| Background:Polycystic ovary syndrome(PCOS)is a heterogeneous disease that affects the reproductive health of women of childbearing age and is one of the main causes of ovulatory infertility.The main pathological manifestations are abnormal hormone level,obstruction of follicular development and disturbance of ovulation,increase of serum androgen,insulin resistance and obesity.It not only affects the reproductive function of women,but also increases the risk of long-term complications such as type 2 diabetes,cardiovascular diseases and endometrial cancer.Although in recent years,numerous researchers have aimed to explore the etiological mechanism of PCOS,but the specific mechanism is still unclear.The amino acid transporter gene Slc7a5(solute carrier family 7,member 5)is the coding gene of L-type amino acid transporter 1(LAT1),whose main function is to transport specific amino acids and provide raw materials for cell growth.Studies indicated that the expression of Slc7a5 is increased in cancerous cells and tissues,including breast cancer,ovarian cancer and endometrial cancer,suggesting that it plays a vital part in the rapid growth and proliferation of tumors.And studies also showed that Slc7a5 is associated with obesity,insulin resistance and inflammation.In the previous study of our group,we searched for the differentially expressed genes in the ovary of rat model of PCOS induced by letrozole,and found that Slc7a5 was increased in the ovary of rat model of PCOS.The significantly increased expression of Slc7a5 indicated that Slc7a5 may play a certain role in the occurrence and progression of PCOS.However,the role of Slc7a5 in many ovarian functions,such as follicular development,oocyte maturation and steroid hormone synthesis has not been reported.Objective:The objective of this study is to explore the expression of Slc7a5 in the ovary of rat model of PCOS and its function in granulosa cells,to study the role of Slc7a5 in the pathogenesis and development of PCOS,and to provide new experimental data and theoretical basis for the diagnosis and treatment of PCOS.Methods and Results:1.Expression of Slc7a5 in the ovary of rat model of PCOS.The rat model of PCOS was established by continuous intragastric administration of letrozole for 23 days.The results showed that the weight of the rats in the PCOS group was significantly higher than that in the control group(P < 0.05).The estrous cycle was identified by vaginal smear.The estrous cycle of the rats in the control group was regular,while the estrous cycle of the rats in the PCOS group was abnormal and always in the interestrous phase.The serum hormone levels of rats were determined by ELISA.The results showed that compared with the control group,the concentrations of LH and T in PCOS group were increased,while the concentration of E2 was decreased(P < 0.05).And there was no significant difference in the concentrations of FSH and TG.The morphological changes of ovary were observed by HE staining.Multiple corpus luteum,follicles at various developmental stages and multiple layers of granulosa cells were observed in the ovaries of control group,while the ovaries of the PCOS group showed typical polycystic changes,with many cystic dilated follicles and a significant decrease in the granulosa cell.Then the expression of Slc7a5 in the ovaries was detected by immunohistochemistry.The results showed that Slc7a5 was expressed in granulosa cells,theca cells and ovarian stroma,and the level of Slc7a5 in PCOS group was higher than that in control group.Furthermore,the expression of Slc7a5 was detected by q RT-PCR and Western blot.Compared with the control group,the mRNA and protein level of Slc7a5 in ovarian tissue of the PCOS group were significantly higher(P < 0.05).2.Effect of Slc7a5 on steroid hormone synthesis in ovarian granulosa cells of ratThe primary granulosa cells were isolated and cultured,and the small interference RNA(si RNA)and over-expression plasmid of Slc7a5 were transfected to explore the effect of Slc7a5 on steroid hormone synthesis in granulosa cells.The expressions of steroid hormone synthesis-related enzymes(Star,Cyp11a1,Hsd3?1)were detected by qRT-PCR and Western blot.The results showed that after interfering with Slc7a5,the expressions of the three enzymes were decreased,but only Cyp11a1 decreased significantly(P < 0.05).And the expressions of three enzymes were increased significantly after over-expression of Slc7a5(P < 0.05).The levels of E2 and P in the supernatant of cell culture were detected by ELISA.The results showed that interference and over-expression of Slc7a5 did not change the levels of E2 and P.In order to explore the effect of androgen on the expression of Slc7a5 in granulosa cells,the level of Slc7a5 in the granulosa cells was detected by q RT-PCR and Western blot after cultured with testosterone for 48 hours.The result showed that the expression of Slc7a5 was significantly increased(P < 0.05).3.Effect and mechanism of Slc7a5 on viability and apoptosis of ovarian granulosa cells of ratThe viability of granulosa cells after interfering with and over-expression of Slc7a5 was detected by observation under microscope and CCK8 test.The results showed that there was no significant change in cell viability after interfering with Slc7a5,but the cell viability after over-expression of Slc7a5 was significantly decreased(P < 0.05).After that,cell apoptosis and cell cycle were detected by flow cytometry.The results showed that after interfering with Slc7a5,the apoptosis rate of granulosa cells did not change significantly.However,after over-expression of Slc7a5,the apoptosis rate of granulosa cells was increased significantly(P < 0.05),meanwhile the S phase of cells was decreased significantly(P < 0.05).In order to explore the mechanism of Slc7a5 promoting apoptosis of granulosa cell,the expressions of apoptosis-related genes and proteins were detected after over-expression of Slc7a5.The results showed that the expressions of apoptosis-related genes Tnf-?,Caspase-3 and Caspase-8 were significantly increased(P < 0.05),and the expressions of apoptosisrelated proteins TNF-?,Caspase-3,Cleaved-Caspase 3,Caspase-8 and CleavedCaspase 8 were also significantly increased(P < 0.05).Furthermore,the activities of Caspase-3 and Caspase-8 in granulosa cells after over-expression of Slc7a5 were further detected.The results showed that the activities of Caspase-3 and Caspase-8 were increased significantly(P < 0.05).In order to explore whether Slc7a5 promotes apoptosis by increasing oxidative stress in granulosa cells,the levels of ROS and superoxide anion after over-expression of Slc7a5 were detected.The results showed that the levels of ROS and superoxide anion were decreased significantly(P < 0.05).Further explore the mechanism of the decrease of intracellular ROS and superoxide anion level,the expressions of antioxidant enzyme-related genes and proteins(Sod2,Hmox1,xCT)were detected.The results showed that the expressions of Sod2,Hmox1,xCT in granulosa cells were increased after over-expression of Slc7a5(P< 0.05).4.Screening of downstream genes of Slc7a5 and its function in granulosa cellsIn order to further explore the function and mechanism of Slc7a5 in granulosa cells,over-expression of Slc7a5 in granulosa cells was followed by transcriptome sequencing.A total of 1943 differentially expressed genes were found after over-expression of Slc7a5 in granulosa cells,of which 998 up-regulated and 945 down-regulated.KEGG and GO enrichment analysis showed that the differential genes were mainly related to cancer pathway,TNF signal pathway,cell proliferation,protein binding and inflammatory response.Through transcriptome sequencing and further verification,Mgst3 was selected as the downstream gene of Slc7a5.After over-expression of Slc7a5 in granulosa cells,the expression of Mgst3 was decreased,so the effect of decreased expression of Mgst3 on granulosa cells was studied.Firstly,the viability of granulosa cells interfered with Mgst3 was detected by CCK8 test.The results showed that the cell viability decreased significantly(P < 0.05).Then the cell apoptosis and cell cycle of granulosa cells after interfering with Mgst3 were detected by flow cytometry.The results showed that the apoptosis rate was increased and the S phase was decreased significantly(P < 0.05).In order to explore the mechanism of Mgst3 promoting apoptosis of granulosa cell,the expression of apoptosis-related genes and proteins were detected by q RT-PCR and Western blot.After interfering with Mgst3,the expressions of apoptosis-related genes Tnf-?,Caspase-3 and Caspase-8 were significantly increased(P < 0.05),and the expressions of apoptosis-related proteins TNF-?,Caspase-3,Cleaved-Caspase 3,Caspase-8 and Cleaved-Caspase 8 were also significantly increased(P < 0.05).After that,the activities of Caspase-3 and Caspase-8 of granulosa cells after interfering with Mgst3 were further detected.The results showed that the activities of Caspase-3 and Caspase-8 were increased significantly(P < 0.05).Conclusion:1.The expression of Slc7a5 was significantly increased in the ovary of rat model of PCOS.2.At the cellular level,testosterone promotes the expression of Slc7a5 in granulosa cells.3.The increased expression of Slc7a5 could affect the expression of steroid hormone synthesis related enzymes.4.Moreover,the increased expression of Slc7a5 could reduce cell viability,activate apoptosis pathways to promote granulosa cell apoptosis through reduced the expression of Mgst3,and thus participate in the occurrence of PCOS. |
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