本文選題：乳腺癌 + 增殖細胞核抗原�。� 參考：《吉林大學》2015年博士論文

【摘要】：增殖細胞核抗原（proliferating cell nuclear antigen，PCNA）是一種獨特的核內(nèi)蛋白質(zhì)。在細胞核內(nèi)，三個PCNA分子首尾相連構(gòu)成圍繞DNA雙螺旋的滑動環(huán)狀結(jié)構(gòu)，借此招募DNA聚合酶和多種不同功能蛋白質(zhì)與其結(jié)合，在DNA合成和損傷修復、細胞周期調(diào)控中發(fā)揮關(guān)鍵作用，被視為細胞增殖指標。不同組織學來源的腫瘤細胞均在核內(nèi)表達高水平PCNA，表明其增殖的不可控性，核內(nèi)PCNA由此成為腫瘤診斷和不良預后的分子標志，也成為腫瘤治療新靶標。然而有研究顯示PCNA不僅表達于細胞核內(nèi)，也在胞質(zhì)中表達，并且能夠和胞質(zhì)內(nèi)多種蛋白質(zhì)相互作用，表明其具有更廣泛的生物學活性。在某些腫瘤細胞系中，PCNA部分在胞質(zhì)表達并和多種腫瘤相關(guān)蛋白結(jié)合，提示胞質(zhì)PCNA可能參與了腫瘤的某些病理過程，但尚缺乏相關(guān)研究證據(jù)。既往有學者在用雷帕霉素和饑餓方法分別處理急性T淋巴細胞白血病細胞和小鼠胚胎成纖維細胞時發(fā)現(xiàn)，這兩種方法可以改變PCNA的細胞內(nèi)定位及蛋白表達水平。由于雷帕霉素及營養(yǎng)缺乏是最常見的細胞自噬誘導劑，尤其在腫瘤細胞中，因此我們推測PCNA在細胞內(nèi)的重新定位可能與細胞自噬活性相關(guān)。 自噬作為細胞內(nèi)長效蛋白質(zhì)降解途徑已被證實參與腫瘤的疾病進程。首先，自噬可以抑制發(fā)生惡性轉(zhuǎn)化的細胞增殖，由此促進細胞衰老而抑制腫瘤產(chǎn)生。一旦腫瘤形成，腫瘤細胞則增強自噬以求在代謝和治療應激中存活，此時自噬成為避免腫瘤細胞損傷的保護機制。如果應激持續(xù)存在，自噬被過度激發(fā)，則可介導腫瘤細胞進入非凋亡性細胞死亡模式，同時釋放炎癥因子，調(diào)動機體的腫瘤相關(guān)免疫活性。自噬在腫瘤發(fā)病機制中的這種復雜作用使其成為目前腫瘤研究的焦點。乳腺癌作為威脅世界女性健康的第二大腫瘤，始終受到極大關(guān)注，近期研究顯示自噬參與了乳腺癌的發(fā)生、進展和轉(zhuǎn)移、治療耐受性產(chǎn)生等病理過程，但其作用及調(diào)控機制尚未完全闡明。深入研究自噬在乳腺癌中的功能及調(diào)控機制，可以準確調(diào)節(jié)乳腺癌細胞自噬水平，從而促進腫瘤治療。 目前已知腫瘤細胞的自噬過程可由多種蛋白及信號通路調(diào)節(jié)，除自噬相關(guān)蛋白及PI3K/Akt/mTOR和AMPK信號通路外，近期研究發(fā)現(xiàn)在各種腫瘤尤其乳腺癌中異常表達的microRNA及p53等均可改變腫瘤細胞自噬活性，提示可能有更多分子或蛋白參與了腫瘤細胞的自噬調(diào)節(jié)過程。根據(jù)前述研究，我們推測在某種條件下PCNA可能在乳腺癌細胞的胞質(zhì)表達并涉及細胞自噬活性調(diào)節(jié)。 為證實該假設(shè)，在本實驗中我們選擇人乳腺癌SK-BR-3細胞系作為靶細胞，首先證實乳腺癌細胞胞質(zhì)中存在PCNA表達，繼而采用不同自噬誘導方法處理細胞，驗證PCNA在乳腺癌細胞胞質(zhì)內(nèi)定位表達與自噬活性相關(guān)；在此基礎(chǔ)上用不同方法改變PCNA胞質(zhì)表達水平，確定PCNA的胞質(zhì)表達具有調(diào)節(jié)乳腺癌細胞自噬活性的功能，為在乳腺癌細胞中準確調(diào)控自噬提供新的研究方向。 研究方法： 1．PCNA的亞細胞定位分析：雷帕霉素處理乳腺癌SK-BR-3細胞系，Western blot及免疫熒光檢測PCNA在胞質(zhì)中表達狀態(tài)；同時用雷帕霉素處理子宮頸癌HeLa細胞系及肺癌A549細胞系，相同實驗分析PCNA在這兩種細胞中定位改變。 2．雷帕霉素誘導乳腺癌SK-BR-3細胞自噬活性：雷帕霉素處理細胞不同時間，單丹磺酰尸胺（monodansylcadaverin, MDC）染色對細胞進行自噬活性分析，Western blot分析Beclin1蛋白表達水平。 3．其他自噬誘導方法對乳腺癌SK-BR-3細胞自噬水平及胞質(zhì)PCNA表達的影響：分別用過氧化氫模擬氧化應激及EBSS緩沖液制造饑餓應激方式誘導細胞自噬，MDC染色確定細胞自噬水平，Western blot分析胞質(zhì)中PCNA及Beclin1蛋白表達水平。 4．胞質(zhì)PCNA表達與乳腺癌SK-BR-3細胞自噬水平的相關(guān)性：用RNAi或出核轉(zhuǎn)運抑制劑leptomycin B改變PCNA在SK-BR-3細胞中的表達及亞細胞定位，MDC染色分析細胞自噬，Western blot檢測Beclin1表達水平。 5．自噬抑制對乳腺癌SK-BR-3細胞PCNA亞細胞定位的影響：自噬抑制劑3-甲基腺嘌呤（3-methyladenine,3-MA）預處理細胞后雷帕霉素誘導細胞自噬，MDC染色分析細胞自噬水平，Western blot分析檢胞質(zhì)中PCNA蛋白表達。 研究結(jié)果： 1．隨雷帕霉素對SK-BR-3細胞作用時間延長，PCNA在胞質(zhì)中表達水平呈先升高后降低改變；HeLa細胞及A549細胞在雷帕霉素處理時也可見PCNA胞核向胞質(zhì)轉(zhuǎn)運，證實PCNA在不同腫瘤細胞接受外界刺激后均可在胞質(zhì)重新定位表達。 2．雷帕霉素作用于SK-BR-3細胞后自噬活性呈先增強后減弱改變，自噬相關(guān)蛋白Beclin1表達先增高后降低，與胞質(zhì)PCNA表達變化趨勢一致。 3．饑餓和氧化應激誘導SK-BR-3細胞，，胞質(zhì)PCNA、細胞自噬活性及自噬相關(guān)蛋白Beclin1表達均呈現(xiàn)先增高后降低改變。 4．用RNAi干擾PCNA表達或用leptomycin B抑制PCNA向胞質(zhì)轉(zhuǎn)運后，雷帕霉素誘導SK-BR-3細胞自噬活化被抑制，Beclin1蛋白表達減少。 5．3-MA預處理SK-BR-3細胞抑制了雷帕霉素誘導的細胞自噬水平，但對PCNA胞質(zhì)表達無影響。 結(jié)論： PCNA可定位于各腫瘤細胞質(zhì)中；乳腺癌細胞PCNA胞質(zhì)表達水平在各種應激條件下與細胞自噬活性協(xié)同改變；胞質(zhì)PCNA可調(diào)節(jié)乳腺癌細胞自噬活性及自身相關(guān)蛋白Beclin1表達，表明胞質(zhì)PCNA可能是乳腺癌細胞自噬調(diào)節(jié)因子，其對自噬的調(diào)節(jié)作用是通過Beclin1實現(xiàn)的，本實驗為研究PCNA和自噬在乳腺癌中的作用及調(diào)控機制提供新思路。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.9

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