MiR-134下调ABCC1的表达增强乳腺癌细胞对阿霉素敏感性的实验研究

发布时间：2018-04-27 09:49

本文选题：乳腺癌 + 阿霉素　；参考：《青岛大学》2016年博士论文

【摘要】：背景和目的:mi R-134在某些肿瘤中表达上调,但在包括乳腺癌在内的多种恶性肿瘤中表达下调,已证实mi R-134能在非小细胞肺癌细胞中下调多药耐药相关蛋白MRP1/ABCC1的表达来增加细胞对阿霉素的敏感性。但mi R-134和ABCC1在阿霉素耐药性乳腺癌中的表达情况及对细胞耐药性的影响还未十分明确。本研究旨在检测mi R-134和ABCC1在阿霉素耐药性乳腺癌病理标本和耐药性细胞株中的表达及相互关系,并探讨mi R-134在乳腺癌细胞阿霉素耐药过程中的作用。方法:1、实时荧光定量PCR(q RT-PCR)法检测非耐药癌旁组织(ASPT)、非耐药乳腺癌组织(ASBC)以及阿霉素耐药癌旁组织(ARPT)和阿霉素耐药性乳腺癌组织(ARBC)中mi R-134的表达。同时检测阿霉素耐药性乳腺癌细胞株MCF-7/ADR与非耐药乳腺癌细胞株MCF-7中mi R-134的表达。2、采用q RT-PCR和Western Blot法评估ASPT、ASBC、ARPT和ARBC组织中ABCC1的表达变化。同时检测MCF-7/ADR细胞和MCF-7细胞中ABCC1的表达情况。3、mi R-134 mimics和NC分别转染入MCF-7/ADR细胞,q RT-PCR验证mi R-134的过表达情况。q RT-PCR、Western Blot和免疫组化检测MCF-7/ADR细胞中ABCC1的表达。4、MTT法明确转染mi R-134 mimics的MCF-7/ADR细胞的IC50值,在IC50浓度阿霉素的作用下,MTT法检测过表达mi R-134的MCF-7/ADR细胞增殖情况。5、在IC50浓度阿霉素的作用下,TUNEL法及流式细胞术检测过表达mi R-134的MCF-7/ADR细胞的凋亡情况。结果:1、乳腺癌组织中mi R-134的表达均显著低于癌旁组织(p0.001)。mi R-134在ARBC组织中的表达显著低于ASBC组织(p0.001),同时mi R-134在MCF-7/ADR细胞中的表达显著低于MCF-7细胞(p0.01)。2、ABCC1在乳腺癌组织中的m RNA水平和蛋白表达水平均显著高于相应癌旁组织(p0.001)。ABCC1在ARBC组织中的m RNA水平和蛋白表达水平均显著高于ASBC组织(p0.05),同时ABCC1在MCF-7/ADR细胞中的m RNA水平和蛋白水平均显著高于MCF-7细胞(p0.001)。3、与NC组MCF-7/ADR细胞相比,mi R-134 mimics组细胞中mi R-134的表达明显增加(p0.001)。过表达mi R-134后,MCF-7/ADR细胞中ABCC1表达在m RNA水平上无明显变化,但在蛋白水平上显著降低(p0.001)。4、过表达mi R-134的MCF-7/ADR细胞的IC50值为226ng/ml。过表达mi R-134的MCF-7/ADR细胞在226ng/ml阿霉素作用下,细胞增殖能力较对照组明显下降,在48h时即与对照组形成显著性差异(p0.05),并随着时间推移,抑制程度更加明显(p0.01)。5、过表达mi R-134的MCF-7/ADR细胞在226ng/ml阿霉素作用下,凋亡的细胞较对照组显著增加(p0.01)。结论:1、mi R-134在乳腺癌组织中表达下调,在阿霉素耐药性乳腺癌组织和细胞中表达水平最低。2、ABCC1在乳腺癌组织中表达上调,在阿霉素耐药性乳腺癌组织和细胞中表达水平最高。3、mi R-134能下调ABCC1的表达。4、MCF-7/ADR细胞中过表达mi R-134能通过下调ABCC1的表达来促进阿霉素诱导的抑制增殖和促凋亡效应,增强MCF-7/ADR细胞对阿霉素的敏感性。
[Abstract]:Background and objective: mi R-134 is up-regulated in some tumors, but down-regulated in many malignant tumors, including breast cancer. It has been proved that mi R-134 can down-regulate the expression of multidrug resistance-associated protein MRP1/ABCC1 in non-small cell lung cancer cells to increase the sensitivity of the cells to adriamycin. However, the expression of miR-134 and ABCC1 in adriamycin resistant breast cancer and its effect on cell resistance are still unclear. The purpose of this study was to investigate the expression and correlation of miR-134 and ABCC1 in adriamycin resistant breast cancer and to explore the role of miR-134 in adriamycin resistance in breast cancer cells. Methods the expression of miR-134 was detected by real-time fluorescence quantitative PCR(q RT-PCRmethod in non-drug-resistant breast cancer tissues, adriamycin resistant tissues and adriamycin resistant breast cancer tissues. Meanwhile, the expression of miR-134 in adriamycin resistant breast cancer cell line MCF-7/ADR and non-drug-resistant breast cancer cell line MCF-7 was detected. The expression of ABCC1 was evaluated by Q RT-PCR and Western Blot methods. Meanwhile, the expression of ABCC1 in MCF-7/ADR cells and MCF-7 cells was detected. 3mil R-134 mimics and NC were transfected into MCF-7/ADR cells Q RT-PCR to verify the overexpression of mi R-134. Q RT-PCRG Western Blot and immunohistochemistry to detect the expression of ABCC1 in MCF-7/ADR cells. The IC50 value of MCF-7/ADR cells, The proliferation of MCF-7/ADR cells overexpression of miR-134 was detected by IC50 assay. The apoptosis of MCF-7/ADR cells overexpression of miR-134 was detected by Tunel and flow cytometry under the action of doxorubicin at the concentration of IC50. Results the expression of miR-134 in breast cancer tissues was significantly lower than that in ARBC tissues, and the expression of miR-134 in MCF-7/ADR cells was significantly lower than that in MCF-7 cells. The level of RNA and protein expression were significantly higher than that of the corresponding adjacent tissues (p0.001. ABCC1) in ARBC tissues, and the level of m RNA and protein expression in ASBC tissues were significantly higher than those in ASBC tissues. Meanwhile, the level of m RNA and protein of ABCC1 in MCF-7/ADR cells were significantly higher. Compared with NC MCF-7/ADR cells, the expression of miR-134 in MCF-7 cells increased significantly compared with that in NC MCF-7/ADR cells. The expression of ABCC1 in MCF-7 / ADR cells was not significantly changed at m RNA level, but decreased significantly at protein level. The IC50 value of MCF-7/ADR cells with overexpression of miR-134 was 226ng / ml. The proliferative ability of MCF-7/ADR cells overexpression of miR-134 was significantly lower than that of the control group under the action of 226ng/ml adriamycin, and the difference was significant at 48 h after treatment with the control group (P 0.05). The inhibition degree was more obvious than that of the control group. MCF-7/ADR cells overexpression of miR-134 increased the number of apoptotic cells significantly compared with the control group under the action of 226ng/ml adriamycin. Conclusion the expression of R134 is down-regulated in breast cancer, and the lowest in adriamycin resistant breast cancer tissues and cells. The expression of ABCC1 is up-regulated in breast cancer. The overexpression of miR-134 could down-regulate the expression of ABCC1 in MCF-7 / ADR cells by down-regulating the expression of ABCC1 to promote the inhibition of proliferation and apoptosis induced by adriamycin. To enhance the sensitivity of MCF-7/ADR cells to adriamycin.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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