miR-124靶向STAT3基因对子宫内膜癌细胞恶性生物学表型的影响

发布时间：2018-03-16 20:04

本文选题：子宫内膜癌　切入点：miR-124　出处：《南昌大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究目的：探究miR-124在子宫内膜癌中的表达及其对子宫内膜癌细胞恶性生物学表型的影响及其机制，为今后的内膜癌早期诊断及生物治疗提供实验依据。方法：我们首先利用stem-loop RT-PCR法检测miR-124在35对子宫内膜癌组织及其对应的瘤旁正常组织的表达；分别运用CCK8法、流式细胞技术、细胞划痕实验以及transwell实验，检测细胞增殖、周期、凋亡、迁移及侵袭的变化；通过进一步的生物信息学靶基因的预测miR-124的靶基因，并运用双萤光报告以及Western Blotting实验验证；进一步运用Rescue实验验证该靶基因在miR-124介导的抑癌功能中的作用。结果：miR-124在子宫内膜癌组织中的表达呈明显下调趋势,提示miR-124可能在子宫内膜癌发生发展中发挥抑癌基因的功能；恢复子宫内膜癌来源的HEC-1B细胞中miR-124表达能够明显抑制细胞的增殖、侵袭和迁移，抑制细胞周期进程，诱导细胞早期凋亡的发生；通过进一步的生物信息学靶基因的预测提示信号转导与转录激活因子(signal transducer and transcription activator3, STAT3)是miR-124的靶基因之一。双萤光报告以及Western Blotting实验证实，miR-124能够明显抑制HEC-1B细胞内STAT3基因的转录，，转染后HEC-1B细胞内STAT3的mRNA及蛋白水平均得到明显抑制；进一步敲低HEC-1B细胞中STAT3的表达与过表达miR-124结果一致，能够明显抑制HEC-1B细胞的恶性生物学表型，提示miR-124介导的抑癌功能可能是部分依赖于靶向调控STAT3通路产生的；同时运用IL-6处理后能够部分性的恢复细胞中STAT3的表达，以及减弱miR-124对HEC-1B细胞恶性表型的抑制作用，这一结果表明在子宫内膜癌细胞中，miR-124介导的抑癌功能是直接通过靶向STAT3产生的；结论：我们的结果进一步确定了miR-124在EC发生发展中的抑癌基因功能及其机制，同时为miR-124用于EC的诊断和治疗奠定了生物学基础。
[Abstract]:Objective: to investigate the expression of miR-124 in endometrial carcinoma and its effect on the malignant biological phenotype of endometrial cancer cells and its mechanism. Methods: stem-loop RT-PCR method was used to detect the expression of miR-124 in 35 pairs of endometrial carcinoma and its adjacent normal tissues, respectively, using CCK8 method and flow cytometry. Cell scratch assay and transwell assay were used to detect the changes of cell proliferation, cell cycle, apoptosis, migration and invasion. The target genes of miR-124 were predicted by further bioinformatics, and verified by double fluorescence report and Western Blotting experiment. Rescue assay was used to verify the role of the target gene in the tumor suppressor function mediated by miR-124. Results the expression of 10 miR-124 was down-regulated in endometrial carcinoma, suggesting that miR-124 might play a role as a tumor suppressor gene in the carcinogenesis and development of endometrial carcinoma. The expression of miR-124 in HEC-1B cells derived from endometrial carcinoma can significantly inhibit cell proliferation, invasion and migration, inhibit the progress of cell cycle and induce early apoptosis. The prediction of further bioinformatics target genes suggested that signal transducer and transcription activator 3 (STAT3) was one of the target genes of miR-124. Double fluorescence reports and Western Blotting experiments showed that the signal transduction and transcription activator 3 (STAT3) could significantly inhibit the transcription of STAT3 gene in HEC-1B cells. After transfection, the mRNA and protein levels of STAT3 in HEC-1B cells were significantly inhibited, and the expression of STAT3 in HEC-1B cells was consistent with the results of overexpression of miR-124, which could inhibit the malignant biological phenotype of HEC-1B cells. The results suggest that the inhibition function of miR-124 may be partly dependent on the targeting regulation of STAT3 pathway, at the same time, it can partially restore the expression of STAT3 in the cells treated with IL-6, and attenuate the inhibitory effect of miR-124 on the malignant phenotype of HEC-1B cells. These results suggest that the inhibitory effect of miR-124 is directly mediated by targeted STAT3 in endometrial cancer cells. Conclusion: our results further confirm the function and mechanism of tumor suppressor gene of miR-124 in the development of EC and lay a biological foundation for the diagnosis and treatment of EC by miR-124.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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