MiR-184 Regulates Apoptosis Of Endometrial Epithelial Cells In Dairy Goats Via STC2-IGF1

Non coding micro RNAs(miRNAs)are expressed in various mammalian tissues,especially in uterus and regulate various physiological activities of organisms.The content of miRNAs in uterus is also very rich.The results of high-throughput sequencing of miRNA in the prophase of endometrial receptivity(RE)showed that the expression of miR-184 in the receptive period(RE)was 31 times higher than that in the pre-receptive period(PE)(P < 0.01).In this study,we predicted and screened STC2 as the target gene of miR-184 by bioinformatics analysis,and STC2 played a key regulatory role during embryo implantation.However,the molecular mechanism of STC2 in dairy goat EECs(Endometrial epithelial cells)is not clear.We take dairy goat EECs as our research object.This experiment used dairy goat EECs as the research object.The expressions of miR-184 and STC2 in RE and PE was were detected by stem-loop RT-qPCR and RT-qPCR.The wild-type(WT)and mutant(MUT)psicheck2-STC2-3?UTR vectors were constructed,and the regulation of miR-184 on dairy goat EECs by STC2-IGF1 was studied by RNA interference,RT-qPCR,Western blot,CCK8,Flow cytometry,EDU and other experimental techniques.The main results are as follows:1.The abundance of miR-184 in RE increased,but the abundance of STC2 mRNA decreasedIn this study,stem-loop RT-qPCR and RT-qPCR were used to verify the difference of miR-184 and STC2 mRNA levels between RE and PE in dairy goats.The results showed that the abundance of miR-184 in RE was higher than that in PE(P < 0.01),while the abundance of STC2 mRNA was lower(P < 0.01).2.STC2 is the target gene of miR-184In this study,we successfully constructed the wild-type(WT)and mutant(MUT)psi CHECK2-STC2-3?UTR vectors,and detected the luciferase activities of miR-184 and wild-type or mutant psi CHECK2-STC2-3?UTR vector plasmid co-transfer treatment group.The results of two-tier luciferase reporter assay showed that miR-184 reduced the ratio of renilla and firefly luciferase activities in wild-type,but had no significant effect on the mutant-type,which indicated that STC2 was the target gene of miR-184.In addition,Western blot showed that miR-184 significantly reduced the expression of STC2 at protein level.Above results demonstrate that miR-184 targets STC2 directly and down-regulates its expression in EECs.3.MiR-184 promotes the apoptosis of EECs and inhibit EECs proliferation through STC2-IGF1 axisIn EECs,Western blot showed that miR-184 inhibited the activity of RAS/ERK pathway,and down-regulate the ratio of BCL2/BAX at protein level.miR-184 could inhibit the proliferation of EECs and induce the apoptosis of EECs by CCK8,Flow cytometry,EDU and other tests.Further study showed that STC2 activated the phosphorylation levels of proteins on Ras/ERK pathway and increased the BCL2/BAX ratio significantly.Meanwhile,CCK8,EDU and Flow cytometry showed that STC2 promoted the proliferation of EECs and inhibited the apoptosis of EECs.It was found that STC2 promoted IGF1 protein level in EECs,while siSTC2 inhibited IGF1 expression;IGF1 activated Ras/ERK signaling pathway and up-regulate BCL2/BAX ratio,which indicates that STC2 can promote EECs proliferation through RAS/ERK signaling pathway by IGF1.In conclusion,miR-184 affects RAS/ERK signaling pathway through STC2-IGF1 axis to inhibit EECs proliferation and induce EECs apoptosis.