【摘要】：乳腺癌是女性常見的惡性腫瘤,發(fā)病率呈上升趨勢,且趨向于年輕化。雖然,近年來,乳腺癌的早期診斷、手術(shù)方式、放射治療以及靶向治療方面已取得很大進(jìn)展,但預(yù)后仍然較差,靶向藥物效率低且價(jià)格昂貴。研究乳腺癌發(fā)生和發(fā)展的分子機(jī)制,探索新的分子靶標(biāo)仍然是乳腺癌研究和臨床中需要解決的難題之一。胱天蛋白酶募集域蛋白9(caspase recruitment domain containing protein 9,CARD9)是一個重要的銜接蛋白,通過蛋白間的相互作用調(diào)節(jié)NF-κB等信號通路,在胃B細(xì)胞淋巴瘤、腎癌、丙型肝炎病毒相關(guān)的肝細(xì)胞癌及結(jié)腸癌的發(fā)生和發(fā)展中扮演著重要角色,但其在乳腺癌中的表達(dá)及對乳腺癌發(fā)生發(fā)展的影響尚未見相關(guān)報(bào)道。本實(shí)驗(yàn)利用實(shí)時(shí)熒光定量PCR和Western-Blotting技術(shù)分別檢測乳腺癌癌組織、癌旁組織、乳腺癌細(xì)胞、正常乳腺細(xì)胞中CARD9 mRNA和蛋白的表達(dá)情況,分析CARD9表達(dá)與乳腺癌患者臨床病理特征間的關(guān)系。通過WST-1法、平板克隆形成實(shí)驗(yàn)、流式細(xì)胞術(shù)、劃痕實(shí)驗(yàn)研究CARD9高表達(dá)對乳腺癌細(xì)胞增殖、凋亡、周期、侵襲轉(zhuǎn)移等生物學(xué)行為的影響。分析CARD9表達(dá)和NF-κB信號通路激活的相關(guān)性,初步探討CARD9在乳腺癌中的作用機(jī)制。結(jié)果表明:乳腺癌組織中CARD9 mRNA(p0.01)和蛋白(p=0.012)表達(dá)水平顯著高于癌旁組織;乳腺癌T-47D細(xì)胞(p=0.021)和MCF-7細(xì)胞(p=0.014)中CARD9 mRNA表達(dá)水平高于正常乳腺細(xì)胞Hs 578Bst;乳腺癌ZR-75-1細(xì)胞中CARD9 mRNA(p=0.003)和蛋白(p=0.015)表達(dá)水平均高于正常乳腺細(xì)胞Hs 578Bst。CARD9蛋白表達(dá)與腫瘤大小和雌激素受體(ER)表達(dá)有關(guān)(p值均0.05)。CARD9高表達(dá)可促進(jìn)乳腺癌細(xì)胞的增殖(p0.05),但對細(xì)胞凋亡、周期、侵襲和轉(zhuǎn)移未見顯著性影響。乳腺癌組織中P50(p0.05)和p-IKKα(p0.01)蛋白的表達(dá)水平高于癌旁組織,而且乳腺癌組織中CARD9 mRNA表達(dá)與P50(r=0.622,p0.001)、P65(r=0.392,p=0.005)、IL-1β(r=0.685,p0.001)和IL-12(r=0.556,p0.001)mRNA表達(dá)間呈正相關(guān)。本實(shí)驗(yàn)的研究結(jié)果揭示了CARD9在乳腺癌組織中的表達(dá)情況及其與乳腺癌臨床病理學(xué)的關(guān)系。同時(shí)發(fā)現(xiàn),CRAD9很可能通過激活NF-κB信號通路促進(jìn)乳腺癌細(xì)胞增殖從而參與乳腺癌發(fā)生發(fā)展進(jìn)程。這些研究結(jié)果有助于深入了解CRAD9在乳腺癌發(fā)生發(fā)展及治療中的潛在價(jià)值,為CARD9成為乳腺癌新的治療靶點(diǎn)提供了理論依據(jù)。
[Abstract]:Breast cancer is a common malignant tumor in women. The incidence of breast cancer is increasing and tends to be younger. Although great progress has been made in early diagnosis, surgical methods, radiotherapy and targeted therapy for breast cancer in recent years, the prognosis is still poor, targeting drugs are inefficient and expensive. To study the molecular mechanism of breast cancer development and explore new molecular targets is still one of the difficult problems to be solved in breast cancer research and clinical practice. Caspase-9 (caspase recruitment domain containing protein-9) is an important binding protein that regulates NF- 魏 B signaling pathways in gastric B-cell lymphoma and renal cancer through protein-to-protein interactions. Hepatitis C virus (HCV) plays an important role in the occurrence and development of hepatocellular carcinoma (HCC) and colon cancer. However, the expression of HCV in breast cancer and its effect on the development of breast cancer have not been reported. The expression of CARD9 mRNA and protein in breast cancer tissues, paracancerous tissues, breast cancer cells and normal breast cells were detected by real-time fluorescence quantitative PCR and Western-Blotting, respectively. To analyze the relationship between the expression of CARD9 and the clinicopathological features of breast cancer patients. The effects of high expression of CARD9 on proliferation, apoptosis, cell cycle, invasion and metastasis of breast cancer cells were studied by WST- 1 assay, plate cloning assay, flow cytometry and scratch assay. To analyze the correlation between the expression of CARD9 and the activation of NF- 魏 B signaling pathway, and to explore the mechanism of CARD9 in breast cancer. The results showed that the expression level of CARD9 mRNA (p0.01) and protein (pA0.012) in breast cancer was significantly higher than that in adjacent tissues. The expression of CARD9 mRNA was higher in breast cancer cells than that in normal breast cells (Hs 578Bst). The expression level of CARD9 mRNA was higher in breast cancer cells than that in normal breast cells (Hs 578BST). The expression level of CARD9 mRNA (p < 0. 0003) and protein (p < 0. 015) in breast cancer ZR-75-1 cells was higher than that in normal breast cells (P < 0. 05). The expression of Hs 578Bst.CARD9 protein in breast cancer cells was correlated with tumor size and estrogen receptor (ER) expression (p < 0. 05). .05). High expression of CARD9 can promote the proliferation of breast cancer cells (p0.05), However, there was no significant effect on cell apoptosis, cell cycle, invasion and metastasis. The expression levels of P50 (p0.05) and p-IKK 偽 (p0.01) protein in breast cancer tissues were higher than those in paracancerous tissues, and the expression of CARD9 mRNA in breast cancer tissues was associated with P50 (r = 0.622, p0.001), P65 (r = 0.392, p < 0.005). There was a positive correlation between the expression of IL-1 尾 (r = 0.685, p0.001) and IL-12 (r = 0.556, p0.001) mRNA. The results of this study revealed the expression of CARD9 in breast cancer and its relationship with the clinicopathology of breast cancer. It was also found that CRAD9 may play an important role in the development of breast cancer by activating NF- 魏 B signaling pathway to promote the proliferation of breast cancer cells. These results are helpful to understand the potential value of CRAD9 in the development and treatment of breast cancer and provide a theoretical basis for CARD9 to become a new therapeutic target for breast cancer.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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