Research On Expression Regulation Of Let-7 In Wnt Signaling Pathway

Planarias are bilaterally symmetric metazoans which are presently considered to belong to turbellaria of phylum Plathelminthes. Planarians possess enormous regenerative ability, the regenerative prowess of planarians has been attributed to abundant and collectively totipotent adult stem cells called neoblasts which is important for the regeneration or replace of missing target tissues and organs.When injure occurs, neoblasts will differentiate into new organs and tissues. So, planarians have become a molecularly tractable model system to study regeneration, tissue homeostasis, and stem-cell biology.Wnt signal pathway plays a key role for the re-establishment of antero-posterior axis polarity during planarians regeneration.We suggest that Wnt/b-catenin functions as a critical molecular switch to specify and maintain antero-posterior identity.Wnt signaling pathway which as key signaling pathways in the animal development, involved in many physiological processes such as animal embryo differentiation;in addition,it is closely related to stem cell self-renewal and proliferation.miRNAs are short non-coding RNA(19–24 nt) moleculeswhich regulate gene expression by binding to sequences in the3’UTR of the target mRNA. miRNAshave been shown to play critical roles in many physiological processes including development, differentiation, regeneration, tumour formation, metabolism and disease development. Let-7 family is presently one class of microRNAs which are deeply researched. Let-7 involves in regulating various biological processes such as cell development, apoptosis, differentiation and tumorigenesis.In order to to study the regulation of Wnt signal pathway by let-7,we first researched the function of the let–7. The planarian regenerated disfigurement after overexpression of let-7,suggesting that let-7 is involved in the regeneration of planarian.Then experiment we found let- 7 almost distributed in planaria whole body by using in situ hybridization, consisted with the distribution of deputy layer stem cells, which namely studied the relationship of let- 7 and planaria regeneration.In situ hybridization of marker genes and immunohistochemical experiment marker genes in situ hybridization and immunohistochemical experiment further verifies that the RNA interference appeared double head, and illustrates the mechanism. So we predicted target genes of let-7 which participate in planarian regeneration,and verified the correctness of the target gene prediction by using fluorescence quantitative PCR and construction of fluorescent protein expression vector at mRNA level; Western blot detection experiments further confirmed the experimental results at Protein levels. To predict the target gene by RNA interference found it and let-7 over expressing was similar to that induced phenotypic defects, indicating that let-7 through the regulation of beta catenin to regulate nerve regeneration in planarians, and as a negative regulatory factor in Wnt signal pathway, involved in planarian regeneration.RNA interference of predicted target gene appeared similar phenotype defect with over-expression of let-7, suggesting that let- 7 regulated nerve regeneration in planarians through regulating the beta-catenin, which involved in planaria regenerate as negative regulatory factors in the wnt.Here,we use planarians as model organism. By researching the regulation of let-7 on Wnt signal which is related to planarians regeneration,and the relationship between let-7 and Wnt genes,our study further provide new research ideas and useful information for the study of regulation mechanism on miRNA and its target genes in planarian regeneration and maintain of stem cells stability.