Let-7c-5p在乳腺癌中的表达、功能及其分子机制的研究

发布时间：2018-05-04 13:55

本文选题：乳腺癌 + Let-7c-5p　；参考：《浙江理工大学》2017年硕士论文

【摘要】：乳腺癌是女性最普遍的恶性疾病之一,它的高发生率及死亡率严重威胁女性身心健康。随着精准医疗的提出,乳腺癌的治疗方法将更加多样化,治疗效果也将更加令人满意。在精准医疗中基因工程将发挥重要作用,所以探究基因在乳腺癌中扮演的角色以及其发挥作用的分子机制能够为精准医疗奠定理论基础。Let-7c-5p是抑癌基因let-7家族中的一员,它在前列腺癌、非小细胞肺癌以及结肠癌中都起着一定的抑癌作用,在乳腺癌患者血清中检测到其表达量呈明显下降趋势,但是let-7c-5p在乳腺癌中的生物学功能尚未明了。本研究选取9对乳腺癌以及相对应癌旁组织的石蜡包埋样本为实验对象,提取其总RNA后,进行RT-qPCR检测let-7c-5p在乳腺癌及癌旁组织中的表达情况;同时在乳腺癌细胞MCF-7中过表达let-7c-5p,采用MTT的方法测试let-7c-5p对MCF-7细胞增殖能力的影响,利用流式细胞仪探测let-7c-5p对MCF-7细胞凋亡的影响;然后利用生物信息学软件预测let-7c-5p潜在的靶标基因,并用双荧光素酶实验加以验证,再采用荧光定量PCR和Western blotting实验深入探索其调控方式。结果显示let-7c-5p在9例乳腺癌组织中的表达量均低于对应的癌旁组织,其在癌组织中的表达量约为癌旁组织的0.23。对乳腺癌细胞MCF-7转染let-7c-5p mimics后,细胞中let-7c-5p表达量较对照组显著升高(P0.01),转染后细胞的增殖能力受到明显抑制(P0.01),且抑制效果与let-7c-5p mimics终浓度呈正相关;流式细胞仪检测结果表明过表达let-7c-5p后细胞凋亡率明显上升(P0.01)。生物信息学软件预测ERCC6为let-7c-5p潜在的靶标基因,双荧光素酶实验、Western blotting和荧光定量PCR结果表明let-7c-5p能够结合至ERCC6 mRNA的3’UTR,从而抑制ERCC6蛋白的合成,但是ERCC6 mRNA的表达水平并不受let-7c-5p影响。综上,本研究结果表明let-7c-5p在乳腺癌组织中呈低表达,在乳腺癌细胞中过表达let-7c-5p能明显抑制细胞增殖,并且引发细胞凋亡,ERCC6为let-7c-5p的靶基因,let-7c-5p可能通过抑制ERCC6蛋白表达发挥其抑癌作用。
[Abstract]:Breast cancer is one of the most common malignant diseases in women. Its high incidence and mortality seriously threaten women's physical and mental health. With the development of precise medicine, the treatment of breast cancer will be more diversified and the therapeutic effect will be more satisfactory. Genetic engineering will play an important role in precision medicine, so exploring the role of genes in breast cancer and its molecular mechanisms could lay the theoretical foundation for precision medicine. Let-7c-5p is a member of the let-7 family of tumor suppressor genes. It plays a certain role in the inhibition of prostate cancer, non-small cell lung cancer and colon cancer. The expression of let-7c-5p in serum of breast cancer patients shows a downward trend, but the biological function of let-7c-5p in breast cancer is not clear. In this study, 9 pairs of paraffin embedded samples of breast cancer and corresponding adjacent tissues were selected as experimental objects. The total RNA was extracted and the expression of let-7c-5p in breast cancer and adjacent tissues was detected by RT-qPCR. At the same time, let-7c-5p was overexpressed in MCF-7 of breast cancer cells. The effect of let-7c-5p on the proliferation of MCF-7 cells was tested by MTT method, the effect of let-7c-5p on MCF-7 cell apoptosis was detected by flow cytometry, and the potential target gene of let-7c-5p was predicted by bioinformatics software. The double luciferase experiment was used to verify it, and then fluorescence quantitative PCR and Western blotting experiments were used to explore its regulation mode. The results showed that the expression of let-7c-5p in 9 breast cancer tissues was lower than that in the corresponding paracancerous tissues, and the expression level in the cancer tissues was about 0.23 of that in the paracancerous tissues. The expression of let-7c-5p in breast cancer cells transfected with let-7c-5p mimics by MCF-7 was significantly higher than that in the control group (P 0.01). The proliferation ability of breast cancer cells was significantly inhibited after transfection, and the inhibitory effect was positively correlated with the final concentration of let-7c-5p mimics. The results of flow cytometry showed that the rate of apoptosis increased significantly after overexpression of let-7c-5p. The bioinformatics software predicted that ERCC6 was a potential target gene for let-7c-5p. The results of double luciferase assay and fluorescent quantitative PCR showed that let-7c-5p could bind to the 3- UTRs of ERCC6 mRNA, thereby inhibiting the synthesis of ERCC6 protein. However, the expression of ERCC6 mRNA was not affected by let-7c-5p. In conclusion, the results showed that the expression of let-7c-5p in breast cancer tissues was low, and overexpression of let-7c-5p in breast cancer cells could significantly inhibit the proliferation of breast cancer cells. ERCC6, a target gene of let-7c-5p, may play an inhibitory role by inhibiting the expression of ERCC6 protein.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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