结直肠癌中LRG1基因的表达及其调控血管生成的作用和机制研究

发布时间：2018-06-22 05:00

本文选题：LRG1 + 结直肠癌　；参考：《中国人民解放军医学院》2017年博士论文

【摘要】：结直肠癌(Colorectal Cancer, CRC )是世界范围内常见的实体肿瘤之一,严重威胁人类的健康。我国2000年至2011年结直肠癌的发病率和死亡率均逐年上升,据国家癌症登记中心公布的最新数据表明,2015年我国结直肠癌共发病37.63万人,死亡19.10万人。目前手术切除仍然是根治结直肠癌的主要手段,但大部分晚期结直肠癌患者或术后复发转移的结直肠癌患者,失去手术治疗的机会,积极地内科治疗是抑制肿瘤生长和延长生命的首选方法。结直肠癌的抗血管生成治疗在内科治疗当中具有举足轻重的地位。因此进一步探索结直肠癌血管生成的机制,寻找抗血管生成的有效靶点具有重要的理论意义和潜在的临床价值。富亮氨酸 α-2 糖蛋白 1 ( Leucine-rich-alpha-2-glycoprotein 1,LRG1 )是 1977 年发现的,在血液中和组织中表达的蛋白。近年来,研究发现LRG1在呼吸系统、消化系统和妇科肿瘤中的表达升高,且与肿瘤的恶性程度相关;也有研究发现LRG1促进肿瘤和其他疾病中的新生血管形成。但LRG1在结直肠癌发生发展和血管生成中的研究较少。基于以上研究背景,本课题通过临床大样本验证,探索LRG1在结直肠癌发生发展和血管生成中的作用,并通过体外细胞实验和体内成瘤实验进一步验证LRG1的促血管生成能力。最后通过信号通路研究,尝试揭示LRG1促进血管生成功能的相关作用机制。实验方法1.临床样本验证:收集中国人民解放军总医院2005年至2009年接受根治性手术治疗的III期结直肠癌的癌和癌旁标本,建立组织芯片和临床信息数据库。应用免疫组织化学染色技术检测LRG1、CD34-微血管密度(Micro-vessel Density, MVD ),分析LRG1与临床病理特征和患者预后的相关性,分析LRG1是否是影响患者生存的独立预后因素。分析LRGI与MVD-CD34的关系,判断LRG1对血管生成的影响。2. LRG1影响结直肠癌血管生成的体外研究:利用慢病毒构建基因过表达基因敲降稳转细胞株。通过Transwell侵袭实验和划痕试验检测LRG1对各稳转癌细胞株的侵袭和迁移能力的影响;并通过MTT实验、Transwell迁移实验和小管形成实验判断LRG1对人脐静脉血管内皮细胞(HUVEC )的增殖、迁移和成管能力的影响。3. LRG1影响结直肠癌血管生成的体内研究:利用过表达和对照细胞株建立裸鼠皮下成瘤模型。测量瘤体体积变化,第30天取出瘤体称重,并制作组织切片进行HE染色、CD34和VEGFA检测,判断LRG1对体内血管生成的影响。4. LRG1促进结直肠癌血管生成机制的初步研究:检测LRG1过表达和敲降细胞株VEGFA的表达和分泌VEGFA的水平;检测PI3K/AKT和MEK/ERK通路相关分子蛋白水平;检测HIF-1α细胞质和细胞核内的分布情况,判断LRG1是否介导HIF-1a移位入核发挥转录因子作用;检测在信号通路抑制剂作用下,细胞中VEGFA表达水平是否改变,以及HUVECs成管能力是否下降,进一步确认LRG1通过AKT、ERK双通路激活VEGFA诱导血管生成。实验结果:1.共312例Ⅲ期结直肠癌患者纳入本课题。免疫组化染色结果显示60.9%病例癌组织内LRG1高表达,只有11.9%患者的癌旁正常组织内LRG1高表达,LRG1在癌组织内表达水平高于癌旁正常组织(X2=35.412, P 0.001 )。单因素分析显示LRG1与肿瘤分化较差、T4分期和血管浸润明显相关(P=0.035,0.028,0.007)。多因素分析显示LRG1是影响312例Ⅲ期结直肠癌患者总生存OS和无疾病生存的独立预后因素(HR=1.517, P=0.020;HR=1.754,P=0.013)。并且 LRG1 水平与肿瘤 MVD 正相关(t=-8.603,P0.001)。2.选择LRG1表达最低的DLD1和表达量最高的HT29细胞系,分别建立LRG1过表达和敲降细胞株。Real-Time PCR证实过表达组LRG1的转录水平升高6.7倍,敲降组与对照组相比LRG1转录水平下降了 35%～96%。体外实验证明LRG1促进癌细胞的侵袭和迁移。进一步利用共培养HUVECs细胞进行体外功能实验,LRG1基因过表达表达促进血管内皮细胞的细胞增殖,迁移和成管功能(P0.01)。3.裸鼠成瘤模型的肿瘤生长曲线显示LRG1促进裸鼠体内移植瘤生长(P0.01 )。过表达组瘤体平均重量大于对照组(P0.05)。瘤体制成组织切片并进行HE染色,结果发现过表达组的移植瘤浸润肌层,LRG1促进肿瘤的生长和浸润。CD34-MVD和VEGFA荧光染色证明过表达组促进为血管形成和VEGFA表达。4.DLD1细胞中过表达组与对照组相比VEGFA表达和分泌水平均升高;HT29细胞中敲降组和对照组相比VEGFA表达和分泌水平下降。Western Blot结果显示LRG1促进 PI3K/AKT/HIF-1α 和 Ras/MEK1/ERK1/2 通路的激活。并且 LRG1 诱导 HIF-1α从细胞质移位至细胞核。利用通路抑制剂分别抑制AKT和ERK通路后,DLD1-LRG1组的VEGFA表达量均有所下降,且LRG1诱导内皮细胞的小管形成能力被抑制。结论LRG1在结直肠癌肿瘤组织内高表达,且其表达水平明显高于对应癌旁正常组织。并与结直肠癌的T分期、分化程度和血管浸润明显相关。LRG1是影响结直肠癌OS和DFS的独立预后因素。LRG1与MVD成明显的正相关。LRG1不仅能够促进肿瘤细胞的侵袭和迁移能力,且能够促进血管内皮细胞的增殖、迁移和成管能力。体内成瘤实验证明LRG1促使移植瘤向肌层浸润,也使CD34-MVD和VEGFA表达增加。肿瘤细胞中LRG1可通过PI3K/AKT/HIF1α和MEK/ERK双通路上调VEGFA分泌介导的血管生成。
[Abstract]:Colorectal Cancer (CRC) is one of the most common solid tumors in the world. It is a serious threat to human health. The incidence and mortality of colorectal cancer in China from 2000 to 2011 are increasing year by year. According to the latest data published by the National Cancer Registration Center, there are 376 thousand and 300 cases of colorectal cancer in China in 2015 and 19.10 deaths in 2015. Surgical excision is still the main cure for colorectal cancer, but most advanced colorectal cancer patients or recurrent colorectal cancer patients have lost the chance of surgical treatment. Active internal medicine is the first choice to suppress tumor growth and prolong life. Antiangiogenic treatment for colorectal cancer is treated in internal medicine. It is of great importance. Therefore, it is of great theoretical significance and potential clinical value to further explore the mechanism of angiogenesis in colorectal cancer and to find effective targets for anti angiogenesis. The leucine alpha -2 glycoprotein 1 (Leucine-rich-alpha-2-glycoprotein 1, LRG1) is discovered in 1977 and is in the blood and tissue. The protein expressed. In recent years, studies have found that LRG1 has increased in the respiratory system, digestive system and gynecologic tumors, and is associated with the malignancy of the tumor. There are also studies found that LRG1 promotes neovascularization in tumors and other diseases. But LRG1 is less studied in the development and angiogenesis of colorectal cancer. Background, this topic is to explore the role of LRG1 in the development and angiogenesis of colorectal cancer by large clinical samples, and to further verify the angiogenesis ability of LRG1 through in vitro cell experiment and in vivo tumorigenesis experiment. Finally, through the study of signal pathway, we try to reveal the relevant mechanism of LRG1 promoting angiogenesis. Test method 1. clinical sample validation: collect cancer and paracancerous specimens of III stage colorectal cancer in General Hospital of PLA from 2005 to 2009, establish tissue chip and clinical information database. Use immunohistochemical staining technique to detect LRG1, CD34- microvascular density (Micro-vessel Density, MVD), and analyze L The correlation between the RG1 and the clinicopathological features and the prognosis of patients, and to analyze whether LRG1 is an independent prognostic factor that affects the survival of the patients. Analysis of the relationship between LRGI and MVD-CD34, and to determine the effect of LRG1 on angiogenesis, the effect of.2. LRG1 on the angiogenesis of colorectal cancer in vitro: the use of lentivirus construction gene overexpressed genes to knock down cell lines. The effect of LRG1 on the invasion and migration of each stable cancer cell line was detected by Transwell invasion test and scratch test, and the effects of LRG1 on the proliferation, migration and angiogenic ability of human umbilical vein endothelial cells (HUVEC) were determined by MTT experiment, Transwell migration and tubule formation, and the effect of.3. LRG1 on the angiogenesis of colorectal cancer was influenced by.3. LRG1. In vivo study: using overexpressed and controlled cell lines to establish a subcutaneous tumor model in nude mice. Measure the volume changes of the tumor, take thirtieth days to take out the weight of the tumor, and make tissue sections for HE staining, CD34 and VEGFA, to determine the effect of LRG1 on the angiogenesis of.4. LRG1 to promote the angiogenesis of colorectal cancer: detection of LRG1 Overexpression and knockdown cell line VEGFA expression and secretion of VEGFA levels; detection of PI3K/AKT and MEK/ERK pathway related molecular protein levels; detection of the distribution of HIF-1 alpha cytoplasm and nucleus; determine whether LRG1 mediates the role of the HIF-1a shift into the nucleus to play a transcription factor; detection of VEGFA expression in cells under the action of signaling pathway inhibitors Whether or not the level changes, and whether the HUVECs tube ability decreased, further confirmed that LRG1 through AKT, ERK double pathway activation of VEGFA induced angiogenesis. Experimental results: 1. a total of 312 cases of stage III colorectal cancer were included in this subject. The immunohistochemical staining results showed that the high expression of LRG1 in the carcinoma tissues of 60.9% cases, only 11.9% patients in the normal para cancerous tissue The expression of LRG1 was higher than that of normal tissue adjacent to the cancer tissue (X2=35.412, P 0.001). Single factor analysis showed that LRG1 was poorly differentiated from tumor, and T4 staging was associated with vascular infiltration (P=0.035,0.028,0.007). The multivariate analysis showed that LRG1 was independent of the survival of OS and disease free survival in 312 patients with stage III colorectal cancer. Prognostic factors (HR=1.517, P=0.020; HR=1.754, P=0.013). And LRG1 level with MVD positive correlation (t=-8.603, P0.001).2. selection LRG1 expressed the lowest DLD1 and the highest expression of HT29 cell lines. In vitro, LRG1 transcriptional level decreased by 35% ~ 96%. in vitro experiments showed that LRG1 promoted invasion and migration of cancer cells. Further functional experiments were carried out by co cultured HUVECs cells. The overexpression of LRG1 gene promoted the proliferation of vascular endothelial cells, migration and tubular function (P0.01) tumor growth curve of.3. nude mice. The results showed that LRG1 promoted the growth of transplanted tumor in nude mice (P0.01). The average weight of the overexpressed group was greater than that of the control group (P0.05). The tumor body was made into tissue section and stained with HE. The results showed that the transplanted tumor infiltrated the muscle layer in the overexpressed group. LRG1 promoted the growth of the tumor and the infiltration of.CD34-MVD and VEGFA fluorescence staining demonstrated that the overexpression group promoted the formation of blood vessels. The expression and secretion of VEGFA in the.4.DLD1 cells expressed in the.4.DLD1 cells and the control group were all higher than those in the control group; the VEGFA expression and secretion level decreased by.Western Blot results in the control group compared with the control group. The results showed that LRG1 promoted PI3K/AKT/HIF-1 alpha and Ras/MEK1/ERK1/2 pathway activation. And LRG1 induced HIF-1 alpha from cytoplasm to cytoplasm. After the inhibition of AKT and ERK pathway, the expression of VEGFA in DLD1-LRG1 group decreased, and the formation ability of LRG1 induced endothelial cells was inhibited. Conclusion LRG1 was highly expressed in colorectal cancer tissues, and the expression level was significantly higher than that of normal tissues adjacent to the carcinoma. And the T score of colorectal cancer was compared with that of colorectal cancer. .LRG1 is an independent prognostic factor affecting OS and DFS in colorectal cancer. The positive correlation between.LRG1 and MVD is a significant positive correlation between.LRG1 and MVD, which not only promotes the invasion and migration of tumor cells, but also promotes the proliferation, migration and angiogenic ability of vascular endothelial cells. In vivo tumorigenesis experiments show that LRG1 promotes transplanted tumor. Infiltration to the myometrium also increases the expression of CD34-MVD and VEGFA. In tumor cells, LRG1 can increase the angiogenesis mediated by VEGFA secretion through the PI3K/AKT/HIF1 - and MEK/ERK double pathways.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34

【参考文献】