乳腺癌耐药细胞中miR-217、miR-375的功能研究

发布时间：2018-03-16 06:08

本文选题：miR-375　切入点：miR-217　出处：《广东药科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过高通量测序分析,筛选出人乳腺癌耐阿霉素细胞与野生型的乳腺癌细胞中差异表达的miRNAs,研究乳腺癌耐阿霉素细胞中低表达的miRNA-375和高表达的miRNA-217对人乳腺癌耐阿霉素细胞增殖以及侵袭、迁移能力的影响。方法:通过WST-1实验先验证得到的乳腺癌耐药细胞是否为稳定的耐药细胞。通过对野生型乳腺癌细胞和耐阿霉素的乳腺癌细胞的样品进行Small RNA高通量测序分析,获取正常乳腺癌细胞和耐阿霉素的乳腺癌细胞差异表达miRNA信息;选择一些差异表达的miRNA,用Real-time RT-PCR实验对筛选出差异表达显著的mi RNA进行验证。将miR-217 inhibitor和inhibitor NC、miR-375模拟物(miR-375 mimics)和mimics NC分别转染MCF-7/Adr;阿霉素处理后用WST-1法检测耐阿霉素细胞的增殖情况;流式细胞仪检测MCF-7/Adr细胞周期和凋亡;细胞划痕和transwell迁移、侵袭实验检测MCF-7/Adr细胞的迁移和侵袭能力。结果:1、通过WST-1实验验证得到的乳腺癌耐药细胞为稳定的耐药细胞,对不同药物浓度的阿霉素,耐药的乳腺癌细胞比野生型的乳腺癌细胞有更明显的耐受性。2、高通量测序结果显示:与配对的野生型的乳腺癌细胞相比,有多个miRNAs在耐阿霉素的乳腺癌细胞中表达异常。3、Real-time RT-PCR结果显示:miR-375在耐阿霉素的乳腺癌细胞中表达显著降低,miR-217在耐阿霉素的乳腺癌细胞中表达显著提高。4、WST-1实验显示在乳腺癌耐药(MCF-7/adr)细胞中分别转染高表达的miR-217、阴性对照组inhibitor NC和低表达的miR-375、模拟物对照组mimics NC,发现表达72h后,与对照组相比转染小分子的乳腺癌耐药细胞的生长速度均明显减慢。5、流式细胞仪测细胞周期、凋亡结果显示:上调miR-375和下调mi R-217的表达后,可以提高MCF-7/Adr细胞对阿霉素的敏感性并促进其凋亡;可以将MCF-7/Adr细胞周期阻滞在G1期。6、Tranwell迁移实验结果显示:上调miR-375和下调miR-217的表达均可以使乳腺癌耐阿霉素细胞的迁移能力降低。7、Tranwell侵袭实验结果显示:上调miR-375和下调miR-217的表达均可以使乳腺癌耐阿霉素细胞的侵袭能力降低。8、细胞划痕结果显示:上调miR-375和下调miR-217的表达会使MCF-7/Adr细胞的迁移能力降低。结论:1、miR-217在乳腺癌耐阿霉素细胞中表达水平显著高于野生型的乳腺癌细胞;miR-375在乳腺癌耐阿霉素细胞中表达水平显著低于野生型的乳腺癌细胞。2、上调乳腺癌耐阿霉素细胞中miR-375的表达,下调乳腺癌耐阿霉素细胞中miR-217的表达水平,均可以增加MCF-7/Adr细胞对阿霉素的敏感性。
[Abstract]:Objective: to analyze by high throughput sequencing. The differentially expressed miRNAss in human breast cancer doxorubicin resistant cells and wild type breast cancer cells were screened. The effects of low expression miRNA-375 and high expression miRNA-217 on the proliferation and invasion of human breast cancer doxorubicin resistant cells were studied. Methods: WST-1 assay was used to determine whether the breast cancer cells were stable drug resistant cells. Small RNA high throughput sequencing was performed on the samples of wild type breast cancer cells and adriamycin resistant breast cancer cells. The differentially expressed miRNA information was obtained between normal breast cancer cells and adriamycin resistant breast cancer cells. Some differentially expressed miRNAs were selected, and the differentially expressed miRNA was screened by Real-time RT-PCR assay. The miR-217 inhibitor and inhibitor NCncfmiR-375 mimicswere transfected into MCF-7 / Adr.After treated with adriamycin, the proliferation of adriamycin-resistant cells was detected by WST-1 method. Flow cytometry was used to detect the cell cycle and apoptosis of MCF-7/Adr cells, cell scratch and transwell migration, invasion assay to detect the migration and invasion ability of MCF-7/Adr cells. For different concentrations of adriamycin, drug-resistant breast cancer cells were significantly more tolerant than wild type breast cancer cells. High-throughput sequencing results showed that: compared with matched wild type breast cancer cells, Abnormal expression of miRNAs in adriamycin-resistant breast cancer cells. 3 Real-time RT-PCR results showed that the expression of 1 miR-375 in adriamycin resistant breast cancer cells decreased significantly, and the expression of mmiR-217 in adriamycin-resistant breast cancer cells increased significantly. Breast cancer MCF-7 / adr cells were transfected with high expression miR-217, negative control group inhibitor NC and low expression miR-375, and mimic control group mimics NC2 h. Compared with the control group, the growth rate of drug-resistant breast cancer cells transfected with small molecules was significantly slower than that of the control group. Flow cytometry was used to measure the cell cycle and apoptosis showed that the expression of miR-375 was up-regulated and the expression of miR-217 was down-regulated. It can increase the sensitivity of MCF-7/Adr cells to adriamycin and promote its apoptosis. The results of tranwell migration assay showed that up-regulation of miR-375 and down-regulation of miR-217 could decrease the migration ability of adriamycin-resistant breast cancer cells. The results showed that both up-regulation of miR-375 and down-regulation of miR-217 were observed. The results of cell scratch showed that up-regulation of miR-375 and down-regulation of miR-217 expression could decrease the migration ability of MCF-7/Adr cells. Conclusion\% 1 + miR-217 can reduce the migration ability of MCF-7/Adr cells in adriamycin-resistant breast cancer cells. The expression level of miR-375 was significantly higher than that of wild type breast cancer cells, and the expression level of miR-375 was significantly lower than that of wild type breast cancer cells. The expression of miR-375 was up-regulated in adriamycin resistant breast cancer cells. Down-regulation of miR-217 expression in adriamycin resistant breast cancer cells increased the sensitivity of MCF-7/Adr cells to adriamycin.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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