Grp94及miR-3652在乳腺癌中的作用及機制研究
發(fā)布時間：2018-08-25 14:02
【摘要】：雌激素受體陽性(estrogen receptor positive,ER+)的乳腺癌常用治療法是內分泌治療,但近來發(fā)現(xiàn)越來越多的患者對內分泌藥物出現(xiàn)耐藥性,因此分子靶向藥物研究更加值得關注。大量研究發(fā)現(xiàn),內質網(wǎng)應激(endoplasmic reticulum stress,ERS)與腫瘤細胞凋亡密切相關,針對ERS的藥物已成為潛在的抗腫瘤藥物,但關于ERS在乳腺癌中的研究還相對較少。葡萄糖調節(jié)蛋白94(glucose regulated protein94,Grp94)是ERS的標志分子,已有文獻報道Grp94在乳腺癌等多種癌細胞異常高表達,其表達水平與腫瘤的發(fā)生發(fā)展及不良預后均有關系。因此,本實驗選擇研究Grp94沉默與ERS介導的凋亡在ER+的乳腺癌細胞MCF-7中的關系,并探索相關分子機制,為Grp94應用于ERS介導的乳腺癌治療提供了理論依據(jù)與新的思路。在對Grp94進行研究的同時,我們發(fā)現(xiàn)有一個micro RNA基因位于Grp94基因5'外顯子,名為miR-3652(micro RNA-3652)。我們也對miR-3652進行了相關研究,通過過表達miR-3652,研究其對MCF-7細胞生物學特性的影響。第一部分Grp94干擾后通過內質網(wǎng)應激誘導人乳腺癌MCF-7細胞凋亡的機制研究目的:研究人乳腺癌MCF-7細胞中Grp94干擾與ERS介導的凋亡的關系并探索相關的分子機制。方法:用衣霉素(tunicamycin,Tm)處理MCF-7細胞,建立ERS體外模型。在ERS狀態(tài)下,設計、合成靶向Grp94的si RNA,通過流式細胞術(flow cytometry,FCM)檢測干擾Grp94對細胞凋亡的影響,并由Western Blotting檢測ERS標志物及未折疊蛋白反應(unfolded protein response,UPR)相關通路分子的表達。結果:在ERS狀態(tài)下,Grp94被干擾后,FCM顯示細胞凋亡率上升;Western Blotting顯示Bip、IRE1、p-JNK表達增加,Bcl-2水平降低。結論:Grp94干擾后,細胞應激狀態(tài)加劇,凋亡增多,且促凋亡效應可能是通過IRE1-JNK-Bcl-2途徑來實現(xiàn)的。第二部分過表達miR-3652對人乳腺癌MCF-7細胞生物學特性的影響目的:研究人乳腺癌MCF-7細胞中過表達miR-3652對其生物學特性的影響。方法:在乳腺癌細胞系MCF-7、MDA-MB-231及SK-BR-3中檢測miR-3652的表達情況,選定適合后續(xù)研究的細胞。通過Real-time PCR在m RNA水平檢測miR-3652在Grp94被敲降后的表達變化,明確它們在轉錄水平之間的聯(lián)系。設計并合成miR-3652模擬物(miR-3652mimics),轉染MCF-7細胞,驗證過表達效率。通過FCM檢測過表達miR-3652對細胞周期及凋亡的影響;通過MTT檢測轉染后細胞增殖能力的變化。結果:miR-3652在MCF-7細胞中表達水平最低。Grp94被si RNA敲降后,miR-3652表達也同樣下降,且差異具有統(tǒng)計學意義(P0.01)。miR-3652 mimics可明顯增加細胞內miR-3652的m RNA水平。過表達miR-3652對MCF-7細胞的周期、凋亡無明顯影響,但在48h后能顯著促進細胞的增殖(P0.05)。結論:miR-3652與宿主基因Grp94表達一致,初步證明它們屬于同一轉錄單元,且miR-3652受宿主基因Grp94的啟動子調控。過表達miR-3652對MCF-7細胞的周期、凋亡無明顯影響,但對細胞的增殖能力具有促進作用。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9
本文編號：2203091
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9
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