【摘要】：腫瘤轉(zhuǎn)移是癌癥病人死亡的主要原因。腫瘤轉(zhuǎn)移過程由幾個獨(dú)立的步驟組成,包括腫瘤細(xì)胞從原發(fā)灶脫離,腫瘤細(xì)胞侵襲到血管內(nèi)皮或淋巴管從而進(jìn)入循環(huán)系統(tǒng),最后,在新的宿主環(huán)境中,粘附并再侵襲,增殖成為一個轉(zhuǎn)移瘤。大量研究顯示,在腫瘤轉(zhuǎn)移的多步驟過程中,許多信號分子被激活并發(fā)生相互作用,形成復(fù)雜的信號網(wǎng)絡(luò)。研究和破解控制腫瘤轉(zhuǎn)移的信號網(wǎng)絡(luò)和關(guān)鍵調(diào)節(jié)機(jī)制,阻止腫瘤轉(zhuǎn)移,是提高腫瘤治療效果的根本所在,也是當(dāng)前國內(nèi)外腫瘤學(xué)家企圖攻克的最主要的堡壘。 細(xì)胞遷移是腫瘤細(xì)胞轉(zhuǎn)移的重要基礎(chǔ),細(xì)胞骨架的重組是細(xì)胞發(fā)生遷移的必要條件,因此,調(diào)節(jié)細(xì)胞骨架重組的分子可能在腫瘤細(xì)胞轉(zhuǎn)移中發(fā)揮重要作用。JWA是新近發(fā)現(xiàn)的微管和微絲結(jié)合蛋白,可調(diào)節(jié)兩種細(xì)胞骨架的重組。本課題組研究證明,JWA表達(dá)缺陷可以阻斷As2O3對HeLa細(xì)胞遷移的抑制,但是可以加強(qiáng)PMA(佛波酯)對細(xì)胞遷移的作用。進(jìn)一步研究發(fā)現(xiàn)JWA對細(xì)胞遷移的調(diào)節(jié)作用是通過激活MAPK信號通路不同下游分子以及對微絲重組的作用而實(shí)現(xiàn)的。本研究發(fā)現(xiàn),JWA在黑色素瘤轉(zhuǎn)移過程中起到非常重要的作用。我們的結(jié)果證明JWA敲減(knock down)可以增加黑色素瘤細(xì)胞的遷移、粘附和侵襲能力;體內(nèi)實(shí)驗(yàn)證實(shí)在小鼠黑色素瘤B16-F10細(xì)胞和人黑色素瘤A375細(xì)胞中敲減JWA后顯著促進(jìn)轉(zhuǎn)移瘤的形成和生長;此外,在黑色素瘤病人的腫瘤組織中,JWA在惡性黑色素瘤的表達(dá)水平顯著低于良性痣。最后,我們的研究證實(shí)JWA的上述作用是通過整合素信號通路來實(shí)現(xiàn)的,當(dāng)干涉轉(zhuǎn)錄因子Sp1后,就破壞了JWA對整合素分子integrinα_Vβ_3及其下游分子的調(diào)節(jié)作用,從而逆轉(zhuǎn)JWA對腫瘤轉(zhuǎn)移過程的影響。這些發(fā)現(xiàn)表明JWA是抑制黑色素瘤轉(zhuǎn)移的重要靶分子。 目標(biāo):探討JWA在調(diào)節(jié)黑色素瘤轉(zhuǎn)移過程多個環(huán)節(jié)中的作用及其分子機(jī)制。 方法:本研究使用有轉(zhuǎn)移性的小鼠黑色素瘤細(xì)胞株B16-F10和人黑色素瘤細(xì)胞株A375來進(jìn)行體內(nèi)外研究。通過穩(wěn)定篩選得到JWA低表達(dá)的細(xì)胞株(B16-As和A375-As)和載體對照的細(xì)胞株(B16-Vec和A375-Vec)。首先,我們使用小鼠尾靜脈腫瘤轉(zhuǎn)移實(shí)驗(yàn)來檢驗(yàn)JWA在黑色素瘤體內(nèi)轉(zhuǎn)移中的作用。接著我們通過劃痕實(shí)驗(yàn)、Transwell遷移實(shí)驗(yàn)和免疫熒光等來研究JWA表達(dá)對腫瘤細(xì)胞轉(zhuǎn)移過程多個環(huán)節(jié)的影響及其機(jī)制。用粘附實(shí)驗(yàn)、侵襲實(shí)驗(yàn)和明膠酶譜實(shí)驗(yàn)檢測JWA是否調(diào)節(jié)細(xì)胞的粘附和侵襲能力。用免疫印跡和RNA干涉等技術(shù)探討了JWA調(diào)節(jié)腫瘤轉(zhuǎn)移過程的信號通路分子變化規(guī)律。最后,我們收集了13例良性痣、5例惡變痣和13例惡性黑色素瘤,用免疫組化方法檢測JWA、integrinαV和β3在組織中的表達(dá),初步驗(yàn)證這些分子的表達(dá)水平與人黑色素瘤惡性程度的關(guān)系。 結(jié)果: 1.在體內(nèi)模型中,將JWA表達(dá)敲減的B16-F10細(xì)胞和載體對照細(xì)胞通過尾靜脈注入小鼠21天后,發(fā)現(xiàn)JWA表達(dá)缺陷組動物肺轉(zhuǎn)移灶形成數(shù)顯著高于載體對照組;將JWA表達(dá)缺陷和載體對照的人黑色素瘤細(xì)胞株A375-As和A375-Vec兩組細(xì)胞注射入免疫缺陷小鼠的尾靜脈中,30天后發(fā)現(xiàn)在JWA表達(dá)缺陷組中40%的小鼠頸部淋巴結(jié)有明顯的轉(zhuǎn)移瘤,但在載體對照組未發(fā)現(xiàn)明顯的頸部轉(zhuǎn)移瘤。通過細(xì)胞增殖實(shí)驗(yàn)檢測了幾組細(xì)胞的增殖能力,結(jié)果發(fā)現(xiàn)JWA表達(dá)缺陷組和載體對照組的細(xì)胞增長速度基本相同。提示JWA缺陷組腫瘤轉(zhuǎn)移灶的增加可能不是由于轉(zhuǎn)染了pEGFP-C1-As JWA質(zhì)粒造成的細(xì)胞增殖能力的增加,而是由于JWA低表達(dá)增強(qiáng)了腫瘤轉(zhuǎn)移能力所致。 2.通過劃痕實(shí)驗(yàn)和Transwell遷移實(shí)驗(yàn),發(fā)現(xiàn)JWA表達(dá)缺陷的細(xì)胞與載體對照的細(xì)胞相比遷移能力顯著增強(qiáng)。免疫熒光的結(jié)果顯示JWA表達(dá)缺陷組細(xì)胞周圍的肌動蛋白絲明顯多于載體對照組,細(xì)胞表現(xiàn)出一種更強(qiáng)的移動能力。 3.粘附實(shí)驗(yàn)發(fā)現(xiàn)在纖維粘連蛋白和玻璃粘連蛋白包被的板子上,B16-As細(xì)胞粘附能力分別升高了103%和69%,A375細(xì)胞粘附能力分別提高了75%和58%。 4.細(xì)胞侵襲能力試驗(yàn)發(fā)現(xiàn)在JWA表達(dá)缺陷B16-As和A375-sh細(xì)胞中,通過侵襲作用穿過Matrigel包被的Boyden小室的細(xì)胞數(shù)目分別增加了173%和79%。明膠酶譜法檢測黑色素瘤細(xì)胞中基質(zhì)金屬蛋白酶的活性的結(jié)果顯示,B16-As和B16-Vec組細(xì)胞的MMP-9活性水平并沒有明顯改變,但是在B16-As組細(xì)胞中MMP-2的活性比載體對照上升了4.5倍。 5.在機(jī)制研究中發(fā)現(xiàn)當(dāng)干涉JWA表達(dá)后,integrinαVβ3信號通路被激活。進(jìn)一步研究發(fā)現(xiàn),在JWA表達(dá)缺陷的B16和A375細(xì)胞中通過siRNA和LM609阻斷integrinαVβ3信號通路可以降低JWA調(diào)節(jié)的細(xì)胞粘附和侵襲能力。在探討JWA如何調(diào)節(jié)integrinαVβ3信號通路時(shí)發(fā)現(xiàn),盡管integrinαVβ3啟動子區(qū)存在很多轉(zhuǎn)錄因子的結(jié)合位點(diǎn),但我們分別干涉后卻發(fā)現(xiàn)只有將B16-As和A375-sh細(xì)胞中的Sp1核轉(zhuǎn)錄因子干涉后,才能削弱JWA對integrinαVβ3信號通路的調(diào)節(jié)作用。隨著integrinαVβ3蛋白表達(dá)水平的降低,腫瘤細(xì)胞粘附和侵襲的能力也顯著下降。 6.用免疫組織化學(xué)方法檢測了良性痣、惡變痣和黑色素瘤組織中JWA、integrinαV和β3的表達(dá)水平。結(jié)果發(fā)現(xiàn),在良性痣中JWA的表達(dá)水平較高,而在惡變痣和黑色素瘤中JWA的表達(dá)卻非常低。而integrinαV和β3在惡變痣和黑色素瘤中的的表達(dá)水平則正好與此相反,即JWA的染色強(qiáng)度與integrinαV和β3的染色強(qiáng)度存在一種非常顯著的負(fù)相關(guān)關(guān)系。 結(jié)論:總而言之,在本研究中,我們發(fā)現(xiàn)黑色素瘤細(xì)胞在干涉JWA以后,腫瘤細(xì)胞的遷移、粘附、侵襲和體內(nèi)的轉(zhuǎn)移能力都顯著增強(qiáng)。這可能是由于JWA通過轉(zhuǎn)錄因子Sp1來調(diào)節(jié)integrinαVβ3信號通路,從而增加腫瘤細(xì)胞的轉(zhuǎn)移潛能。我們的工作對以后研究integrinαVβ3信號通路在腫瘤轉(zhuǎn)移中的作用提供了重要的線索。同時(shí),JWA也有可能成為抗腫瘤轉(zhuǎn)移的靶點(diǎn)或觀察腫瘤患者預(yù)后和轉(zhuǎn)移的生物標(biāo)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R739.5

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本文編號：2224883