8-Cl-Ado對乳腺癌MDA-MB-231和SK-BR-3細(xì)胞的增殖抑制作用及其與ADAR1蛋白表達(dá)的相關(guān)性研究
發(fā)布時間：2018-08-29 15:20
【摘要】：目的:探討8-氯腺苷(8-chloro-adenosine,8-Cl-Ado)對乳腺癌MDA-MB-231和SK-BR-3細(xì)胞增殖和細(xì)胞周期的影響,及其與RNA編輯酶1(adenosine deaminases acting on RNA 1,ADAR1)蛋白的相關(guān)性。方法:通過蛋白免疫印跡法(western blotting,WB)檢測人乳腺癌癌組織、癌旁組織及不同乳腺癌細(xì)胞系中ADAR1的表達(dá)情況;通過MTT法檢測不同濃度的8-Cl-Ado(0、1、2、4、6、8、10、20μmol/L)作用乳腺癌MDA-MB-231和SK-BR-3細(xì)胞48 h后對細(xì)胞活力的影響;通過WB檢測不同濃度的8-Cl-Ado(0、2、4、6、8、10、20μmol/L)處理乳腺癌MDA-MB-231和SK-BR-3細(xì)胞48 h后ADAR1蛋白的表達(dá)情況;乳腺癌細(xì)胞轉(zhuǎn)染ADAR1-p110和ADAR1-p150質(zhì)粒后,加10μmol/L的8-Cl-Ado處理0、48 h,通過MTT法檢測乳腺癌MDA-MB-231和SK-BR-3細(xì)胞各轉(zhuǎn)染組的48 h抑制率;10μmol/L的8-Cl-Ado處理乳腺癌MDA-MB-231和SK-BR-3細(xì)胞不同時間后,通過流式細(xì)胞術(shù)(Flow cytometre,FCM)檢測細(xì)胞周期的變化以及WB檢測細(xì)胞中ADAR1、P53、P21和Cyclin D1蛋白的表達(dá)情況;乳腺癌細(xì)胞轉(zhuǎn)染ADAR1-p110和ADAR1-p150質(zhì)粒后,WB檢測ADAR1、P53、P21和Cyclin D1蛋白的表達(dá)情況;乳腺癌細(xì)胞轉(zhuǎn)染野生型ADAR1-p110質(zhì)粒、ADAR1-p150質(zhì)粒和突變型ADAR1-△E/A質(zhì)粒、ADAR1-△R質(zhì)粒以及p CMV空載質(zhì)粒,加10μmol/L的8-Cl-Ado處理0、24、48 h,通過MTT法檢測各質(zhì)粒組的抑制率;乳腺癌細(xì)胞轉(zhuǎn)染各質(zhì)粒,加10μmol/L的8-Cl-Ado處理48 h,通過FCM檢測細(xì)胞周期G1期百分比的變化,以及通過傷口愈合實驗檢測各質(zhì)粒組遷移率。結(jié)果:1.ADAR1蛋白的表達(dá)量在人乳腺癌癌組織中明顯比癌旁組織高;ADAR1蛋白在人乳腺癌MDA-MB-231和SK-BR-3細(xì)胞中表達(dá)相對較高;2.10μmol/L的8-Cl-Ado能明顯抑制乳腺癌MDA-MB-231和SK-BR-3細(xì)胞活力;ADAR1蛋白表達(dá)量隨8-Cl-Ado濃度的增加而降低;轉(zhuǎn)染ADAR1-p110和ADAR1-p150質(zhì)粒后,8-Cl-Ado對乳腺癌細(xì)胞的抑制率明顯降低;3.10μmol/L的8-Cl-Ado處理乳腺癌MDA-MB-231和SK-BR-3細(xì)胞不同時間后,細(xì)胞周期G1期百分比隨時間增加逐漸升高,細(xì)胞內(nèi)P53、P21蛋白表達(dá)也逐漸升高,ADAR1、Cyclin D1蛋白表達(dá)逐漸降低;轉(zhuǎn)染ADAR1-p110和ADAR1-p150質(zhì)粒后,P53、P21蛋白表達(dá)降低,ADAR1、Cyclin D1蛋白表達(dá)升高;4.與空白對照組和pCMV空載組相比,野生型ADAR1質(zhì)粒組和突變型ADAR1-△E/A組的增殖抑制率及G1期百分比明顯降低,突變型ADAR1-△R質(zhì)粒無明顯差異;與空白對照組和p CMV空載組相比,野生型ADAR1質(zhì)粒組和突變型ADAR1-△E/A組遷移率明顯升高,突變型ADAR1-△R質(zhì)粒無明顯差異。結(jié)論:1.ADAR1蛋白在人乳腺癌組織與人乳腺癌細(xì)胞系中高表達(dá),表明人乳腺癌的發(fā)生發(fā)展可能與ADAR1蛋白密切相關(guān)。2.10μmol/L的8-Cl-Ado可能通過下調(diào)ADAR1蛋白表達(dá)來引起乳腺癌MDA-MB-231和SK-BR-3細(xì)胞的增殖抑制以及細(xì)胞周期G1期阻滯。3.8-Cl-Ado對乳腺癌MDA-MB-231和SK-BR-3細(xì)胞的增殖抑制及G1期阻滯的作用,可能與ADAR1蛋白雙鏈RNA結(jié)合域(double-strand RNA binding domain,ds-RBD)有關(guān)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9
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