ATP1A1基因沉默对胶质瘤细胞U251侵袭性的影响及其机制的初步探究

发布时间：2018-05-10 13:11

本文选题：ATP1A1 + RNA干扰　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的ATP1A1属于Na+/K+ATPase酶家族之一,ATP1A1过表达已在多种癌症中有所报道,包括食管癌、黑色素瘤、肝癌等。据报道它在与癌症相关的各生物学过程起到关键作用,而沉默ATP1A1后对肿瘤的生长、转移抑制也有所报道,但尚未见在胶质瘤的侵袭性方面有单独报道。为了证明这一假想,我们借助RNAi(RNA干扰技术),定向沉默胶质瘤U251细胞的ATP1A1基因,观察细胞增殖、迁移、侵袭以及成瘤能力的改变,并探讨MMP-2/9(基质金属蛋白酶2/9)表达的调节是否为其关键的作用靶点。方法构建ATP1A1/sh RNA质粒三对和阴性对照一对,通过三质粒系统包装慢病毒并转染U251细胞系从而抑制ATP1A1基因的表达。通过嘌呤霉素筛选,获取嘌呤霉素抗性单克隆进行培养,扩增后分别用免疫荧光技术观察荧光量,RT-q PCR(实时定量PCR技术)和Western blot(免役印迹技术)各自检测ATP1A1 m RNA和蛋白的表达,从而验证获得的稳定转染细胞株。后续实验分为空白组、对照组、实验组。空白组:未作任何干预的U251胶质瘤细胞,对照组:空载质粒转染的U251胶质瘤细胞,实验组:选出沉默效果最佳的一组。MTT法观察细胞体外的增殖能力,细胞划痕实验观察细胞的迁移能力,Transwell小室观察细胞的侵袭能力,裸鼠皮下成瘤观察体内成瘤能力,Western blot检测MMP-2、MMP-9的表达。结果经转染和嘌呤霉素筛选后,荧光显微镜下见有明显表达抑制ATP1A1基因的细胞,RT-q PCR、Western blot分别检测稳定株ATP1A1的m RNA和蛋白表达,均显著降低(P0.05),通过RNAi技术成功建立U251低表达ATP1A1的稳定株。与对照组相比,实验组细胞的增殖和迁移、侵袭以及成瘤能力也显著受抑(P0.05);MMP-2和MMP-9的表达也明显降低(P0.05)。数据有统计学意义。结论靶向沉默ATP1A1基因能够明显抑制胶质瘤U251细胞的体外增殖、迁移、侵袭及体内成瘤性,其机制可能与MMP-2、MMP-9的下调相关,ATP1A1可能作为胶质瘤治疗的一个潜在靶点。
[Abstract]:Objective the overexpression of ATP1A1 belongs to the Na / K ATPase family has been reported in many cancers, including esophageal cancer, melanoma, liver cancer and so on. It is reported that it plays a key role in various biological processes related to cancer, and the inhibition of tumor growth and metastasis after silencing ATP1A1 has been reported, but there has not been a separate report on the invasiveness of glioma. To prove this hypothesis, we used the RNAi(RNA interference technique to target the ATP1A1 gene of U251 glioma cells and observe the changes in cell proliferation, migration, invasion and tumorigenesis. And to investigate whether the regulation of MMP-2 / 9 (matrix metalloproteinase 2 / 9) expression is a key target. Methods three pairs of ATP1A1/sh RNA plasmids and a pair of negative controls were constructed. The lentivirus was packaged with three plasmids and transfected into U251 cell line to inhibit the expression of ATP1A1 gene. Purine mycin resistant monoclonal clone was obtained by purine mycin screening, and the expression of ATP1A1 m RNA and protein were detected by immunofluorescence technique using RT-q PCR (real-time quantitative PCR) and Western blot (no-service blotting), respectively. The stable transfection cell line was verified. The follow-up experiment was divided into blank group, control group and experimental group. Blank group: U251 glioma cells without any intervention, control group: U251 glioma cells transfected with empty plasmids, experimental group: select the best silencing effect group. MTT method to observe the proliferation of cells in vitro. The cell migration ability was observed by cell scratch assay and the invasion ability was observed by Transwell chamber, and the expression of MMP-2 and MMP-9 was detected by Western blot in nude mice by subcutaneous tumorigenesis. Results after transfection and purine mycin screening, the expression of m RNA and protein in the stable ATP1A1 strain was detected by RT-PCR blot. The stable strain of U251 with low expression of ATP1A1 was successfully established by RNAi. Compared with the control group, the proliferation and migration, invasion and tumorigenesis of the cells in the experimental group were also significantly inhibited by the expression of MMP-2 and MMP-9. The data are statistically significant. Conclusion silencing ATP1A1 gene can significantly inhibit the proliferation, migration, invasion and tumorigenesis of U251 glioma cells in vitro, and its mechanism may be related to the down-regulation of MMP-2 / MMP 9. It may be a potential target for the treatment of glioma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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