The Study Of MiR-122 On The Regulation Of FOXO3 Gene In Dairy Goat Endometrial Epithelium Cell

Micro RNA(miRNA)is a kind of single-stranded RNA molecule with a length of about19-25 nucleotides.It contains a specific seed sequence that can recognize the corresponding gene sequence,and then participates in regulating the post-transcriptional expression of genes in order to regulate various physiological activities of animals and plants.In this study,by comparing the high-throughput sequencing data made by our laboratory,it was found that the relative expression of miR-122 during the endometrial receptive phase of dairy goats was significantly different from the relative expression during the non-receptive phase(P<0.01).And then by searching for genes with complementary sequences in the gene library of goat based on the seed sequence of miR-122,that FOXO3 was the target gene of miR-122 was speculated.Studies have shown that miR-122 plays an important role in the development,differentiation,and stress of liver cells,but there is no research on its regulation mechanism in dairy goat endometrial epithelial cells(EEC).FOXO3(forkhead box O3 or FOXO3a)belongs to the O subtype of the forkhead transcription factor family.The family is characterized by a distinctive forkhead DNA binding domain,which plays an important role in the process of cell transcription,proliferation and differentiation.Therefore,studying the targeted regulation of mir-122 on FOXO3 in dairy goat endometrial epithelial cells is helpful to establish a molecular regulatory network for the related physiological activities of dairy goat endometrial cells.In this study we use the endometrial epithelial cells of Xinong Saanen dairy goats as the research object.First of all,we use RT-qPCR technology to detect the expression of miR-122 and FOXO3 in endometrial epithelial cells,and then complete the construction of the wild type(WT)and mutant type(MUT)pmirGLO-FOXO3-3’UTR vector in order to perform dual luciferase detection to determine the targeting relationship between miR-122 and FOXO3,and finally use RT-qPCR,CCK8,EdU,Annexin V-APC/7-AAD cell apoptosis detection,Western blot and other techniques to study the regulation of miR-122 on dairy goat EEC through FOXO3.The main results of this research are as follows:1.In this study,the wild-type pmirGLO-FOXO3-3’UTR-WT and mutant pmirGLO-FOXO3-3’UTR-MUT dual luciferase vectors were constructed at first,and then combined with miR-122 mimic,miR-122 inhibitor,Negative The control(NC)and Negative control inhibitor(NCH)were co-transfected into HEK293 T cells,and the intensity of their respective renilla fluorescence and firefly fluorescence was measured.The results of dual fluorescence detection showed that compared with the negative control group,the luciferase activity of the wild-type test group co-transfected with miR-122 was significantly lower(P<0.01),but there was no significant effect in the mutant test group,indicating that miR-122 interacts with FOXO3-3’UTR to reduce luciferase activity.This result preliminarily indicates that FOXO3 is the target gene of miR-122.2.We transfect miR-122 into EEC cells,and detect the expression of FOXO3 at the transcription level and protein level by RT-qPCR and Western blot.The results show that miR-122 can inhibit the target gene FOXO3 at the transcription level and protein level.The expression of FOXO3 further shows that miR-122 can negatively regulate the expression of the target gene FOXO3.3.In EEC,Western blot results showed that miR-122 can promote the activity of the RAS/ERK pathway,and up-regulate the ratio of BCL2/BAX at the protein level.And the result of CCK8,EdU and Annexin V-APC/7-AAD cell apoptosis detection technology showed that miR-122 can inhibit apoptosis of EEC.4.After the FOXO3 overexpression vector was transfected into EEC,it was found that FOXO3 can induce the apoptosis of EEC through CCK8,EdU and Annexin V-APC/7-AAD cell apoptosis detection technology processing and WB analysis.In conclusion,this experiment successfully verified that miR-122 can inhibiting cell apoptosis by targeting FOXO3 in the endometrial epithelial cell of Xinong Saanen dairy goat.