EFEMP2对子宫内膜癌侵袭转移作用的研究

发布时间：2018-03-11 14:38

本文选题：EFEMP2　切入点：子宫内膜癌　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究依据子宫内膜癌(endometrial carcinoma,EC)是在女性生殖系统发生的恶性肿瘤,尽管早期子宫内膜癌患者的预后相对理想,但是超过30%的患者在发现时已处于肿瘤晚期,并且部分患者伴随局部、盆腔甚至远端转移,因此预后不良。肿瘤转移是造成患者死亡的主要原因,因此探索子宫内膜癌的侵袭、转移机制,进一步阐明肿瘤的发展过程显得尤为迫切。近年来,多种生物标记物已被证实与子宫内膜癌的发展有关,但EFEMP2在子宫内膜癌细胞侵袭转移过程中的作用尚未见报道。EFEMP2(EGF containing fibulin-like extracellular matrix protein 2)是一种细胞外基质蛋白,主要参与弹性纤维的合成与排列。EFEMP2与皮肤松弛症、主动脉夹层、骨关节炎、恶性肿瘤等多种疾病有关。为了探索EFEMP2在子宫内膜癌增殖、侵袭、转移中的作用,本研究首次探讨EFEMP2在子宫内膜癌组织及细胞系中的表达情况及相关机制。研究目的1.探究EFEMP2在子宫内膜癌组织、细胞中的表达及其意义。2.探究慢病毒转染后不同侵袭能力子宫内膜癌细胞的功能转变。3.探究EFEMP2对子宫内膜癌细胞侵袭转移的作用机制。研究方法1.免疫组化(IHC)探究EFEMP2在正常子宫内膜组织及子宫内膜癌组织中的表达情况;2.单细胞克隆技术,从KLE、ISK中分离出具有高侵袭能力的克隆细胞亚系KLE-1、ISK-1和低侵袭能力的克隆细胞亚系KLE-28、ISK-23;3.逆转录-聚合酶链反应(RT-qPCR)、免疫印迹(Western blot)、免疫细胞化学(ICC)检测EFEMP2在正常子宫内膜、子宫内膜癌细胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆细胞亚系中的表达情况;4.生长曲线、软琼脂克隆形成实验检测子宫内膜癌细胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆细胞亚系的增殖能力;5.Transwell侵袭转移实验检测子宫内膜癌细胞系(KLE、ISK、HEC-1A、HEC-1B)以及克隆细胞亚系的侵袭转移能力;6.慢病毒介导EFEMP2在高侵袭克隆细胞亚系过表达及RNA干扰沉默EFEMP2在低侵袭克隆细胞亚系表达,并验证转染效率;7.建立裸鼠移植瘤模型,体内模拟子宫内膜癌发展过程,进一步探究EFEMP2在子宫内膜癌细胞增殖、侵袭、转移过程中的作用;8.Western blot、RT-qPCR检测EFEMP2与子宫内膜癌细胞上皮间质转化(EMT)之间的关系。研究结果1.EFEMP2在正常子宫内膜组织中阳性表达率高,在子宫内膜癌组织中阳性表达率低(P0.05);EFEMP2的表达情况与子宫内膜癌组织分化、淋巴结转移、以及不良预后密切相关(P0.05);2.EFEMP2在正常子宫内膜细胞中高表达,在子宫内膜癌细胞中低表达;在高侵袭克隆细胞亚系中低表达,在低侵袭克隆细胞亚系高表达(P0.05);3.慢病毒介导EFEMP2过表达后高侵袭克隆细胞增殖能力、侵袭和转移能力减弱;RNA干扰沉默EFEMP2表达后低侵袭克隆细胞增殖能力、侵袭和转移能力增强(P0.05);4.裸鼠移植瘤模型:高侵袭克隆细胞亚系组肿瘤生长速度快,体积大,低侵袭克隆细胞亚系组肿瘤生长速度慢,体积小;EFEMP2过表达组成瘤率较低,瘤体体积小,肺转移率低,EFEMP2干扰组成瘤率高,瘤体体积大,肺转移率高(P0.05);5.EFEMP2 过表达组 E-cadherin 表达升高,N-cadherin、Vimentin、Snail、Slug、Twist的表达降低,抑制EMT;EFEMP2干扰组,E-cadherin表达降低,Vimentin、Snail、Slug、Twist 的表达升高,促进 EMT(P0.05)。研究结论1.EFEMP2是子宫内膜癌的一个抑癌因子,能够抑制子宫内膜癌细胞侵袭、转移;2.EFEMP2抑制子宫内膜癌细胞EMT,进一步抑制子宫内膜癌细胞侵袭、转移。
[Abstract]:On the basis of endometrial carcinoma (endometrial, carcinoma, EC) in the occurrence of the female reproductive system malignant tumors, although the early prognosis of patients with endometrial cancer is relatively ideal, but more than 30% of patients in the discovery has been in advanced cancer, and some patients with local, pelvic even distant metastasis, poor prognosis. Therefore tumor metastasis is the result of the main cause of death in patients, so the exploration of endometrial cancer invasion, metastasis, and further clarify the process of tumor development is particularly urgent. In recent years, a variety of biological markers have been linked to the development of endometrial cancer, but EFEMP2 in endometrial cancer cell invasion and metastasis process has not been reported in.EFEMP2 (EGF containing fibulin-like extracellular matrix protein 2) is an extracellular matrix protein, mainly involved in the synthesis and arrangement of elastic fibers and skin.EFEMP2 Skin relaxation, aortic dissection, osteoarthritis, malignant tumors and other diseases. In order to explore EFEMP2 invasion in endometrial carcinoma, proliferation, metastasis, this is the first study to investigate the expression of EFEMP2 in endometrial cancer tissues and cells and the related mechanism. The purpose of the study is to explore 1. EFEMP2 in endometrial carcinoma the organization, function change of.3..2. expression and significance of the research of lentiviral transfection after invasion of endometrial cancer cells to explore the mechanism of action of EFEMP2 on invasion and metastasis of endometrial carcinoma cells. Methods: 1. immunohistochemical (IHC) expression of EFEMP2 in normal endometrium and endometrial carcinoma; 2. single cell cloning technique from KLE, isolation and cloning of KLE-1 cells with high invasive ability of ISK, cloning cell lines KLE-28, ISK-1 and ISK-23 3. low invasion; reverse transcriptase Polymerase chain reaction (RT-qPCR) and immunoblotting (Western blot), immunocytochemistry (ICC) detection of EFEMP2 in normal endometrium, endometrial carcinoma cell lines (KLE, ISK, HEC-1A, HEC-1B) and cloned cell lines in expression; 4. growth curve, soft agar colony formation assay of cancer cells endometrial lines (KLE, ISK, HEC-1A, HEC-1B) and clonal cell sublines proliferation of cancer cells; experimental detection of endometrial 5.Transwell invasion and metastasis (KLE, ISK, HEC-1A, HEC-1B) and cloning cell line invasion and metastasis ability; 6. lentivirus mediated EFEMP2 overexpression and RNA interference to silence EFEMP2 in highly invasive cell cloning expression in low invasive clone cell line, and to verify the transfection efficiency; 7. nude mice in vivo, simulation and development of endometrial cancer, further research on the EFEMP2 in the invasion and proliferation of endometrial cancer cells, metastasis The role of 8.Western blot, RT-qPCR EFEMP2; and the detection of endometrial cancer cell epithelial mesenchymal transition (EMT). The relationship between the results of 1.EFEMP2 in normal endometrial tissues. The positive expression rate is high, the positive expression in endometrial carcinoma was low (P0.05); the expression and differentiation of EFEMP2 in endometrial carcinoma the lymph node metastasis, and the prognosis is closely related (P0.05); the expression of 2.EFEMP2 in normal endometrial cells, low expression in endometrial cancer cells; low expression in highly invasive clonal cell sublines, high expression in low invasive clone cell line (P0.05); 3. lentivirus mediated overexpression of EFEMP2 after the high invasive cell proliferation clone, weaken the ability of invasion and metastasis; RNA interference silencing EFEMP2 expression after low invasive clonal cell proliferation ability, enhance the ability of invasion and metastasis (P0.05); xenograft model in nude mice: 4. grams of high invasion Lung cell line tumor growth speed, large volume, low invasive clonal cell line tumor growth is slow, the volume is small; the over expression of EFEMP2 of tumor formation rate is low, the tumor volume is small, the low rate of lung metastasis, EFEMP2 interference of tumor formation rate, tumor volume and lung metastasis rate (P0.05); 5.EFEMP2 overexpression increased the expression of E-cadherin group N-cadherin, Vimentin, Snail, Slug, Twist expression decreased, the inhibition of EMT; EFEMP2 interference group, decreased expression of E-cadherin, Vimentin, Snail, Slug, increased the expression of Twist, promote EMT (P0.05). Conclusions 1.EFEMP2 is a tumor suppressor gene in endometrial cancer, can inhibit the invasion of endometrial cancer cell metastasis; 2.EFEMP2 inhibits endometrial cancer cell EMT and further inhibit the endometrial cancer cell invasion and metastasis.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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