| BackgroundThe von Hippel-Lindau (VHL) gene which was discovered by positional cloning technique from the VHL disease patients has been identified as the tumor suppressor gene. The VHL disease is a rare multisystem familial tumor syndrome of autosomal dominant inheritance. The VHL protein encoded by the VHL gene is a multifunctional protein, which induces cell cycle, differentiation and growth arrest through integration of cell-cell and cell-extracellular matrix signaling. It also regulates placental vasculogenesis and embryonic development. Deficiency or inactivation of the VHL gene causes defective placental vasculogenesis and reduced growth of the placental labyrinthine layer, which result in embryonic necrosis and death. The VHL gene can be detected in many human organs. Mutations or aberrant expression of the VHL gene are associated with diseases of various organs such as central nervous system, kidney, adrenal, parorchis. Meanwhile, hypermethylation, loss of heterozygosity and gene silencing of the VHL tumor suppressor gene are frequently present in breast cancer, ovarian tumor and uterine cervix cancer. Endometrial carcinoma is one kind of human female reproductive system tumors, and it associated mortality rate seems to increase in relation with the gradually rising annual incidence. Ectopic pregnancy is the gynaecological emergency par excellence, and the fallopian tube is a common site of ectopic implantation. However, whether the VHL gene may contribute to endometrial carcinogenesis and tubal pregnancy genesis is presently unknown. Aberrant expression or mutations of the VHL gene should be deeply reseached in human endometrial carcinoma and tubal pregnancy tissues. The study should carry out to investigate the association between the expression or mutations of the VHL gene and human endometrial carcinoma or tubal pregnancy, which may elucidate a novel acknowledgement concerning the mechanism of diseases and a new method for clinical diagnosis.Objectives1. To investigate the expression of the VHL gene in tissues of human fallopian tube and tubal pregnancy, and to elucidate the association between the expression of the VHL gene and tubal pregnancy.2. Automation sequenator was performed to detect three exons'DNA sequences of the VHL gene. To compare DNA sequences between tubal pregnancy and normal fallopian tube tissues, and to evaluate the mechanism of the VHL gene in tubal pregnancy development.3. To observe the expression of the VHL gene in normal, hyperplastic and malignant endometrial tissues, and to determine whether aberrant expression of the VHL gene in hyperplastic and malignant endometrial tissues was involved in endometrial carcinogenesis.Materials and MethodsExperiment 1:Expressions of VHL mRNA and protein in human tubal pregnancy tissuesTwenty samples of tubal tissues were obtained from patients undergoing salpingectomy for tubal pregnancy. Each sample was separated into both the implantation site (the area within 5 mm of the gestational mass) and the non-implantation site (more than 10 mm beyond the gestational mass) as the implantation group and the non-implantation group, respectively. Twenty samples of ampullary tubal tissues during midsecretory phase were obtained from patients with tubal ligation or benign uterine disease as the control group. One part of each sample in three groups was immediately frozen in liquid nitrogen and subsequently stored at -70℃until further process for real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The other part was fixed in 10% formalin and embedded in paraffin for histopathological examination and immunohistochemistry. Real-time RT-PCR, Western blotting and immunohistochemistry analysis were performed to detect expressions of VHL mRNA and protein.Experiment 2:DNA sequence analysis of the VHL gene in human tubal pregnancy tissuesTwenty samples of tubal tissues were obtained from patients undergoing salpingectomy for tubal pregnancy as the study group. Ten samples of ampullary tubal tissues during midsecretory phase were obtained from patients with tubal ligation or benign uterine disease as the control group. Automation sequenator was performed to detect three exons'DNA sequences of the VHL gene. DNA sequences were compared between the study group and the control group.Inclusion criteria for the study group were nonruptured tubal pregnancy in the ampullary region and a history of regular monthly menstrual cycles. The gestational age was determined by the date of commencement of the last menstrual period with 6 to 9 weeks. The diagnosis of tubal pregnancy was suspected because of the presence of an adnexal mass on pelvic ultrasound, and there was no evidence of intrauterine pregnancy. Exclusion criteria were that patients in the study group used intrauterine device or receive methotrexate treatment before the operation, and patients in two groups received exogenous hormone treatment or chemotherapy within the last 6 months before surgery.Experiment 3:Expressions of VHL mRNA and protein in tissues of human normal endometrium and endometrial hyperplasiaParaffin embedded sections of endometrial hyperplasia consisting of simple hyperplasia (n=30), complex hyperplasia (n=30) and atypical hyperplasia (n=30), and normal endometrium consisting of proliferative phase (n=30) and secretory phase (n=30) were detected by immunohistochemistry. Fresh tissue samples of endometrial hyperplasia consisting of simple hyperplasia (n=26), complex hyperplasia (n=23) and atypical hyperplasia (n=20), and normal endometrium (n=40) were measured using real-time RT-PCR and Western blotting.Samples of human normal and hyperplastic endometrial tissues were obtained at diagnostic curettage or hysterectomy for benign reasons unrelated to endometrial dysfunction (e.g., leiomyoma, cervical dysplasia, uterine prolaps).Experiment 4:Expressions of VHL mRNA and protein in adenocarcinoma endometrium tissuesSamples of human malignant endometrial tissues were obtained form patients who were operated for endometrial carcinoma. Histology of all malignant samples was endometrioid adenocarcinoma. VHL mRNA and protein were measured using real-time RT-PCR and Western blotting in fresh tissue samples of endometrioid adenocarcinoma (n=17). The VHL staining were detected by immunohistochemistry in paraffin embedded sections of endometrioid adenocarcinoma (n=30).Statistical analysisStatistical analysis was performed using SPSS software, Windows version 13.0 (SPSS, Chicago, IL, USA). Values were expressed as mean±SD, and One-way analysis of variance test was used. The number of subjects in this study was calculated assuming a=0.05. Differences were considered as statistically significant for P<0.05.Antibody-associated staining intensity was described according to positive unit (PU) value by Leica Q550 image analytical system (Leica, Wetzlar, Germany). The formula was PU=100(Ga-Gβ)/Gmax. Gαand Gβwere the average gray scale of positive staining and background, respectively. Gmax was the maximum gray scale of image analytical system.Comparison of RNA assay methods was used to normalize cDNA for quantitative real-time RT-PCR. The PCR amplification was related to a standard curve. The amplification plot, the graph of the increment of fluorescence reporter signal versus cycle number during PCR, was examined early in the reaction, at a point that represents the logarithmic phase of product accumulation. Band intensities of Western blotting were quantified by scanning densitometry utilizing Bio-Rad Quantity One. Bioedit software was used to analyze DNA sequence.Results1. Expressions of VHL mRNA and protein in human tubal pregnancy tissuesVHL staining mainly appeared in the cytoplasm of tubal epithelial cells including secretory cells and ciliated cells, and was faint in stromal cells. The positive staining was also shown in cilia of ciliated cells. VHL staining was strong in the implantation site (1.53±0.98) and weak in the non-implantation site (1.29±0.79) or in the control group (1.21±0.67). A significant difference on VHL staining was found between the implantation group and the non-implantation group or the control group (P<0.01), but no difference was found on VHL staining between the non-implantation group and the control group (P>0.05).The expression of VHL mRNA in the implantation group (2.79±0.74) was higher than that in the non-implantation group (2.11±0.52) or in the control group (2.08±0.69). There was difference on the expression of VHL mRNA between the implantation group and the non-implantation group or the control group (P<0.01), but no difference was found on the expression of VHL mRNA between the control group and the non-implantation group (P>0.05).Bands of VHL protein by Western blotting analysis were observed in the implantation group, the non-implantation group and the control group. Intensity of VHL protein in the implantation group was increased compared with that in the non-implantation group or in the control group (P<0.05), but no difference on VHL protein level between the non-implantation group and the control group (P>0.05).2. DNA sequence analysis of VHL gene in human tubal pregnancy tissuesTotal DNA was extracted from fresh tissue samples of human fallopian tube and tubal pregnancy, and bands of specificity PCR amplification by agarose gel electrophoresis were observed. Amplicon sizes of VHL exon-1, exon-2 and exon-3 were 409bp,208bp and 271bp, respectively. Automation sequenator was performed to detect three exons' DNA sequences of the VHL gene. DNA sequences of VHL exon-1, exon-2 and exon-3 in tissues of tubal pregnancy were coincident with these in normal fallopian tube tissues, but no mutation was found.3. Expressions of VHL mRNA and protein in tissues of human normal endometrium and endometrial hyperplasiaVHL staining appeared in the cytoplasm of epithelial cells and stroma. It was consistently stronger in the superficial layers than that in the basal layers. Staining intensity in proliferative endometrium (5.66±1.77) or simple hyperplasia (5.56±1.76) was stronger than that in secretory endometrium (4.86±1.63) (P<0.01), and no difference was found between proliferative endometrium and simple hyperplasia (P>0.05). Complex hyperplasia (4.74±1.82) had weaker staining compared to simple hyperplasia (P<0.01), but similar to secretory endometrium (P>0.05). VHL staining in atypical hyperplasia (2.61±1.18) was much weaker than that in complex hyperplasia (P<0.01),VHL mRNA expression levels were high in normal endometrium (2.65±0.56) and simple hyperplasia (2.43±0.37), and were low in complex hyperplasia (1.80±0.26) and atypical hyperplasia (1.21±0.12). No difference was found on the expression of VHL mRNA between normal endometrium and simple hyperplasia (P>0.05). A decreased expression of VHL mRNA in endometrial hyperplasia from simple hyperplasia, complex hyperplasia to atypical hyperplasia was observed, and there were differences among simple hyperplasia, complex hyperplasia and atypical hyperplasia (P<0.01).Western blotting analysis was performed for VHL protein. Single band with a molecular mass of 24kd was observed. VHL protein levels were decreased in atypical hyperplasia compared with normal endometrium, simple hyperplasia or complex hyperplasia (P<0.01), respectively. There was no difference among normal endometrium, simple hyperplasia or complex hyperplasia (P>0.05).4. Expressions of VHL mRNA and protein in human adenocarcinoma endometrium tissuesVHL staining appeared in the epithelial cells of adenocarcinoma endometrium. Staining intensity in adenocarcinoma endometrium (2.53±1.27) was much weaker than that in proliferative endometrium, secretory endometrium, simple hyperplasia or complex hyperplasia (.P<0.01). No difference was found on VHL staining between atypical hyperplasia and adenocarcinoma endometrium (P>0.05). VHL mRNA and protein levels in adenocarcinoma endometrium were significantly lower than those in normal endometrium, simple hyperplasia or complex hyperplasia (P<0.01), but similar to those in atypical hyperplasia (P>0.05).Conclusions1. VHL mRNA and protein were present in tissues of human fallopian tube and tubal pregnancy. The VHL gene expression in the implantation site of tubal pregnancy was increased compared with that in the non-implantation site or in normal fallopian tube. The VHL gene could regulate vascular endothelial growth factor mRNA expression at a post-transcriptional level, and the VHL protein was required for proper assembly of an extracellular fibronectin matrix. The VHL gene might be involved in embryonic development and implantation, and locally elevated expression of the VHL gene might be associated with human tubal pregnancy.2. Bands of specificity PCR amplification in tissues of human fallopian tube and tubal pregnancy were the same with designed amplicon size of VHL exon-1, exon-2 and exon-3. DNA sequences of VHL exon-1, exon-2 and exon-3 in tissues of tubal pregnancy were coincident with these in normal fallopian tube tissues, but no mutation was found.3. VHL positive staining appeared in the cytoplasm of epithelial cells and stroma, and VHL mRNA and protein were present in tissues of human normal endometrium and endometrial hyperplasia. VHL staining intensity had been shown to be strong in proliferative endometrium and simple hyperplasia, but weak in secretory endometrium. A decreased expression of the VHL gene in endometrial hyperplasia from simple, complex to atypical hyperplasia was observed. It might suggest that the VHL gene down-regulates vascular endothelial growth factor in proliferative endometrium and simple hyperplasia of endometrium in order to prevent an excess of vascular proliferation. 4. Expressions of VHL mRNA and protein were greatly diminished in adenocarcinoma endometrium, and a novel acknowledgement concerning the mechanism of carcinogenesis was elucidated. Meanwhile, significant VHL gene decrease had been present in atypical hyperplasia which indicates that aberrant expression of the VHL gene was associated with the risk of endometrial hyperplasia progressing to adenocarcinoma endometrium, and its expression levels were useful as a predictive indicator for adenocarcinoma endometrium.
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