本文選題：結(jié)直腸癌轉(zhuǎn)移　切入點：失巢凋亡　出處：《浙江大學》2017年博士論文　論文類型：學位論文

【摘要】：研究背景結(jié)直腸癌是常見的消化道惡性腫瘤,發(fā)病率和死亡率在我國分別位居惡性腫瘤第四位和第五位,嚴重威脅著人類的健康。結(jié)直腸癌的擴散和轉(zhuǎn)移是其引起患者高死亡率的主要原因之一。研究顯示,約有四分之一的患者在初次診斷結(jié)直腸癌時已發(fā)生肝轉(zhuǎn)移,而近一半的患者最終都會發(fā)生肝轉(zhuǎn)移,另外腸癌也常發(fā)生肺轉(zhuǎn)移、骨轉(zhuǎn)移、腦轉(zhuǎn)移等。HNF6(HepatocyteNuclearFactors-6)是肝細胞核因子家族的成員之一,作為轉(zhuǎn)錄因子參與多種基因的調(diào)控。目前研究發(fā)現(xiàn)HNF6在胰腺、內(nèi)分泌腺、膽管的形成與分化,以及肝臟損傷修復等過程中具有重要作用。同時近幾年有大量研究發(fā)現(xiàn),HNF6在多種惡性腫瘤的發(fā)生、發(fā)展和轉(zhuǎn)移過中也都發(fā)揮了重要的作用,比如肝癌、乳腺癌和胰腺癌等。而HNF6在結(jié)直腸癌中的作用和機制的研究目前仍非常有限。本課題組前期構(gòu)建了 HNF6過表達的SW620腸癌細胞系,并對其進行了初步的生物學行為影響研究,發(fā)現(xiàn)HNF6過表達的SW620細胞相對于普通SW620細胞在細胞成瘤和遷移功能上有明顯差異,但具體機制尚不明確。研究內(nèi)容本研究通過GEO(Gene Expression Omnibus)數(shù)據(jù)庫分析比較HNF6在轉(zhuǎn)移性結(jié)直腸癌中的表達變化,并結(jié)合我中心腸癌術(shù)后病理標本進行蛋白水平的驗證,以證明HNF6在結(jié)直腸癌轉(zhuǎn)移中發(fā)揮著一定的作用;然后利用本課題組前期構(gòu)建的HNF6過表達的SW620腸癌細胞模型,通過細胞功能實驗說明HNF6對腸癌細胞轉(zhuǎn)移能力的影響;最后,通過盲腸原位裸鼠肝轉(zhuǎn)移模型結(jié)合芯片分析初步探索HNF6引起結(jié)直腸癌轉(zhuǎn)移的分子機制。本研究擬通過以上研究加深對HNF6功能的認識,進一步豐富腸癌轉(zhuǎn)移的分子機制。研究結(jié)果1、HNF6在腸癌轉(zhuǎn)移瘤的表達明顯高于腸癌原發(fā)腫瘤,且提示不良預(yù)后對GEO基因表達數(shù)據(jù)和腸癌病理組織免疫組化結(jié)果分別進行HNF6差異表達分析,發(fā)現(xiàn)HNF6在腸癌轉(zhuǎn)移瘤的表達明顯高于腸癌原發(fā)瘤,而與原發(fā)瘤位置、大小、分化程度、分期,以及腫瘤標志物CEA水平等均無明顯相關(guān)。同時,HNF6高表達提示患者不良預(yù)后。2、HNF6抵抗細胞失巢凋亡,促進結(jié)直腸癌肝轉(zhuǎn)移利用本課題組前期構(gòu)建的HNF6過表達的SW620腸癌細胞系SW620-HNF6,及對照組細胞SW620-control。第一部分結(jié)果表明,HNF6可能與腸癌的轉(zhuǎn)移相關(guān)。因此我們首先進行裸鼠肝轉(zhuǎn)移模型研究,發(fā)現(xiàn)SW620-HNF6組裸鼠肝轉(zhuǎn)移較對照組明顯增多,說明HNF6促進了腸癌肝轉(zhuǎn)移。繼而,通過細胞功能實驗表明,SW620-HNF6細胞遷移能力下降,克隆形成能力提高,而細胞增殖能力較對照組無明顯差異。進一步我們通過懸浮培養(yǎng)模擬細胞失巢環(huán)境,發(fā)現(xiàn)SW620-HNF6細胞的凋亡明顯比SW620-control細胞少,表明HNF6促進了 SW620細胞抵抗失巢凋亡。抵抗失巢凋亡是惡性腫瘤轉(zhuǎn)移的基礎(chǔ),我們推測HNF6通過抵抗細胞失巢凋亡,促進了腸癌肝轉(zhuǎn)移。3、HNF6抵抗細胞失巢凋亡促進轉(zhuǎn)移的機制初探通過Western Blot實驗進行分析發(fā)現(xiàn),SW620-HNF6細胞中,Claudin-1和ZO-1和Src表達明顯上調(diào),而其他轉(zhuǎn)移相關(guān)的分子標記物如β-catenin、E-cadherin、Vimentin、Snail等與對照組細胞相比無明顯差異。根據(jù)文獻報導,在腸癌細胞中,Claudin-1可以與ZO-1、Src形成復合體,通過Src-Akt-Bcl-2抑制腸癌細胞的失巢凋亡。然后我們敲低SW620-HNF6細胞的Claudin-1表達,通過Transwell實驗表明,敲低Claudin-1的表達可明顯逆轉(zhuǎn)HNF6對SW620細胞遷移能力的抑制作用。結(jié)論HNF6在腸癌轉(zhuǎn)移瘤的表達明顯高于腸癌原發(fā)腫瘤,且HNF6高表達提示不良預(yù)后。HNF6的過表達可激活Claudin-1、ZO-1和Src復合體,增強腸癌細胞抵抗失巢凋亡的能力,促進轉(zhuǎn)移的發(fā)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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.34

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