Expression Of MiR-29b In Bovine Corpus Luteum At Different Developmental Stages And Its Effect On Cells Apoptosis And Secretion Of Progesterone

The corpus luteum(CL)is a transient ovarian structure that develops rapidly after ovulation.The molecular mechanism of the development and degradation of the corpus luteum has been a research hotspot in the field of animal reproduction,in which apoptosis and angiogenesis are accompanied by the whole process of corpus luteum development,and more and more evidence shows that mi RNA plays an important role in the process of corpus luteum function.MiR-29 b is an effective regulator of tumorigenesis,targeting the regulation of cell proliferation,cycle,aging,differentiation,apoptosis and metastasis.At present,it has been demonstrated that miR-29 b is differentially expressed in bovine corpus luteum at different developmental stages.However,the specific function and molecular regulation mechanism of miR-29 b in the corpus luteum is not yet clear.In order to explore the function of miR-29 b in bovine corpus luteum,we first analyzed the expression level of miR-29 b at different developmental stages of corpus luteum by RT-qPCR.The results showed that the expression of miR-29 b gradually decreased from the early corpus luteum to the degressed corpus luteum and the difference between the late corpus luteum and degressed corpus luteum was not significant.To further study the function of miR-29 b in bovine corpus luteum,we predicted the target gene of miR-29 b by using TargetScan and RNA22,demonstrated that oxytocin receptor(OXTR)is a target gene of mi R-29 b in bovine corpus luteum.We used western blot and immunohistochemical methods to explore the expression and location of OXTR at different developmental stages of corpus luteum.The results showed that the OXTR protein expression level gradually increasedfrom the early to the degenerative corpus luteum.The results of immunohistochemistry showed that OXTR was expressed not only in the four stages of corpus luteum,but also in both large and small corpus luteum cells.The expression was mainly in the plasma membrane and cytoplasm of large and small corpus luteum cells.To verify the effect of miR-29 b on bovine corpus luteum cell proliferation and progesterone secretion,we isolated and cultured corpus luteum cells in vitro.MiR-29 b mimics / mi R-29 b inhibitor was designed to overexpress or inhibit miR-29 b in bovine corpus luteum cells.RT-qPCR,western blot,ELISA and flow cytometry were used to detect the expression of OXTR,cell proliferation and the effect of progesterone secretion.The results showed that overexpression of miR-29 b significantly reduced the expression of OXTR,while inhibition of mi R-29 b hardly affected the expression of OXTR.Similarly,overexpression of miR-29 b can promote progesterone secretion in bovine somatic cells,while inhibition of miR-29 b does not affect progesterone synthesis.In view of the above experimental results,we only designed the miR-29 b overexpression group to study the impact of miR-29 b on the proliferation of bovine corpus luteum cells.,flow cytometry results showed that overexpression of miR-29 b can inhibit the apoptosis of bovine corpus luteum cells.,CCK8 experiments demonstrated that overexpression of miR-29 b can promote the proliferation ofbovine corpus luteum cells in vitro.In summary,in this experiment,we analyzed the expression of miR-29 b in bovine corpus luteum at different developmental stages and predicted and verified that OXTR is a target gene of miR-29 b in bovine corpus luteum.We confirmed that miR-29 b can reduce OXTR expression,influence progesterone secretion,and inhibit corpus luteum apoptosis and regulatecorpus luteum function.In this study,by clarifying the expression of miR-29 b and its target gene in the corpus luteum and its effect on the proliferation and apoptosis of corpus luteum cells,we analyzed the mechanism of miR-29 b and its target gene regulating the function of corpus luteum,The role in the estrous cycle provides a theoretical basis,and provides a new direction for the treatment of luteinic-related diseases and improving animal fertility.