Effects Of RBP-4 On Proliferation Of Porcine Granulosa Mediated By MiRNAs

RBP-4 is a newly discovered adipokine that is widely distributed in the body.A large number of studies have shown that a series of metabolic diseases such as obesity,type 2 diabetes,and insulin resistance are associated with the expression of RBP-4.It also plays an important role in the development of reproductive-related diseases like follicular cysts,PCOS,gestational diabetes,hypertensive disorders of pregnancy,and abnormal embryonic development.At present,the research on RBP-4 mainly focuses on physiological changes and dominant diseases,however,regarding the puzzles about the relationship between RBP-4 and fertility,follicular development,and its correlation with gonadotropin,especially the downstream expression of RBP-4 in porcine follicular granulosa cells,such as miRNAs and their signaling pathways and regulatory networks,has hardly been involved.To clarify these issues,we did the following research.First,RBP-4 protein is located in follicular granulosa cells by immunohistochemistry.Our results have shown that there was a large amount of RBP-4 in the porcine follicular fluid,which was expressed by granulosa cells There was also some in theca cells in the follicles while negative around oocytes.This finding was also supported by PCR results.The expression of RBP-4 in granulosa cells,theca cells and oocytes was detected with RT-PCR.We also categorized ovaries by the size of follicles into 3categories,big(8~12 mm)(n=3),medium(5~7 mm)(n=3)and small(2~4 mm)(n=3)follicles and then performed qRT-PCR to detect the expression level of RBP-4.Results from qRT-PCR indicated that the expression level between big follicles and medium follicles,big follicles and small follicles were significantly different(P<0.01).However,the expression level between medium follicles and small follicles was not significantly different(P>0.05).We performed Western Blotting to detect the expression of RBP-4 in these 3 categories.The results demonstrated that as the size of the follicles increased,the intensity of the bands increased as well,although the intensity difference of the bands between medium and small follicles was not distinct.These results indicated that RBP-4 is mainly produced by granulosa cells,and as the follicle developed,the content of RBP-4 increased.When RBP-4 concentration was10 ng/mL,it promoted granulosa cell growth,but high concentration of RBP-4(?100ng/mL)inhibited granulosa cell proliferationSecond,we treated primary cultured porcine follicular granulosa cells with FSH and LH for 24 h,and detected the mRNA level of RBP-4 by qRT-PCR.FSH and LH were externally increased by 5 gradients of 0.5 IU/mL,2 IU/mL,4 IU/mL,8 IU/mL,and 32 IU/mL.When the concentration of FSH reached 4 IU/mL and the concentration of LH reached 2 IU/mL,the expression of RBP-4 was the highest in granulosa cells.Even if the concentration reached 32 IU/mL,the expression of RBP-4was no longer increased.This demonstrated that a low concentration of gonadotropin(FSH,LH)could stimulate the expression of RBP-4 in swine ovarian granulosa cells.We also treated granulosa cells with RBP-4 to detect the differential expression level of FSHR,LHR by Western Blotting.The results showed that RBP-4 could increase the expression of FSHR and LHR.These experiments indicated that RBP-4 interacted with gonadotropin to coordinate the growth of granulosa cells to regulate the functions of swine ovaries.What's more,the large,medium and small follicular granulosa cells were treated with RBP-4 at 10 ng/mL,50 ng/mL and 100 ng/mL,respectively.The expression of FSHR and LHR were detected by Western blotting.The results showed that the expression of FSHR and LHR increased significantly from 10 ng/mL to 50ng/mL,and when it continued to increase to 100 ng/mL,there was no increase.This suggested that RBP-4 interacts with gonadotropins,which synergistically promote granulosa cell growth.Third,we transfected granulosa cells with porcine RBP-4 gene-loaded lentivirus to overexpress RBP-4.Total RNA was extracted and sequenced.The results showed that there were 81 miRNAs in porcine follicular granulosa cells that were differentially expressed,of which 39 were up-regulated and 42 were down-regulated.We selected and verified porcine differential miRNAs associated with regulation of cell growth function: ssc-miR-148a-5p_R1-21L22,ssc-miR-142-3p_R2-22L22,mmu-miR-1927_R1-15L23,efu-miR-423-9A-G,ssc-miR-193a-3p and bta-miR-149-3p.Among them ssc-miR-193a-3p,bta-miR-149-3p,and ssc-miR-148a-5p_R1-21L22 weredown-regulated.efu-miR-423-9A-G,ssc-miR-142-3p_R2-22L22 and mmu-miR-1927_R1-15L23 were up-regulated,consistent with high-throughput sequencing of miRNAs.Selected the down-regulated gene of ssc-miR-193a-3p,and verified its function using flow cytometry and qRT-PCR.The result showed CCK-8 is down-regulated after transfection with mimic,up-regulated with inhibitor,which were the same with the cell cycle experiments.Besides,four genes were selected for quantitative analysis.Two steroid synthesis genes,CYP19a1 and HSD3? were down-regulated after transfection with mimics and up-regulated after transfection with inhibitor.Two proliferating genes,CCNB1 and PCNA,were down-regulated after transfection with mimic and up-regulated after transfection with inhibitor.These experiments manifest that miR-193 a regulates follicular granulosa cell proliferation and steroid hormone synthesis.RBP-4 affects granulocyte function by regulating the expression of miRNAsIn conclusion,RBP-4 is mainly expressed in granulosa cells and it has a significant interaction with Gonadotropins(FSH and LH).Low concentration of RBP-4 promotes granulosa cell proliferation,but high concentration of RBP-4 inhibits granulosa cell proliferation.Meanwhile,miRNAs also plays an important role in RBP-4-regulated proliferation and steroid hormone synthesis of granulosa cells in the ovary.