PLK1在结肠炎相关结肠癌发生中的作用

发布时间：2018-06-25 14:32

本文选题：结肠炎相关结肠癌 + PLK1　；参考：《北京协和医学院》2017年硕士论文

【摘要】：结直肠癌(Colorectalcancer,CRC)是最常见的消化道肿瘤之一。近年,我国结直肠癌的发病率呈逐年上升趋势。炎性肠病(inflammatory bowel disease,IBD)作为一种反复发作的慢性炎症,是结肠癌发生的重要高危因素之一,约20%的结肠炎患者最终会发展为结肠炎相关结肠癌(Colitis-associated Cancer,CAC)。结肠炎向结肠癌转化是一个涉及到多个信号通路和分子的复杂过程,目前相关的机制尚不明确。丝氨酸/苏氨酸蛋白激酶PLKl(polo-likekinasel)是哺乳动物有丝分裂进程中的关键调节因子。大量研究结果显示PLK1在包括结直肠癌(colorectal cancer,CRC)在内的多种恶性肿瘤组织中表达升高,但迄今为止PLK1与CAC之间的关系尚无文献报道。因此本研究旨在探索PLK1在炎症、免疫及结肠癌之间的潜在联系并初步探讨PLK1在CAC发生的作用及相关分子机制。为探索PLK1与CAC发生之间的关系,本研究利用氧化偶氮甲烷(azoxymethane,AOM)和葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导 PLK1基因敲入小鼠(PLK1fl/+小鼠,实验组)及同窝野生型小鼠(WT小鼠,对照组)发生CAC,结果显示PLK1fl/+小鼠结肠肿瘤数量和肿瘤负荷显著低于WT对照小鼠,提示PLK1过表达可有效抑制AOM/DSS诱导的CAC的发生。以往研究发现PLK1在多种实体肿瘤中发挥促癌作用,因此亟需揭示PLK1过表达抑制CAC发生的作用机制。我们使用免疫组织化学(IHC)染色法检测了AOM/DSS诱导野生型小鼠形成的CAC病灶部位中PLK1的表达情况,发现其在肠道上皮间质细胞中有高水平表达。据此,我们在小鼠结肠炎相关结肠癌肿瘤组织的连续切片中进行了 PLK1和Gr1(标记髓系来源细胞)的IHC染色,发现PLK1和Gr1阳性细胞在结肠病灶组织中有一致的定位。免疫荧光(IF)共定位检测结果显示,PLK1与Gr1标记的髓系细胞及F4/80标记的巨噬细胞存在共定位现象。上述结果显示CAC发生过程中PLK1主要表达于肠道炎症微环境中的髓系细胞。肠炎/肠癌微环境中的巨噬细胞等髓系细胞可通过分泌促炎细胞因子及趋化因子促进结肠炎向结肠癌的转化。为进一步揭示PLK1在肠道炎症微环境髓系细胞中表达抑制CAC发生的分子机制,我们使用细胞因子芯片检测经DSS处理的PLK1fl/+和WT小鼠的结肠组织培养上清中炎性因子的表达情况。芯片检测结果显示PLK1高表达可以明显抑制小鼠结肠病灶组织表达和分泌包括TNF-α在内的多个细胞因子和趋化因子。ELISA和qRT-PCR检测证实了PLK1fl/+小鼠结肠组织中TNF-α表达和分泌的改变。我们进一步使用PLK1特异性的siRNA或小分子抑制剂处理体外培养的巨噬细胞J774.1,发现抑制PLK1的表达可明显上调TNF-α表达。上述结果提示,PLK1可能通过抑制肠道炎症微环境中髓系细胞的促炎和趋化功能进而抑制CAC的发生。综上所述,本研究结果提示PLK1可能参与调控间质髓系细胞的促炎/促癌功能,其可能通过抑制TNF-α等炎性因子的表达和分泌进而抑制AOM/DSS诱导的CAC的发生。本研究不仅有助于阐明PLK1在肠炎/肠癌相关免疫细胞中新的作用及分子机制,而且可能为今后开发针对免疫微环境的治疗策略提供理论依据和实验基础。结直肠癌作为常见恶性肿瘤,极大的威胁着人类的健康和生命。结直肠癌术后复发转移是导致患者死亡的主要原因之一,因此寻找能够用于预测结直肠癌复发转移或准确判断预后的分子标志物对于提高结直肠癌患者的生存率具有重要意义。本实验室在前期工作中发现KIAA1522在食管鳞状细胞癌、肺癌等肿瘤中异常表达,并与患者的预后相关。但迄今为止,KIAA1522在结直肠癌中的表达改变尚不清楚。本研究应用组织芯片-免疫组织化学染色技术,对96例结直肠癌组织及其配对癌旁正常组织中KIAA1522蛋白的表达情况进行检测,并对其表达改变与结直肠癌临床病理指标及患者预后的关系进行统计分析。免疫组织化学分析结果显示结直肠上皮细胞中KIAA1522蛋白主要定位于细胞浆。在结直肠癌组织中,KIAA1522蛋白表达的阳性率为81%(78/96);而在配对的癌旁正常组织中其表达的阳性率为13%(12/96),两者间差异显著(P0.05)。进一步的统计分析结果显示,KIAA1522蛋白过表达与结直肠癌患者术后三年无瘤生存期短显著正相关(P = 0.017),并且与远处转移正相关(P = 0.012)。多因素Cox回归分析结果表明,KIAA1522过表达是结直肠癌患者预后不良的独立预测因素(P= 0.020)。综上所述,KIAA1522在结直肠癌组织中表达上调,并与肿瘤的远处转移及患者术后生存期短密切相关,可能作为预测结直肠癌患者预后及转移的潜在分子标志。
[Abstract]:Colorectalcancer (CRC) is one of the most common digestive tract tumors. In recent years, the incidence of colorectal cancer in China is increasing year by year. Inflammatory bowel disease (IBD), as a recurrent and chronic inflammation, is one of the most important risk factors for colon cancer, and about 20% of the patients with colitis will eventually develop. Colitis-associated Cancer (CAC). The transformation of colitis to colon cancer is a complex process involving multiple signaling pathways and molecules. The related mechanisms are not yet clear. Serine / threonine protein kinase PLKl (polo-likekinasel) is a key regulator in the process of mammalian mitosis. The results show that PLK1 is expressed in a variety of malignant tumor tissues, including colorectal cancer (CRC), but the relationship between PLK1 and CAC has not been reported so far. Therefore, the purpose of this study is to explore the potential of PLK1 in inflammation, immune and colon cancer and to explore the role of PLK1 in CAC and to explore the role of PLK1 in CAC. In order to explore the relationship between PLK1 and CAC, this study used oxidative azo methane (azoxymethane, AOM) and sodium dextran sulfate (dextran sulfate sodium, DSS) to induce the PLK1 gene to knock into mice (PLK1fl/+ mice, experimental group) and the same nest wild type mice (WT mice, control groups). The number and load of intestinal tumor are significantly lower than that of WT control mice. It is suggested that overexpression of PLK1 can effectively inhibit the occurrence of CAC induced by AOM/DSS. Previous studies have found that PLK1 plays a role in promoting cancer in various solid tumors. Therefore, it is urgent to reveal the mechanism of PLK1 overexpression to inhibit the occurrence of CAC. We use immunohistochemistry (IHC) staining method to detect the mechanism of CAC. The expression of PLK1 in the CAC foci of the wild type mice induced by AOM/DSS was found to be highly expressed in the intestinal epithelial mesenchymal cells. Accordingly, we carried out IHC staining of PLK1 and Gr1 (labeled myeloid derived cells) in the continuous sections of the tumor tissue of colitis related colon cancer in mice, and found the positive cells of PLK1 and Gr1. There was a consistent location in the lesions of the colon. The results of immunofluorescence (IF) Co localization showed that there was a co localization phenomenon between PLK1 and Gr1 labelled medullary cells and F4/80 labeled macrophages. The results showed that PLK1 was mainly expressed in myeloid cells in the intestinal microenvironment during the occurrence of CAC. The macrophages in the microenvironment of enteritis / colon cancer Cell lines can promote the transformation of colitis to colon by secreting proinflammatory cytokines and chemokines. To further reveal the molecular mechanism of inhibiting the occurrence of CAC in the microenvironmental myeloid cells of intestinal inflammation, we use cytokine microarray to detect the colonic tissue culture of DSS treated PLK1fl/+ and WT mice. The expression of inflammatory factors in the colon was detected. The results of microchip detection showed that high expression of PLK1 could obviously inhibit the expression and secretion of multiple cytokines including TNF- alpha, including TNF- alpha and.ELISA and qRT-PCR, which confirmed the changes in the expression and secretion of TNF- alpha in the colon tissue of PLK1fl/+ mice. We further use PLK1 Specific siRNA or small molecule inhibitors treat the cultured macrophage J774.1 in vitro, and it is found that inhibition of the expression of PLK1 can obviously increase the expression of TNF- alpha. These results suggest that PLK1 may inhibit the pathogenesis and chemotaxis of myeloid cells in the intestinal inflammation microenvironment and inhibit the occurrence of CAC. The results of this study suggest that PLK1 can be used in this study. It can regulate the proinflammatory / carcinogenic function of interstitial myeloid cells, which may inhibit the occurrence of AOM/DSS induced CAC by inhibiting the expression and secretion of TNF- alpha and other inflammatory factors. This study not only helps to elucidate the new role and molecular mechanism of PLK1 in enteritis / colon related immune cells, but also may be developed for immunization in the future. The treatment strategy of environment provides theoretical basis and experimental basis. Colorectal cancer, as a common malignant tumor, is a great threat to human health and life. The recurrence and metastasis of colorectal cancer is one of the main causes of death. Therefore, it is found that the molecular markers can be used to predict the recurrence or prognosis of colorectal cancer. It is important to improve the survival rate of patients with colorectal cancer. In our laboratory, we found that KIAA1522 was abnormal expression in squamous cell carcinoma of the esophagus, lung cancer and other tumors, and was related to the prognosis of the patients. But so far, the expression changes of KIAA1522 in colorectal cancer are not clear. This study applied the tissue chip immuno group. The expression of KIAA1522 protein in 96 cases of colorectal cancer tissue and its paired cancerous normal tissues was detected by chemical staining, and the relationship between the expression changes and the clinicopathological indexes of colorectal cancer and the prognosis of the patients were statistically analyzed. The results of immunohistochemical analysis showed the main KIAA1522 protein in the colorectal epithelial cells. The positive rate of KIAA1522 protein expression was 81% (78/96) in colorectal cancer tissues and 13% (12/96) in normal tissues adjacent to the paired cancerous tissues (P0.05). Further statistical analysis showed that the overexpression of KIAA1522 protein and the three year tumor free survival of the colorectal cancer patients were three years after operation. A short and significant positive correlation (P = 0.017) and a positive correlation with distant metastasis (P = 0.012). Multiple factor Cox regression analysis showed that KIAA1522 overexpression was an independent predictor of poor prognosis in colorectal cancer patients (P= 0.020). To sum up, KIAA1522 was up-regulated in colorectal cancer tissue and distant metastasis of tumor and postoperative patients. Short term survival may be a potential molecular marker for predicting prognosis and metastasis of colorectal cancer.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62;R735.35

【参考文献】