E3泛素连接酶DTL促进乳腺癌细胞增殖的研究

发布时间：2018-01-28 23:27

本文关键词： E3泛素连接酶DTL 乳腺癌细胞周期细胞增殖　出处：《浙江大学》2016年硕士论文　论文类型：学位论文

【摘要】：乳腺癌是世界范围内严重威胁女性健康,最常见并且致死率很高的肿瘤。尽管乳腺癌的治疗疗效不断提高,但仍有相当一部分患者复发或对常规治疗耐药。进一步筛选和发现乳腺癌治疗的新靶标,可能为乳腺癌的诊治提供新的思路。我们对乳腺癌细胞系基因表达谱芯片结果进行分析后,筛选出与乳腺癌生长相关的一个E3泛素连接酶DTL (denticleless E3 ubiquitin protein ligase),并对其在乳腺癌细胞中的表达和功能进行了初步研究。为了探究DTL在乳腺癌细胞中可能发挥的调控作用,我们首先通过荧光定量PCR (Q-PCR)和Western Blot的方法确定了DTL在几种乳腺癌细胞系中的表达情况,发现其在乳腺癌细胞中的表达较正常细胞高。用免疫荧光与激光共聚焦显微镜观察了DTL细胞亚定位情况,DTL定位于细胞质和细胞核。然后我们利用RNA干扰技术在MCF-7, MDA-MB-231中下调DTL的表达,通过MTT比色法和Brdu标记法检测细胞增殖情况,发现干扰DTL可以明显抑制乳腺癌细胞MCF-7,MDA-MB-231的增殖。因此我们推测DTL可能是通过影响细胞周期来发挥其调控作用的。接着我们在MCF-7, MDA-MB-231这两种细胞中转染siRNA-DTL后利用流式细胞术观察细胞周期的变化,发现干扰DTL后使得乳腺癌细胞周期G1期比例减少,G2/M期细胞比例增多。接着,我们通过RT-qPCR检测了几种常见的细胞周期蛋白、蛋白激酶以及蛋白激酶抑制因子的表达情况,发现在MCF-7中干扰DTL后可显著促进细胞周期蛋白激酶抑制因子P21 mRNA的表达, Western Blot提示干扰DTL后P21的蛋白水平也增加。说明DTL对细胞周期的作用可能通过调节P21的表达来发挥作用,但具体如何发挥作用,是否通过泛素相关的途径还是其他机制还有待于进一步研究。但在MDA-MB-231中siRNA-DTL后P21的mRNA和蛋白质水平没有变化,可能在不同的细胞中DTL的作用机制不同,有待于后续进一步验证。综上,在本研究中,我们发现DTL在各种乳腺癌细胞中高表达,干扰DTL可以抑制乳腺癌细胞增殖,使细胞周期阻滞在G2/M期。DTL可能通过抑制P21的表达,促进MCF-7细胞的增殖,有可能作为乳腺癌的潜在治疗靶点。
[Abstract]:Breast cancer is a worldwide threat to women's health, the most common and with a high mortality rate, despite the increasing therapeutic efficacy of breast cancer. However, a considerable number of patients still recur or are resistant to conventional therapy. New targets for breast cancer therapy are further screened and identified. It may provide a new idea for the diagnosis and treatment of breast cancer. We analyze the results of gene expression microarray of breast cancer cell line. An E3-ubiquitin ligase DTL denticleless E3 ubiquitin protein associated with breast cancer growth was screened. In order to explore the possible regulatory role of DTL in breast cancer cells, the expression and function of DTL in breast cancer cells were studied. We first determined the expression of DTL in several breast cancer cell lines by fluorescence quantitative PCR Q-PCR and Western Blot. It was found that its expression in breast cancer cells was higher than that in normal cells. The sublocalization of DTL cells was observed by immunofluorescence and confocal laser microscopy. DTL was located in cytoplasm and nucleus. Then we down-regulated the expression of DTL in MCF-7 and MDA-MB-231 by RNA interference technique. The proliferation of breast cancer cells was detected by MTT colorimetry and Brdu labeling. It was found that interfering with DTL could significantly inhibit MCF-7 of breast cancer cells. The proliferation of MDA-MB-231. Therefore, we speculate that DTL may play a regulatory role by affecting cell cycle. We then work in MCF-7. The changes of cell cycle were observed by flow cytometry after transfection of siRNA-DTL into MDA-MB-231 cells. It was found that interfering with DTL reduced the proportion of G _ 2 / M phase cells in breast cancer cell cycle. Then, we detected several common cell cycle proteins by RT-qPCR. The expression of protein kinase and protein kinase inhibitor showed that interfering with DTL in MCF-7 could significantly promote the expression of cyclin kinase inhibitor P21 mRNA. Western Blot suggested that the protein level of P21 increased after interfering with DTL, suggesting that the effect of DTL on cell cycle might be mediated by regulating the expression of P21. But how it works. Whether the mechanism is related to ubiquitin or not remains to be further studied, but the mRNA and protein levels of P21 do not change after siRNA-DTL in MDA-MB-231. It is possible that the mechanism of DTL in different cells is different, which needs to be further verified. In summary, we found that DTL is highly expressed in various breast cancer cells in this study. Interfering with DTL can inhibit the proliferation of breast cancer cells and arrest the cell cycle in G _ 2 / M phase. DTL may promote the proliferation of MCF-7 cells by inhibiting the expression of P21. It may be a potential therapeutic target for breast cancer.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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