| Adenomyosis is a nontumorous condition characterized by the presence of ectopic endometrium in the myometrium with hyperplasia of adjacent smooth muscle. This probably occurs by invagination of the basalis endometrium into the myometrium. The process of invagination and intramyometrial dissemination may be facilitated by the non-cyclic, anti-apoptotic activity of the basalis associated with relative hyper-oestrogenic states. Most cases of adenomyosis are discovered in multiparous women during the transitional years (40-50 years). It can result in debilitating pelvic pain (both cyclical and non-cyclical) and abnormal uterine bleeding. Magnetic resonance imaging establishes the diagnosis in cases of equivocal or nondiagnostic ultrasounds. Definite diagnosis and treatment of adenomyosis are obtained by hysterectomy. Vessel embolization, hormonal treatments (progestagens, continuous oral contraceptive pills, anti-estrogens and gonadotrophin-releasing hormone (GnRH) analogues) and high-intensity focused ultrasound can be tried in infertile women, however, the effect of these treatments is limited. Surgical extirpation has been the best therapeutic option for symptom relief so far. The etiology, pathogenesis and the better conservative treatment are still unknown to date.Autophagy, characterized by autophagic vacuoles accumulating, is considered as programmed cell death typeⅡwhich is known as autophagic cell death. Autophagy is involved not only in the balance between protein synthesis and degradation but also in the balance between cell survival and cell death. Beclin 1, a haploinsufficient tumor suppressor gene on chromosome 17q21 and a mammalian homolog of yeast Atg6/Vps30, is monoallelically deleted in up to 75% of ovarian cancers,50% of breast cancers and 40% of prostate cancers. Reduction of Beclin 1 is also observed in other cancers including human brain tumors and cervical carcinoma. Overexpression of Beclin 1 not only promotes nutrient deprivation-induced autophagy but also inhibits clonigenicity and tumorigenesis in human breast carcinoma cells. Structural studies display that Beclin 1 has a BH3-only domain, a central coiled-coil domain (CCD), and an evolutionarily conserved domain (ECD). The ECD of Beclin 1 is required for Vps34 binding, autophagy and tumor suppression. The BH3-only domain can interact with Bcl-2 and Bcl-XL. The Beclin 1 protein is an important convergence point of autophagy and apoptosis; it interacts with the anti-apoptotic and anti-autophagic Bcl-2 protein.Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. HIF-1 transcriptional activity is determined by regulated expression of the HIF-la subunit. HIF-1a is a transcriptional factor that regulates genes involved in response to hypoxia and promotes neoangiogenesis, which are considered essential for tumor growth and progression. Under normoxic conditions it is constitutively produced and degraded by the ubiquitin-proteasome system. But under hypoxic conditions it becomes stabilized.Hypoxia is associated with autophagy. Mitochondrial autophagy is induced by hypoxia, this process requires the HIF-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1. In cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death.Researchers have launched a new area of febrile investigations on the autophagy-related gene Beclin 1. However, no study has previously shown the contribution of Beclin 1 to adenomyosis and the correlation between Beclin 1 and HIF-1a. This study investigated the expression of Beclin 1 in human adenomyosis, its association with clinical characteristics and its regulation in endometrial stromal cells by hypoxia. Cell cultures, immunohistochemical technique, RT-PCR, and western blotting were used in this study. The study was divided into three parts:1. Decreased expression of Beclin 1 in eutopic endometrium of women with adenomyosis and its association with clinical characteristics.2. Beclin 1, HIF-1αexpression in eutopic endometrium and adenomyotic foci of women with adenomyosis and the correlation between Beclin 1 and HIF-1α.3. Beclin 1 expression in endometrial stromal cells from adenomyois and its regulation by hypoxia.Part one Decreased expression of Beclin 1 in eutopic endometrium of women with adenomyosis and its association with clinical characteristics.Objective:To investigate whether Beclin 1 expression is altered in eutopic endometrium of women with adenomyosis.Methods:We collected tissue samples from the eutopic endometria of 30 women with adenomyosis and 32 healthy women undergoing surgery for benign indications. Beclin 1 expression of eutopic endometrial tissues was assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis.Results:Beclin 1 mRNA expression level in tissue samples of women with adenomyosis (0.71±0.10) was significantly lower than that of control (0.93±0.10) (P<0.001). The average level of Beclin 1 protein expression of women with adenomyosis in tissue samples (0.82±0.22) was also significantly lower than that of control (1.22±0.36) (P<0.001). Beclin 1 protein expression in eutopic endometrial tissues was negatively correlated with serum CA125 (r=-0.307, P=0.015), and pelvic pain (r=-0.542, P=0.000).Part two Beclin 1, HIF-la expression in eutopic endometrium and adenomyotic foci of women with adenomyosis and the correlation between Beclin 1 and HIF-1α.Objective:To investigate whether Beclin 1 and HIF-la were expressed in eutopic endometrium and adenomyotic foci of women with adenomyosis and the correlation between Beclin 1 and HIF-1α.Methods: Beclin 1 and HIF-1αexpression were assessed by immunohistochemistry.Results:1. Beclin 1 immunoreactive staining was present in the cytoplasm of glandular epithelial and stromal cells. HIF-1αimmunoreactive staining was present in the cytoplasm and nucleus of glandular epithelial and stromal cells in eutopic endometrium, mainly in the cytoplasm of glandular epithelial cells in adenomyotic foci. 2. Beclin 1 expression in eutopic endometrium of adenomyosis (3.62±2.69) was significantly lower than that in control (10.32±4.12)(P<0.01). Beclin 1 expression in adenomyotic foci (0.27±0.52) was significantly lower than that in eutopic endometrium of adenomyosis or in control (P<0.01). HIF-1αexpression in eutopic endometrium of adenomyosis (8.70±2.13) was significantly higher than that in control (4.49±2.55)(P<0.01). HIF-1αexpression in adenomyotic foci(7.97±2.54)was significantly higher than that in control(P<0.01), and not different from that in eutopic endometrium of adenomyosis. There were no differences of Beclin 1 expression between proliferative period and secretory period in adenomyosis or control. Beclin 1 expression of proliferative period or secretory period in adenomyosis was significantly lower than that in control(P<0.01). There were no differences of HIF-1αbetween proliferative period and secretory period in adenomyosis. There were differences of HIF-1αexpression between proliferative period and secretory period in control (P<0.01). HIF-1αexpression of proliferative period in adenomyosis was significantly higher than that in control(P<0.01). There were no differences of HIF-1αbetween adenomyosis and control in secretory period. Beclin 1 expression in adenomyotic foci was significantly lower than HIF-1αexpression(P<0.01).3. Beclin 1 expression was negatively correlated with serum CA125 (n=140, r2=0.232, P=0.000), and pelvic pain (n=140, r2=0.508, P=0.000). HIF-1αexpression was positively correlated with serum CA125 (n=140, r2 =0.268, P=0.000), and pelvic pain (n=140, r2=0.574, P=0.000). Beclin 1 expression was negatively correlated with HIF-1αexpression (n=140, r2=0.201, P=0.000).Part three Beclin 1 expression in endometrial stromal cells from adenomyois and its regulation by hypoxiaObjective:To investigate whether Beclin 1 expression is altered in endometrial stromal cells from adenomyosis and its regulation by hypoxia.Methods:Beclin 1 expression was assessed by RT-PCR and western blot analysis. The capability of proliferation and metastasis of endometrial stromal cells were assessed by MTT and migration assay.Results:1. Beclin 1 mRNA expression level in cultured stromal cells of women with adenomyosis (0.72±0.19) was significantly lower than that of without (1.17±0.44) (P<0.05). Beclin 1 protein expression of women with adenomyosis in cultured stromal cells (0.20±0.03) was significantly lower compared with that of control (0.71±0.12)(P<0.01).2. The growth rate by MTT assay and the migrated distance of endometrial stromal cells in adenomyosis (0.33±0.03,0.31±0.02mm, respectively) were significantly higher than those in the control group (0.24±0.04,0.28±0.02mm, respectively) (P<0.01). The growth rate by MTT assay and the migrated distance of endometrial stromal cells after 24h hypoxia in adenomyosis (0.46±0.03,0.37±0.02mm, respectively) were significantly higher than those under normoxia in adenomyosis (0.33±0.03,0.31±0.02mm, respectively) (P<0.01). The growth rate by MTT assay and the migrated distance of endometrial stromal cells after 24h hypoxia in control (0.20±0.02,0.14±0.02mm, respectively) were significantly lower than those under normoxia in control (0.24±0.04,0.28±0.02mm, respectively) (P<0.05).3. Beclin 1 protein expression of endometrial stromal cells after 24h hypoxia in adenomyosis (0.30±0.01) was significantly lower than that in adenomyosis under normoxia (0.49±0.01) (P<0.01)。Beclin 1 protein expression of endometrial stromal cells after 24h hypoxia in adenomyosis (0.30±0.01) was significantly lower than that in control under hypoxia (0.65±0.02) (P<0.01)。Beclin 1 protein expression of endometrial stromal cells from in adenomyosis (0.49±0.01) was significantly lower than that in control (0.70±0.03) (P<0.01)。There was no difference between the control group under hypoxia (0.65±0.02) and the control group under normoxia (0.70±0.03).4. In endometrial stromal cells after 24h hypoxia from adenomyosis, Beclin 1 protein level was negatively related with the cell proliferation and migration(r2=0.564,0.469;P=0.004,0.042). Conclusion:1. Beclin 1 immunoreactive staining was present in the cytoplasm of glandular epithelial and stromal cells. Expression of Beclin 1 was decreased in eutopic endometrium and adenomyotic foci of women with adenomyosis, and the Beclin 1 expression was negatively correlated with the serum CA125 and pelvic pain, which might be related to the pathogenesis and progress of adenomyosis.2. HIF-1αimmunoreactive staining was present in the cytoplasm and nucleus of glandular epithelial and stromal cells in eutopic endometrium, mainly in the cytoplasm of glandular epithelial cells in adenomyotic foci. Expression of HIF-1αwas increased in eutopic endometrium and adenomyotic foci of women with adenomyosis. Its expression level was positively correlated with the serum CA125 and pelvic pain, which might be related to the pathogenesis and progress of adenomyosis.3. Beclin 1 expression was negatively correlated with HIF-1αexpression. If the expression of HIF-1αis higher and the expression of Beclin 1 is lower, the adenomyosis is more severe.4. The growth rate and the migrated distance of endometrial stromal cells in adenomyosis were significantly higher than those in control. The growth rate and the migrated distance of endometrial stromal cells after 24h hypoxia in adenomyosis were significantly higher than those under normoxia in adenomyosis. The endometrial stromal cells of adenomyosis had the greater capacity of proliferation and migration induced by hypoxia.5. Beclin 1 protein expression of endometrial stromal cells under hypoxia in adenomyosis was significantly lower than that under normoxia in adenomyosis。Beclin 1 protein expression of endometrial stromal cells under hypoxia in adenomyosis was significantly lower than that under hypoxia in control.6. In endometrial stromal cells after 24h hypoxia from adenomyosis, Beclin 1 protein level was negatively related with the cell proliferation and migration, hypoxia-induced change of Beclin 1 might be related to the pathogenesis and progress of adenomyosis.
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