| miRNAs are short length RNAs that affect gene regulation by binding to the 3'UTR regions of the m RNAs of certain target genes.A growing number of studies show that miRNAs participate in various physiological processes,including the growth and development of plants and animals,signal transduction and immune response.In recent years,with the need of the fur industry to obtain greater quantities of high quality fur,and the research of hair follicle development attracting greater attention,the role of miRNAs in hair follicle development has become a field of intense interest.Based on previous studies,we found that the expression level of miR-202 was higher in white alpaca skin tissue than that in brown alpaca skin tissue.Target gene predicted and found one of the target genes was kit for the dominant regulation of white hairs.With the aid of Illumina sequencing technology,some miRNAs were founded,which expressed differently in alpaca skin tissue with different hair color,and in which,miR-202 was included.These researches have demonstrated that miR-202 affected the heredity of the coat color,but no further researches were done to demonstrate that how miR-202 acts on the coat color?In order to illuminate the mechanism of the miR-202,which acts on the hair color,we selected the skin tissue of C57BL/6 mice and BALB/c mice as the samples,detected the expression of miR-202,and predicted its' target genes by some bioinfermation softwares.The expression of miR-202 target genes(wnt5a,kit and tcf7)were examined by q PCR(Real-time Quantitative PCR)and Western blot in the mouse skin tissue.Target points were detected by Dual-luciferase report gene assay,and analyzed the relationship of miR-202 and target genes.RNA and protein of transfected cells(Human Embryonic Kidney 293 T cells)was extracted to detection by q PCR and Western blot.We hope that these results would provide new basic insights into the mutation breeding by the research.The results of research are as follows:1.In vivo,the results showed that miR-202 were expressed in skin of C57BL/6 mice and BALB/c mice.The q PCR detection showed that the expression level of miR-202 was lower in BALB/c mice skin tissue than that in C57BL/6 mice skin tissue(P<0.05).2.Target genes of miR-202 were predicted by some bioinfermation softwares.The target genes have screened,wnt5 a,kit,tcf7,which were related to coat color.3.In vivo,q PCR and Western blot detection showed that the expression levels of wnt5 a,kit and tcf7 in the skin tissue of BALB/c mice were higher than that in C57BL/6 mice.The results of western blot were consistent with the results of fluorescence quantitative(P<0.05).4.Immunohistochemical results show that wnt5 a,kit,tcf7 have expressed in epidermis(base)and the dermis(sebaceous glands and hair follicles)of C57BL/6 mice and BALB/c mice with skin tissues.5.The binding sites of wnt5 a,kit and tcf7 for miR-202 were determined by Dual-luciferase reporter gene assay.6.Overexpression of miR-202 in HEK293 T cells.The results showed that the expression of miR-202 of the overexpressing miR-202 group was higher than that of the negative control group.However,the levels of expression of wnt5 a,kit,tcf7 in the overexpressing miR-202 and negative group were reversed to the expression of miR-202.Interestingly,the expression of miR-202,wnt5 a,kit,and tcf7 in the overexpressing miR-202 group and the negative control group were lower than that in the normal group(P<0.05).7.Overexpression of miR-202 in HEK293 T cells.The results of protein expression experiments showed that the expression levels of wnt5 a,kit,and tcf7 in the overexpressing miR-202 group were lower than that in the negative control group and the blank group,but no significant difference was found between the negative control group and the normal group(P<0.05).To sum up,wnt5 a,kit and tcf7 was target genes of miR-202.miR-202 down-regulates the expression of target genes,and that involved the formation of mice with coat color. |
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