摘要
目的探讨微小RNA-145(miR-145)对性别决定区Y框蛋白11(SOX11)的靶向调控作用及对乳腺癌细胞增殖、凋亡的影响。方法采用实时荧光定量PCR(QPCR)检测正常乳腺上皮细胞HBL-100和乳腺癌MDA-MB-231细胞中的miR-145的表达水平;选取对数生长期MDA-MB-231细胞,采用脂质体法分别转染miR-145模拟物mimics(过表达组)或阴性对照质粒NC(阴性对照组),以未行转染的细胞为空白对照组;采用QPCR检测转染48 h后各组miR-145的表达水平,采用CCK-8和AnnexinⅤ-FITC/PI法检测各组细胞的增殖率和凋亡率,QPCR和Western blotting检测各组转染48 h后SOX11 mRNA和蛋白水平,采用荧光素酶报告实验验证miR-145与SOX11的靶向调控关系。结果 HBL-100细胞的miR-145表达水平为1.047±0.026,高于MDA-MB-231细胞的0.218±0.019(P<0.05)。空白对照组、阴性对照组和过表达组的miR-145表达水平分别为1.022±0.034、0.987±0.028和3.408±0.097,过表达组的miR-145表达水平高于其余两组(P<0.05);过表达组转染48、72 h后的增殖率均低于空白对照组和阴性对照组(P<0.05)。空白对照组、阴性对照组和过表达组的凋亡率分别为(6.12±0.79)%、(7.12±0.82)%和(26.11±1.95)%,SOX11 mRNA水平分别为1.066±0.074、1.105±0.091和0.126±0.023,SOX11蛋白水平分别为0.524±0.048、0.492±0.056和0.173±0.038,过表达组的凋亡率高于其余两组,SOX11 mRNA和蛋白水平低于其余两组(P<0.05)。荧光素酶活性检测结果显示miR-145可抑制野生型SOX11 3’UTR报告基因载体的荧光素酶活性,而对突变型SOX11 3’UTR的荧光素酶活性无影响。结论 miR-145在乳腺癌细胞中表达降低,可与SOX11 3’UTR特异结合,调控乳腺癌细胞的增殖和凋亡,有可能成为乳腺癌新的生物治疗靶点。
Objective To explore the effect of microRNA-145（miR-145） on the target regulation of sex determining region Y-box 11（ SOX11） and the effect on the proliferation and apoptosis of breast cancer cells. Methods Real-time fluorescence quantitative PCR（ QPCR） was used to detect the expression level of miR-145 in normal mammary epithelial cells（ HBL-100） and breast cancer MDA-MB-231 cells. MDA-MB-231 cells in growth phase were transfected with miR-145 mimics（ overexpression group） or negative control plasmid NC（ negative control group） by liposome method,and the cells without transfection were used as blank control group.QPCR was used to detect the miR-145 level of each group at 48 h after transfection. CCK-8 and Annexin V-FITC/PI were used to detect the proliferative rate and apoptosis rate of each group. QPCR and Western blotting were used to detect mRNA and protein levels of SOX11 at 48 h after transfection. The targeting regulatory relationship between miR-145 and SOX11 was verified by the luciferase report test. Results The level of miR-145 in HBL-100 cells was 1. 047 ± 0. 026,higher than 0. 218 ± 0. 019 of MDA-MB-231 cells（ P〈0. 05）. The levels of miR-145 in blank control group,negative control group and overexpression group were 1. 022 ± 0. 034,0. 987± 0. 028 and 3. 408 ± 0. 097,and the miR-145 level in overexpression group was higher than those in the other two groups（ P〈0. 05）. The proliferative rates of the overexpression group were lower than those in the blank control group and the negative control group at 48 and 72 h after transfection（ P〈0. 05）. As for the blank control group,negative control group and overexpression group,apoptotic rates were（6. 12 ± 0. 79） %,（7. 12 ± 0. 82） % and（26. 11 ± 1. 95） %,SOX11 mRNA levels were 1. 066 ± 0. 074,1. 105± 0. 091 and 0. 126 ± 0. 023,and SOX11 protein levels were 0. 524 ± 0. 048,0. 492 ± 0. 056 and 0. 173 ± 0. 038. Compared with other groups,there were higher apoptotic rate and lower mRNA and protein levels of SOX11 in overexpression group（ P〈0. 05）. Luciferase activity assay showed that miR-145 inhibited the luciferase activity of wild-type SOX11 3＇UTR reporter vector,but had no effect on the luciferase activity of mutant SOX11 3＇UTR. Conclusion The decreased expression of miRNA-145 in breast cancer cells may regulate the proliferation and apoptosis of breast cancer cells and bind with SOX11 3＇UTR,possibly becoming a new target for breast cancer therapy.
作者
郭辉
张斌
胡利民
GUO Hui;ZHANG Bin;HU Limin(Department of General Surgery, the First People＇s Hospital of Tianmen, Tianmen 431700, China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2018年第3期200-205,共6页
Chinese Clinical Oncology
