molecules can be involved in a variety of signaling pathways,and a number of studies have confirmed that the extracellular signal-regulated kinase（ERK1/2）phosphorylation is a crucial signaling pathway in the cell differentiation,we sought to investigate the association between ERK1/2 signaling and let-7b expression in psoriasis development.Firstly,We investigated p-ERK1/2 activation in the epidermis derived from lesioned skin of human psoriasis patients.Notably,by immunohistochemistry we found that levels of p-ERK1/2were significantly increased in the epidermis of psoriatic lesions compared to healthy controls.In addition,Western Blotting showed that p-ERK expression markedly decreased in skin tissue of transgenic mice compared to wild-type mice.In accordance with these results,phosphorylation of ERK1/2 was suppressed in normal skin and in 3and 5 days after treatment with IMQ in wild-type and let-7b^TG mice.Further study we found cell differentiation was induced by treatment with inhibitors of MEK/ERK（PD98059）,however,knockdown of let-7b can rescue the differentiation by regulation of phosphorylated ERK in HaCaT.At last,our study directly proved that blocking ERK signaling could alleviate psoriasis pathology in vivo using ERK inhibitor（PD98059）treatment.We demonstrated that PD98059 treatment in the epidermis resulted in a pronounced decrease in skin thickness in mice treated with IMQ,and a striking decrease of skin inflammation as reflected by mPASI.The present study has shown that let-7b overexpression promote keratinocyte differentiation and attenuates disease severity in mouse models of psoriasis.Psoriasis and scleroderma are two common autoimmune diseases and they are consistency in clinical manifestation.It is known that hardening of the skin could lead to skin cell keratin process changed,including the cutin cell keratin latency,and abnormal differentiation.The study we comfired let-7b regulated cell differentiation contributes to the process of psoriasis by target IL-6,so it give us a hypothesis that whether let-7b involved in regulation of scleroderma?Firstly,we constructed scleroderma model with mice by injected bleomycin,the mice skin appear lesion including with obvious thickening in the dermis,collagen fiber bundle of bulky homogenization,populations phenotypes after treated with drug three weeks.However,the skin phenotypic of wild type mice is more serious than let-7b^TG mice.In addition,we detected the expression of IL-6 was increased in injury of scleroderma by Real-time PCR,but let-7b target IL-6 lead to IL-6 expression decreased and attenuates disease severity in let-7b^TG mice.Skin fibrosis is a characteristic feature of scleroderma,it is closely related to the collagen synthesis.In order to explore whether let-7b participate in the synthesis of collagen,we detected the gene related with collagen synthesis both in normal and pathological conditions of mice by RT-PCR,and found COL1A1expression was declined.At the same time,we used Targetscan software to predicted gene and found only COLA1 has a complementary sequence of bases with let-7b.COL1A1 may as the let-7b molecular targets involved in regulation of scleroderma pathogenesis.In summary,the present study provided the first and critical in vivo and vitro genetic evidence showing a role of let-7b in psoriasis.Fisrtly,the results showed the let-7b promote cell differentiation,but not affect cell proliferation and apoptosis.In addition,we explore the meachanism of let-7b in the process of development psoriasis more directly and in-depth by construct transgenic mice with the keratinocytes-specific over-expression of let-7b.Let-7b promotes keratinocyte differentiation by targeting IL-6 mediated ERK signaling and attenuates disease severity in mouse models of psoriasis.At the same time,scleroderma model was constructed on mice,we preliminary explore let-7b could regulate the expression of COL1A1 and influence the development of scleroderma.