Effect And Mechanism Of Retinol Binding Protein 4 On The Function Of Porcine Granulosa Cells

Follicular development is an extremely complex biological process,regulated by the classical"hypothalamic-pituitary-sexual"secretion of hormones,paracrine,and autocrine factors.Follicular granulosa cells are the main type of somatic cells in follicles.They play a vital function in synthesizing and secreting hormones and cytokines,and transmiting nutrients between the oocytes and the peripheral tissues.Cytokines,which secreted by granulosa cells,can regulate follicular development through complex signal transduction and participate in the selection of dominant follicles and follicular atresia.Several studies have confirmed that the apoptosis of granulosa cell is the potential mechanism of follicular atresia.Altogether,it is significant to explore the mechanism of the apoptosis of granulosa cell and the potential molecular markers affecting follicular development for improving female animal fertility and ovarian diseases.RBP4,one member of the lipocalin family,is expressed mainly in the liver and is a specific transporter of retinol,responsible for the transport of retinol from the liver to peripheral tissues.Many studies have confirmed that RBP4 is expressed in many mammalian ovaries.Moreover,RBP4 is associated with insulin resistance and polycystic ovary syndrome in humans,and RBP4 may be associated with the occurrence of ovarian diseases.Previous studies have shown that a high concentration of RBP4 protein has been dectected in the follicular fluid of porcine cysts,and we speculate that RBP4may play an important role in the process of granulosa cell proliferation and apoptosis.However,current available information on the function and regulatory mechanism of RBP4 in GCs is limited.In order to explore the function of RBP4,the expression of RBP4 in porcine follicular granulosa cells was increased and decreased extremely by constructing RBP4 overexpression vector to package overexpressed lentivirus and designing interfering RNA,and molecular biological technologies such as flow cytometry,RT-qPCR and Western blotting were used.The effects of RBP4 on apoptosis and progesterone synthesis and secretion of porcine granulosa cells were detected by gene expression level.High-throughput sequencing analysis showed that the differentially expressed genes in the RBP4 treatment group were enriched in the insulin pathway,and combined with the treatment of the inhibitor(U0126)at the ERK1/2 site of the insulin downstream pathway,elucidating that RBP4 affects granulosa cells apoptosis and progesterone secretion through the ERK1/2 signaling pathway.The results are as follows:1.The expression vector of porcine RBP4 gene lentivirus was constructed and the recombinant lentivirus of PLVX-RBP4 was packaged,and the virus titer reached3×10~8TU/m L.The recombinant lentivirus successfully infected primary cultured porcine follicular granulocyte cells and effectively enhanced the expression of RBP4gene in the cells.The results showed that overexpression of RBP4 could decrease the mRNA expression of anti-apoptotic genes(BCL2 and XIAP),increase the mRNA expression of apoptotic gene(BAX),promote the apoptosis of porcine follicular granulosa cells and inhibit the secretion of progesterone.2.The interference sequence of RBP4 was designed and synthesized,and it was confirmed that siRBP4?01 interference fragment could significantly reduce the expression level of RBP4 after transfection of granulocyte cells for 72 h.Interfering with RBP4 can increase cell viability and inhibit the apoptosis of granulosa cells by inhibiting the expression of apoptotic genes.3.Porcine follicular granulosa cells were treated with recombinant lentivirus for72h,and the changes of mRNAs expression pattern were detected by high-throughput sequencing.The results showed that compared with the control group,a total of 113 differential genes were detected in the RBP4 overexpression group,among which 71 genes were up-regulated and 42 genes were down-regulated.There were 16 different gene enrichment pathways including oxidative phosphorylation pathway,insulin pathway and AMPK signaling pathway.4.RBP4 promotes the apoptosis of granulosa cells and inhibits the synthesis and secretion of progesterone by inhibiting the ERK1/2 pathway.To illustrate the molecular pathways associated with the effects of RBP4 on GCs,we used RT-qPCR,WB,and molecular net analysis in GCs overexpressing RBP4.We find that RBP4 blocks the ERK1/2 signaling pathway and promotes the apoptotic pathway in granulosa cells.Thereby reducing the vitality of granulosa cells whereas the apoptotic pathway is enhanced.The expression of progesterone synthesis gene was blocked resulting in decreased progesterone secretion in granulosa cells.