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目的:探讨基于催乳素(prolactin,PRL)反应的人乳腺癌细胞microRNA(miRNA)表达谱变化,以了解其在乳腺癌发生和发展中的潜在作用。方法:人重组PRL处理高表达催乳素受体(PRLR)的人乳腺癌T-47D细胞系,MTT法检测细胞增殖能力变化,流式细胞术检测细胞周期和凋亡变化情况。利用Solexa高通量测序技术检测PRL处理组和未处理组的T-47D细胞miRNAs表达谱,荧光定量PCR法进行初步验证,同时进行相关的生物信息学分析。结果:PRL作用于T-47D细胞后,MTT结果显示细胞增殖能力增强,细胞周期检测结果表明PRL处理后,G1期的细胞比例减少,S期的细胞比例升高,而G2/M期没有变化。细胞凋亡分析结果则显示PRL可抑制细胞凋亡,导致细胞凋亡率下降。通过Solexa高通量测序技术成功检测了PRL处理和未处理组的人乳腺癌T-47D细胞miRNA表达谱,两组中分别检测到821个和798个miRNAs成熟体,其中428个共表达,42个为两组间明显差异表达。选取的4个miRNAs分子经荧光定量PCR法证实其细胞内的表达与测序结果一致。两组miRNA表达谱中同时也检测到86个和115个novel miRNAs成熟体,其中46个novel miRNAs在两组细胞中共表达。结论:在初期实验明确PRL对人乳腺癌T-47D细胞产生促增殖作用的基础上,利用Solexa高通量测序技术检测两类T-47D细胞miRNAs表达谱,获得了一系列与PRLR信号通路高度相关的miRNAs分子,为进一步研究miRNAs分子在人乳腺癌发生发展中的作用奠定重要的研究基础。
Objective: To investigate the changes of microRNA (miRNA) expression profile of human breast cancer cells based on prolactin (PRL) response in order to understand its potential role in the development and progression of breast cancer. METHODS: Human breast cancer T-47D cell line with high prolactin receptor (PRLR) expression was treated with recombinant human PRL. Cell proliferation was assessed by MTT assay, and changes in cell cycle and apoptosis were assessed by flow cytometry. The Solexa high-throughput sequencing technology was used to detect the miRNAs expression profile of T-47D cells in PRL-treated and untreated groups. The fluorescence quantitative PCR method was used for preliminary verification and related bioinformatics analysis was performed. RESULTS: After PRL acted on T-47D cells, the MTT results showed that the cell proliferation was enhanced. The cell cycle test results showed that after PRL treatment, the proportion of cells in G1 phase decreased, the proportion of cells in S phase increased, but there was no change in G2/M phase. . Apoptosis analysis showed that PRL can inhibit apoptosis, leading to decreased apoptosis rate. The miRNA expression profiles of human breast cancer T-47D cells in PRL-treated and untreated groups were successfully detected by Solexa high-throughput sequencing technology. 821 and 798 mature miRNAs were detected in the two groups, of which 428 were co-expressed,42 Two were significantly different between the two groups. The four selected miRNAs were verified by fluorescent quantitative PCR to confirm their intracellular expression and sequencing results. In the miRNA expression profiles of both groups, 86 and 115 novel mature miRNAs were also detected, of which 46 novel miRNAs were co-expressed in the two groups of cells. CONCLUSIONS: Based on the initial experiments to clarify the pro-proliferative effects of PRL on human breast cancer T-47D cells, Solexa high-throughput sequencing technology was used to detect the expression profiles of miRNAs in two types of T-47D cells, and a series of signals with the PRLR signal pathway were obtained. Related miRNAs lay an important foundation for the further study of the role of miRNAs in the development of human breast cancer.