敲除CLCA2基因对宫颈癌Siha细胞侵袭的影响及对紫杉醇药物敏感性的相关研究

发布时间：2018-01-30 11:16

本文关键词： 宫颈癌 SiHa细胞 CLCA2 侵袭上皮-间质转化(EMT)　出处：《兰州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的:宫颈癌是我国妇女最常见的恶性肿瘤,也是全世界导致妇女死亡的最主要恶性肿瘤之一,严重危害着妇女的健康。肿瘤的药物耐药、侵袭和转移、术后复发是晚期宫颈癌患者常见的死亡原因,同时也是影响患者治疗和临床预后的重要因素。尽管晚期宫颈癌患者不能够得到彻底的治愈,但有很多治疗方式可以提高患者的生命长度和质量。近年来,精准医学在的癌症治疗中的迅速发展以及对肿瘤生物学理解的逐渐加深为寻找治疗癌症的新方法提供了新的方向。CLCA2已被证实是p53基因的靶基因之一,其在调控细胞增殖、迁移,肿瘤细胞浸润中起着重要的作用,目前发现其与多种肿瘤侵袭和转移的发生关系密切,但其在宫颈癌中的作用尚不清楚,我们实验组在前期的研究中通过应用基因芯片技术来研究异紫堇碱(Isocorydine)作用于宫颈癌SiHa细胞后基因谱的变化时发现了CLCA2基因在异紫堇碱作用48h后明显上调。因此,本实验旨在研究CLCA2基因在宫颈癌中的作用及其可能的机制,并初步探讨CLCA2对临床常用化疗药物紫杉醇的敏感性,以期对宫颈癌的诊治及预防提供一定的实验依据。方法:构建特异性CLCA2-shRNA,并借助慢病毒载体将其转入宫颈癌Siha细胞中,实验分为Siha组(空白对照组)、Siha-NC组(阴性对照组)、CLCA2-shRNA-1组(CLCA2-shRNA-1转染的宫颈癌Siha细胞,下文以K1组表示)和CLCA2-shRNA-2组(CLCA2-shRNA-2转染的宫颈癌Siha细胞,下文以K2表示),采用Western blot验证CLCA2基因的敲除效率,随后采用Transwell侵袭实验观察敲除CLCA2基因的宫颈癌Siha细胞侵袭力的变化,并用Western blot检测各组细胞上皮-间质转化标志物E-钙黏蛋白(E-cadherin)及波形蛋白(Vimentin)的表达水平,并检测基质金属蛋白酶-2(matrix metalloproteinase2,MMP-2)和基质金属蛋白酶-9(matrix metalloproteinase9,MMP-9)的表达。最后,利用MTS实验初步探讨敲除CLCA2基因的宫颈癌Siha细胞对紫杉醇的药物敏感性。结果:1、.成功构建了慢病毒表达载体并且能够高效转入宫颈癌Siha细胞内。Western blot检测显示:与Siha组和Siha-NC组相比,K1组和K2组中CLCA2蛋白的表达水平明显下降(P0.05);2、Transwell侵袭实验显示:与Siha组和Siha-NC组相比,K1组和K2组侵袭至Transwell孔下室的细胞数量明显增多,敲除CLCA2基因的细胞的侵袭力明显增加,差异具有统计学意义(P0.05);3、Western blot检测显K1和K2组中的E-cadherin蛋白的表达较Siha组和Siha-NC组均显著下降,差异具有统计学意义(p0.05),而Vimentin蛋白的表达K1组仅与Siha组相比明显上调,差异具有统计学意义(P0.05),而与Siha-NC组相比,差异无统计学意义(P0.05),K2组较Siha组和Siha-NC组均明显升高,差异具有统计学意义(p0.05);4、Western blot的进一步检测显示K1组和K2组中MMP-2蛋白和MMP-9蛋白的表达较Siha组和Siha-NC组均显著升高,差异具有统计学意义(p0.05);5.MTS实验结果显示:与Siha组和Siha-NC组相比,K1和K2组在不同浓度的紫杉醇(0.5,1,1.5,2,3,4,5nmol/l)作用下的抑制率明显增高,差异具有统计学意义(P0.05)。结论:1、CLCA2基因的敲除能够增强宫颈癌Siha细胞的侵袭能力;2、CLCA2基因的敲除能够促进宫颈癌Siha细胞上皮-间质的转化;3、CLCA2基因的敲除后能够通过促进MMP-2和MMP-9的表达来增强肿瘤细胞的侵袭能力;4、CLCA2基因的敲除能够增强紫杉醇对宫颈癌Siha细胞的敏感性。
[Abstract]:Background and objective: cervical cancer is the most common malignant tumors of women in our country, but also the whole world to one of the major malignant tumor death in women, seriously endanger the health of women. The drug resistance of tumor, invasion and metastasis, recurrence is common in patients with advanced cervical cancer deaths, but also an important factor affecting patients the treatment and clinical prognosis. Although patients with advanced cervical cancer can not be completely cured, but there are a lot of treatment can improve the patients' quality of life and length. In recent years, precision medicine in cancer treatment in the rapid development and gradually deepening understanding of tumor biology in order to find new method for the treatment of cancer provides a new the direction of.CLCA2 has been proven to be one of the target genes of p53 gene and its migration in the regulation of cell proliferation, tumor cells plays an important role in the invasion, and several There is a close relationship between tumor invasion and metastasis in cervical cancer, but its role is not clear, our experimental group in the previous study by using gene chip technology to study different Corydaline (Isocorydine) of CLCA2 gene was found in different Corydaline after 48h was significantly increased in the change of gene expression profile of human cervical cancer SiHa cells after the time. Therefore, the aim of this study is to investigate the role of CLCA2 gene in cervical cancer and its possible mechanism, and to investigate the sensitivity of CLCA2 to clinical commonly used chemotherapy drug taxol, provide some experimental basis for the diagnosis and prevention of cervical cancer. Methods: to construct specific CLCA2-shRNA, and put it into the cervix cancer cell Siha by slow virus vector, the experiments were divided into Siha group (control group), Siha-NC group (negative control group), CLCA2-shRNA-1 group (CLCA2-shRNA-1 transfected Siha cell, below in the K1 group Said) and group CLCA2-shRNA-2 (cervical cancer Siha cells transfected with CLCA2-shRNA-2 below K2), using Western blot to verify CLCA2 gene knockdown efficiency, followed by Transwell invasion assay. Knockout invasion of cervical cancer Siha cells CLCA2 gene, and Western blot to detect the expression of epithelial mesenchymal transition mark E- cadherin (E-cadherin) and vimentin (Vimentin) expression and detection of matrix metalloproteinase -2 (matrix metalloproteinase2 MMP-2) and matrix metalloproteinase -9 (matrix Metalloproteinase9 MMP-9) expression of MTS. Finally, study the knockout drug of cervical cancer Siha cells CLCA2 gene to paclitaxel sensitivity use. Results: 1. Successfully constructed the lentiviral expression vector and can be efficiently transferred to the display of cervical cancer Siha cells in.Western blot detection with Siha group and Siha-NC group. Than, the expression level of CLCA2 protein in K1 group and K2 group decreased significantly (P0.05); 2, Transwell invasion assay showed that: compared with Siha group and Siha-NC group, the number of cells in K1 group and K2 group to the Transwell invasion chamber was significantly increased, CLCA2 knockout cell invasiveness has increased significantly statistically significant difference (P0.05); 3, the expression of Western blot and K2 K1 detection of E-cadherin protein in the group than in Siha group and Siha-NC group were significantly decreased, the difference was statistically significant (P0.05), and the expression of K1 protein in Vimentin group was significantly increased compared with Siha group, the difference was statistically significant (P0.05). Compared with Siha-NC group, the difference was not statistically significant (P0.05), K2 group than in Siha group and Siha-NC group were significantly higher, the difference was statistically significant (P0.05); 4, further detection of Western blot showed that the expression of MMP-2 protein and MMP-9 protein in K1 group and K2 group than in Siha group And the Siha-NC group were significantly increased, the difference was statistically significant (P0.05); 5.MTS results showed: compared with Siha group and Siha-NC group, K1 group and K2 in different concentrations of paclitaxel (0.5,1,1.5,2,3,4,5nmol/l) inhibited the rate increased significantly, the difference was statistically significant (P0.05). Conclusion: 1. CLCA2 gene knockout of cervical carcinoma can enhance the invasion ability of Siha cell; 2, CLCA2 gene knockout can promote cervical cancer Siha cell epithelial mesenchymal transformation; 3, CLCA2 gene knockout can promote the expression of MMP-2 and MMP-9 to enhance the ability of tumor cell invasion; 4, CLCA2 gene knockout to enhance paclitaxel on human cervical cancer Siha cell sensitivity.

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

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