ERK5信号通路在吸烟暴露诱导的肾癌细胞上皮间质转化中的作用研究

发布时间：2018-05-05 16:08

本文选题：肾细胞癌 + 吸烟　；参考：《安徽医科大学》2017年硕士论文

【摘要】：背景与研究目的肾癌是泌尿生殖系统常见的恶性肿瘤,发生率排名居人类恶性肿瘤第十四位。它起源于肾小管上皮细胞,以透明细胞癌最常见,占肾细胞癌的70%-80%,另外还有乳头状癌、嫌色细胞癌等。目前肾癌病因尚未明确,有资料显示其发病与吸烟、解热镇痛药、激素、病毒、咖啡镉等有关。其中吸烟是已知最明确的危险因素,据推测20%-30%的男性和10%-20%的女性肾癌的发生是由吸烟引起。我国是世界上最大的烟草生产和消费国,吸烟引发的肿瘤及相关疾病,已成为国民健康的严重威胁。上皮间质转化在细胞恶转及肿瘤转移过程中起到至关重要的作用。本课题通过体外实验研究吸烟暴露对人肾癌细胞上皮间质转化(Epithelial-mesenchymal transition,EMT)的影响,并探讨ERK5信号通路在吸烟暴露诱导肾癌细胞的EMT中的作用。方法建立体外细胞吸烟处理模型;MTT法筛选对肾癌细胞ACHN、A498细胞活力无明显影响的香烟悬液(cigarette smoking extract,CSE)浓度,后根据筛选结果选择相应浓度CSE处理肾癌细胞。(1)通过显微镜观察CSE对肾癌细胞形态的影响并记录;(2)通过transwell实验、划痕实验观察CSE能否引起肾癌细胞侵袭、转移能力改变;(3)运用western blotting、免疫荧光检测上皮间质转化相关分子标志物的变化;q RT-PCR方法检测CSE对肾癌细胞上皮间质转化相关标志物m RNA表达水平的影响;(4)用western blotting检测吸烟处理对肾癌细胞ERK5通路及AP-1通路活性的影响;(5)ERK5通路特异性抑制剂XMD8-92处理细胞,检测其对吸烟诱导的肾癌细胞ACHN、A498上皮间质转化的干预效果。结果(1)通过MTT法筛选适宜浓度的CSE作为实验浓度(0%、0.1%、0.25%、0.5%),当CSE浓度在0.75%及以上时,细胞活力明显受到影响,并具有统计学差异(P0.05);(2)CSE处理肾癌细胞6天后,镜下观察细胞由原来的聚集变的更为分散,形态由原来的扁椭圆形变为长梭形,部分细胞伸出伪足;划痕实验、transwell实验表明,经CSE处理后肾癌细胞转移侵袭能力明显增强。(3)western blotting、免疫荧光检测发现肾癌细胞经CSE处理后,上皮标志物E-Cadherin、ZO-1表达水平呈上调趋势,间质标志物vimentin、N-cadherin表达水平呈下调趋势;同时,经q RT-PCR证实,上述相关标志物的m RNA表达水平亦表现相同的变化趋势。(4)通过western blotting检测发现,磷酸化ERK5(phosphorylated ERK5,P-ERK5)水平呈明显上升趋势,而总ERK5(total ERK5,T-ERK5)呈明显下降趋势;AP-1通路中磷酸化c-jun(phosphorylated c-jun,p-c-jun)、磷酸化c-fos(phosphorylated c-fos,p-c-fos)表达水平呈上升趋势。可见,吸烟处理可激活ERK5通路。(5)ERK5通路特异性抑制剂XMD8-92可逆转吸烟诱导的肾癌细胞上皮间质转化过程,表现如下:细胞形态上,由间质表型向上皮表型转变,且细胞重新表现相互聚集状态;划痕实验及transwell实验见细胞转移侵袭能力出现逆转;EMT相关标志物,上皮标志物E-cadherin、ZO-1表达水平上升,而间质标志物间质标志物vimentin、N-cadherin表达水平下调,相关标志物m RNA表达水平也表现相应的变化趋势。结论吸烟暴露可诱导肾癌细胞ACHN、A498发生上皮间质转化,ERK5信号通路在这一变化过程中起重要的正调控作用,ERK5通路的抑制可阻止这样变化的发生。这也进一步证实了,ERK5在吸烟引起的肾癌转移侵袭过程中起重要作用。
[Abstract]:Background and research objective renal cancer is a common malignant tumor of the genitourinary system. The incidence of the fourteenth human malignant tumor is in the human malignant tumor. It originated from the renal tubular epithelial cells. It is the most common type of clear cell carcinoma, which accounts for 70%-80% of renal cell carcinoma. There are also papillary carcinoma and chromophobe cell carcinoma. The etiology of renal cancer is not yet clear. It is associated with smoking, antipyretic analgesics, hormones, viruses, and coffee and cadmium. Smoking is the most clear risk factor known. It is presumed that the incidence of kidney cancer in 20%-30% men and 10%-20% is caused by smoking. China is the largest tobacco producing and consumer country in the world, smoking caused cancer and related diseases, and has become the national health. The transformation of epithelial mesenchymal transition plays a vital role in the process of malignant transformation of cells and tumor metastasis. In this study, the effects of smoking exposure on Epithelial-mesenchymal transition (EMT) in human renal cell carcinoma cells were studied in vitro, and the EMT of ERK5 signaling pathway in the induced EMT of renal cancer cells in smoking exposure was explored. Methods to establish an in vitro cell smoking treatment model, the MTT method was used to screen the concentration of cigarette smoking extract (CSE), which had no significant influence on the activity of ACHN and A498 cells in renal cell carcinoma cells, and then selected the corresponding concentration CSE to treat the renal cell carcinoma cells according to the screening results. (1) the effects of CSE on the morphology of renal cell carcinoma cells were observed and recorded. (2) through the Transwell experiment, the scratch test was used to observe whether CSE could cause the invasion of renal cell carcinoma cells and change the metastasis ability; (3) the changes of epithelial mesenchymal transition markers were detected by Western blotting and immunofluorescence; and the Q RT-PCR method was used to detect the effect of CSE on the RNA expression level of the epithelial mesenchymal transition related markers of renal cell carcinoma cells; (4) weste. RN blotting was used to detect the effects of smoking treatment on ERK5 pathway and AP-1 pathway activity in renal cell carcinoma cells; (5) ERK5 pathway specific inhibitor XMD8-92 cells were used to detect the interference effect on ACHN and A498 epithelial mesenchymal transition in renal cancer cells induced by smoking. Results (1) the concentration of CSE was screened by MTT method as the experimental concentration (0%, 0.1%, 0.25%, 0.5%). When the concentration of CSE was 0.75% or more, the cell vitality was significantly affected and had statistical difference (P0.05). (2) after 6 days of CSE treatment of renal cell carcinoma cells, the cells were observed to be more dispersed from the original aggregation, the shape changed from the original oblate ellipsoid to the long spindle shape, the part of the cells extended to the pseudo foot; the scratch test, the Transwell experiment showed that CSE was found. The metastasis and invasion ability of the renal cell carcinoma cells was obviously enhanced. (3) Western blotting, the immunofluorescence detection showed that the epithelial markers E-Cadherin, the expression level of ZO-1 were up trend, the interstitial marker vimentin, the N-cadherin expression level downregulated, and the m RNA table of the above related markers was confirmed by Q RT-PCR. (4) through Western blotting detection, the level of phosphorylated ERK5 (phosphorylated ERK5, P-ERK5) showed an obvious upward trend, while the total ERK5 (total ERK5, T-ERK5) showed a significant downward trend, and the AP-1 pathway was phosphorylated. The expression level showed an upward trend. Smoking treatment can activate the ERK5 pathway. (5) the ERK5 pathway specific inhibitor XMD8-92 can reverse the process of epithelial mesenchymal transition in renal carcinoma cells induced by smoking, as follows: cell morphology changes from interstitial phenotype to epitheliality, and cell reacts with mutual aggregation; scratch test and Transwell It was found that the invasion ability of cell metastasis was reversed, EMT related markers, epithelial markers E-cadherin, ZO-1 expression level increased, the interstitial marker vimentin, N-cadherin expression level decreased, and the related markers m RNA expression level also showed the corresponding change trend. Conclusion smoking exposure can induce renal cancer cell ACHN, A498 hair. The ERK5 signaling pathway plays an important positive role in the transformation of epithelial mesenchymal transition, and the inhibition of ERK5 pathway can prevent such changes. This further confirms that ERK5 plays an important role in the process of metastasis and invasion of kidney cancer.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11

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