The Expression Of Von Hippel-Lindau Gene In Human Endometrium And Fallopian Tube And VHL Gene Methylation Status In Human Endometrial Tissues

Objectives1. The expression and change of von Hippel-Lindau (VHL) gene was investigated in human endometrium during the menstrual cycle to analyze the function of VHL gene.2. The change of P300 and cAMP responsive element-binding protein binding protein (P300/CBP) and methylation status of VHL gene was investigated in human endometrium to explore the status of epigenetics in physiological function of human endometrium.3. The change of hypoxia inducible factor-1?(HIF-1?) was detected in human endometrium. The correlation of HIF-1?and VHL protein, HIF-la and P300/CBP was analyzed to explore the regulation and control mechanisms of HIF-1?.4. The expression of VHL mRNA and protein was investigated in human fallopian tube.Materials and Methods1. Expression of the VHL protein and VHL mRNA in human endometriumEndometrial samples were collected from 30 childbearing age women underwent abdominal hysterectomy for benign disease in the pelvic cavity. These women had normal menstrual cycles and history of normal pregnancy. The collected specimens were divided into 4 groups based on their menstrual cycles:early-proliferative stage (n=7), mid- and late-proliferative stage (n=8), early-secretory stage (n=6), and rnid-and late-secretory stage (n=9). The expression of VHL protein and VHL mRNA in endometrium was determined by laser cofocal scanning microscopy (LCSM), immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and western blotting methods.2. Expression of the P300/CBP and VHL gene methylation in human endometriumFresh endometrial samples were collected from 15 childbearing age women and divided into proliferative stage (n=7) and secretory stage (n=8). These samples were used to detect P300/CBP by western blotting method and methylation status of VHL gene by methylating-specific polymerase chain reaction method.3. The correlation among VHL protein, P300/CBP and HIF-1?Endometrial samples were collected from 30 childbearing age women underwent abdominal hysterectomy for benign disease in the pelvic cavity. These women had normal menstrual cycles and history of normal pregnancy. The collected specimens were divided into 4 groups based on their menstrual cycles:early-proliferative stage (n=7), mid- and late-proliferative stage (n=8), early-secretory stage (n=6), and mid-and late-secretory stage (n=9). The expression of HIF-1?protein and HIF-1?mRNA in the endometrium was determined by immunohistochemistry, RT-PCR and western blotting methods. The correlation among VHL protein, P300/CBP and HIF-la was analyzed by Pearson analyze of SPSS.4. Expression of the VHL protein and VHL mRNA in human fallopian tubeFallopian tubes were collected from 27 childbearing age women underwent abdominal hysterectomy with adnexectomy for benign disease in the pelvic cavity. These women had normal menstrual cycles and history of normal pregnancy. Isthmus, ampullary and umbrella segments of fallopian tubes were respectively collected. The collected specimens were divided into 4 groups based on their menstrual cycles:early-proliferative stage (n=6), mid- and late-proliferative stage (n=8), early-secretory stage (n=4), and mid- and late-secretory stage (n=9). The expression of VHL protein and VHL mRNA in fallopian tube was determined by LCSM, RT-PCR and western blotting methods. The change of VHL protein was determined by immunohistochemistry.5. Statistical analysisImmunohistochemical results were indicated by the positive unit value (PU). The densitometric values were used to calculate the ratios between target and GAPDH or?-actin bands. Results were expressed as mean±standard derivation (SD). SPSS 11.5 statistics software was employed to process the data.Results1. Expression of the VHL mRNA and VHL protein in human endometriumUsing RT-PCR and western blotting method VHL mRNA and VHL protein appeared in human endometrium. Positive VHL protein staining was observed in the glandular and luminal epithelial and stromal cells of the endometrial tissues. In the menstrual cycle, the expression level of VHL protein in the epithelium was higher in the proliferative stage (the early-proliferative stage:3.99±1.68; the mid- and late-proliferative stage:3.68±1.31), and decreased in the secretory stage (the early-secretory stage:2.29±0.73; the mid- and late-secretory stage 2.02±0.59). The difference between the proliferative and secretory stages was significant (P<0.05). The level of VHL mRNA was significantly higher in proliferative stage (1.26±0.46) than that in secretory stage (0.69±0.28) (P<0.05).2. Expression of the P300/CBP and VHL gene methylation in human endometriumThe level of P300/CBP protein was significantly higher in proliferative stage (0.72±0.27) than secretory stage (0.24±0.19) (P<0.05). There was unmethylated stasus of VHL gene in human endometrium.3. The correlation among VHL protein, P300/CBP and HIF-laPositive expression of HIF-la protein and HIF-la mRNA was observed in the cytoplasm of the epithelial and stromal cells. In the menstrual cycle, the expression level of HIF-la protein in the epithelium was higher in the proliferative stage (the early-proliferative stage:2.81±1.01; the mid- and late-proliferative stage:3.21±0.86), and decreased in the secretory stage (the early-secretory stage:1.83±0.73; the mid-and late-secretory stage:1.63±0.53). The difference between the proliferative and secretory stages was significant (P<0.05). The level of HIF-1?mRNA was significantly higher in proliferative stage (0.86±0.43) than that in secretory stage (0.32±0.23) (P<0.05).The expression of HIF-1?in human endometrium had positive correlation with the expression of VHL protein and P300/CBP.4. Expression of the VHL mRNA and VHL protein in human fallopian tubePositive expression of VHL mRNA and VHL protein was detected in human fallopian tube by RT-PCR and western blotting methods. The VHL protein appeared, in the cytoplasm of the ciliated and nonciliated cells. Stromal cells showed weak positive expression of VHL protein. In the menstrual cycle, the expression level of VHL protein in the epithelium was not changed during the early-proliferative stage (isthmus:5.21±4.17; ampullary:4.27±2.26; umbrella:3.58±1.31), mid- and late-proliferative stage (isthmus:2.99±1.00; ampullary:2.98±0.83; umbrella: 4.26±1.03), early-secretory stage (isthmus:3.69±1.80; ampullary:3.87±2.78; umbrella:3.33±1.22), and mid- and late-secretory stage (isthmus:3.44±1.66; ampullary:3.84±2.52; umbrella:2.88±1.26). In the same phase of menstrual cycle, there was no si gnificant difference in the expression level of VHL protein in the epithelium among isthmus, ampulla and umbrella segments (P>0.05). VHL protein expression in the same part of tube was unchanged in the different phases of menstrual cycle (P>0.05).Conclusions1. The VHL protein and gene appeared in human endometrium and its expression changed cyclically. VHL gene might be involved in regulating the balance of angiogenesis in the proliferating endometrium.2. The expression of P300/CBP in human endometrium had cyclic change. P300/CBP was perhaps essential for estrogen-induced endometrial growth. The demethylation of VHL gene would regulate the transcription of VHL gene to participate in physiological activity of human endometrium.3. The HIF-la protein and gene existed in human endometrium and their expression had cyclic change in the menstrual cycle. Perhaps HIF-la was essencial for induction of genes that were necessary to basal physiological action in normal human endometrium. P300/CBP and VHL protein participated in the regulation of HIF-la expression in the endometrium.4. The VHL protein and gene appeared in human fallopian tube. The expression of VHL protein was unchanged during the menstrual cycle and might involve in the differentiation of ciliated and secretory cells.