| OBJECTIVE:Polycystic ovary syndrome(PCOS)is the most common endocrine disorders in women of childbearing age,and most of the patients develop hyperinsulinemia and insulin resistance as the disease progresses.Studies have found that miR-320 involves in the regulation of insulin sensitivity in adipose tissues,the overexpression of miR-320 could reduce insulin sensitivity and lead to the occurrence of IR.It can therefore be inferred that the expression abnormalities and dysfunction of miR-320 play an important role in the occurrence and development of IR in PCOS patients.In addition,studies have confirmed that the PI3K/AKT signaling pathway is vital in the PCOS insulin resistance,therefore,this study is to investigate the role of miRNA-320 in the regulation of insulin resistance in PCOS rats through PI3K/Akt signaling pathway.METHODS:The PCOS rat model was established by intragastric administration of Letrozole,and the control group was established at the same time,continuous detections of vaginal smear were carried out in both the control and experimental groups.Based on the test results,it was determined which estrous cycle the rats were in,graphs of estrus cycle were drawn for comparison.HE staining was used to detect and compare morphological characteristics of ovarian tissue in rats.The expressions of rats' serum sex hormones(LH,FSH,T)were detected and compared by enzyme-linked immunosorbent assay(ELISA),The ovarian granulosa cells were extracted,cultured,and were then transfected with miR-320 mimic and miR-320 inhibitor respectively,while the control group was established at the same time.The expression of miR-320 of ovarian granulosa cells in both groups were detected and compared by RT-PCR,while the protein expression of PI3K/Akt signaling pathway(PI3K,AKT,GLUT4,Foxo3a,P27)was detected and compared by western blot.24 Wistar rats were randomly divided into 3 groups:miR-320 mimic group,miR-320 inhibitor group and PCOS model group,with 8 in each group and PCOS modelled.Wistar rats in miR-320 mimic group were injected with MiR-320 mimic(2 mg/kg),Wistar rats in miR-320 inhibitor group were injected with miR-320 inhibitor(2 mg/kg),and in the PCOS model group,rats were injected with normal saline solution of the same volume,which lasted for 30 days.The HE staining was used to detect and compare the ovarian tissue between groups,the serum FINS,TG,FPG and HOMA-IR of each group were detected and compared by radioimmunoassay,and western blot was applied for the detection and comparison of the protein expression of PI3K/Akt pathway.RESULTS:There were obvious regularities in terms of the estrous cycles of rats in control group,but rats in the model group were always in the estrous cycle with no obvious regularity.Light microscopy observation revealed that corpus luteum and follicle tissues of varioous phases were detected in the ovarian tissue sections of rats in control group,pathological changes were observed in the anatomical structure of the ovarian tissue of rats in the model group,which was marked by a significant increase of the number of follicles in the ovary,and a significant decrease of the number of corpus luteum and follicle tissues in the developmental stage.The serum expression levels of LH,FSH and T of rats in the model group were significantly higher than those in the control group(P<0.05),while the levels of E2 in the serum of rats in model group were significantly lower than those of rats in the control group(P<0.05);the expression of 320 mRNA of ovarian granule cells of rats in the control group was significantly lower than that of rats in miR-320 mimic group,but significantly higher than that of rats in the miR-320 inhibitor group(P<0.05);in terms of the protein expression(PI3K?AKT?GLUT4?Foxo3a?P27)of PI3K/Akt signaling pathway,rats in the control group had a significantly higher level than those from the miR-320 mimic group,but significantly lower then the level of rats from miR-320 inhibitor group(P<0.05).It had been found in the model group that many small follicles and atresia follicles were in early development,the cystic dilatation was more obvious,the granulocyte layer was reduced or even disappeared,there was less of the corpus luteum and the ovary displayed typical polycysticoid changes.In contrast,there was a clear increase of small follicles and atresia follicles in the miR-320 mimic group,but no obvious granulosa cells or corpus luteum were observed,and there was a clear decrease of small follicles and atresia follicles in the miR-320 inhibitor group,obvious granulosa cells and corpus luteum were observed,and polycystic changes were detected in part of the ovary.In terms of the TG and FPG levels of serum,there was no significant difference between groups(P>0.05),in comaparison with rats from the control group,the FINS and HOMA-IR level of serum of rats in miR-320 mimic group was significantly higher(P<0.05),but the FINS and HOMA-IR level of serum of rats in miR-320 inhibitor group was significantly lower(P<0.05).The expression of PI3K/Akt signaling pathway proteins(PI3K,AKT,GLUT4,Foxo3a,P27)in ovarian tissues of rats in miR-320 mimic group was significantly lower than that of rats in model group(P<0.05),but rats in the miR-320 inhibitor group showed a significantly higher level in this regard(P<0.05).CONCLUSION:Letrozole successfully constructed PCOS rat model and the primary culture of rat ovarian granulosa cells was also a success,when miR-320 mimic and miR-320 inhibitor were respectively transfected into rat ovarian granulosa cells,there was a clear corresponding rise and inhibition of the expression of miR-320 mRNA.The overexpression of miR-320 could significantly reduce the expression of PI3K/Akt signaling pathway proteins(PI3K,AKT,GLUT4,Foxo3a,P27)in ovarian granulosa cells and the ovary of PCOS rats,which resulted in the inhibition of PI3K/Akt Signaling pathway,through the PI3K/Akt signaling pathway,the overexpression of miR-320 might thereby inhibit the PI3K/Akt signaling pathway,and eventually leads an increased IR in PCOS rats. |
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