JWA通过整合素信号通路调节黑色素瘤转移的分子机制研究
发布时间：2018-09-05 17:01
【摘要】：肿瘤转移是癌症病人死亡的主要原因。肿瘤转移过程由几个独立的步骤组成,包括肿瘤细胞从原发灶脱离,肿瘤细胞侵袭到血管内皮或淋巴管从而进入循环系统,最后,在新的宿主环境中,粘附并再侵袭,增殖成为一个转移瘤。大量研究显示,在肿瘤转移的多步骤过程中,许多信号分子被激活并发生相互作用,形成复杂的信号网络。研究和破解控制肿瘤转移的信号网络和关键调节机制,阻止肿瘤转移,是提高肿瘤治疗效果的根本所在,也是当前国内外肿瘤学家企图攻克的最主要的堡垒。 细胞迁移是肿瘤细胞转移的重要基础,细胞骨架的重组是细胞发生迁移的必要条件,因此,调节细胞骨架重组的分子可能在肿瘤细胞转移中发挥重要作用。JWA是新近发现的微管和微丝结合蛋白,可调节两种细胞骨架的重组。本课题组研究证明,JWA表达缺陷可以阻断As2O3对HeLa细胞迁移的抑制,但是可以加强PMA(佛波酯)对细胞迁移的作用。进一步研究发现JWA对细胞迁移的调节作用是通过激活MAPK信号通路不同下游分子以及对微丝重组的作用而实现的。本研究发现,JWA在黑色素瘤转移过程中起到非常重要的作用。我们的结果证明JWA敲减(knock down)可以增加黑色素瘤细胞的迁移、粘附和侵袭能力;体内实验证实在小鼠黑色素瘤B16-F10细胞和人黑色素瘤A375细胞中敲减JWA后显著促进转移瘤的形成和生长;此外,在黑色素瘤病人的肿瘤组织中,JWA在恶性黑色素瘤的表达水平显著低于良性痣。最后,我们的研究证实JWA的上述作用是通过整合素信号通路来实现的,当干涉转录因子Sp1后,就破坏了JWA对整合素分子integrinα_Vβ_3及其下游分子的调节作用,从而逆转JWA对肿瘤转移过程的影响。这些发现表明JWA是抑制黑色素瘤转移的重要靶分子。 目标:探讨JWA在调节黑色素瘤转移过程多个环节中的作用及其分子机制。 方法:本研究使用有转移性的小鼠黑色素瘤细胞株B16-F10和人黑色素瘤细胞株A375来进行体内外研究。通过稳定筛选得到JWA低表达的细胞株(B16-As和A375-As)和载体对照的细胞株(B16-Vec和A375-Vec)。首先,我们使用小鼠尾静脉肿瘤转移实验来检验JWA在黑色素瘤体内转移中的作用。接着我们通过划痕实验、Transwell迁移实验和免疫荧光等来研究JWA表达对肿瘤细胞转移过程多个环节的影响及其机制。用粘附实验、侵袭实验和明胶酶谱实验检测JWA是否调节细胞的粘附和侵袭能力。用免疫印迹和RNA干涉等技术探讨了JWA调节肿瘤转移过程的信号通路分子变化规律。最后,我们收集了13例良性痣、5例恶变痣和13例恶性黑色素瘤,用免疫组化方法检测JWA、integrinαV和β3在组织中的表达,初步验证这些分子的表达水平与人黑色素瘤恶性程度的关系。 结果: 1.在体内模型中,将JWA表达敲减的B16-F10细胞和载体对照细胞通过尾静脉注入小鼠21天后,发现JWA表达缺陷组动物肺转移灶形成数显著高于载体对照组;将JWA表达缺陷和载体对照的人黑色素瘤细胞株A375-As和A375-Vec两组细胞注射入免疫缺陷小鼠的尾静脉中,30天后发现在JWA表达缺陷组中40%的小鼠颈部淋巴结有明显的转移瘤,但在载体对照组未发现明显的颈部转移瘤。通过细胞增殖实验检测了几组细胞的增殖能力,结果发现JWA表达缺陷组和载体对照组的细胞增长速度基本相同。提示JWA缺陷组肿瘤转移灶的增加可能不是由于转染了pEGFP-C1-As JWA质粒造成的细胞增殖能力的增加,而是由于JWA低表达增强了肿瘤转移能力所致。 2.通过划痕实验和Transwell迁移实验,发现JWA表达缺陷的细胞与载体对照的细胞相比迁移能力显著增强。免疫荧光的结果显示JWA表达缺陷组细胞周围的肌动蛋白丝明显多于载体对照组,细胞表现出一种更强的移动能力。 3.粘附实验发现在纤维粘连蛋白和玻璃粘连蛋白包被的板子上,B16-As细胞粘附能力分别升高了103%和69%,A375细胞粘附能力分别提高了75%和58%。 4.细胞侵袭能力试验发现在JWA表达缺陷B16-As和A375-sh细胞中,通过侵袭作用穿过Matrigel包被的Boyden小室的细胞数目分别增加了173%和79%。明胶酶谱法检测黑色素瘤细胞中基质金属蛋白酶的活性的结果显示,B16-As和B16-Vec组细胞的MMP-9活性水平并没有明显改变,但是在B16-As组细胞中MMP-2的活性比载体对照上升了4.5倍。 5.在机制研究中发现当干涉JWA表达后,integrinαVβ3信号通路被激活。进一步研究发现,在JWA表达缺陷的B16和A375细胞中通过siRNA和LM609阻断integrinαVβ3信号通路可以降低JWA调节的细胞粘附和侵袭能力。在探讨JWA如何调节integrinαVβ3信号通路时发现,尽管integrinαVβ3启动子区存在很多转录因子的结合位点,但我们分别干涉后却发现只有将B16-As和A375-sh细胞中的Sp1核转录因子干涉后,才能削弱JWA对integrinαVβ3信号通路的调节作用。随着integrinαVβ3蛋白表达水平的降低,肿瘤细胞粘附和侵袭的能力也显著下降。 6.用免疫组织化学方法检测了良性痣、恶变痣和黑色素瘤组织中JWA、integrinαV和β3的表达水平。结果发现,在良性痣中JWA的表达水平较高,而在恶变痣和黑色素瘤中JWA的表达却非常低。而integrinαV和β3在恶变痣和黑色素瘤中的的表达水平则正好与此相反,即JWA的染色强度与integrinαV和β3的染色强度存在一种非常显著的负相关关系。 结论:总而言之,在本研究中,我们发现黑色素瘤细胞在干涉JWA以后,肿瘤细胞的迁移、粘附、侵袭和体内的转移能力都显著增强。这可能是由于JWA通过转录因子Sp1来调节integrinαVβ3信号通路,从而增加肿瘤细胞的转移潜能。我们的工作对以后研究integrinαVβ3信号通路在肿瘤转移中的作用提供了重要的线索。同时,JWA也有可能成为抗肿瘤转移的靶点或观察肿瘤患者预后和转移的生物标志物。
[Abstract]:Tumor metastasis is the leading cause of death in cancer patients. The process of tumor metastasis consists of several independent steps, including the detachment of tumor cells from the primary focus, the invasion of tumor cells into the vascular endothelium or lymphatic vessels and into the circulatory system. Finally, in the new host environment, adhesion and re-invasion, proliferation into a metastatic tumor. It is indicated that many signal molecules are activated and interacted in the multistep process of tumor metastasis to form complex signal networks. To study and decipher the signal networks and key regulatory mechanisms controlling tumor metastasis and prevent tumor metastasis is the fundamental to improve the therapeutic effect of tumor, and is also the current attempt of oncologists at home and abroad to conquer it. The most important fortress.
Cell migration is an important basis for tumor cell metastasis, and cytoskeleton reorganization is a necessary condition for cell migration. Therefore, molecules regulating cytoskeleton reorganization may play an important role in tumor cell metastasis. JWA is a recently discovered microtubule and microfilament binding protein that can regulate the reorganization of two cytoskeletons. It was found that JWA deficiency could block the migration inhibition of HeLa cells by As2O3, but could enhance the migration of HeLa cells by PMA (verbose ester). Our results demonstrate that knock down of JWA can increase the migration, adhesion and invasion of melanoma cells; knock down of JWA in murine melanoma B16-F10 cells and human melanoma A375 cells can significantly promote the formation and growth of metastatic tumors. In melanoma patients, the expression of JWA in malignant melanoma was significantly lower than that in benign nevus. Finally, our study confirmed that the above effect of JWA was achieved through the integrin signaling pathway. When interfering with the transcription factor Sp1, the regulation of JWA on integrin alpha_Vbeta_3 and its downstream molecules was disrupted. These findings suggest that JWA is an important target molecule for inhibiting the metastasis of melanoma.
Objective: To explore the role and molecular mechanism of JWA in regulating multiple stages of melanoma metastasis.
Methods: In this study, metastatic murine melanoma cell lines B16-F10 and human melanoma cell line A375 were used for in vitro and in vivo studies. JWA low-expression cell lines (B16-As and A375-As) and vector-controlled cell lines (B16-Vec and A375-Vec) were obtained by stable screening. First, we used the mouse tail vein tumor metastasis test to detect the tumor. To investigate the role of JWA in the metastasis of melanoma in vivo.Then we studied the effect of JWA expression on several links of metastasis and its mechanism by scratch test,transwell migration test and immunofluorescence.Adhesion test,invasion test and gelatinase zymogram test were used to determine whether JWA regulated the adhesion and invasion of melanoma cells. Finally, we collected 13 benign nevus, 5 malignant nevus and 13 malignant melanoma, detected the expression of JWA, integrin alpha V and beta 3 in tissues by immunohistochemistry, and preliminarily verified the expression level of these molecules in human melanoma. The relationship between malignant degree of melanoma.
Result:
1. In vivo model, B16-F10 cells with JWA knock-down and vector-control cells were injected into tail vein of mice for 21 days, and the number of lung metastases in JWA-deficient group was significantly higher than that in vector-control group. In the caudal vein of the trapped mice, 40% of the cervical lymph nodes in the JWA-deficient mice were found to have metastatic tumors 30 days later, but no metastatic tumors were found in the carrier control group. It is suggested that the increase of metastasis in JWA deficiency group may not be due to the increase of cell proliferation induced by transfection of pEGFP-C1-As JWA plasmid, but to the enhancement of tumor metastasis caused by low expression of JWA.
2. Scratch test and Transwell migration test showed that the migration ability of JWA-deficient cells was significantly enhanced compared with that of vector-control cells.
3. Adhesion test showed that the adhesion ability of B16-As cells increased by 103% and 69% respectively, and that of A375 cells increased by 75% and 58% respectively.
4. Cell invasiveness test showed that the number of cells penetrating Matrigel-coated Boyden cells increased by 173% and 79% respectively in JWA-deficient B16-As and A375-sh cells. Matrix metalloproteinase activity in B16-As and B16-Vec melanoma cells was detected by gelatin zymography. The level of MMP-2 did not change significantly, but the activity of MMP-2 in group B16-As was 4.5 times higher than that in vector control.
5. Integrin alpha V beta 3 signaling pathway was activated when JWA was interfered with. Further studies showed that blocking integrin alpha V beta 3 signaling pathway by siRNA and LM609 in B16 and A375 cells with JWA deficiency could reduce JWA-regulated cell adhesion and invasion. It was found that although there were many binding sites of integrin alpha V beta 3 promoter, only by interfering with SP1 nuclear transcription factors in B16-As and A375-sh cells could the regulation of JWA on integrin alpha V beta 3 signaling pathway be weakened. The ability of cell adhesion and invasion also decreased significantly.
6. Immunohistochemistry was used to detect the expression of JWA, integrin alpha V and beta 3 in benign nevus, malignant nevus and melanoma. The results showed that the expression of JWA was high in benign nevus, but very low in malignant nevus and melanoma. On the contrary, there is a very significant negative correlation between the dyeing strength of JWA and that of integrin alpha V and beta 3.
CONCLUSION: In conclusion, in this study, we found that the migration, adhesion, invasion and in vivo metastasis of melanoma cells were significantly enhanced after JWA intervention. This may be due to the fact that JWA regulates the integrin alpha V beta 3 signaling pathway through the transcription factor Sp1, thereby increasing the metastatic potential of tumor cells. Posterior studies on the role of integrin alpha V beta 3 signaling pathway in tumor metastasis provide important clues. At the same time, JWA may also become a target of anti-tumor metastasis or a biomarker of tumor prognosis and metastasis.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.5
本文编号：2224883
[Abstract]:Tumor metastasis is the leading cause of death in cancer patients. The process of tumor metastasis consists of several independent steps, including the detachment of tumor cells from the primary focus, the invasion of tumor cells into the vascular endothelium or lymphatic vessels and into the circulatory system. Finally, in the new host environment, adhesion and re-invasion, proliferation into a metastatic tumor. It is indicated that many signal molecules are activated and interacted in the multistep process of tumor metastasis to form complex signal networks. To study and decipher the signal networks and key regulatory mechanisms controlling tumor metastasis and prevent tumor metastasis is the fundamental to improve the therapeutic effect of tumor, and is also the current attempt of oncologists at home and abroad to conquer it. The most important fortress.
Cell migration is an important basis for tumor cell metastasis, and cytoskeleton reorganization is a necessary condition for cell migration. Therefore, molecules regulating cytoskeleton reorganization may play an important role in tumor cell metastasis. JWA is a recently discovered microtubule and microfilament binding protein that can regulate the reorganization of two cytoskeletons. It was found that JWA deficiency could block the migration inhibition of HeLa cells by As2O3, but could enhance the migration of HeLa cells by PMA (verbose ester). Our results demonstrate that knock down of JWA can increase the migration, adhesion and invasion of melanoma cells; knock down of JWA in murine melanoma B16-F10 cells and human melanoma A375 cells can significantly promote the formation and growth of metastatic tumors. In melanoma patients, the expression of JWA in malignant melanoma was significantly lower than that in benign nevus. Finally, our study confirmed that the above effect of JWA was achieved through the integrin signaling pathway. When interfering with the transcription factor Sp1, the regulation of JWA on integrin alpha_Vbeta_3 and its downstream molecules was disrupted. These findings suggest that JWA is an important target molecule for inhibiting the metastasis of melanoma.
Objective: To explore the role and molecular mechanism of JWA in regulating multiple stages of melanoma metastasis.
Methods: In this study, metastatic murine melanoma cell lines B16-F10 and human melanoma cell line A375 were used for in vitro and in vivo studies. JWA low-expression cell lines (B16-As and A375-As) and vector-controlled cell lines (B16-Vec and A375-Vec) were obtained by stable screening. First, we used the mouse tail vein tumor metastasis test to detect the tumor. To investigate the role of JWA in the metastasis of melanoma in vivo.Then we studied the effect of JWA expression on several links of metastasis and its mechanism by scratch test,transwell migration test and immunofluorescence.Adhesion test,invasion test and gelatinase zymogram test were used to determine whether JWA regulated the adhesion and invasion of melanoma cells. Finally, we collected 13 benign nevus, 5 malignant nevus and 13 malignant melanoma, detected the expression of JWA, integrin alpha V and beta 3 in tissues by immunohistochemistry, and preliminarily verified the expression level of these molecules in human melanoma. The relationship between malignant degree of melanoma.
Result:
1. In vivo model, B16-F10 cells with JWA knock-down and vector-control cells were injected into tail vein of mice for 21 days, and the number of lung metastases in JWA-deficient group was significantly higher than that in vector-control group. In the caudal vein of the trapped mice, 40% of the cervical lymph nodes in the JWA-deficient mice were found to have metastatic tumors 30 days later, but no metastatic tumors were found in the carrier control group. It is suggested that the increase of metastasis in JWA deficiency group may not be due to the increase of cell proliferation induced by transfection of pEGFP-C1-As JWA plasmid, but to the enhancement of tumor metastasis caused by low expression of JWA.
2. Scratch test and Transwell migration test showed that the migration ability of JWA-deficient cells was significantly enhanced compared with that of vector-control cells.
3. Adhesion test showed that the adhesion ability of B16-As cells increased by 103% and 69% respectively, and that of A375 cells increased by 75% and 58% respectively.
4. Cell invasiveness test showed that the number of cells penetrating Matrigel-coated Boyden cells increased by 173% and 79% respectively in JWA-deficient B16-As and A375-sh cells. Matrix metalloproteinase activity in B16-As and B16-Vec melanoma cells was detected by gelatin zymography. The level of MMP-2 did not change significantly, but the activity of MMP-2 in group B16-As was 4.5 times higher than that in vector control.
5. Integrin alpha V beta 3 signaling pathway was activated when JWA was interfered with. Further studies showed that blocking integrin alpha V beta 3 signaling pathway by siRNA and LM609 in B16 and A375 cells with JWA deficiency could reduce JWA-regulated cell adhesion and invasion. It was found that although there were many binding sites of integrin alpha V beta 3 promoter, only by interfering with SP1 nuclear transcription factors in B16-As and A375-sh cells could the regulation of JWA on integrin alpha V beta 3 signaling pathway be weakened. The ability of cell adhesion and invasion also decreased significantly.
6. Immunohistochemistry was used to detect the expression of JWA, integrin alpha V and beta 3 in benign nevus, malignant nevus and melanoma. The results showed that the expression of JWA was high in benign nevus, but very low in malignant nevus and melanoma. On the contrary, there is a very significant negative correlation between the dyeing strength of JWA and that of integrin alpha V and beta 3.
CONCLUSION: In conclusion, in this study, we found that the migration, adhesion, invasion and in vivo metastasis of melanoma cells were significantly enhanced after JWA intervention. This may be due to the fact that JWA regulates the integrin alpha V beta 3 signaling pathway through the transcription factor Sp1, thereby increasing the metastatic potential of tumor cells. Posterior studies on the role of integrin alpha V beta 3 signaling pathway in tumor metastasis provide important clues. At the same time, JWA may also become a target of anti-tumor metastasis or a biomarker of tumor prognosis and metastasis.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.5
【参考文献】
相关期刊论文 前4条
1 ;Integrin betal mediates hepatocellular carcinoma cells chemotaxis to laminin[J];Hepatobiliary & Pancreatic Diseases International;2004年04期
2 ;Role of JWA in acute promye-locytic leukemia cell differentiation and apoptosis triggered by retinoic acid,12-tetradecanoylphorbol-13- acetate and arsenic trioxide[J];Chinese Science Bulletin;2002年10期
3 ;JWA,a novel microtubule-associated protein,regulates homeostasis of intracellular amino acids in PC12 cells[J];Chinese Science Bulletin;2003年17期
4 ;JWA protein binds to α-tubulin in PC12 cells[J];Chinese Science Bulletin;2004年05期
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