Effect Of All-trans Retinoic Acid On Synthesis And Secretion Of Progesterone In Murine Granulosa Cell Line KK-1

All-trans-retinoic acid(at RA)derived from retinol is the main form of retinoic acid,which plays a biological role through retinoic acid receptors.It has been confirmed that synthetases of RA and retinoic acid receptors were expressed in many tissues.The important roles of at RA are not only in the maintenance of vision,but also in cell proliferation and differentiation,growth,embryonic development.It has effects on immune system,nervous system,digestive system,respiratory system and reproductive system.It is used to prevent and treat cancer also.RA is involved in sex determination,spermatogenesis and sperm energy metabolism,and influences oocyte maturation,fertilization,and early embryonic development.Previous studies have shown that granulosa cells are the major sites for retinol uptaken and at RA synthesis.The main function of granulosa cells is to secrete hormones and cytokines,participates in the selection of dominant follicles and follicular atresia,and regulates follicular development.Granulosa cells are the targets of retinol in ovary and can synthesize at RA.It is thought that at RA may affect the function of the granulosa cells.This study aims to investigate the effect of at RA on synthesis and secretion of progesterone in murine granulosa cell line KK-1 including mechanism and the effect of at RA on m RNAs and mi RNAs expression profiles.The results are as follows:1.Expression of genes related to retinol uptaken,RA synthesis and retinoic acid receptors in KK-1QPCR was used to detect the relative expression of m RNAs related to retinol uptaken: STRA6,CRBP1 and CRBP2;synthetases of RA: ADH1,ADH2 and ADH7;retinoic acid receptor:RAR?,RAR? and RAR?.All the m RNAs mentioned were detected in KK-1 cells.2.Effect of at RA on synthesis and secretion of progesterone of KK-1 cellsKK-1 cells were treated with at RA at different concentrations(0?M?0.01?M?0.1?M?1?M?10?M?30?M)for 6h,12 h,18h and 24 h,and the effects of at RA on the cell viability and progesterone synthesis and secretion of KK-1 cells were detected.The results showed that treatment with 0.01?M?0.1?M?1?M?10?M at RA for 6h,12 h,18h and 24 h increased the cell viability significantly(P?0.05).Treatment with 0.1?M,1?M and 10?M for 18 h showed the best effect,and there was no significant difference among three concentrations(P?0.05).Compared with the control group,the expression of St AR m RNA was significantly increased when treated with 0.1?M,1?M and 10?M at RA for 18h(P?0.05).The expression of St AR protein was significantly increased when treatment with at RA for 6h,12 h,18h and 24h(P?0.05).The secretion of progesterone was significantly increased when treatment with 0.1?M,1?M and 10?M at RA for 6h,12 h,18h and 24h(P?0.05).3.Effect of at RA on expression of m RNAs and mi RNAs of KK-1 cellsKK-1 cells were treated with 0.1 M at RA for 18 h and blank control was set up.Expression of m RNAs and mi RNAs were detected.Some different expressed m RNA and mi RNA were selected to verified by q PCR.MRNA sequencing showed that 569 genes were differentially expressed in at RA treated group.Expression levels of 433 genes were incresed and expression levels of 136 genes were decreased.RAR,VDR,CYP26B1,CD36,Paqr7,AK5(up)and BMP4,Ass1,Id1,Ssc5,Tle6 and Nos2(down)were selected for q PCR verification,and the results were consistent with the sequencing results.Mi RNA sequencing showed that 81 mi RNAs were differentially expressed in at RA treated group.Expression levels of 34 mi RNAs were incresed and expression levels of 47 mi RNAs were decreased.Mi RNA-6900-5p,mi RNA-181c-3p,mi RNA-6945-3p and mi RNA-3109-5p were selected for q PCR verification,and the results were consistent with the sequencing results.4.Effect of at RA on synthesis and secretion of progesterone of KK-1 cells through c AMP-PKA-CREB singal pathwayKK-1 cells were treated with at RA,c AMP activator(forskolin)and PKA inhibitor(H-89)seperately or together,and then detect CREB phosphorylation level and progesterone synthesis.The results showed that,compared with the control group,the phosphorylation level of CREB was increased significantly when treated with at RA,forskolin,at RA+forskolin,the phosphorylation level of CREB was decreased significantly when treated with H-89,at RA+H-89;at RA;the expression of St AR m RNA was significantly increased when treated with forskolin,the expression of St AR m RNA was significantly decreased when treated with at RA+forskolin,H-89,at RA+H-89;the expression of CYP11A1 was significantly increased when treated with forskolin,at RA forskolin,the expression of CYP11A1 was significantly decreased when treated with H-89,at RA+H-89;the expression of St AR was increased when treated with forskolin,at RA+forskolin,the expression of St AR was significantly decreased when treated with H-89,at RA+H-89;the expression of CYP11A1 was when treated with at RA,forskolin,at RA+forskolin,the expression of CYP11A1 was significantly decreased when treated with H-89,at RA+H-89;the level of progesterone was significantly increased when treated with at RA+forskolin,the level of progesterone was significantly decreased when treated with H-89,at RA+H-89.5.Effect of at RA on synthesis and secretion of progesterone of KK-1 cells through RARKK-1 cells were treated with at RA and RA against(AGN193109)seperately or together,and the levels of progesterone and CREB phosphorylation were detected.The results showed that,compared with the control group,the level of St AR m RNA was significantly decreased when treater with AGN193109 and at RA+AGN193109,the level of CYP11A1 m RNA was significantly decreased when treater with at RA,AGN193109 and at RA+AGN193109;level of St AR and CYP11A1 were significantly decreased when treater with AGN193109 and at RA+AGN193109;level of progesterone was significantly increased in the at RA group,but was significantly decreased in the AGN193109 group and at RA+AGN193109 group.The phosphorylation level of CREB in the at RA group was significantly increased,while was significantly decreased in the AGN193109 and at RA + AGN193109 groups.In conclusion,retinoic acid was synthesized in KK-1 cells.At RA affected the expression of St AR and CYP11A1 and the synthesis and secretion of progesterone through RAR/c AMP-PKA-CREB signal pathway in KK-1 cells.At RA affected the expression profiles of m RNAs and mi RNAs in KK-1 cells.