利拉鲁肽对人乳腺癌MCF-7细胞增殖、凋亡的影响及其机制研究

发布时间：2018-06-26 18:28

本文选题：2型糖尿病 + 乳腺癌　；参考：《天津医科大学》2016年博士论文

【摘要】：目的:近来人们发现糖尿病与部分肿瘤的发生发展密切相关。在女性2型糖尿病患者中,乳腺癌发病率显著高于非糖尿病患者。已有文献报道,二甲双胍可通过激活AMPK信号转导通路改善乳腺癌患者的预后。利拉鲁肽是GLP-1的类似物,已广泛应用于糖尿病患者的降糖治疗。利拉鲁肽可通过激活AMPK信号转导通路发挥降糖以外生物学作用。那么,利拉鲁肽是否亦可通过激活AMPK信号转导通路影响乳腺癌细胞的增殖、凋亡尚不清楚。miR-27a有原癌基因活性,在乳腺癌,胃癌,结肠癌,子宫内膜癌等多种肿瘤细胞中呈高表达,具有调控乳腺癌细胞凋亡及细胞周期等功能。Targetscan网站预测AMPKa2可能是miR-27a的靶基因。AMPKa2表达在乳腺癌细胞MCF-7中广泛受到抑制,具有肿瘤抑制因子的作用。综上所述,利拉鲁肽是否可以通过影响miR-27a表达,进而上调AMPKa2表达,发挥抗肿瘤作用目前并不清楚。因此在我们研究中,以MCF-7(人乳腺癌)细胞为研究对象,探讨利拉鲁肽对乳腺癌细胞增殖、凋亡的影响,并从miRNA层面探索其可能机制。方法:1.不同浓度利拉鲁肽(10n M、100n M、1000n M、10000n M)干预MCF-7细胞48h,应用CCK-8方法检测MCF-7细胞增殖抑制率。2.给予MCF-7细胞1000n M利拉鲁肽干预24h、48h、72h,应用CCK-8方法检测MCF-7细胞增殖抑制率。3.给予MCF-7细胞1000n M利拉鲁肽干预48h,应用平板克隆形成实验检测人乳腺癌MCF-7细胞克隆形成率;应用流式细胞仪检测MCF-7细胞早期和晚期凋亡率。4.不同浓度利拉鲁肽(10n M、100n M、1000n M)干预MCF-7细胞48h,应用实时荧光定量PCR方法检测MCF-7细胞miR-27a及AMPKα2 m RNA表达水平;应用Western Blotting方法检测AMPKα2蛋白表达水平。5.miR-27a mimics和inhibitor转染MCF-7细胞48h,应用实时荧光定量PCR和Western Blotting方法检测MCF-7细胞AMPKα2表达水平。6.双荧光素报告酶基因分析实验验证miR-27a可结合AMPKα2 3’-UTR。7.miR-27a mimics和inhibitor转染MCF-7细胞48h,应用CCK-8方法检测MCF-7细胞光密度值,并计算细胞增殖抑制率;应用流式细胞仪检测MCF-7细胞早期和晚期凋亡率;应用Western Blotting方法检测PCNA、caspase-3蛋白表达水平。结果:1.不同浓度利拉鲁肽(10n M、100n M、1000n M、10000n M)干预MCF-7细胞48h,MCF-7细胞增殖抑制率分别为10.3%,37.34%,58.8%,以及89.27%,具有统计学差异(P0.05)。2.给予MCF-7细胞1000n M利拉鲁肽干预24h、48h、72h,MCF-7细胞增殖抑制率分别为13.11%,45.3%以及53.59%,具有统计学差异(P0.05)。3.给予MCF-7细胞1000n M利拉鲁肽干预48h,MCF-7细胞克隆形成率为对照组的69.37%(P0.05);早期、晚期凋亡率分别为1.26%±0.18%、6.06%±0.32%,与空载体组(0.81%±0.13%、4.27%±0.26%)比较,两组晚期凋亡率存在统计学差异(P0.05)。4.不同浓度利拉鲁肽(10n M、100n M、1000n M)干预MCF-7细胞48h,miR-27a表达较对照组分别降低15.5%、44.5%、61.8%;AMPKα2 m RNA表达较对照组分别增加1.61倍、6.63倍和9.25倍;AMPKα2蛋白表达分别增加1.5倍、4.0倍和6.8倍,差别具有统计学意义(P0.05)。5.双荧光素酶报告基因实验显示,AMPKα2野生型3'UTR基因报告质粒与miR-27a共转染后,荧光素酶活性明显降低(P0.05)。6.miR-27a mimics转染MCF-7细胞48h,miR-27a表达量约为阴性对照组3.33倍;AMPKα2 m RNA、蛋白表达分别下调57.77%、52.55%(P0.05);miR-27a inhibitor转染MCF-7细胞48h,miR-27a表达量为阴性对照组的43.19%;AMPKα2m RNA、蛋白表达分别增加1.61倍、2.27倍(P0.05)。7.miR-27a mimics转染MCF-7细胞48h,两组早期、晚期凋亡率分别为0.79%±0.09%、2.16%±0.41%,与空载体组(0.88%±0.11%、4.71%±0.23%)比较,晚期凋亡百分比差别具有统计学意义(P0.05);PCNA蛋白表达增加2倍、caspase-3蛋白表达降低28.56%(P0.05)。miR-27a inhibitor转染MCF-7细胞48h,miR-27a inhibitor组的增殖抑制率为56.78%±2.24%。两组早期、晚期凋亡率分别为1.31%±0.07%、6.91%±0.27%,与空载体组(0.88%±0.11%、4.71%±0.23%)比较,两组晚期凋亡百分比差别具有统计学意义(P0.05);PCNA蛋白表达减低43.22%、caspase-3蛋白表达增加2.1倍(P0.05)。结论:利拉鲁肽抑制人乳腺癌MCF-7细胞增殖作用呈浓度依赖性和时间依赖性,且能促进MCF-7细胞凋亡,其机制可能与利拉鲁肽下调miR-27a表达,通过miR-27a靶基因AMPKα2表达水平上调来抑制PCNA表达,及促进caspase-3表达来发挥抗乳腺癌作用相关。
[Abstract]:Objective: Recently, people have found that diabetes is closely related to the development of some tumors. In women with type 2 diabetes, the incidence of breast cancer is significantly higher than that in non diabetic patients. It has been reported that metformin can improve the prognosis of breast cancer patients by activating the AMPK signal transduction pathway. It is used in diabetic patients with hypoglycemic therapy. By activating the AMPK signal transduction pathway, it can play a biological role other than hypoglycemic. Then, whether Leila Lou can also affect the proliferation of breast cancer cells by activating the AMPK signal transduction pathway, it is not clear that.MiR-27a has the proto oncogene activity in breast, gastric and colon cancer. A variety of tumor cells, such as endometrial cancer, are highly expressed, and the.Targetscan site that regulates the apoptosis and cell cycle of breast cancer cells predicts that AMPKa2 may be the target gene of miR-27a, which is widely suppressed in the MCF-7 of breast cancer cells and has the role of tumor suppressor. It is not clear that the expression of miR-27a, and then up regulation of AMPKa2 expression, and the antitumor effect is not clear. Therefore, in our study, the effects of rrrru on the proliferation and apoptosis of breast cancer cells were studied with MCF-7 (human breast cancer) cells, and the possible mechanisms were explored from the miRNA level. Methods: 1. different concentrations of rrrru (10N M, 100N) M, 1000N M, 10000n M) interfered MCF-7 cell 48h, and CCK-8 method was used to detect the proliferation inhibition rate of MCF-7 cells. The rate of cell clone formation was detected by using flow cytometry to detect the early and late apoptosis rate of MCF-7 cells with different concentrations of Liraru peptide (10N M, 100N M, 1000N M) in MCF-7 cell 48h. The expression level of MCF-7 cells and alpha 2 was detected by real-time fluorescent quantitative PCR method, and the expression level of alpha 2 protein was detected by using the method of real-time fluorescent quantitative PCR. .5.miR-27a mimics and inhibitor transfected MCF-7 cells 48h, real-time fluorescent quantitative PCR and Western Blotting methods were used to detect the expression level of AMPK alpha 2 in MCF-7 cells,.6. double fluorescein reporter gene analysis experiment verified miR-27a can be combined with alpha 23 'and transfected cells The cell light density value and the cell proliferation inhibition rate were calculated. The early and late apoptosis rates of MCF-7 cells were detected by flow cytometry, and the expression of PCNA and caspase-3 protein was detected by Western Blotting. Results: 1. the proliferation inhibition rate of 10N M, 100N M, 1000N M, 10000n cells were 10, respectively. .3%, 37.34%, 58.8%, and 89.27%, with statistical difference (P0.05).2. gave MCF-7 cells 1000N M Li La Lu peptide intervention 24h, 48h, 72h, MCF-7 cell proliferation inhibition rate was 13.11%, 45.3% and 53.59%, respectively. (P0.05); the early stage of apoptosis was 1.26% + 0.18% and 6.06% + 0.32% respectively. Compared with the unloaded body group (0.81% + 0.13%, 4.27% + 0.26%), the late apoptotic rate of the two groups was statistically different (P0.05).4. different concentrations of alalu peptide (10N M, 100N M, 1000N M) intervened MCF-7 cell 48h, miR-27a expression decreased 15.5%, 44.5%, 61.8%, respectively. The expression of NA was 1.61 times, 6.63 times and 9.25 times more than that of the control group, and the expression of AMPK alpha 2 protein increased by 1.5 times, 4 times and 6.8 times respectively. The difference was statistically significant (P0.05).5. double luciferase reporter gene experiment showed that the luciferase activity of AMPK a 2 wild type 3'UTR gene reporter plasmid was significantly reduced (P0.05).6.miR-27a (P0.05).6.miR-27a after CO transfection. Mimics transfected MCF-7 cells 48h, the expression of miR-27a was about 3.33 times that of negative control group, AMPK alpha 2 m RNA, protein expression down 57.77%, 52.55% (P0.05), miR-27a inhibitor transfected to MCF-7 cell 48h, and the expression amount was 43.19% of negative control group. 7 cell 48h, early stage of two group, the late apoptosis rate was 0.79% + 0.09%, 2.16% + 0.41% respectively. Compared with the unloaded body group (0.88% + 0.11%, 4.71% + 0.23%), the percentage of late apoptosis was statistically significant (P0.05), the expression of PCNA protein increased by 2 times, the expression of caspase-3 protein decreased (P0.05).MiR-27a inhibitor transfection to MCF-7 cells 48h, miR-27a inhibi. The proliferation inhibition rate of group tor was 56.78% + 2.24%. two groups, the late apoptosis rate was 1.31% + 0.07%, 6.91% + 0.27% respectively. Compared with the no-load group (0.88% + 0.11%, 4.71% + 0.23%), the percentage of late apoptosis in group two was statistically significant (P0.05); PCNA protein expression decreased 43.22%, caspase-3 protein expression increased significantly (P0.05). Conclusion: lila The inhibitory effect of RUP on the proliferation of human breast cancer MCF-7 cells is both concentration dependent and time dependent, and can promote the apoptosis of MCF-7 cells. The mechanism may be related to the downregulation of miR-27a expression with ralalurin, the up-regulation of AMPK alpha 2 expression level of miR-27a target gene to inhibit the expression of PCNA and promote the expression of Caspase-3 to play the role of anti breast cancer.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

【相似文献】