8-Cl-Ado对乳腺癌MDA-MB-231和SK-BR-3细胞的增殖抑制作用及其与ADAR1蛋白表达的相关性研究
发布时间：2018-08-29 15:20
【摘要】：目的:探讨8-氯腺苷(8-chloro-adenosine,8-Cl-Ado)对乳腺癌MDA-MB-231和SK-BR-3细胞增殖和细胞周期的影响,及其与RNA编辑酶1(adenosine deaminases acting on RNA 1,ADAR1)蛋白的相关性。方法:通过蛋白免疫印迹法(western blotting,WB)检测人乳腺癌癌组织、癌旁组织及不同乳腺癌细胞系中ADAR1的表达情况;通过MTT法检测不同浓度的8-Cl-Ado(0、1、2、4、6、8、10、20μmol/L)作用乳腺癌MDA-MB-231和SK-BR-3细胞48 h后对细胞活力的影响;通过WB检测不同浓度的8-Cl-Ado(0、2、4、6、8、10、20μmol/L)处理乳腺癌MDA-MB-231和SK-BR-3细胞48 h后ADAR1蛋白的表达情况;乳腺癌细胞转染ADAR1-p110和ADAR1-p150质粒后,加10μmol/L的8-Cl-Ado处理0、48 h,通过MTT法检测乳腺癌MDA-MB-231和SK-BR-3细胞各转染组的48 h抑制率;10μmol/L的8-Cl-Ado处理乳腺癌MDA-MB-231和SK-BR-3细胞不同时间后,通过流式细胞术(Flow cytometre,FCM)检测细胞周期的变化以及WB检测细胞中ADAR1、P53、P21和Cyclin D1蛋白的表达情况;乳腺癌细胞转染ADAR1-p110和ADAR1-p150质粒后,WB检测ADAR1、P53、P21和Cyclin D1蛋白的表达情况;乳腺癌细胞转染野生型ADAR1-p110质粒、ADAR1-p150质粒和突变型ADAR1-△E/A质粒、ADAR1-△R质粒以及p CMV空载质粒,加10μmol/L的8-Cl-Ado处理0、24、48 h,通过MTT法检测各质粒组的抑制率;乳腺癌细胞转染各质粒,加10μmol/L的8-Cl-Ado处理48 h,通过FCM检测细胞周期G1期百分比的变化,以及通过伤口愈合实验检测各质粒组迁移率。结果:1.ADAR1蛋白的表达量在人乳腺癌癌组织中明显比癌旁组织高;ADAR1蛋白在人乳腺癌MDA-MB-231和SK-BR-3细胞中表达相对较高;2.10μmol/L的8-Cl-Ado能明显抑制乳腺癌MDA-MB-231和SK-BR-3细胞活力;ADAR1蛋白表达量随8-Cl-Ado浓度的增加而降低;转染ADAR1-p110和ADAR1-p150质粒后,8-Cl-Ado对乳腺癌细胞的抑制率明显降低;3.10μmol/L的8-Cl-Ado处理乳腺癌MDA-MB-231和SK-BR-3细胞不同时间后,细胞周期G1期百分比随时间增加逐渐升高,细胞内P53、P21蛋白表达也逐渐升高,ADAR1、Cyclin D1蛋白表达逐渐降低;转染ADAR1-p110和ADAR1-p150质粒后,P53、P21蛋白表达降低,ADAR1、Cyclin D1蛋白表达升高;4.与空白对照组和pCMV空载组相比,野生型ADAR1质粒组和突变型ADAR1-△E/A组的增殖抑制率及G1期百分比明显降低,突变型ADAR1-△R质粒无明显差异;与空白对照组和p CMV空载组相比,野生型ADAR1质粒组和突变型ADAR1-△E/A组迁移率明显升高,突变型ADAR1-△R质粒无明显差异。结论:1.ADAR1蛋白在人乳腺癌组织与人乳腺癌细胞系中高表达,表明人乳腺癌的发生发展可能与ADAR1蛋白密切相关。2.10μmol/L的8-Cl-Ado可能通过下调ADAR1蛋白表达来引起乳腺癌MDA-MB-231和SK-BR-3细胞的增殖抑制以及细胞周期G1期阻滞。3.8-Cl-Ado对乳腺癌MDA-MB-231和SK-BR-3细胞的增殖抑制及G1期阻滞的作用,可能与ADAR1蛋白双链RNA结合域(double-strand RNA binding domain,ds-RBD)有关。
[Abstract]:Aim: to investigate the effects of 8-chloro-adenosine 8-Cl-Ado on the proliferation and cell cycle of breast cancer MDA-MB-231 and SK-BR-3 cells, and the relationship between 8-chloro-adenosine 8-chloro-adenosine (8-chloro-adenosine 8-Cl-Ado) and RNA editing enzyme 1 (adenosine deaminases acting on RNA 1 ADAR1 protein. Methods: the expression of ADAR1 in human breast cancer tissues, adjacent tissues and different breast cancer cell lines was detected by Western blotting (western blotting,WB). The expression of ADAR1 protein in MDA-MB-231 and SK-BR-3 cells of breast cancer treated with different concentrations of 8-Cl-Ado (01020 渭 mol/L) for 48 h was detected by MTT assay, and the expression of ADAR1 protein in MDA-MB-231 and SK-BR-3 cells of breast cancer was detected by WB after 48 h treatment with different concentrations of 8-Cl-Ado. Breast cancer cells were transfected with ADAR1-p110 and ADAR1-p150 plasmids and treated with 10 渭 mol/L 8-Cl-Ado for 48 h. The 48 h inhibition rate of breast cancer MDA-MB-231 and SK-BR-3 cells transfected with 10 渭 mol/L 8-Cl-Ado was detected by MTT assay. The changes of cell cycle were detected by flow cytometry (Flow cytometre,FCM) and the expression of ADAR1,P53,P21 and Cyclin D1 protein were detected by WB, and the expression of ADAR1,P53,P21 and Cyclin D1 protein was detected by wideband after transfection of ADAR1-p110 and ADAR1-p150 plasmids in breast cancer cells. Breast cancer cells were transfected with wild type ADAR1-p110 plasmids ADAR1-p150, mutant ADAR1- E / A plasmids ADAR1R and p CMV empty plasmids, and then treated with 10 渭 mol/L 8-Cl-Ado for 48 h. The inhibition rates of each plasmid group were detected by MTT assay, and the plasmids were transfected by breast cancer cells. After treatment with 10 渭 mol/L 8-Cl-Ado for 48 h, the percentage change of G1 phase of cell cycle was detected by FCM, and the mobility of plasmid groups was detected by wound healing experiment. Results 1. The expression of ADAR1 protein in human breast cancer tissues was significantly higher than that in the adjacent tissues of human breast cancer. 8-Cl-Ado with a relatively high expression of 2.10 渭 mol/L in human breast cancer MDA-MB-231 and SK-BR-3 cells could significantly inhibit the expression of ADAR1 protein in breast cancer MDA-MB-231 and SK-BR-3 cells. The concentration of 8-Cl-Ado decreased with the increase of 8-Cl-Ado concentration. After transfection of ADAR1-p110 and ADAR1-p150 plasmids, the inhibition rate of 8-Cl-Ado on breast cancer cells was significantly decreased after treated with 8-Cl-Ado of 3.10 渭 mol/L for different time, the percentage of G1 phase of cell cycle gradually increased with the increase of time. The expression of P53 / P21 protein also increased gradually and decreased gradually after transfection of ADAR1-p110 and ADAR1-p150 plasmids, while the expression of P53 / P21 protein decreased after transfection of ADAR1 and ADAR1-p150 plasmids, and the expression of ADAR1 / Cyclin D1 protein increased gradually. Compared with the blank control group and the pCMV no-load group, the inhibition rate of proliferation and the percentage of G1 phase in the wild-type ADAR1 plasmid group and the mutant ADAR1- E / A group were significantly decreased, but there was no significant difference between the mutant ADAR1- R plasmid and the blank control group and the p CMV no-load group. The mobility of wild-type ADAR1 plasmid group and mutant ADAR1- E / A group was significantly increased, but there was no significant difference in mutant ADAR1- R plasmid. Conclusion: 1. ADAR1 protein is highly expressed in human breast cancer tissues and human breast cancer cell lines. It suggests that the occurrence and development of human breast cancer may be closely related to ADAR1 protein. 2.10 渭 mol/L 8-Cl-Ado may induce the proliferation inhibition of MDA-MB-231 and SK-BR-3 cells and the G1 phase arrest of cell cycle. 3.8-Cl-Ado on MDA-MB-231 and SK-BR-3 of breast cancer by down-regulating the expression of ADAR1 protein. Inhibition of cell proliferation and arrest of G1 phase, It may be related to the double stranded RNA binding domain (double-strand RNA binding domain,ds-RBD) of ADAR1 protein.
【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
[Abstract]:Aim: to investigate the effects of 8-chloro-adenosine 8-Cl-Ado on the proliferation and cell cycle of breast cancer MDA-MB-231 and SK-BR-3 cells, and the relationship between 8-chloro-adenosine 8-chloro-adenosine (8-chloro-adenosine 8-Cl-Ado) and RNA editing enzyme 1 (adenosine deaminases acting on RNA 1 ADAR1 protein. Methods: the expression of ADAR1 in human breast cancer tissues, adjacent tissues and different breast cancer cell lines was detected by Western blotting (western blotting,WB). The expression of ADAR1 protein in MDA-MB-231 and SK-BR-3 cells of breast cancer treated with different concentrations of 8-Cl-Ado (01020 渭 mol/L) for 48 h was detected by MTT assay, and the expression of ADAR1 protein in MDA-MB-231 and SK-BR-3 cells of breast cancer was detected by WB after 48 h treatment with different concentrations of 8-Cl-Ado. Breast cancer cells were transfected with ADAR1-p110 and ADAR1-p150 plasmids and treated with 10 渭 mol/L 8-Cl-Ado for 48 h. The 48 h inhibition rate of breast cancer MDA-MB-231 and SK-BR-3 cells transfected with 10 渭 mol/L 8-Cl-Ado was detected by MTT assay. The changes of cell cycle were detected by flow cytometry (Flow cytometre,FCM) and the expression of ADAR1,P53,P21 and Cyclin D1 protein were detected by WB, and the expression of ADAR1,P53,P21 and Cyclin D1 protein was detected by wideband after transfection of ADAR1-p110 and ADAR1-p150 plasmids in breast cancer cells. Breast cancer cells were transfected with wild type ADAR1-p110 plasmids ADAR1-p150, mutant ADAR1- E / A plasmids ADAR1R and p CMV empty plasmids, and then treated with 10 渭 mol/L 8-Cl-Ado for 48 h. The inhibition rates of each plasmid group were detected by MTT assay, and the plasmids were transfected by breast cancer cells. After treatment with 10 渭 mol/L 8-Cl-Ado for 48 h, the percentage change of G1 phase of cell cycle was detected by FCM, and the mobility of plasmid groups was detected by wound healing experiment. Results 1. The expression of ADAR1 protein in human breast cancer tissues was significantly higher than that in the adjacent tissues of human breast cancer. 8-Cl-Ado with a relatively high expression of 2.10 渭 mol/L in human breast cancer MDA-MB-231 and SK-BR-3 cells could significantly inhibit the expression of ADAR1 protein in breast cancer MDA-MB-231 and SK-BR-3 cells. The concentration of 8-Cl-Ado decreased with the increase of 8-Cl-Ado concentration. After transfection of ADAR1-p110 and ADAR1-p150 plasmids, the inhibition rate of 8-Cl-Ado on breast cancer cells was significantly decreased after treated with 8-Cl-Ado of 3.10 渭 mol/L for different time, the percentage of G1 phase of cell cycle gradually increased with the increase of time. The expression of P53 / P21 protein also increased gradually and decreased gradually after transfection of ADAR1-p110 and ADAR1-p150 plasmids, while the expression of P53 / P21 protein decreased after transfection of ADAR1 and ADAR1-p150 plasmids, and the expression of ADAR1 / Cyclin D1 protein increased gradually. Compared with the blank control group and the pCMV no-load group, the inhibition rate of proliferation and the percentage of G1 phase in the wild-type ADAR1 plasmid group and the mutant ADAR1- E / A group were significantly decreased, but there was no significant difference between the mutant ADAR1- R plasmid and the blank control group and the p CMV no-load group. The mobility of wild-type ADAR1 plasmid group and mutant ADAR1- E / A group was significantly increased, but there was no significant difference in mutant ADAR1- R plasmid. Conclusion: 1. ADAR1 protein is highly expressed in human breast cancer tissues and human breast cancer cell lines. It suggests that the occurrence and development of human breast cancer may be closely related to ADAR1 protein. 2.10 渭 mol/L 8-Cl-Ado may induce the proliferation inhibition of MDA-MB-231 and SK-BR-3 cells and the G1 phase arrest of cell cycle. 3.8-Cl-Ado on MDA-MB-231 and SK-BR-3 of breast cancer by down-regulating the expression of ADAR1 protein. Inhibition of cell proliferation and arrest of G1 phase, It may be related to the double stranded RNA binding domain (double-strand RNA binding domain,ds-RBD) of ADAR1 protein.
【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
【参考文献】
相关期刊论文 前7条
1 周晶晶;周艳;聂勇战;李小安;;ADAR1与非编码RNAs的研究进展[J];成都医学院学报;2016年02期
2 马俊;侯小强;郑秋生;何涛;;DNA修复酶基因NEIL1的A-I RNA编辑水平在白血病和乳腺癌细胞中的下调[J];军事医学;2014年06期
3 左描绘;胡婷秀;杨土保;;乳腺癌影响因素研究进展[J];实用预防医学;2013年09期
4 王岩;冯子s，
本文编号：2211605
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2211605.html
