【作者】 王婷；

【导师】 黄立中；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 目的筛选受黄芩苷调控的差异miRNA,从miRNA角度探讨黄芩苷对乳腺癌的抑制可能的途径和机制。方法体外实验首选通过不同浓度黄芩苷作用于乳腺癌细胞MCF-7、MDA-MB-231细胞,免疫组化分析筛选出药物最低有效作用浓度,运用miRNA芯片筛选受药物调控的差异miRNA, qRT-PCR对芯片结果中上调的miRNA进行验证,再通过MTT法检测细胞生存率、Transwell实验检测细胞侵袭与转移、蛋白印迹技术检测凋亡相关蛋白表达变化及流式细胞术检测细胞凋亡情况。临床实验检验癌组织和正常组织细胞因子免疫组化差异及qRT-PCR检测miRNA表达的差异。结果体外实验1)黄芩苷作用于乳腺癌细胞后转化生长因子β (transforming growth factor-β, TGF-β)、血管内皮生长因子(vascular endothelial growth factors VEGF)免疫组化评分下降,最佳作用浓度为50μmol/L；2)黄芩苷作用于乳腺癌细胞,芯片筛选出miR-126表达上调最明显；3)黄芩苷和miR-126作用于乳腺癌细胞,细胞生长率和侵袭力显著降低；4)黄芩苷和miR-126作用后乳腺癌细胞中半胱氨酸天冬氨酸酶-3(Cysteinyl aspartate-specific proteinase-3, Caspase-3)、半胱氨酸天冬氨酸酶-9(Cysteinyl aspartate-specific proteinase-9, Caspase-9)、Bcl-2相关X蛋白(Bcl-2Associated X Protein, Bax)和p53基因的表达增加而B细胞淋巴瘤基因-2(B cell lymphoma2,Bcl-2)表达下降,同时丝裂原活化蛋白激酶(mitogen-activated protein kinase,p-38MAPK)活化亦增加；5)黄芩苷和miR-126作用后乳腺癌细胞凋亡增加。临床实验1)乳腺癌患者组织中TGF-β、VEGF的免疫组化评分与正常乳腺腺体组织相比,明显升高；2)乳腺癌组织中miR-126的相对表达较正常乳腺组织明显降低。结论1)乳腺癌细胞中miR-126表达下调；2)黄芩苷可上调miR-126等抑癌作用的miRNA基因；3)黄芩苷通过上调miR-126的表达抑制乳腺癌细胞的增殖和侵袭,并可能抑制癌细胞的转移。更多还原

【Abstract】 Objective Screen differential miRNA which is regulated by the baicalin, from the perspective of miRNA,to explore the approaches and mechanism of the inhibitory effect of Baicalin on breast cancer.Methods In vitro experiments effect on breast cancer cell MCF-7、 MDA-MB-231by different concentrations of baicalin, Immunohistochemical analysis showed that the lowest effective drug concentration, by using miRNA chip,screening differentially miRNA by drug regulation. qRT-PCR for upregulated miRNA microarray results validate, and then detect cell survival by MTT assay, detection of cell invasion and metastasis by transwell experiment, detection of apoptosis-related protein expression by western blot and flow cytometry to detect apoptosis.clinical trials testing cancer tissue and normal tissue cytokine’s immunohistochemistry difference and qRT-PCR to detect differences in miRNA expression.ResultsIn vitro experiments1) After baicalin effect on breast cancer cell, TGF-β(transform growth factorp), VEGF(vascular endothelial growth factors), immunohistochemical score decreased, the best concentration was50μmol/l;2) baicalin effect on breast cancer cell, microarray screens miR-126expression increased most;3) baicalin and miR-126effect on breast cancer cell, the rate of cell growth and invasiveness was significantly lower;4) baicalin and miR-126effect on breast cancer cell,the expression of Caspase-3(Cysteinyl aspartate-specific proteinase-3), Caspase-9(Cysteinyl aspartate-specific proteinase-9), Bax (Bcl-2Associated X Protein) and P53increased and the expression of Bcl-2(B cell lymphoma2) decrease, at the same time P-38(mitogen-activated protein kinase) activation also increased;5) baicalin and miR-126effect on breast cancer cell, apoptosis of breast cancer cells increased;Clinical trials1) compared with normal mammary gland tissue, immunohisto-chemical score of TGF-β, VEGF significantly increased in breast tissue;2) The relative expression of miR-126in breast cancer tissue was significantly lower than normal breast tissue;Conclusion1) Expression of miR-126in breast cancer cells decrease;2) Baicalin upregulates tumor suppressor miRNA genes such as miR-126;3) By upregulating the expression of miR-126, baicalin inhibits the proliferation and invasion, probably inhibit metastasis of breast cancer cells.更多还原