IL-13在结直肠癌细胞上皮间质转化中的作用及相关机制研究
发布时间：2018-08-04 14:07
【摘要】：转移是恶性肿瘤的的主要特征之一,也是恶性肿瘤患者死亡的主要原因。肿瘤的转移是一个多阶段,多步骤的复杂过程。越来越多的研究证据表明上皮-间质转化(epithelial-mesenchymal transition, EMT)在肿瘤的侵袭和转移过程中扮演着关键和复杂的角色。EMT是指具有上皮表型的细胞通过特定的程序转化成为具有间质表型的生物学过程。上皮细胞通过EMT过程失去细胞极性,与基底膜的连接消失,从而逐渐获得间质细胞的形态特征以及迁移侵袭能力提高和干性特征增强等生物学功能。EMT发生的分子机制非常复杂,目前仍未阐明。为了在结直肠癌(colorectal cancer, CRC)中筛选鉴定出新的调控基因,我们实验室前期对癌细胞主体和正常肠粘膜上皮细胞进行基因表达谱芯片(Affymetrix人全基因表达谱芯片GeneChip(?) Human Genome U133 Plus 2.0)检测,成功筛选出了381个差异表达基因。其中,白介素-13(interleukin-13, IL-13)在肿瘤细胞中的表达比正常细胞表达高出2.5倍。IL-13主要是由活化的Th2 (CD4+)细胞分泌的细胞因子,广泛参与器官纤维化、炎症和肿瘤的发生发展,但它在结直肠癌中的作用和机制还不是很清楚。因此,本研究旨在明确IL-13在结直肠癌细胞中的作用,并探讨相关分子机制。我们体外模拟旁分泌途径,运用人重组IL-13蛋白刺激HT29和SW480细胞后观察到这两株细胞的形态发现了明显的变化,从之前的椭圆扁平的多边形转变为松散的梭形,这表明IL-13刺激可能促进了结直肠癌细胞上皮间质转化过程的发生。然后免疫荧光结果显示IL-13处理HT29和SW480两株细胞系都能够下调上皮标志物E-cadherin的表达,其主要分布在细胞膜；而间质标记物Vimentin的表达上调,其主要分布在细胞浆。进一步分析表明,IL-13处理可在mRNA水平和蛋白水平上显著下调上皮标志物E-cadherin和ZO-1的表达,上调间质标志物MMP9和Vimentin的表达。另外,调控EMT的核心转录因子ZEB1, ZEB2和Snail的表达在IL-13诱导之后也有不同程度的增加,其中ZEB1的mRNA和蛋白水平变化最明显。IL-13还能够明显促进HT29和SW480细胞的迁移侵袭能力。由此可见,IL-13可以诱导结直肠癌细胞EMT过程的发生和促进细胞恶性衍化。为了进一步研究参与IL-13诱导EMT的具体通路,我们在IL-13刺激的HT29和SW480细胞中检测了IL-13下游的常见通路,结果显示细胞的STAT6和AKT蛋白的磷酸化水平表达显著增加,而STAT3和Erkl/2的磷酸化水平没有明显变化。运用JAK抑制剂JAK inhibitor 1 (JAKi 1)和P13K抑制剂LY294002分别抑制了IL-13处理引起的STAT6和AKT的磷酸化,其中只有JAKi 1能够有效阻断IL-13所引起的EMT标志物变化。此外,JAKi 1还可以有效逆转IL-13引起的细胞迁移和侵袭能力增强。为了进一步分析STAT6与EMT的关系,我们在HT29细胞和SW480细胞中运用shRNA干扰技术沉默STAT6的表达。结果发现STAT6干扰后的细胞在IL-13刺激前后都维持上皮样的特征,EMT标志物和ZEB1的表达不再改变,细胞迁移和侵袭能力也不再增加。以上结果表明STAT6在IL-13诱导EMT的发生过程和迁移侵袭性增强中是必须的。另外,在另一株STAT6null的结直肠癌细胞Caco2中发现,IL-13不能使该细胞系的EMT指标发生变化。然而向Caco2细胞中转入STAT6的表达载体后再用IL-13刺激即可引起EMT指标发生变化,这就再次证明IL-13引起EMT标志物表达的改变是依赖STAT6通路的激活来完成的。很多研究发现EMT过程可以使肿瘤细胞具有干细胞的特征,并且肿瘤干细胞被认为是肿瘤侵袭和转移的主要细胞,是肿瘤复发的根源。本实验结果显示IL-13可以使HT-29和SW480细胞的干性标志物nanog等的表达上调,并且可以被JAKi 1逆转。在STAT6干扰的HT29和SW480细胞株中IL-13也不能诱导干性标志物的表达增加。这说明IL-13可以引起干细胞标志表达的上调,并且也同样是受STAT6通路调控的。随后,我们在实验室所有的结直肠癌细胞株中检测了IL-13的受体IL-13Rα1和IL-13Rα2 mRNA水平的表达。结果表明,IL-13Rα2除了在RKO细胞株中高表达外,在其它的细胞株中都呈现低表达或者不表达,而IL-13Rα1在所有的细胞株中都高表达。然后我们在33例结直肠癌患者的肿瘤组织和配对正常组织cDNA中对IL-13Rα1和IL-13Rα2进行mRNA水平上的表达检测。结果显示IL-13Rα1在肿瘤组织中的表达明显高于其配对的正常组织,而IL-13Rα2在所有样本中表达都偏低,且肿瘤和正常组织之间表达没有差异。同时,我们发现前面细胞实验用到的HT29和SW480细胞只表达IL-13Rα1,并且JAK/STAT6通路是IL-13Rα1受体介导的主要通路,这也就可以揭示IL-13主要通过IL-13Rα1受体激活STAT6通路促进EMT的发生和细胞恶性衍化。此外,我们发现TGF-β和TNF-α诱导的EMT模型可以使IL-13Rα1表达上调,而对IL-13Rα2没有影响,这就暗示了IL-13和其它的经典EMT诱导因子可能协同参与结直肠癌EMT的发生,具体机制还需要进一步研究。综上,本研究揭示了IL-13/IL-13Rα1/STAT6通路在结直肠癌细胞中可以促进EMT的发生和细胞迁移侵袭能力及干性特征的增加,并藉此提供了新的潜在结直肠癌转移相关生物标志物和治疗药物分子靶点。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34
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