zeste基因增强子同源物2对胃癌细胞衰老的影响及调控机制

发布时间：2018-04-09 13:28

本文选题：胃癌　切入点：阿霉素　出处：《华中科技大学》2015年博士论文

【摘要】：第一部分抑制EZH2表达对阿霉素诱导的胃癌细胞衰老的影响及相关机制目的：检测阿霉素对胃癌细胞系的衰老诱导作用,及抑制EZH2表达对该作用的影响,并探讨相关机制。方法：使用阿霉素诱导AGS和MKN28两种胃癌细胞系衰老,采用SA-β-gal染色、：DAPI染色、流式周期检测方法检测衰老诱导效果。观察siRNA或特异性EZH2抑制剂EGCG联合阿霉素的作用对胃癌细胞的影响。使用Western-blot检测p53/p21通路活化程度。结果：阿霉素能够有效诱导AGS和MKN28两种胃癌细胞系衰老,从而抑制胃癌细胞增殖。沉默EZH2能够促进阿霉素对细胞的增殖抑制作用,但就其机制而言,p53野生型与突变型细胞涉及的机制不同。对p53突变型胃癌细胞MKN28,抑制EZH2在诱导衰老及促进凋亡两方面均与阿霉素存在协同作用,但对于p53野生型胃癌细胞系AGS无此协同作用。结论：实验结果表明阿霉素能够促进胃癌细胞衰老。并揭示了EZH2与阿霉素联合使用对胃癌细胞衰老和凋亡进程的影响与细胞p53背景密切相关。第二部分抑制EZH2表达对胃癌细胞系的衰老诱导机制目的：研究抑制EZH2表达对胃癌细胞系MKN28、SGC-7901中INK4/ARF位点的影响,从而探索衰老诱导机制。方法：采用MTT、细胞增殖实验及克隆形成实验检测shRNA-EZH2通过抑制EZH2表达对胃癌细胞增殖的影响。SA-β-gal染色、流式周期检测方法检测细胞衰老情况。通过Western blot及ChIP验证抑制EZH2表达对胃癌细胞系MKN28、SGC-7901中INK4/ARF位点的影响,从而探索衰老诱导机制。结果： shRNA-EZH2能够有效抑制EZH2及其下游产物H3K27me3的表达,并能抑制对胃癌细胞的增殖及克隆形成能力。对于p21、p16均不表达的MKN28细胞系,抑制p15表达能够阻碍shRNA-EZH2的衰老诱导作用。最后,通过Western blot及ChIP验证EZH2可通过表观遗传学途径影响INK4/ARF位点的转录,从而调控周期影响衰老。结论：揭示了EZH2在调控INK4/ARF位点表达及诱导胃癌细胞衰老中的重要作用,为以原癌基因EZH2为靶点的肿瘤治疗策略的应用奠定了一定理论基础。
[Abstract]:The first part: inhibition of EZH2 expression on adriamycin-induced senescence of gastric cancer cells and its related mechanismsObjective:To detect the senescence induction of adriamycin on gastric cancer cell line and the effect of inhibiting the expression of EZH2 on this effect, and to explore the related mechanism.Methods:Adriamycin was used to induce senescence of AGS and MKN28 gastric cancer cell lines. SA- 尾 -gal staining was used to detect the senescence induction of gastric cancer cell lines.To observe the effect of siRNA or specific EZH2 inhibitor EGCG combined with adriamycin on gastric cancer cells.The activation of p53/p21 pathway was detected by Western-blot.Results:Adriamycin can effectively induce senescence of AGS and MKN28 gastric cancer cell lines and inhibit the proliferation of gastric cancer cells.Silencing EZH2 can promote the proliferation inhibition of adriamycin, but the mechanisms involved in p53 wild type and mutant type cells are different.On p53 mutant gastric cancer cell line MKN28, inhibition of EZH2 has synergistic effect with doxorubicin on inducing senescence and promoting apoptosis, but it has no synergistic effect on p53 wild-type gastric cancer cell line AGS.Conclusion:The results showed that adriamycin could promote the aging of gastric cancer cells.The effects of EZH2 combined with adriamycin on the senescence and apoptosis of gastric cancer cells were closely related to the background of p53.The second part of the mechanism of senescence induced by inhibition of EZH2 expression in gastric cancer cell lineObjective:To investigate the effect of inhibition of EZH2 expression on the INK4/ARF site in gastric cancer cell line MKN28 SGC-7901, and to explore the mechanism of senescence induction.Methods:MTT, cell proliferation and clone formation assay were used to detect the effect of shRNA-EZH2 on the proliferation of gastric cancer cells by inhibiting the expression of EZH2. SA- 尾 -gal staining and flow cytometry were used to detect the senescence of gastric cancer cells.The inhibition of EZH2 expression in gastric cancer cell line MKN28SGC-7901 was verified by Western blot and ChIP to explore the mechanism of senescence induction.Results:ShRNA-EZH2 can effectively inhibit the expression of EZH2 and its downstream product H3K27me3, and inhibit the proliferation and clone formation of gastric cancer cells.Inhibition of p15 expression in MKN28 cell lines without p21 p16 expression could inhibit the senescence induction of shRNA-EZH2.Finally, Western blot and ChIP showed that EZH2 could affect the transcription of INK4/ARF site through epigenetic pathway, thus regulating the cycle of senescence.Conclusion:This study revealed the important role of EZH2 in regulating the expression of INK4/ARF sites and inducing the senescence of gastric cancer cells, which laid a theoretical foundation for the application of tumor therapy strategies targeting proto-oncogene EZH2.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

【相似文献】