Grp94及miR-3652在乳腺癌中的作用及机制研究

发布时间：2018-08-25 14:02

【摘要】：雌激素受体阳性(estrogen receptor positive,ER+)的乳腺癌常用治疗法是内分泌治疗,但近来发现越来越多的患者对内分泌药物出现耐药性,因此分子靶向药物研究更加值得关注。大量研究发现,内质网应激(endoplasmic reticulum stress,ERS)与肿瘤细胞凋亡密切相关,针对ERS的药物已成为潜在的抗肿瘤药物,但关于ERS在乳腺癌中的研究还相对较少。葡萄糖调节蛋白94(glucose regulated protein94,Grp94)是ERS的标志分子,已有文献报道Grp94在乳腺癌等多种癌细胞异常高表达,其表达水平与肿瘤的发生发展及不良预后均有关系。因此,本实验选择研究Grp94沉默与ERS介导的凋亡在ER+的乳腺癌细胞MCF-7中的关系,并探索相关分子机制,为Grp94应用于ERS介导的乳腺癌治疗提供了理论依据与新的思路。在对Grp94进行研究的同时,我们发现有一个micro RNA基因位于Grp94基因5'外显子,名为miR-3652(micro RNA-3652)。我们也对miR-3652进行了相关研究,通过过表达miR-3652,研究其对MCF-7细胞生物学特性的影响。第一部分Grp94干扰后通过内质网应激诱导人乳腺癌MCF-7细胞凋亡的机制研究目的:研究人乳腺癌MCF-7细胞中Grp94干扰与ERS介导的凋亡的关系并探索相关的分子机制。方法:用衣霉素(tunicamycin,Tm)处理MCF-7细胞,建立ERS体外模型。在ERS状态下,设计、合成靶向Grp94的si RNA,通过流式细胞术(flow cytometry,FCM)检测干扰Grp94对细胞凋亡的影响,并由Western Blotting检测ERS标志物及未折叠蛋白反应(unfolded protein response,UPR)相关通路分子的表达。结果:在ERS状态下,Grp94被干扰后,FCM显示细胞凋亡率上升;Western Blotting显示Bip、IRE1、p-JNK表达增加,Bcl-2水平降低。结论:Grp94干扰后,细胞应激状态加剧,凋亡增多,且促凋亡效应可能是通过IRE1-JNK-Bcl-2途径来实现的。第二部分过表达miR-3652对人乳腺癌MCF-7细胞生物学特性的影响目的:研究人乳腺癌MCF-7细胞中过表达miR-3652对其生物学特性的影响。方法:在乳腺癌细胞系MCF-7、MDA-MB-231及SK-BR-3中检测miR-3652的表达情况,选定适合后续研究的细胞。通过Real-time PCR在m RNA水平检测miR-3652在Grp94被敲降后的表达变化,明确它们在转录水平之间的联系。设计并合成miR-3652模拟物(miR-3652mimics),转染MCF-7细胞,验证过表达效率。通过FCM检测过表达miR-3652对细胞周期及凋亡的影响;通过MTT检测转染后细胞增殖能力的变化。结果:miR-3652在MCF-7细胞中表达水平最低。Grp94被si RNA敲降后,miR-3652表达也同样下降,且差异具有统计学意义(P0.01)。miR-3652 mimics可明显增加细胞内miR-3652的m RNA水平。过表达miR-3652对MCF-7细胞的周期、凋亡无明显影响,但在48h后能显著促进细胞的增殖(P0.05)。结论:miR-3652与宿主基因Grp94表达一致,初步证明它们属于同一转录单元,且miR-3652受宿主基因Grp94的启动子调控。过表达miR-3652对MCF-7细胞的周期、凋亡无明显影响,但对细胞的增殖能力具有促进作用。
[Abstract]:Endocrine therapy is commonly used to treat breast cancer with estrogen receptor positive (estrogen receptor positive,ER). Recently, more and more patients have been found to be resistant to endocrine drugs, so the study of molecular targeted drugs is more worthy of attention. A large number of studies have found that endoplasmic reticulum stress (endoplasmic reticulum stress,ERS) is closely related to apoptosis of tumor cells. Drugs directed against ERS have become potential antitumor drugs, but there are relatively few studies on ERS in breast cancer. Glucose-regulated protein 94 (glucose regulated protein94,Grp94) is a marker of ERS. It has been reported that Grp94 is highly expressed in many kinds of cancer cells, such as breast cancer, and its expression level is related to the occurrence, development and poor prognosis of the tumor. Therefore, this experiment selected to study the relationship between Grp94 silencing and ERS mediated apoptosis in ER breast cancer cell MCF-7, and explore the molecular mechanism, which provides a theoretical basis and a new idea for the application of Grp94 in ERS mediated breast cancer therapy. While studying Grp94, we found that there is a micro RNA gene located in the 5 'exon of Grp94 gene, named miR-3652 (micro RNA-3652). We also studied the effects of miR-3652 on the biological characteristics of MCF-7 cells by overexpression of miR-3652,. Part I the mechanism of endoplasmic reticulum stress inducing apoptosis of human breast cancer MCF-7 cells after Grp94 interference objective: to study the relationship between Grp94 interference and ERS mediated apoptosis in human breast cancer MCF-7 cells and to explore the molecular mechanism. Methods: MCF-7 cells were treated with tunicamycin,Tm and ERS model was established in vitro. Under the condition of ERS, si RNA, targeting Grp94 was designed and synthesized to detect the effect of interfering Grp94 on apoptosis by flow cytometry (flow cytometry,FCM), and Western Blotting was used to detect the expression of ERS markers and (unfolded protein response,UPR pathway molecules. Results: in the presence of ERS, the apoptotic rate was increased and the expression of Bip,IRE1,p-JNK increased and the level of Bcl-2 decreased. Conclusion the stress state and apoptosis of the cells increased after the interference of W Grp94, and the effect of promoting apoptosis may be realized by IRE1-JNK-Bcl-2 pathway. The second part: the effect of overexpression of miR-3652 on the biological characteristics of human breast cancer MCF-7 cells objective: to study the effects of overexpression of miR-3652 on the biological characteristics of human breast cancer MCF-7 cells. Methods: the expression of miR-3652 was detected in breast cancer cell line MCF-7,MDA-MB-231 and SK-BR-3. The changes of miR-3652 expression after Grp94 knock down were detected by Real-time PCR at m RNA level, and the relationship between them at transcription level was clarified. MiR-3652 mimics (miR-3652mimics) were designed and synthesized and transfected into MCF-7 cells to verify the overexpression efficiency. The effects of overexpression of miR-3652 on cell cycle and apoptosis were detected by FCM and the changes of cell proliferation after transfection by MTT. Results the expression of miR-3652 in MCF-7 cells was the lowest. The expression of miR-3652 in MCF-7 cells was also decreased after si RNA knocked down, and the difference was statistically significant (P0.01) .miR-3652 mimics could significantly increase the m RNA level of miR-3652 in the cells. Overexpression of miR-3652 had no effect on the cell cycle and apoptosis of MCF-7 cells, but it could significantly promote the proliferation of MCF-7 cells after 48 hours (P0.05). Conclusion the Grp94 expression of the host gene is consistent with that of the host gene, which preliminarily proves that they belong to the same transcription unit, and miR-3652 is regulated by the promoter of the host gene Grp94. Overexpression of miR-3652 had no effect on the cell cycle and apoptosis of MCF-7 cells, but it promoted the proliferation of MCF-7 cells.
【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】：R737.9

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