||The functional role of hypoxia-induced MXI1 in endometriosis
||Department of Physiology
子宮內膜異位症，泛指子宮內膜腺體及基質細胞生長於子宮外，是一種極為常見的婦女疾病，並大大影響了病患的生活品質。子宮內膜異位症被廣泛接受是起因於子宮內膜組織遭遇了經血逆流的壓力，在此過程中，剝落下來之子宮內膜組織因獲得細胞抗凋亡及細胞增生能力，以至於這些細胞能夠生存在像是缺氧、細胞不貼附、以及營養不足等許多逆境中。缺氧在子宮內膜異位症中調控許多細胞生長反應，像是發炎反應、類固醇形成、血管新生等。但在子宮內膜異位症中，缺氧是否提高細胞增生及抗凋亡能力的機制是尚未被闡明的。因此，我們假設在經血逆流過程中，剝落下來的子宮內膜原位基質細胞會遭遇到缺氧逆境、進而提高細胞抗凋亡及細胞增生能力。首先，經由西方墨點分析凋亡蛋白酶三，以及溴化去氧尿苷的滲入培養來偵測增生中細胞等實驗下，我們證實了在缺氧及缺氧誘導物DMOG的前處理後，子宮內膜原位基質細胞不但對於喜樹鹼所引發的細胞凋亡能力顯著下降，並且其細胞增生能力顯著提升，本結果支持我們提出之缺氧誘導子宮內膜原位基質抗凋亡及細胞增生現象之假說。為了更進一步探討在基質細胞中有什麼因子調控缺氧誘導的保護作用，我們利用GEO生物資訊分析成對的子宮內膜原位及異位基質細胞中的基因表現 (GSE7305)，發現MXI1這個抗凋亡轉錄調控因子在子宮內膜異位組織的表現量是上升的。而利用定量聚合酶連鎖反應以及西方墨點分析下，我們首次證實了MXI1的信使核糖核酸以及蛋白質量確實在人類子宮內膜異位基質細胞中表現上升。我們更進一步利用生物資訊工具The Best探討人類MXI1基因內部的結合元件，釐清其啟動子區域內具有兩個典型的缺氧誘導元件，可被缺氧誘導因子所接合。在藉由缺氧及DMOG的培養下，子宮內膜原位基質細胞的MXI1信使核糖核酸以及蛋白質的表現量上升。然而，缺氧前處理的子宮內膜原位基質細胞其細胞增生能力上升現象在給予微小干擾MXI1核苷酸後是被抑制的。綜上所述，缺氧誘導MXI1在子宮內膜異位症致病機轉中可能是一個重要的細胞增生調控因子。
Endometriosis, defined as the presence of endometrial glandular and stromal tissues outside of the uterine cavity, is one of the most common gynecological diseases and greatly reduces the quality of patients’ life. It is well-accepted that ectopic endometriotic tissues are derived and established from the retrograded endometrial fragments. During retrogradation, the cast-off endometrial tissues have to gain the capacities of anti-apoptosis and proliferation to survive under numerous stresses such as hypoxia, cell detachment, and nutritional deprivation. Hypoxia modulates several cellular processes involved in the pathological developments of endometriosis such as inflammation, steroidogenesis, and angiogenesis, but it still remains elusive whether hypoxia indeed promotes anti-apoptosis and proliferation in endometriosis. Therefore, we hypothesized that the hypoxic stress promotes anti-apoptosis and cell proliferation in eutopic stromal cells during retrograded menstruation. First, the western blotting results of caspase 3 activation and the BrdU incorporation assay, which directly detects proliferating cells showed that pretreatment with hypoxia and hypoxia mimetic Dimethyloxaloylglycine (DMOG) in eutopic stromal cells significantly not only reduced camptothecin (CPT)-induced apoptosis but also promoted cell proliferation, supporting the hypothesis that hypoxia induces anti-apoptosis and proliferation in eutopic stromal cells. To further investigate what factor mediates the hypoxia-induced protective effect in the stromal cells, we analyzed the differentially expressed genes in paired eutopic and ectopic stromal cells by using Gene Expression Omnibus (GEO) data (GSE7305), and found that MAX interactor 1, dimerization protein (MXI1), an anti-apoptotic transcription factor, was elevated in ectopic stromal cells. By using quantitative PCR and western blotting, we first confirmed that MXI1 is elevated in human ectopic stromal cells compared to the paired eutopic stromal cells. Moreover, the Binding Elements Searching Tools (The BEST) identified two typical hypoxia-responsive elements (HREs), the DNA sequence recognized by hypoxia-inducible factors, in human MXI1 promoter. Treatment with hypoxia or DMOG in eutopic stromal cells induced the expression of MXI1 mRNA and protein. Nevertheless, the hypoxia pretreatment-induced cell proliferation was suppressed by MXI1 knockdown in eutopic stromal cells. Taken together, the evidence suggests that hypoxia-induced MXI1 may be an important regulator for cell proliferation during the pathogenesis of endometriosis.
Table of Content VII
Material & Methods 10
Clinical patients 10
Isolation of stromal cells 10
Cell culture and hypoxia treatment 11
DNA damage-induced apoptosis 12
RNA isolation, reverse transcription and quantitative real-time PCR (RT-qPCR) 12
Western blotting analysis 13
Transfection, RNA interference, and overexpression construct 13
BrdU incorporation assay 14
Statistical analysis 14
Hypoxia pretreatment protected eutopic endometrial stromal cells from CPT-induced apoptosis 16
Hypoxia pretreatment promoted cell proliferation in eutopic endometrial stromal cells 16
MXI1 was elevated in human endometriotic tissues compared with normal endometrium 19
Upregulation of MXI1 was induced by hypoxia treatment in eutopic endometrial stromal cells 26
MXI1-SRβ was not involved in hypoxia-suppressed apoptosis in eutopic endometrial stromal cells 26
Hypoxia-induced cell proliferation was inhibited by siMXI1 knockdown in eutopic endometrial stromal cells 32
1. Primer list 54
2. The siRNA oligonucleotides sequences 54
3. Antibodies 55
4. Reagents and buffers used in cell culture 56
5. Reagents used in cell treatment 57
6. Reagents and buffers used in RNA isolation 57
7. Reagents and buffers used in PCR 58
8. Reagents and buffers used in Western Blotting 58
9. Reagents and buffers used in BrdU incorporation assay 60
10. Protocols 60
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