CDC25B，C的mRNA的选择性剪接变化与宫颈癌细胞抗顺铂的关系研究

发布时间：2018-07-15 12:24

【摘要】：宫颈癌是女性最多见的恶性肿瘤,严重威胁着女性的健康与生活。顺铂是治疗宫颈癌的常用化疗药物,但癌细胞对顺铂的抗药性使其治疗效果大为减弱。在用顺铂处理的宫颈癌细胞SiHa,C33A中,CDC25B 与 CDC25C基因的mRNA的选择性剪接发生了改变,由于CDC25B 与 CDC25C在正常生理条件下和应对DNA损伤时对细胞周期的检查点功能有重要的调控作用,因此推测CDC25B与CDC25C基因的mRNA的剪接变化很可能与细胞耐受顺铂有关。本研究拟探究CDC25B与CDC25C的各主要剪接isoforms具有抗凋亡还是促凋亡的作用?这一剪接变化是否增加了宫颈癌细胞对顺铂的抗性?得到的结果和信息将有助于阐明CDC25B 与 CDC25C在肿瘤发生与发展中的作用,解读肿瘤细胞对放化疗‘抗性的机制。如果CDC25B 与 CDC25C 的 mRNA的剪接变化会影响其checkpoint的功能,则对其的调节有可能会提高顺铂的疗效,调节的途径和基因将成为新的治疗靶点,为癌症的个体化治疗提供重要的信息和依据。目的：探究CDC25B与CDC25C 的 mRNA的选择性剪接变化对宫颈癌细胞SiHa和C33A的生长、增殖、凋亡以及顺铂敏感性的影响。方法：应用MTT比色法确定宫颈癌细胞SiHa, C33A的顺铂半致死量浓度；RT-PCR检测相同时间、不同浓度顺铂干预及不同时间、半致死量顺铂干预对各组宫颈癌细胞CDC25B, C的mRNA的选择性剪接的影响；将CDC25B1,B3及CDC25C1.C5基因转染宫颈癌SiHa, C33A细胞,RT-PCR 及 Western Blot分别检测转染细胞中CDC25B1, B3, C1, C5 的 mRNA和蛋白表达水平;MTT比色法测量CDC25B1, B3, C1, C5基因过表达的宫颈癌SiHa, C33A细胞的顺铂半致死量浓度,与之前比较得出结论。结果：SiHa细胞顺铂的半致死量浓度约为40μmol/L,C33A细胞顺铂的半致死量浓度约为22.5 μmol/L。选取差异最为明显的CDC25B1,B3, C1, C5四种剪接体进行转染、上调,RT-PCR及Western Blot分别检测到相应的mRNA和蛋白表达均上调；MTT比色法测量顺铂半致死量浓度发现,与之前所得到的顺铂半致死量浓度相比,分别上调了CDC25B1与CDC25C1的细胞组的顺铂半致死量浓度显著升高,差异具有统计学意义(P0.01)；而分别上调了CDC25B3与CDC25C5的细胞组的顺铂半致死量浓度显著降低,差异具有统计学意义(P0.01)。结论：CDC25B的mRNA的剪接体CDC25B1能够增强宫颈癌细胞系SiHa, C33A对顺铂的抗性；剪接体CDC25B3能够减弱宫颈癌细胞系SiHa,C33A对顺铂的抗性。CDC25C的mRN A的剪接体CDC25C1能够增强宫颈癌细胞系SiHa,C33A对顺铂的抗性；剪接体CDC25C5能够减弱宫颈癌细胞系SiHaC33A对顺铂的抗性。
[Abstract]:Cervical cancer is the most common malignant tumor in women, which seriously threatens the health and life of women. Cisplatin is a commonly used chemotherapeutic agent for cervical cancer, but the resistance of cancer cells to cisplatin weakens its therapeutic effect. The selective splicing of CDC25B and CDC25C mRNA in the cervical cancer cell line SiHaC33A treated with cisplatin has been changed, because CDC25B and CDC25C play an important role in regulating the checkpoint function of cell cycle under normal physiological conditions and in response to DNA damage. It is suggested that the splicing changes of CDC25B and CDC25C mRNA may be related to cell tolerance to cisplatin. The purpose of this study is to investigate whether major splicing isoforms of CDC25B and CDC25C have anti-apoptotic or pro-apoptotic effects. Does this splicing change increase the resistance of cervical cancer cells to cisplatin? The results and information obtained will be helpful to clarify the role of CDC25B and CDC25C in carcinogenesis and development, and to interpret the mechanism of radio-chemotherapeutic resistance of tumor cells. If the splicing changes of CDC25B and CDC25C mRNA affect the function of checkpoint, the regulation of CDC25B may improve the efficacy of cisplatin, and the pathway and gene of regulation will become a new therapeutic target. To provide important information and basis for individualized treatment of cancer. Aim: to investigate the effects of selective splicing of CDC25B and CDC25C mRNA on the growth, proliferation, apoptosis and cisplatin sensitivity of cervical cancer cells SIHA and C33A. Methods: MTT colorimetric assay was used to determine the semi-lethal concentration of cisplatin in SiHaand C33A cells. RT-PCR was used to detect the same time, different concentration of cisplatin and different time. Effects of semi-lethal cisplatin intervention on selective splicing of CDC25B, C mRNA in cervical cancer cells in each group; The mRNA and protein expression levels of CDC25B1, B3, C1, C5 gene were detected by RT-PCR and Western Blot. MTT colorimetric assay was used to measure the cisplatin semi-lethal concentration of CDC25B1, B3, C1, C5 gene overexpression in cervical cancer SiHaand C33A cells. Compare with the previous conclusion. Results the semi-lethal concentration of cisplatin was about 40 渭 mol 路L ~ (-1) 路L ~ (-1) and about 22.5 渭 mol 路L ~ (-1) 路L ~ (-1) of cisplatin. Four splices CDC25B1B3, C1, C5 were selected for transfection. The up-regulated mRNA and protein expression were detected by RT-PCR and Western Blot respectively. MTT colorimetric assay was used to measure the semi-lethal concentration of cisplatin. Compared with the previously obtained semi-lethal concentration of cisplatin, the CDC25B1 and CDC25C1 cell groups increased the CDC25B1 and CDC25C1 semi-lethal concentration significantly (P0.01). The semi-lethal concentration of cisplatin in CDC25B3 and CDC25C5 cells was significantly lower than that in CDC25B3 and CDC25C5 cells (P0.01). Conclusion the splicing of CDC25B mRNA can enhance the resistance of cervical cancer cell line SiHaC33A to cisplatin. CDC25B3 could attenuate the resistance of SiHaC33A to cisplatin. CDC25C1 of mRNA of CDC25C could enhance the resistance of cervical cancer cell line SiHaC33A to cisplatin, and CDC25C5 could weaken the resistance of cervical cancer cell line SiHaC33A to cisplatin.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.33

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