【摘要】：轉移是惡性腫瘤的的主要特征之一,也是惡性腫瘤患者死亡的主要原因。腫瘤的轉移是一個多階段,多步驟的復雜過程。越來越多的研究證據(jù)表明上皮-間質(zhì)轉化(epithelial-mesenchymal transition, EMT)在腫瘤的侵襲和轉移過程中扮演著關鍵和復雜的角色。EMT是指具有上皮表型的細胞通過特定的程序轉化成為具有間質(zhì)表型的生物學過程。上皮細胞通過EMT過程失去細胞極性,與基底膜的連接消失,從而逐漸獲得間質(zhì)細胞的形態(tài)特征以及遷移侵襲能力提高和干性特征增強等生物學功能。EMT發(fā)生的分子機制非常復雜,目前仍未闡明。為了在結直腸癌(colorectal cancer, CRC)中篩選鑒定出新的調(diào)控基因,我們實驗室前期對癌細胞主體和正常腸粘膜上皮細胞進行基因表達譜芯片(Affymetrix人全基因表達譜芯片GeneChip(?) Human Genome U133 Plus 2.0)檢測,成功篩選出了381個差異表達基因。其中,白介素-13(interleukin-13, IL-13)在腫瘤細胞中的表達比正常細胞表達高出2.5倍。IL-13主要是由活化的Th2 (CD4+)細胞分泌的細胞因子,廣泛參與器官纖維化、炎癥和腫瘤的發(fā)生發(fā)展,但它在結直腸癌中的作用和機制還不是很清楚。因此,本研究旨在明確IL-13在結直腸癌細胞中的作用,并探討相關分子機制。我們體外模擬旁分泌途徑,運用人重組IL-13蛋白刺激HT29和SW480細胞后觀察到這兩株細胞的形態(tài)發(fā)現(xiàn)了明顯的變化,從之前的橢圓扁平的多邊形轉變?yōu)樗缮⒌乃笮?這表明IL-13刺激可能促進了結直腸癌細胞上皮間質(zhì)轉化過程的發(fā)生。然后免疫熒光結果顯示IL-13處理HT29和SW480兩株細胞系都能夠下調(diào)上皮標志物E-cadherin的表達,其主要分布在細胞膜；而間質(zhì)標記物Vimentin的表達上調(diào),其主要分布在細胞漿。進一步分析表明,IL-13處理可在mRNA水平和蛋白水平上顯著下調(diào)上皮標志物E-cadherin和ZO-1的表達,上調(diào)間質(zhì)標志物MMP9和Vimentin的表達。另外,調(diào)控EMT的核心轉錄因子ZEB1, ZEB2和Snail的表達在IL-13誘導之后也有不同程度的增加,其中ZEB1的mRNA和蛋白水平變化最明顯。IL-13還能夠明顯促進HT29和SW480細胞的遷移侵襲能力。由此可見,IL-13可以誘導結直腸癌細胞EMT過程的發(fā)生和促進細胞惡性衍化。為了進一步研究參與IL-13誘導EMT的具體通路,我們在IL-13刺激的HT29和SW480細胞中檢測了IL-13下游的常見通路,結果顯示細胞的STAT6和AKT蛋白的磷酸化水平表達顯著增加,而STAT3和Erkl/2的磷酸化水平?jīng)]有明顯變化。運用JAK抑制劑JAK inhibitor 1 (JAKi 1)和P13K抑制劑LY294002分別抑制了IL-13處理引起的STAT6和AKT的磷酸化,其中只有JAKi 1能夠有效阻斷IL-13所引起的EMT標志物變化。此外,JAKi 1還可以有效逆轉IL-13引起的細胞遷移和侵襲能力增強。為了進一步分析STAT6與EMT的關系,我們在HT29細胞和SW480細胞中運用shRNA干擾技術沉默STAT6的表達。結果發(fā)現(xiàn)STAT6干擾后的細胞在IL-13刺激前后都維持上皮樣的特征,EMT標志物和ZEB1的表達不再改變,細胞遷移和侵襲能力也不再增加。以上結果表明STAT6在IL-13誘導EMT的發(fā)生過程和遷移侵襲性增強中是必須的。另外,在另一株STAT6null的結直腸癌細胞Caco2中發(fā)現(xiàn),IL-13不能使該細胞系的EMT指標發(fā)生變化。然而向Caco2細胞中轉入STAT6的表達載體后再用IL-13刺激即可引起EMT指標發(fā)生變化,這就再次證明IL-13引起EMT標志物表達的改變是依賴STAT6通路的激活來完成的。很多研究發(fā)現(xiàn)EMT過程可以使腫瘤細胞具有干細胞的特征,并且腫瘤干細胞被認為是腫瘤侵襲和轉移的主要細胞,是腫瘤復發(fā)的根源。本實驗結果顯示IL-13可以使HT-29和SW480細胞的干性標志物nanog等的表達上調(diào),并且可以被JAKi 1逆轉。在STAT6干擾的HT29和SW480細胞株中IL-13也不能誘導干性標志物的表達增加。這說明IL-13可以引起干細胞標志表達的上調(diào),并且也同樣是受STAT6通路調(diào)控的。隨后,我們在實驗室所有的結直腸癌細胞株中檢測了IL-13的受體IL-13Rα1和IL-13Rα2 mRNA水平的表達。結果表明,IL-13Rα2除了在RKO細胞株中高表達外,在其它的細胞株中都呈現(xiàn)低表達或者不表達,而IL-13Rα1在所有的細胞株中都高表達。然后我們在33例結直腸癌患者的腫瘤組織和配對正常組織cDNA中對IL-13Rα1和IL-13Rα2進行mRNA水平上的表達檢測。結果顯示IL-13Rα1在腫瘤組織中的表達明顯高于其配對的正常組織,而IL-13Rα2在所有樣本中表達都偏低,且腫瘤和正常組織之間表達沒有差異。同時,我們發(fā)現(xiàn)前面細胞實驗用到的HT29和SW480細胞只表達IL-13Rα1,并且JAK/STAT6通路是IL-13Rα1受體介導的主要通路,這也就可以揭示IL-13主要通過IL-13Rα1受體激活STAT6通路促進EMT的發(fā)生和細胞惡性衍化。此外,我們發(fā)現(xiàn)TGF-β和TNF-α誘導的EMT模型可以使IL-13Rα1表達上調(diào),而對IL-13Rα2沒有影響,這就暗示了IL-13和其它的經(jīng)典EMT誘導因子可能協(xié)同參與結直腸癌EMT的發(fā)生,具體機制還需要進一步研究。綜上,本研究揭示了IL-13/IL-13Rα1/STAT6通路在結直腸癌細胞中可以促進EMT的發(fā)生和細胞遷移侵襲能力及干性特征的增加,并藉此提供了新的潛在結直腸癌轉移相關生物標志物和治療藥物分子靶點。
[Abstract]:Metastasis is one of the main features of malignant tumor and the main cause of death in patients with malignant tumors. Metastasis is a multistage, multistep and complex process. More and more evidence shows that epithelial-mesenchymal transition (EMT) plays a key role in the invasion and metastasis of tumor. Complex role.EMT refers to the biological process of transforming cells with epithelial phenotype into an interstitial phenotype through a specific program. Epithelial cells lose the polarity of cells through the EMT process and disappear from the connection of the basement membrane, thus gradually obtaining the morphological characteristics of the stromal cells, the enhancement of migration and invasion, and the enhancement of the dry character. The molecular mechanism of the physical function of.EMT is very complex and is still unexplained. In order to identify new regulatory genes in the colorectal cancer (CRC), the gene expression core (Affymetrix human whole gene expression chip GeneChip (?) Hu) of the cancer cell body and normal intestinal mucosa epithelial cells was carried out in the early laboratory. Man Genome U133 Plus 2) detected 381 differentially expressed genes. Among them, the expression of -13 (interleukin-13, IL-13) in the tumor cells is 2.5 times higher than normal cell expression, which is mainly composed of activated Th2 (CD4+) cells secreted by cells, which are widely involved in organ fibrosis, inflammation and the occurrence of tumor. However, its role and mechanism in colorectal cancer is not clear. Therefore, the purpose of this study is to identify the role of IL-13 in colorectal cancer cells and to explore the related molecular mechanisms. We have observed the paracrine pathway in vitro and the use of recombinant human IL-13 protein to stimulate HT29 and SW480 cells to observe the apparent morphology of these two cells. The change, from the previous Elliptical Flat polygon to loose shuttle shape, indicates that IL-13 stimulation may promote the formation of epithelial mesenchymal transition in rectal cancer cells. Immunofluorescence results show that IL-13 treatment of HT29 and SW480 two cell lines can downregulate the expression of the epithelial marker E-cadherin, which is mainly distributed in the cells. The expression of the interstitial marker Vimentin was up-regulated and mainly distributed in the cytoplasm. Further analysis showed that IL-13 treatment could significantly reduce the expression of E-cadherin and ZO-1 on the mRNA and protein levels, up the expression of the interstitial markers MMP9 and Vimentin. In addition, it regulates the core transcription factors of EMT, ZEB1, ZEB2, and Snail. The expression of ZEB1 is also increased in varying degrees after IL-13 induction, in which the most obvious changes in the level of mRNA and protein of ZEB1 also significantly promote the migration and invasion of HT29 and SW480 cells. Thus, IL-13 can induce the occurrence of EMT process in colorectal cancer cells and promote the proliferation of cell malignant cells. In order to further study the participation of IL-13 Inducing the specific pathway of EMT, we detected the common pathway in the downstream of IL-13 in IL-13 stimulated HT29 and SW480 cells. The results showed that the expression of phosphorylation of STAT6 and AKT proteins increased significantly, while the phosphorylation level of STAT3 and Erkl/2 was not significantly changed. JAK inhibitor JAK inhibitor 1 (1) and the inhibitors were used. 2 inhibits the phosphorylation of STAT6 and AKT caused by IL-13 treatment, and only JAKi 1 can effectively block the change of EMT markers caused by IL-13. In addition, JAKi 1 can also effectively reverse the cell migration and invasion enhancement caused by IL-13. In order to further analyze the relationship between STAT6 and EMT, we use both HT29 and SW480 cells. HRNA interference technique was used to silence the expression of STAT6. The results showed that the cells after the STAT6 interference were all epithelioid before and after the IL-13 stimulation, the expression of EMT markers and ZEB1 no longer changed, and the cell migration and invasion ability no longer increased. The above results indicated that STAT6 was necessary in the progression of EMT induced by IL-13 and the enhancement of migration and invasiveness. In addition, in another STAT6null colon cancer cell Caco2, it was found that IL-13 did not change the EMT index of the cell line. However, the IL-13 stimulation could lead to a change in the EMT index after the transfer of the STAT6 expression vector into the Caco2 cell, which again demonstrated that the change in the expression of EMT markers caused by IL-13 is dependent on the STAT6 pathway. Many studies have found that the EMT process can make the tumor cells have the characteristics of stem cells, and the tumor stem cells are considered to be the main cells of tumor invasion and metastasis, and the root of the tumor recurrence. The results of this experiment show that IL-13 can increase the expression of Nanog, the dry marker of HT-29 and SW480 cells, and can be made by J. AKi 1 was reversed. IL-13 did not induce an increase in the expression of dry markers in STAT6 interfered HT29 and SW480 cells. This indicates that IL-13 can cause up regulation of the expression of stem cell markers, and is also regulated by the STAT6 pathway. Then, we detected IL-13 receptor IL-13R alpha 1 and IL- in all colorectal cancer cell lines in the laboratory. The expression of 13R alpha 2 mRNA level. The results showed that IL-13R alpha 2, in addition to high expression in RKO cell lines, showed low expression or non expression in other cell lines, and IL-13R alpha 1 was highly expressed in all cell lines. Then we were in 33 cases of colorectal cancer and paired normal tissue cDNA for IL-13R a 1 and IL-13R alpha 2. The expression in the mRNA level was detected. The results showed that the expression of IL-13R alpha 1 in the tumor tissues was significantly higher than that of the normal tissue, but the expression of IL-13R alpha 2 was low in all samples, and there was no difference in the expression between the tumor and the normal tissue. At the same time, we found that the HT29 and SW480 cells used in the anterior cells were only expressed as IL-13R alpha 1. And the JAK/STAT6 pathway is the main pathway mediated by IL-13R alpha 1 receptor, which can also reveal that IL-13 promotes the occurrence of EMT and cell malignancy through the activation of the STAT6 pathway by the IL-13R alpha 1 receptor. In addition, we have found that the EMT model induced by TGF- beta and TNF- alpha can make the IL-13R alpha 1 expressed in the IL-13R alpha 1, but there is no effect on IL-13R alpha 2, which implies IL. -13 and other classical EMT inducible factors may be involved in the occurrence of EMT in colorectal cancer. The specific mechanism needs further study. To sum up, this study reveals that the IL-13/IL-13R alpha 1/STAT6 pathway promotes the occurrence of EMT in colorectal cancer cells and the increase of cell migration and invasion and dry characteristics, and provides a new potential. Metastasis related biomarkers and therapeutic molecular targets for colorectal cancer.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.34

【相似文獻】

相關期刊論文 前10條

1 董超;劉迪;馮業(yè)童;劉朋飛;吳璇;吳昊昱;周余來;;結直腸癌細胞外泌體的制備及其促單核細胞增殖作用[J];吉林大學學報(醫(yī)學版);2013年03期

2 張義平;鄭美蓉;殷嫦嫦;吳萍;周許峰;吳建芳;周小鷗;;熱休克誘導結直腸癌細胞外泌體的免疫效應[J];腫瘤防治研究;2011年07期

3 崔濱濱,劉明,趙鵬,趙家宏,董新舒;重組腺病毒介導的野生型p53基因?qū)Y直腸癌細胞的放射治療增敏作用[J];中華外科雜志;2005年15期

4 耿焱;姜泊;王偉;馬強;;四分子交聯(lián)體5表達上調(diào)對結直腸癌細胞轉移能力的促進作用觀察[J];解放軍醫(yī)學雜志;2010年06期

5 楊曉東;邢春根;吳永友;趙奎;龔巍;陳博;何騰飛;;結直腸癌細胞輻射敏感性的定量比較蛋白組學研究[J];原子能科學技術;2010年09期

6 張繼民,劉明姬,n澗托，

本文編號：2164130