慢病毒介导的α-烯醇化酶基因沉默对宫颈癌细胞生长及侵袭转移能力的影响

发布时间：2018-02-27 04:22

本文关键词： ENO1 基因沉默药物敏感性成瘤性侵袭转移　出处：《兰州大学》2014年硕士论文　论文类型：学位论文

【摘要】：肿瘤细胞增殖所需能量主要是通过有氧糖酵解途径代谢产生,糖酵解是细胞恶变过程中最为基础的代谢改变之一,是肿瘤细胞代谢的一个重要特征。Q-烯醇化酶(ENO1)是一种重要的糖酵解酶,在有氧糖酵解过程中起到关键作用。此外,ENO1的功能具有多样性,它既可催化糖酵解过程中2-磷酸甘油酸(PGA)向磷酸烯醇式丙酮酸(PEP)的转化,又可以编码c-myc启动子结合蛋白(MBP-1),下调原癌基因c-myc的活性,发挥抗肿瘤作用。本研究的目的是利用慢病毒介导的RNA干扰技术,构建ENO1基因稳定沉默的Hela和SiHa细胞系,探讨ENO1对人宫颈癌细胞生长,药物敏感性及侵袭转移能力的影响。本研究采用慢病毒介导的RNA干扰技术,建立ENO1基因稳定沉默的Hela和SiHa细胞系,并通过western-blot方法验证ENO1蛋白的表达水平。实验分为ENO1沉默组(pLKO.1shENO1)、阴性对照组(scr)和正常细胞组。采用MTT的方法来观察宫颈癌细胞Hela和SiHa在沉默ENO1基因后对化疗药物敏感性的变化。细胞克隆形成实验观察宫颈癌细胞成瘤能力的变化,同时采用细胞划痕实验、Transwell小室、免疫细胞化学技术检测细胞侵袭转移能力的变化及可能的机制。本研究结果表明,成功建立ENO1基因沉默的宫颈癌细胞系,通过MTT的方法检测三组细胞48h对不同浓度顺铂和紫杉醇的敏感性,结果显示不同浓度药物对ENO1基因沉默组细胞的抑制率要高于对照组,结果具有统计学意义。克隆形成实验结果表明,ENO1基因沉默后细胞的肉眼可见克隆数目显著低于正常细胞和空载体组细胞,Hela pLKO.1shENO1组及SiHa pLKO.1shENO1组克隆形成率分别是5.17%和6.33%,明显低于对照组细胞,P0.05,差异具有统计学意义。细胞划痕实验检测细胞迁移能力的变化,发现ENO1沉默组细胞迁移速率降低。采用无基质胶小室进行细胞迁移能力的检测,ENO1沉默组细胞迁移至下室的细胞明显减少,光镜下每组细胞随机选取5个视野,取平均数后进行统计分析,差异具有统计学意义。上室铺基质胶后进行细胞侵袭能力的检测,同样发现,ENO1沉默组细胞穿过基质胶的数量明显减少,差异具有统计学意义。采用免疫细胞化学技术检测SiHa细胞ENO1基因沉默后,侵袭转移相关因子VEGF, EGFR, MMP9,COX2,Notch2的表达情况,DAB染色后,ENO1沉默组细胞表达均较空载体组明显下降。本研究结论：沉默ENO1基因能够增强宫颈癌细胞Hela和SiHa对化疗药物紫杉醇和顺铂的药物敏感性,减低成瘤能力,抑制细胞的侵袭转移。总之,ENO1与宫颈癌细胞生长及侵袭转移密切相关。
[Abstract]:The energy needed for the proliferation of tumor cells is mainly produced by aerobic glycolysis. Glycolysis is one of the most basic metabolic changes in the process of cell malignancy. Q- enolase ENO1) is an important glycolytic enzyme that plays a key role in aerobic glycolysis. It can not only catalyze the transformation of 2-phosphoglyceric acid PGA to PEP (phosphoenolpyruvate) during glycolysis, but also encode c-myc promoter binding protein MBP-1, which can down-regulate the activity of proto-oncogene c-myc. The aim of this study was to construct Hela and SiHa cell lines with stable silencing of ENO1 gene by lentivirus mediated RNA interference, and to investigate the effects of ENO1 on the growth, drug sensitivity and invasion and metastasis of human cervical cancer cells. In this study, ENO1 gene stable silencing Hela and SiHa cell lines were established by lentivirus-mediated RNA interference technique. The expression level of ENO1 protein was verified by western-blot. The experiment was divided into two groups: ENO1 silencing group (pLK0.1 shENO1) and normal cell group. MTT method was used to observe the sensitivity of Hela and SiHa to chemotherapeutic drugs after silencing ENO1 gene. To observe the change of tumorigenic ability of cervical cancer cells by cell clone formation assay. At the same time, cell scratch test was used to detect the change of cell invasion and metastasis ability and its possible mechanism. The results showed that the cervical cancer cell line silenced by ENO1 gene was successfully established. The sensitivity of three groups of cells to different concentrations of cisplatin and paclitaxel was detected by MTT method. The results showed that the inhibition rate of different concentrations of drugs on ENO1 gene silencing group was higher than that of control group. The results showed that the number of clones in Hela pLKO.1shENO1 group and SiHa pLKO.1shENO1 group was significantly lower than that in normal cells and empty vector group. The clone formation rates of Hela pLKO.1shENO1 and SiHa pLKO.1shENO1 were 5.17% and 6.33, respectively. It was significantly lower than that of control group (P 0.05), the difference was statistically significant. Cell scratch assay was used to detect the change of cell migration ability. It was found that the cell migration rate of the ENO1 silencing group was decreased. The cell migration ability of the ENO1 silencing group was measured by using the non-matrix colloidal chamber. The number of cells migrating to the lower chamber in the ENO1 silencing group was significantly reduced, and 5 visual fields were randomly selected for each group under light microscope. After taking the average, the difference was statistically significant. The number of cells passing through the matrix glue in ENO1 silencing group was significantly decreased. The expression of VEGF, EGFR, MMP9 COX2Notch2 in SiHa cells after ENO1 gene silencing was detected by immunocytochemistry. Conclusion: silencing ENO1 gene can enhance the sensitivity of Hela and SiHa to the chemotherapeutic drugs paclitaxel and cisplatin, and reduce the tumorigenesis. ENO1 is closely related to the growth, invasion and metastasis of cervical cancer cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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