HMGA2在镉诱导的小鼠肺组织和A549细胞迁移和侵袭中的作用研究

发布时间：2018-04-30 03:00

本文选题：镉 + A549细胞　；参考：《大连医科大学》2017年硕士论文

【摘要】：镉(Cadmium)是一种在环境中存在十分广泛的有毒重金属,普遍应用于制造陶瓷釉彩、镉电池、复写纸和照相机及合成纤维印染助剂等。镉被人体吸收后,会通过镉硫蛋白的形式蓄积在肾、肝、脑、肺、膀胱、前列腺以及子宫内膜中,从而对多个器官系统造成巨大的危害,1993年镉被国际癌症研究机构列为Ⅰ类致癌物。除了职业性暴露以外,人们还会通过吸烟和食用被污染的食物这种非职业性暴露的形式吸收镉。目前,镉被发现可以扰乱DNA修复系统,进而促进癌症的形成和发展,其还可以增强一些组织和细胞的迁移、侵袭能力。高迁移率族蛋白A2(HMGA2)在真核生物中大量存在,因为分子质量小,且在凝胶电泳中迁移率高而得名。其自身缺乏转录活性,但具有三个特殊的AT-hooks结构,这些AT-hooks结构可以与DNA中AT富集序列特异性结合,从而改变基因的DNA结构,或直接作用于相关基因,加强或者抑制它们的转录活性,进而影响胚胎形成和组织发育,以及引起肿瘤的发生。因为HMGA2具有这种通过改变染色质的结构来调节很多靶基因表达的特点,它常被称做“构架转录因子”。HMGA2的表达升高在胚胎形成期几乎无处不在,并且对于胚胎发育期细胞的增长繁殖和分化起了必不可少的作用。它同时被发现在很多肿瘤中表达升高。HMGA2的大量表达与结肠癌晚期、浸润型乳腺癌的高组织学分级,以及乳腺癌、结肠癌、肺癌患者的低生存率相关。但是HMGA2是否参与了镉诱导的迁移和侵袭尚不十分清楚。目的:探讨HMGA2在镉诱导的迁移和侵袭中的作用。方法:本研究以人肺腺癌细胞(A549细胞)、人胚肾细胞(HEK 293细胞)和小鼠肺组织为研究对象。按实验设计,对小鼠连续6周皮下注射不同浓度镉(0,0.25,0.5和1mg/kg),提取小鼠肺组织蛋白;将低浓度(2μM)氯化镉作用于A549细胞和HMGA2 siRNA处理后的A549细胞;pc DNA3.1-HMGA2质粒转染HEK 293细胞得到HMGA2表达升高的细胞模型。应用蛋白印迹法(Western blot)检测细胞和组织中HMGA2蛋白和迁移侵袭相关蛋白MMP-9、MMP-2、FAK以及p-FAK水平的变化。通过Transwell小室实验观察低浓度镉作用后A549细胞迁移侵袭能力的变化。实验结果采用独立样本t检验进行分析。结果:每天给小鼠皮下注射不同浓度镉连续6周后,镉处理组HMGA2的表达量与对照组相比明显增高(P0.01),说明低浓度镉可以诱导小鼠肺组织中HMGA2的表达。同时,迁移侵袭相关蛋白MMP-9、MMP-2和p-FAK的表达显著升高(P0.01),提示低浓度镉可以使小鼠肺组织细胞的迁移侵袭能力增强。在人肺腺癌细胞(A549细胞)中,用低浓度(2μM)氯化镉染毒48小时后,检测到HMGA2、MMP-9、MMP-2和p-FAK蛋白表达量显著升高(P0.01),表明低浓度镉可以诱导A549细胞的迁移侵袭相关蛋白的表达,Transwell小室实验中,镉处理组A549细胞迁移和侵袭通过孔径膜和基质胶的数量与对照组相比明显增高(P0.01)。用HMGA2 siRNA处理A549细胞使HMGA2表达下降以后,迁移侵袭相关蛋白MMP-9、MMP-2和p-FAK的表达显著下降(P0.01),并且Transwell小室实验中用HMGA2 siRNA处理后的染镉组迁移和侵袭通过孔径膜和基质胶的A549细胞数量与control siRNA处理后的染镉组相比显著减少(P0.01)。pc DNA3.1-HMGA2质粒转染HEK 293细胞使HMGA2的表达升高后,迁移侵袭相关蛋白MMP-9、MMP-2和p-FAK的表达与阴性对照组相比显著升高(P0.01)。这些结果表明HMGA2在镉诱导的迁移侵袭中发挥了重要作用。结论:镉可以增强小鼠肺组织和A549细胞的迁移、侵袭能力;HMGA2在镉诱导的迁移侵袭中发挥了重要作用。
[Abstract]:Cadmium (Cadmium) is a very widespread toxic heavy metal in the environment. It is widely used in the manufacture of ceramic glaze color, cadmium battery, duplex paper, camera and synthetic fiber printing and dyeing auxiliaries. Cadmium is absorbed by human body and accumulates in the kidney, liver, brain, lung, bladder, prostate and endometrium through the absorption of cadmium sulfide in the form of cadmium sulphide protein. The official system has caused great harm. In 1993, cadmium was classified as a type I carcinogen by the International Cancer Research Institute. In addition to occupational exposure, cadmium can also be absorbed in the form of nonoccupational exposure to smoking and food contaminated food. Cadmium has been found to disrupt the DNA repair system and promote the formation and development of cancer. It can also enhance the migration and invasiveness of some tissues and cells. High mobility group protein A2 (HMGA2) exists in eukaryotes, because of its small molecular weight and high mobility in gel electrophoresis. It lacks transcriptional activity, but has three special AT-hooks structures, and these AT-hooks structures can be enriched with AT in DNA. Let heterosexual binding, which changes the DNA structure of the gene, or directly acts on the related genes, strengthens or inhibits their transcriptional activity, and then affects the formation and development of the embryos, and causes the occurrence of tumors. Because HMGA2 has the characteristics of many target genes by changing the structure of chromatin, it is often called to be called. The expression of "structural transcription factor".HMGA2 is almost ubiquitous in the embryonic period, and is essential for the growth and differentiation of cells in embryonic development. It is also found to be expressed in many tumors and the high expression of.HMGA2 in the late stage of colon cancer and the high histological grade of invasive breast cancer. It is related to the low survival rate of patients with breast cancer, colon cancer and lung cancer. But whether HMGA2 is involved in cadmium induced migration and invasion is not very clear. Objective: To explore the role of HMGA2 in cadmium induced migration and invasion. Methods: This study was conducted with human lung adenocarcinoma cells (A549 cells), human embryonic kidney cells (HEK 293 cells) and mice lung tissue Object. According to the experimental design, mice were subcutaneously injected with different concentrations of cadmium (0,0.25,0.5 and 1mg/kg) for 6 weeks to extract the mouse lung tissue protein; the low concentration (2 M) of cadmium chloride was acted on the A549 cells treated with A549 cells and HMGA2 siRNA; PC DNA3.1-HMGA2 plasmid transfected to HEK 293 cells to obtain the cell model of HMGA2 expression. The changes in the level of HMGA2 protein and migration associated protein MMP-9, MMP-2, FAK and p-FAK in cells and tissues were detected by Western blot. The changes in the migration and invasion of A549 cells after low concentration of cadmium were observed through the Transwell chamber experiment. The experimental results were analyzed by independent sample t examination. Results: the mice were injected subcutaneously every day. After 6 weeks of cadmium concentration, the expression of HMGA2 in the cadmium treatment group was significantly higher than that in the control group (P0.01), indicating that the low concentration of cadmium could induce the expression of HMGA2 in the lung tissue of mice. Meanwhile, the migration and invasion related protein MMP-9, the expression of MMP-2 and p-FAK increased significantly (P0.01), suggesting that low concentration of cadmium could induce migration of lung tissue cells in mice. In human lung adenocarcinoma cells (A549 cells), the expression of HMGA2, MMP-9, MMP-2 and p-FAK was significantly increased (P0.01) after 48 hours of cadmium chloride exposure at low concentration (2 u M), indicating that low concentration of cadmium could induce the expression of migration and invasion of A549 cells, and the migration of A549 cells in the cadmium treatment group in the Transwell lab experiment The number of and invasion through the aperture membrane and matrix gel was significantly higher than that in the control group (P0.01). After the A549 cells were treated with HMGA2 siRNA to decrease the HMGA2 expression, the migration and invasion related protein MMP-9, the expression of MMP-2 and p-FAK decreased significantly (P0.01), and in the Transwell lab experiment, the migration and invasion of the cadmium exposed group after HMGA2 siRNA were passed through the Transwell chamber experiment. The number of A549 cells in the aperture membrane and the matrix gel decreased significantly compared with that of the control siRNA treated group (P0.01). The.Pc DNA3.1-HMGA2 plasmid transfected with HEK 293 cells increased the expression of HMGA2, and the expression of the migration related proteins MMP-9, MMP-2 and p-FAK increased significantly compared with the negative control group (P0.01). These results showed that the cadmium DNA3.1-HMGA2 was induced by cadmium. Conclusion: cadmium can enhance the migration and invasiveness of lung and A549 cells in mice, and HMGA2 plays an important role in the migration and invasion induced by cadmium.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

【参考文献】