HNF6抵抗细胞失巢凋亡促进结直肠癌转移

发布时间：2018-03-06 09:40

本文选题：结直肠癌转移　切入点：失巢凋亡　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景结直肠癌是常见的消化道恶性肿瘤,发病率和死亡率在我国分别位居恶性肿瘤第四位和第五位,严重威胁着人类的健康。结直肠癌的扩散和转移是其引起患者高死亡率的主要原因之一。研究显示,约有四分之一的患者在初次诊断结直肠癌时已发生肝转移,而近一半的患者最终都会发生肝转移,另外肠癌也常发生肺转移、骨转移、脑转移等。HNF6(HepatocyteNuclearFactors-6)是肝细胞核因子家族的成员之一,作为转录因子参与多种基因的调控。目前研究发现HNF6在胰腺、内分泌腺、胆管的形成与分化,以及肝脏损伤修复等过程中具有重要作用。同时近几年有大量研究发现,HNF6在多种恶性肿瘤的发生、发展和转移过中也都发挥了重要的作用,比如肝癌、乳腺癌和胰腺癌等。而HNF6在结直肠癌中的作用和机制的研究目前仍非常有限。本课题组前期构建了 HNF6过表达的SW620肠癌细胞系,并对其进行了初步的生物学行为影响研究,发现HNF6过表达的SW620细胞相对于普通SW620细胞在细胞成瘤和迁移功能上有明显差异,但具体机制尚不明确。研究内容本研究通过GEO(Gene Expression Omnibus)数据库分析比较HNF6在转移性结直肠癌中的表达变化,并结合我中心肠癌术后病理标本进行蛋白水平的验证,以证明HNF6在结直肠癌转移中发挥着一定的作用;然后利用本课题组前期构建的HNF6过表达的SW620肠癌细胞模型,通过细胞功能实验说明HNF6对肠癌细胞转移能力的影响;最后,通过盲肠原位裸鼠肝转移模型结合芯片分析初步探索HNF6引起结直肠癌转移的分子机制。本研究拟通过以上研究加深对HNF6功能的认识,进一步丰富肠癌转移的分子机制。研究结果1、HNF6在肠癌转移瘤的表达明显高于肠癌原发肿瘤,且提示不良预后对GEO基因表达数据和肠癌病理组织免疫组化结果分别进行HNF6差异表达分析,发现HNF6在肠癌转移瘤的表达明显高于肠癌原发瘤,而与原发瘤位置、大小、分化程度、分期,以及肿瘤标志物CEA水平等均无明显相关。同时,HNF6高表达提示患者不良预后。2、HNF6抵抗细胞失巢凋亡,促进结直肠癌肝转移利用本课题组前期构建的HNF6过表达的SW620肠癌细胞系SW620-HNF6,及对照组细胞SW620-control。第一部分结果表明,HNF6可能与肠癌的转移相关。因此我们首先进行裸鼠肝转移模型研究,发现SW620-HNF6组裸鼠肝转移较对照组明显增多,说明HNF6促进了肠癌肝转移。继而,通过细胞功能实验表明,SW620-HNF6细胞迁移能力下降,克隆形成能力提高,而细胞增殖能力较对照组无明显差异。进一步我们通过悬浮培养模拟细胞失巢环境,发现SW620-HNF6细胞的凋亡明显比SW620-control细胞少,表明HNF6促进了 SW620细胞抵抗失巢凋亡。抵抗失巢凋亡是恶性肿瘤转移的基础,我们推测HNF6通过抵抗细胞失巢凋亡,促进了肠癌肝转移。3、HNF6抵抗细胞失巢凋亡促进转移的机制初探通过Western Blot实验进行分析发现,SW620-HNF6细胞中,Claudin-1和ZO-1和Src表达明显上调,而其他转移相关的分子标记物如β-catenin、E-cadherin、Vimentin、Snail等与对照组细胞相比无明显差异。根据文献报导,在肠癌细胞中,Claudin-1可以与ZO-1、Src形成复合体,通过Src-Akt-Bcl-2抑制肠癌细胞的失巢凋亡。然后我们敲低SW620-HNF6细胞的Claudin-1表达,通过Transwell实验表明,敲低Claudin-1的表达可明显逆转HNF6对SW620细胞迁移能力的抑制作用。结论HNF6在肠癌转移瘤的表达明显高于肠癌原发肿瘤,且HNF6高表达提示不良预后。HNF6的过表达可激活Claudin-1、ZO-1和Src复合体,增强肠癌细胞抵抗失巢凋亡的能力,促进转移的发生。
[Abstract]:Background: colorectal carcinoma is a common malignant tumor of digestive tract, the incidence and mortality in our country were ranked fourth and fifth malignant tumor, a serious threat to human health. The proliferation and metastasis of colorectal cancer is the major cause of high mortality of patients. The study showed that about 1/4 of the patients in the the initial diagnosis of colorectal cancer had liver metastasis, while nearly half of the patients will eventually develop liver metastases of colorectal cancer, also often had lung metastasis, bone metastasis, brain metastasis of.HNF6 (HepatocyteNuclearFactors-6) is a member of the hepatocyte nuclear factor family, as a regulatory transcription factor involved in many genes. The study found that HNF6 in pancreatic endocrine. Gland formation and differentiation of the bile duct, and plays an important role in liver injury repair process. At the same time, in recent years, numerous studies have found that HNF6, in a variety of malignant tumor Tumor occurrence, development and metastasis also played an important role, such as liver cancer, breast cancer and pancreatic cancer. The study of role and mechanism of HNF6 in colorectal cancer is still very limited. Ourprevious constructed SW620 colorectal cancer cell lines HNF6 overexpression, and has carried on the study on the influence of the biological behavior of preliminary, the over expression of HNF6 SW620 cells in normal SW620 cells into tumor cells have obvious difference and migration function with respect to, but the specific mechanism is not clear. This research by GEO (Gene Expression Omnibus) expression data analysis and comparison of HNF6 in metastatic colorectal cancer, and I was confirmed by postoperative pathology were center protein level, to prove that HNF6 play a role in the metastasis of colorectal cancer; then the overexpression of ourprevious constructed HNF6 SW620 Model of colon cancer cells, through the cell function experiments show that effect of HNF6 on metastasis of colon cancer cells; finally, combined with the study of HNF6 chip caused by the molecular mechanism of node metastasis of rectal carcinoma by orthotopic liver metastasis in nude mice model. This study through the above research to deepen understanding of the function of HNF6, further enrich the molecular mechanism of metastasis in colorectal cancer. The results of the study in 1, HNF6 in colon cancer metastasis was significantly higher than that in primary colorectal cancer, and the prognostic factors of colorectal cancer GEO gene expression data and immunohistochemistry results were HNF6 differential expression analysis, HNF6 was found in colorectal cancer metastases was significantly higher than that of the primary colorectal tumor and primary tumor location, size and the degree of differentiation, staging, tumor markers and CEA levels were not significantly related. At the same time, the high expression of HNF6 suggests poor prognosis in patients with.2, HNF6 cell anoikis resistance, Promote the liver metastasis of colorectal cancer by using the previous construction of HNF6 over expression of SW620 in human colorectal cancer cell line SW620-HNF6, and the cells in the control group the first part of the SW620-control. results show that HNF6 may transfer and colorectal cancer. Therefore we first model of liver metastases in nude mice, SW620-HNF6 group liver metastasis in nude mice was significantly increased compared with control group, HNF6 promote the hepatic metastasis of colorectal carcinoma. Then, through the cell function experiments show that SW620-HNF6 cell migration ability, colony forming ability and improve the ability of cell proliferation, compared with the control group, no significant difference. We further through suspension culture cell anoikis simulation environment, found that the apoptosis of SW620-HNF6 cells significantly less than SW620-control cells, showed that HNF6 promoted SW620 cells resistance to anoikis. Anoikis resistance is the basis of tumor metastasis, we speculate that HNF6 through the resistance loss of cells Nest apoptosis, promote hepatic metastasis of colorectal carcinoma.3 HNF6 cell anoikis resistance, promote the mechanism of metastasis were analyzed by Western Blot experiment, SW620-HNF6 cells, Claudin-1 and ZO-1 and Src expression was up-regulated, while other metastasis related molecular markers such as beta -catenin, E-cadherin, Vimentin, Snail and control group no significant difference in comparison. According to reports, in colon cancer cells, Claudin-1 and ZO-1, Src complex formation, inhibition of cancer cell anoikis by Src-Akt-Bcl-2 expression. Then we knockdown SW620-HNF6 cells Claudin-1 through Transwell experiments show that knockdown Claudin-1 protein can significantly reverse the HNF6 migration ability of SW620 cells inhibition of metastasis in colorectal cancer. Conclusion HNF6 expression was significantly higher than that of the primary colorectal cancer, and the high expression of HNF6 over expression of prognostic factors of.HNF6 can activate Claud IN-1, ZO-1 and Src complexes enhance the ability of colon cancer cells to resist anoikis and promote metastasis.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34

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