The Expression And Correlation Study Of The PDCD4 Gene In Patients With Polyeystic Ovary Syndrome

BackgroundPolycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders, affecting 5-10% of women of reproductive age. The syndrome is a complex ovarian dysfunction involving multiple organs and systems. Most cardinal clinical features of the syndrome are polycystic ovary (PCO) morphology and hyperandrogenism. Its clinical manifestations include menstrual irregularities (oligomenorrhea and amenorrhea), signs of androgen excess (hirsutism, acne and androgenic alopecia) and ovulation disorders (oligo-or anovulation). The long-term complications include type II diabetes, cardiovascular disease, etc. The incidence of PCOS patients with obesity was 47%, which significantly higher than general healthy population and the degree of obesity is highly positive correlation with Insulin Resistance. Comparative study also showed that PCOS patients with obesity had more serious metabolic disorder than that of without obesity, which intensified IR. It has great significance to inhibit obesity and IR for blocking the process of PCOS and improving endocrine abnormalities and ovulation restoration.Growth, maturity and ovulation of follicles are affected by a variety of factors. There only has a mature follicle ovulation in each menstrual cycle and other follicular appear atresia in different periods. A large number of studies have shown that follicular atresia is associated with granulosa cell apoptosis, which is significantly increased in PCOS patients.Programmed Cell Death 4 (PDCD4) has been initially identified as an apoptosis-related gene. Recently, more researches shows that PDCD4 play an increasingly crucial role in some metabolic diseases. High-fat-diet-fed PDCD^-deficienct mice displayed an absolutely lean phenotype through improving insulin sensitivity, which appeared higher energy expenditure, lower epididymal fat weight, and reduced macrophage infiltration, inflammatory cytokine secretion in white adipose tissue. The data demonstrate that PDCD4 expression might be as a potential treating target for obesity-associated diseases. These also indicate that the obesity and pro-apoptosis effects of PDCD4 expression under certain pathological conditions, while the role of PDCD4 expression in PCOS whether is related with obesity and apoptosis has not been established.This study was designed to analyze the expression of PDCD4 in PCOS patients and normal healthy women, further to explore its effect on the development of granulosa cells apoptosis, and to discuss possible significance of PDCD4 expression in the PCOS pathogenesis.Objective1. Observe the expression state and clinical significance of PDCD4 in PCOS patients.2. Analyse the relation between PDCD4 expression and obesity, IR, lipid metabolism disturbance.3. Explore the effect of PDCD4 expression on development of granulosa cells apoptosis.MethodsClinical samplesA total of 144 Chinese Han women were obtained from Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University. Among them,77 PCOS patients and 67 healthy women as case and control groups according to new Chinese Diagnosis Criteria for PCOS. Written informed consent was obtained from all of women before participation. The Center for Reproductive Medicine Institutional Review Board (IRB) completely approved the study protocol. PCOS subjects should present at least two of the following criteria:oligo-and/or anovulation, clinical or biochemical hyperandrogenism and polycystic ovarian morphology through ultrasonography after exclusion of other etiologies (eg, congenital adrenal hyperplasia, Cushing's syndrome and androgen-secreting tumors). All of fertile women controls have normal androgen levels, regular menstrual cycles and exclude signs of polycystic ovaries by trans-vaginal ultrasound. The body mass index (BMI) > 25 kg/m2 were identified as obese. None of the patients studied had received adjuvant medication or surgery treatments at before.Quantitative real-time PCR (qRT-PCR)Total RNA was extracted from isolated peripheral blood mononuclear cells (PBMCs). The level of mRNA for PDCD4 relative to P-actin was calculated using the ??Ct method.Isolation of Granulosa CellsAn ultrasound scan and serum estradiol assays were performed for monitoring follicular size. If there were three or more follicles with mean diameter?1.8 cm, 8000-10,000 IU human chorionic gonadotropin was given in the time of 36 hours before ultrasound-guided immature oocyte retrieval. After anesthetized, oocyte retrieval was performed through a 17-G double-lumen aspiration needle. GCs around oocytes were collected and washed twice by Dulbecco's Modified Eagle Medium (DMEM) for further study after removal of the oocyte in follicular aspirates using Pasteur pipette.Small interfering RNA transfectionGCs were transfected using siPORT NeoFX Transfection Agent. siRNA interference was performed using Lipofectamine 2000 according to the instruction. Negative control siRNA was used as transfection control.Flowcytometry analysisOvarian GCs were stained with Annexin V-FITC apoptosis detection KIT and/or propidium iodide according to the manufacturer's protocols.TUNEL analysisOvarian granulosa cells of PCOS patients were transfected by PDCD4-SiRNA after isolation and purification and were dyed using TUNEL apoptosis detection kit. Apoptosis changes of GCs were observed by laser confocal microscopy.Western blot assayCells were lysed in SDS buffer, and concentration of protein was determined using BCA Protein Assay. Equal amounts of protein were separated on SDS-PAGE and transferred onto PVDF membranes. Membranes were then blocked with 5% skim milk in TBST containing 0.1% Tween-20 for 1 h, and then filters were incubated at 4? overnight with rabbit monoclonal antibodies against Bax, Bcl-2 and rabbit anti-?-actin polyclonal antibody respectively. Immunoblotting was done by incubating the membranes and followed by secondary antibody conjugated with peroxidase for 1 h at room temperature. After washing, signals were visualized by SuperSignal West Pico Chemiluminescent Substrate. Statistical AnalysisAll calculations were performed using SPSS statistical software package. All statistical analyses were two-sided, and values are presented as mean ± SEM. ANOVA or Student t tests were performed to determine significance. Spearman's correlation and multivariate regression analyses were used to examine the association between PDCD4 expression and clinical characteristics. Differences were considered significant as P<0.05.Results1. Different expression status of PDCD4 in PCOS patients and controls.(1) Patient characteristicsPatients with PCOS had greater E2, Total Testosterone, LH, Insulin 0, Insulin 120 and HOMA-IR (homeostasis model assessment-insulin resistance index) than control subjects (P<0.05). Age, BMI, TSH, Glucose 0, Glucose 120, HOMA-? (homeostasis model assessment-? cell function index), Triglycerides and HDL (high-density lipoprotein) were not significant differences between PCOS women and controls (P> 0.05).(2) PDCD4 expression in PBMCs of controls and women with PCOSPDCD4 levels are significantly higher in PCOS women compared with controls by qRT-PCR in PBMCs (P<0.05). According to BMI, PCOS women and controls were further divided into obese and lean sub-groups. After this classification, PDCD4 levels in obese PCOS patients were more statistically different from those in BMI-matched controls (P<0.01).2. The relationship between PDCD4 expression and obesity, IR, lipid metabolism disturbance in PCOS patients.To explore significance of increased PDCD4 levels in PCOS, we firstly analyzed the relationship between PDCD4 expression and obesity. Results showed that no significant correlation between PDCD4 expression and BMI in control group. However, PDCD4 expression significantly correlated with BMI (r=0.52, P<0.0001) of PCOS group, which indicated that PDCD4 expression was markedly associated with obesity progression of PCOS patients.It was known that HOMA-IR and HOMA-p were respectively most important indexes of homeostasis model assessment for insulin resistance and ?-cell function. PCOS women had significantly higher Insulin 0, Insulin 120 and HOMA-IR compared with controls (P<0.01 or 0.05). The results were similar as these indices with addition of Glucose 120 between BMI-matched obese PCOS women and controls (P<0.01 or 0.05). There was no relation between PDCD4 expression and these indexes in control group. However, PDCD4 expression positively correlated with Insulin 0, Insulin 120, Glucose 120, HOMA-IR and HOMA-? (r=0.34,0.31, 0.29,0.36 and 0.25, separately; P<0.01 or 0.05) in PCOS patients. Interestingly, after metformin 500 mg twice daily for two or three months, PDCD4 expression was distinctly down-regulated for obese PCOS women with insulin resistance (P<0.05). It suggested that PDCD4 levels correlated with IR or ?-cell dysfunction of PCOS patients, which could be improved through oral metformin for decreasing the expression.To determine effect of PDCD4 expression on lipid metabolism for PCOS patients, we further analyzed the relationship between PDCD4 expression and lipid metabolic parameters. Though there were no statistical differences between PCOS women and controls for Triglycerides and HDL, PDCD4 expression positively correlated with Triglycerides (r=0.39;P<0.001) and negatively correlated with HDL (r=-0.41; P< 0.001) in PCOS patients. These results indicated that PDCD4 expression might greatly promote lipid accumulation for PCOS women and lead to lipid metabolic dysfunction.To examine whether PDCD4 expression was an independent risk factor for PCOS patients, we performed multivariate logistic regression analysis to further explore the association between PDCD4 expression and PCOS risk. Our model included potential confounders, such as age, BMI, hormone, IR and lipid metabolic indexes. The level of PDCD4 expression could significantly predict the risk of PCOS patients independent of other clinicopathologic variables after adjusting for E2 and Insulin 120 (OR=1.318; 95% confidence interval,1.021-1.703; P<0.05).3. The effect of PDCD4 expression on the development of granulosa cells apoptosis.It had been recognized that PDCD4 expression could inhibit protein translation by binding with eIF4A and resulted in cell apoptosis. Furthermore, there were more easily apoptotic for ovarian GCs of PCOS patients. Flow cytometry analysis showed that the percentage of apoptotic GCs in PCOS group was much higher than that of controls (P<0.05).To confirm important role of PDCD4 expression on GCs apoptosis, we examined knockdown of PDCD4 expression on effect of GCs apoptosis by PDCD4-specific siRNA sequences. Compared with negative control, apoptosis percentage was statistically diminished in PDCD4-knockdown group through Annexin V-FITC/PI analysis (P<0.05). In addition, TUNEL analysis further revealed that silence of PDCD4 expression significantly decreased number of apoptotic cells after siRNA transfection for 24 h.To determine whether silence of PDCD4 expression impacted signal pathway of apoptosis, we detected both expression of pro-apotosis factor Bax and anti-apoptosis factor Bcl-2. Western blot showed that Bax expression was decreased, Bcl-2 expression was increased and Bax/Bcl-2 ratio was lower by inhibition of PDCD4 expression.Conclusion1. PCOS patients had higher PDCD4 expression, similar with BMI-matched with obese group.2. Higher PDCD4 expression positively was correlated with obesity, IR and lipid metabolic dysfunction.3. PDCD4 could promote GCs apoptosis of PCOS patients, which could be obviously inhibited after gene knockdown by influencing the expression of key signaling molecule of apoptosis pathway.