增殖细胞核抗原胞质表达对乳腺癌细胞自噬调控作用的研究

发布时间：2018-07-02 20:05

本文选题：乳腺癌 + 增殖细胞核抗原　；参考：《吉林大学》2015年博士论文

【摘要】：增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）是一种独特的核内蛋白质。在细胞核内，三个PCNA分子首尾相连构成围绕DNA双螺旋的滑动环状结构，借此招募DNA聚合酶和多种不同功能蛋白质与其结合，在DNA合成和损伤修复、细胞周期调控中发挥关键作用，被视为细胞增殖指标。不同组织学来源的肿瘤细胞均在核内表达高水平PCNA，表明其增殖的不可控性，核内PCNA由此成为肿瘤诊断和不良预后的分子标志，也成为肿瘤治疗新靶标。然而有研究显示PCNA不仅表达于细胞核内，也在胞质中表达，并且能够和胞质内多种蛋白质相互作用，表明其具有更广泛的生物学活性。在某些肿瘤细胞系中，PCNA部分在胞质表达并和多种肿瘤相关蛋白结合，提示胞质PCNA可能参与了肿瘤的某些病理过程，但尚缺乏相关研究证据。既往有学者在用雷帕霉素和饥饿方法分别处理急性T淋巴细胞白血病细胞和小鼠胚胎成纤维细胞时发现，这两种方法可以改变PCNA的细胞内定位及蛋白表达水平。由于雷帕霉素及营养缺乏是最常见的细胞自噬诱导剂，尤其在肿瘤细胞中，因此我们推测PCNA在细胞内的重新定位可能与细胞自噬活性相关。自噬作为细胞内长效蛋白质降解途径已被证实参与肿瘤的疾病进程。首先，自噬可以抑制发生恶性转化的细胞增殖，由此促进细胞衰老而抑制肿瘤产生。一旦肿瘤形成，肿瘤细胞则增强自噬以求在代谢和治疗应激中存活，此时自噬成为避免肿瘤细胞损伤的保护机制。如果应激持续存在，自噬被过度激发，则可介导肿瘤细胞进入非凋亡性细胞死亡模式，同时释放炎症因子，调动机体的肿瘤相关免疫活性。自噬在肿瘤发病机制中的这种复杂作用使其成为目前肿瘤研究的焦点。乳腺癌作为威胁世界女性健康的第二大肿瘤，始终受到极大关注，近期研究显示自噬参与了乳腺癌的发生、进展和转移、治疗耐受性产生等病理过程，但其作用及调控机制尚未完全阐明。深入研究自噬在乳腺癌中的功能及调控机制，可以准确调节乳腺癌细胞自噬水平，从而促进肿瘤治疗。目前已知肿瘤细胞的自噬过程可由多种蛋白及信号通路调节，除自噬相关蛋白及PI3K/Akt/mTOR和AMPK信号通路外，近期研究发现在各种肿瘤尤其乳腺癌中异常表达的microRNA及p53等均可改变肿瘤细胞自噬活性，提示可能有更多分子或蛋白参与了肿瘤细胞的自噬调节过程。根据前述研究，我们推测在某种条件下PCNA可能在乳腺癌细胞的胞质表达并涉及细胞自噬活性调节。为证实该假设，在本实验中我们选择人乳腺癌SK-BR-3细胞系作为靶细胞，首先证实乳腺癌细胞胞质中存在PCNA表达，继而采用不同自噬诱导方法处理细胞，验证PCNA在乳腺癌细胞胞质内定位表达与自噬活性相关；在此基础上用不同方法改变PCNA胞质表达水平，确定PCNA的胞质表达具有调节乳腺癌细胞自噬活性的功能，为在乳腺癌细胞中准确调控自噬提供新的研究方向。研究方法： 1．PCNA的亚细胞定位分析：雷帕霉素处理乳腺癌SK-BR-3细胞系，Western blot及免疫荧光检测PCNA在胞质中表达状态；同时用雷帕霉素处理子宫颈癌HeLa细胞系及肺癌A549细胞系，相同实验分析PCNA在这两种细胞中定位改变。 2．雷帕霉素诱导乳腺癌SK-BR-3细胞自噬活性：雷帕霉素处理细胞不同时间，单丹磺酰尸胺（monodansylcadaverin, MDC）染色对细胞进行自噬活性分析，Western blot分析Beclin1蛋白表达水平。 3．其他自噬诱导方法对乳腺癌SK-BR-3细胞自噬水平及胞质PCNA表达的影响：分别用过氧化氢模拟氧化应激及EBSS缓冲液制造饥饿应激方式诱导细胞自噬，MDC染色确定细胞自噬水平，Western blot分析胞质中PCNA及Beclin1蛋白表达水平。 4．胞质PCNA表达与乳腺癌SK-BR-3细胞自噬水平的相关性：用RNAi或出核转运抑制剂leptomycin B改变PCNA在SK-BR-3细胞中的表达及亚细胞定位，MDC染色分析细胞自噬，Western blot检测Beclin1表达水平。 5．自噬抑制对乳腺癌SK-BR-3细胞PCNA亚细胞定位的影响：自噬抑制剂3-甲基腺嘌呤（3-methyladenine,3-MA）预处理细胞后雷帕霉素诱导细胞自噬，MDC染色分析细胞自噬水平，Western blot分析检胞质中PCNA蛋白表达。研究结果： 1．随雷帕霉素对SK-BR-3细胞作用时间延长，PCNA在胞质中表达水平呈先升高后降低改变；HeLa细胞及A549细胞在雷帕霉素处理时也可见PCNA胞核向胞质转运，证实PCNA在不同肿瘤细胞接受外界刺激后均可在胞质重新定位表达。 2．雷帕霉素作用于SK-BR-3细胞后自噬活性呈先增强后减弱改变，自噬相关蛋白Beclin1表达先增高后降低，与胞质PCNA表达变化趋势一致。 3．饥饿和氧化应激诱导SK-BR-3细胞，，胞质PCNA、细胞自噬活性及自噬相关蛋白Beclin1表达均呈现先增高后降低改变。 4．用RNAi干扰PCNA表达或用leptomycin B抑制PCNA向胞质转运后，雷帕霉素诱导SK-BR-3细胞自噬活化被抑制，Beclin1蛋白表达减少。 5．3-MA预处理SK-BR-3细胞抑制了雷帕霉素诱导的细胞自噬水平，但对PCNA胞质表达无影响。结论： PCNA可定位于各肿瘤细胞质中；乳腺癌细胞PCNA胞质表达水平在各种应激条件下与细胞自噬活性协同改变；胞质PCNA可调节乳腺癌细胞自噬活性及自身相关蛋白Beclin1表达，表明胞质PCNA可能是乳腺癌细胞自噬调节因子，其对自噬的调节作用是通过Beclin1实现的，本实验为研究PCNA和自噬在乳腺癌中的作用及调控机制提供新思路。
[Abstract]:Proliferating cell nuclear antigen (PCNA) is a unique intra nuclear protein. In the nucleus, three PCNA molecules are linked together to form a sliding circular structure around the DNA double helix to recruit DNA polymerase and a variety of different functional proteins to combine with them, in DNA synthesis and damage repair, cell cycle regulation. The tumor cells from different histology sources express high levels of PCNA in the nucleus, indicating that the proliferation is uncontrollable. The PCNA in the nucleus becomes a molecular marker for the diagnosis and poor prognosis of the tumor, and is also a new target for the treatment of cancer. However, some studies have shown that PCNA is not only expressed in the nucleus, but also in the nucleus, It is also expressed in the cytoplasm and can interact with a variety of proteins in the cytoplasm, indicating that it has more extensive biological activity. In some tumor cell lines, the PCNA part is expressed in the cytoplasm and combined with a variety of tumor related proteins, suggesting that cytoplasmic PCNA may be involved in some pathological processes of the tumor, but there is still lack of relevant research evidence. The two methods can change the intracellular localization and protein expression level of PCNA in the treatment of acute T lymphocyte leukemia cells and mouse embryonic fibroblasts using rapamycin and starvation, respectively. The most common cell autophagy inducer, especially in tumor cells, is the most common cell autophagy inducer Therefore, we speculate that the reorientation of PCNA in cells may be related to autophagy activity.
Autophagy has been proved to be involved in the process of cancer in the cell. First, autophagy can inhibit the proliferation of malignant transformed cells, thereby promoting cell aging and inhibiting the production of tumors. Once the tumor is formed, the tumor cells enhance autophagy to survive in metabolism and treat stress, and autophagy becomes an autophagy. The protection mechanism to avoid tumor cell damage. If stress persists and autophagy is excessively stimulated, it can mediate tumor cells into non apoptotic cell death patterns, release inflammatory factors and mobilize the body's tumor related immune activity. The complex role of autophagy in the pathogenesis of tumor makes it a current tumor research. Breast cancer, as the second major tumor that threatens the health of women in the world, has always received great attention. Recent studies show that autophagy has been involved in the occurrence, progression and metastasis of breast cancer and the treatment of tolerance, but its function and mechanism have not been fully elucidated. The function and regulation mechanism of autophagy in breast cancer have been deeply studied. It can accurately regulate the autophagy level of breast cancer cells and promote tumor therapy.
At present, the autophagy process of tumor cells is known to be regulated by a variety of proteins and signaling pathways. In addition to autophagy related proteins and PI3K/Akt/mTOR and AMPK signaling pathways, recent studies have found that the abnormal expression of microRNA and p53 in various tumors, especially breast cancers, can change the autophagy activity of the tumor cells, suggesting that more molecules or proteins may be involved. The autophagy regulates the process of autophagy in tumor cells. According to the previous study, we speculate that under some conditions, PCNA may be expressed in the cytoplasm of breast cancer cells and involved in the regulation of cellular autophagy.
In order to confirm this hypothesis, we chose the human breast cancer SK-BR-3 cell line as a target cell. First, we confirmed that there was PCNA expression in the cytoplasm of breast cancer cells, and then the cells were treated with different autophagy induction methods, and the expression of PCNA in the cytoplasm of breast cancer cells was related to the autophagy activity in the cytoplasm of breast cancer cells. On this basis, different methods were used to modify the cell cytoplasm. Changing the expression level of PCNA cytoplasm and determining the cytoplasmic expression of PCNA have the function of regulating autophagic activity of breast cancer cells and provide a new direction for the accurate regulation of autophagy in breast cancer cells.
Research methods:
Subcellular localization analysis of 1.PCNA: rapamycin treatment of breast cancer SK-BR-3 cell line, Western blot and immunofluorescence detection PCNA expression in cytoplasm; simultaneously using rapamycin to treat cervical cancer HeLa cell line and lung cancer A549 cell line. The same experiment analyzed the localization of PCNA in these two cells.
2. the autophagy induced by rapamycin in breast cancer SK-BR-3 cells: the autophagy was analyzed by MDC (monodansylcadaverin, MDC), and the expression level of Beclin1 protein was analyzed by Western blot.
3. the effect of other autophagy induction methods on the autophagy level and cytoplasmic PCNA expression in SK-BR-3 cells of breast cancer: using hydrogen peroxide to simulate oxidative stress and EBSS buffer to induce autophagy by producing starvation stress, MDC staining to determine the level of autophagy, and Western blot analysis of the expression level of PCNA and Beclin1 protein in the cytoplasm.
The correlation between the expression of 4. cytoplasmic PCNA and the autophagy level of breast cancer SK-BR-3 cells: the expression and subcellular localization of PCNA in SK-BR-3 cells by RNAi or leptomycin B, and MDC staining to analyze the autophagy of cells and Western blot to detect the Beclin1 expression level.
The effect of autophagy inhibition on the PCNA subcellular localization of SK-BR-3 cells in breast cancer SK-BR-3 cells: autophagy inhibitor 3- methyl adenine (3-methyladenine, 3-MA) pretreated the cell autophagy induced by rapamycin, MDC staining analysis of autophagy level, and PCNA egg white expression in the cytoplasm of Western blot.
The results of the study:
1. with the prolongation of the action time of rapamycin to SK-BR-3 cells, the expression level of PCNA in the cytoplasm increased first and then decreased. The transport of PCNA cell nucleus to cytoplasm in HeLa cells and A549 cells was also seen in the treatment of rapamycin, which confirmed that PCNA could relocate in the cytoplasm after different tumor cells were stimulated by external stimulation.
2. after the effect of rapamycin on SK-BR-3 cells, autophagic activity increased first and then weakened, and the expression of autophagy related protein Beclin1 increased first and then decreased, which was consistent with the change trend of cytoplasmic PCNA expression.
3. starvation and oxidative stress induced SK-BR-3 cells, cytoplasmic PCNA, autophagy activity and autophagy related protein Beclin1 expression increased first and then decreased.
4. after interfering with PCNA by RNAi or using leptomycin B to inhibit the transport of PCNA into the cytoplasm, the activation of autophagy in SK-BR-3 cells induced by rapamycin was inhibited and the expression of Beclin1 protein decreased.
Pretreatment of SK-BR-3 cells with 5.3-MA inhibited rapamycin induced autophagy but had no effect on PCNA cytoplasmic expression.
Conclusion:
PCNA can be located in the cytoplasm of all tumor cells, and the cytoplasmic expression of PCNA in breast cancer cells changes in synergy with autophagic activity under various stressful conditions. Cytoplasmic PCNA can regulate autophagy and the expression of autophagy Beclin1 in breast cancer cells, indicating that cytoplasmic PCNA may be an autophagy regulator of breast cancer cells and its regulation of autophagy The action was achieved through Beclin1. This experiment provides a new idea for studying the role and regulation mechanism of PCNA and autophagy in breast cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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