miR-199b-5p在乳腺癌中的研究

发布时间：2018-07-12 12:43

本文选题：miR-199b-5p + 乳腺癌　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]本研究通过检测miR-199b-5p在乳腺癌患者组织(包括癌、癌旁及正常组织)及血浆外泌体中的表达情况,探讨其临床意义及相关性;同时,我们选用三阴性乳腺癌细胞株HCC1937(ER、PR及HER-2均阴性)及MCF-7 (激素ER阳性、PR及HER-2阴性)作为实验对象,通过mimic在这两株细胞中过表达miR-199b-5p,观察 miR-199b-5p 对乳腺癌 HCC1937、MCF-7 细胞增殖、迁移、侵袭及凋亡的影响;最后,探讨miR-199b-5p与DDR1的相互关系,为临床治疗乳腺癌提供新的标志和潜在重要靶点奠定基础。[方法]1、以miR-199b-5p为作用靶点合成miRNAmimic并用脂质体法转染HCC1937、MCF-7细胞,同时设NC作为阴性对照组。2、通过荧光及qRT-PCR筛选稳定细胞转染株。3、用细胞划痕的方法检测过表达miR-199b-5p对乳腺癌细胞HCC1937、MCF-7迁移能力的影响。4、用Transwell小室检测过表达miR-199b-5p对HCC1937、MCF-7细胞迁移及侵袭能力的影响。5、用CCK8法检测过表达miR-199b-5p对HCC1937、MCF-7细胞增殖能力的影响。6、用流式细胞术分析过表达miR-199b-5p对HCC1937、MCF-7细胞凋亡率的影响。7、用 western blotting 检测过表达 miR-199b-5p 后对 HCC1937、MCF-7 细胞中DDR1蛋白表达的影响。8、用试剂盒提取组织及外周血外泌体中的miRNA,并用qRT-PCR方法检测miR-199b-5p在乳腺癌组织中及外周血外泌体中的表达情况。[结果]1、 qRT-PCR 检测显示 miR-199b-5p mimic 能明显提高 HCC1937、MCF-7乳腺癌细胞株中miR-199b-5p表达水平(p0.05);2、 CCK8 检测显示 miR-199b-5pmimic 转染 HCC1937、MCF-7 细胞后细胞增殖受到显著抑制(p0.05);3、细胞划痕实验显示经miR-199b-5p mimic转染HCC1937细胞后迁移速度明显减慢(p 0.05);4、流式细胞术检测结果显示miR-199b-5p mimic转染HCC1937、MCF-7细胞后凋亡率显著增加(p 0.05);5、通过qRT-PCR检测乳腺癌患者癌组织、癌旁组织及正常组织发现,相对于正常组织,乳腺癌组织中miR-199b-5p表达显著下调(p 0.05)6、 Western blotting 结果显示:经 miR-199b-5p mimic 转染后,DDR1 蛋白表达均降低(p0.05)。[结论]1、过表达miR-199b-5p可明显抑制乳腺癌细胞株HCC1937、MCF-7的增殖,减低其迁移速度,促进乳腺癌细胞HCC1937、MCF-7的凋亡。2、在HCC1937、MCF-7乳腺癌细胞株中过表达miR-199b-5p使DDR1表达明显降低。3、相对于正常组织,乳腺癌组织中miR-199b-5p表达显著下调。4、乳腺良恶性肿瘤患者外周血外泌体中miR-199b-5p表达无明显差异。
[Abstract]:[objective] to investigate the clinical significance and correlation of miR-199b-5p expression in breast cancer tissues (including cancer, cancer and normal tissues) and plasma exocrine. Three negative breast cancer cell lines HCC1937 (ER-PR and HER-2 negative) and MCF-7 (hormone ER positive PR and HER-2 negative) were selected as the experimental subjects. The expression of miR-199b-5pin HCC1937 and HER-2 negative breast cancer cell lines was observed by mimic, and the proliferation and migration of MCF-7 cells were observed by miR-199b-5p. Finally, to explore the relationship between miR-199b-5p and DDR1, to provide a new marker and potential important target for clinical treatment of breast cancer. [methods] 1. Using miR-199b-5p as target, miRNAmimic was synthesized and transfected into HCC1937 MCF-7 cells by liposome method. At the same time, NC was used as negative control group. The stable cell transfection cell line. 3 was screened by fluorescence and qRT-PCR. The effect of overexpression of miR-199b-5p on the migration ability of HCC1937 MCF-7 was detected by cell scratch method. The expression of miR-199b-5p in HCC1937 MCF-7 was detected by Transwell chamber. Effect of cell migration and invasion. The effect of overexpression of miR-199b-5p on the proliferation of HCC1937 MCF-7 cells was detected by CCK8 method. Flow cytometry was used to analyze the effect of overexpression of miR-199b-5p on apoptosis rate of HCC1937 MCF-7 cells. Western blotting was used to detect the expression of miR-199b-5p on the apoptosis rate of MCF-7 cells. Effects of DDR1 protein expression on HCC1937 / MCF-7 cells. MiRNAs were extracted from tissues and peripheral blood secretory bodies with kit, and the expression of miR-199b-5p in breast cancer tissue and peripheral blood exocrine was detected by qRT-PCR. [results] 1. The results of qRT-PCR showed that miR-199b-5p mimic could significantly increase the expression level of miR-199b-5p in HCC1937 MCF-7 breast cancer cell line (p0.05). CCK8 assay showed that miR-199b-5pmimic transfection of HCC1977 MCF-7 cells was significantly inhibited (p0.05), and the cell scratch assay showed that miR-199b-5p mimic was transfected into HCC1937 MCF-7 cell line. The migration rate of HCC1937 cells decreased significantly (p0.05). The results of flow cytometry showed that the apoptosis rate of HCC1937 MCF-7 cells transfected with miR-199b-5p mimic was significantly increased (p0.05). The cancer tissues of breast cancer patients were detected by qRT-PCR. The expression of miR-199b-5p was significantly down-regulated (p0.05) in paracancerous tissues and normal tissues. Western blotting showed that the expression of miR-199b-5p mimic was decreased after transfection (p0.05). [conclusion] 1. Overexpression of miR-199b-5p could significantly inhibit the proliferation of MCF-7 cell line HCC1937, decrease its migration speed, and promote apoptosis of MCF-7 cell line HCC1937. Overexpression of miR-199b-5p in HCC1937 / MCF-7 breast cancer cell line significantly decreased DDR1 expression. The expression of miR-199b-5p was significantly down-regulated in breast cancer, but there was no significant difference in the expression of miR-199b-5p in peripheral blood of patients with benign and malignant breast cancer.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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