HMGA2-FOXL2通路调控多药耐药胃癌细胞上皮间质转分化及侵袭转移的研究
发布时间：2018-08-23 09:04
【摘要】：【背景】胃癌严重危害国人健康,其发病率高,死亡率高,预后较差。化疗是胃癌治疗的基础手段之一,在降低肿瘤负荷方面发挥着重要作用。在治疗的初始阶段多数患者体内的胃癌细胞对化疗药物反应敏感。然而,随着治疗的进展,肿瘤细胞逐渐发生多药耐药。值得注意的是,发生多药耐药后的胃癌往往进展迅速,在短时间内就出现局部侵犯和全身广泛转移,提示胃癌耐药与转移之间存在密切关系。上皮间质转分化(Epithelial-mesenchymal transition,EMT)是肿瘤细胞恶性进展的重要驱动因素。近年来的研究表明,EMT作为启动肿瘤细胞转移的关键步骤与肿瘤细胞的耐药有着十分密切的关系,表现为:肿瘤细胞发生EMT后可对多种化疗药物产生耐受,而化疗耐药的肿瘤细胞也常常具有EMT的特征。因此,深入探索耐药细胞发生EMT的关键机制,寻找可以逆转胃癌耐药细胞EMT的靶点可为抑制胃癌多药耐药细胞的侵袭转移和改善患者预后提供新的思路。【目的】1.明确EMT在胃癌耐药细胞侵袭转移中的作用。2.筛选并验证调控胃癌耐药细胞EMT的关键分子并明确其作用机制。3.明确调控胃癌细胞EMT的分子在胃癌组织中表达的临床意义。【方法】1.利用Transwell实验和裸鼠尾静脉注射肺转移实验比较胃癌耐药细胞和亲本细胞侵袭转移能力的差别;利用实时荧光定量PCR(quantitative Real-time PCR,q RT-PCR)技术、western blot技术和免疫荧光技术检测胃癌耐药和亲本细胞中EMT标志物的变化;采用c DNA芯片结合生物信息学分析的方法筛选参与调控胃癌耐药细胞EMT的关键分子;采用免疫组化检测候选分子与EMT标志物E-cadherin在胃癌组织中的表达,并统计分析其相关性。2.合成针对HMGA2和FOXL2的小干扰RNA(small interfering RNA,si RNA),构建HMGA2和FOXL2的过表达和sh RNA干扰载体,分别通过瞬时和稳定转染构建HMGA2和FOXL2的功能缺失和功能获得细胞模型。分别采用Transwell和原位移植瘤转移模型实验评价细胞的体内外侵袭转移能力,采用western blot和q RT-PCR技术检测EMT标志物的变化。采用western blot技术检测化疗药物处理后HMGA2和FOXL2表达的变化。3.干预HMGA2和FOXL2表达后,采用western blot和q RT-PCR分别从蛋白和m RNA水平检测相关分子表达;采用Transwell和原位移植瘤模型检测细胞体内外侵袭转移能力。4.采用瞬时和稳定转染的方法构建功能获得和功能缺失细胞模型;采用western blot和q RT-PCR分别从蛋白和m RNA水平检测相关分子表达;采用Luciferase报告基因检测E2F1对FOXL2的转录调控;采用免疫共沉淀技术联合western blot检测蛋白相互作用。5.采用c DNA芯片筛选FOXL2下游靶基因;在FOXL2功能获得和功能缺失的细胞里采用western blot和q RT-PCR检测ITGA2表达;在ITGA2功能获得和功能缺失的细胞里采用q RT-PCR、western blot和免疫荧光检测EMT标志物的变化,采用Transwell和裸鼠原位移植瘤模型检测细胞侵袭转移能力变化。6.采用免疫组化在胃癌原发灶和转移灶组织中检测HMGA2、FOXL2和ITGA2的表达,并统计分析其表达水平在原发灶、转移灶中差别及其表达水平与患者预后的关系。【结果】1.Transwell和尾静脉注射转移实验结果表明胃癌耐药细胞的体内外侵袭转移能力较亲本细胞显著增强。q RT-PCR和western blot检测发现耐药细胞与亲本细胞相比E-cadherin表达下降,Vimentin表达升高,表明耐药细胞发生了EMT。通过基因芯片筛查我们获得了423个在耐药细胞中表达升高2倍以上的候选基因,通过生物信息学分析我们锁定了8个与基因转录相关的候选分子,通过查阅Kaplan-Meier Plot网站发现其中4个基因表达升高与胃癌预后差相关。最后,通过对胃癌组织进行免疫组化染色发现HMGA2和FOXL2表达与上皮标志物E-cadherin表达呈负相关。2.在耐药细胞中分别下调HMGA2和FOXL2显著抑制细胞的体内外侵袭转移能力,并促进上皮标志物E-cadherin的表达,抑制间质标志物Vimentin的表达;在亲本细胞中分别过表达HMGA2和FOXL2则抑制E-cadherin表达,促进Vimentin表达并促进细胞的体内外侵袭转移,表明HMGA2和FOXL2通过诱导EMT调控了胃癌耐药细胞的侵袭转移。同时,我们发现短时间化疗药物处理能使胃癌细胞中HMGA2和FOXL2表达升高,表明两者可被化疗药物诱导表达。3.内源性HMGA2和FOXL2在多种胃癌细胞系中表达水平不同,但干预HMGA2的表达均可引起FOXL2发生同向表达变化。同时,我们发现抑制FOXL2可阻断HMGA2对胃癌耐药细胞侵袭转移和EMT的调控作用,提示HMGA2可能通过影响FOXL2发挥功能。4.q RT-PCR结果表明E2F1经典下游靶基因在耐药细胞中表达升高,提示耐药细胞中E2F1转录活性增强;在耐药和亲本细胞中干预E2F1表达可以引起FOXL2同向变化,表明FOXL2表达受E2F1调控;启动子Luciferase报告基因结果显示FOXL2启动子区域存在E2F1的结合位点;免疫共沉淀及Luciferase报告基因结果表明HMGA2与p Rb相互作用可以促进E2F1对FOXL2的转录。5.基因芯片筛选结果表明ITGA2是FOXL2潜在的下游基因;在耐药细胞下调FOXL2可以导致ITGA2表达下降,提示ITGA2的表达受FOXL2的调控;在耐药细胞中下调ITGA2显著抑制细胞的体内外侵袭转移能力,并导致E-cadherin表达升高,Vimentin表达降低;在亲本细胞中过表达ITGA2则促进细胞的侵袭转移、抑制E-cadherin表达、促进Vimentin表达,表明ITGA2同可以通过调控EMT促进胃癌耐药细胞的侵袭转移。同时,我们发现ITGA2可以拮抗FOXL2的促EMT作用。6.免疫组化染色结果表明HMGA2、FOXL2和ITGA2在胃癌淋巴结和其他转移灶中的表达水平显著高于原发灶的水平,而且转移癌中HMGA2、FOXL2和ITGA2的表达高于非转移癌,提示HMGA2、FOXL2和ITGA2高表达与胃癌转移相关。通过对HMGA2、FOXL2和ITGA2的表达与胃癌患者生存情况进行分析,发现三个分子中任意一个分子高表达都与胃癌患者预后不良相关,且其中任意两个分子组合的预测效果优于单一分子预测效果。【结论】本课题阐明了一条新的促进胃癌耐药细胞EMT和侵袭转移的信号通路。在化疗药物的作用下,HMGA2表达升高并通过与p Rb相互作用促进E2F1对FOXL2的表达,后者进一步通过促进ITGA2的表达从而导致胃癌细胞发生EMT和侵袭转移能力的增强。通过临床标本验证,我们发现HMGA2、FOXL2和ITGA2高表达与胃癌转移和预后不良相关。综上所述,本课题为认识胃癌耐药和转移的内在联系提供了理论基础,为开发同时靶向胃癌耐药和转移的治疗策略提供了候选靶点,也为评估胃癌患者转移风险提供了新的潜在标志物。
[Abstract]:[BACKGROUND] Gastric cancer is a serious threat to the health of Chinese people. It has a high morbidity, high mortality and poor prognosis. Chemotherapy is one of the basic treatment methods for gastric cancer and plays an important role in reducing tumor burden. It is noteworthy that gastric cancer after multidrug resistance often progresses rapidly, local invasion and extensive metastasis occur in a short time, suggesting a close relationship between drug resistance and metastasis. Epithelial-mesenchymal transition (EMT) is a malignant progression of cancer cells. Recent studies have shown that EMT, as a key step in initiating metastasis of tumor cells, is closely related to drug resistance of tumor cells. EMT can induce tolerance to a variety of chemotherapeutics, and chemotherapeutically resistant tumor cells often have the characteristics of EMT. The key mechanism of EMT in gastric cancer cells and the target of reversing EMT in gastric cancer cells may provide new ideas for inhibiting the invasion and metastasis of multidrug-resistant cells and improving the prognosis of patients with gastric cancer. 3. The clinical significance of regulating the expression of EMT in gastric cancer tissues was clarified. [Methods] 1. Transwell assay and tail vein lung metastasis assay were used to compare the invasion and metastasis ability of drug-resistant gastric cancer cells and parental cells, and real-time fluorescence quantitative real-time PCR (quantitative Real-time PCR). Q RT-PCR, Western blot and immunofluorescence were used to detect the changes of EMT markers in gastric cancer resistance and parental cells; the key molecules involved in regulating EMT in gastric cancer resistant cells were screened by c-DNA chip combined with bioinformatics analysis; and the candidate molecules and EMT marker E-cadherin were detected by immunohistochemistry in gastric cancer tissues. The expression of HMGA 2 and FOXL 2 was analyzed statistically. 2. Small interfering RNA (si RNA) was synthesized for HMGA 2 and FOXL 2, and the over-expression and sh RNA interference vectors of HMGA 2 and FOXL 2 were constructed. Western blot and Q RT-PCR were used to detect the changes of EMT markers. Western blot was used to detect the changes of HMGA2 and FOXL2 expression after chemotherapy. 3. After intervention of HMGA2 and FOXL2 expression, Western blot and Q RT-PCR were used to detect protein and m RNA levels respectively. Transwell and proto-displaced tumor model were used to detect the invasion and metastasis ability in vitro and in vivo. 4. Functional acquisition and functional deletion cell models were constructed by transient and stable transfection methods. Western blot and Q RT-PCR were used to detect the expression of related molecules at protein and m RNA levels, respectively. Luciferase reporter gene was used to detect the expression of related molecules. E2F1 was used to detect the transcriptional regulation of FOXL2. Immunocoprecipitation and Western blot were used to detect protein interaction. 5. The downstream target gene of FOXL2 was screened by c-DNA chip; the expression of ITGA2 was detected by Western blot and Q RT-PCR in the cells with FOXL2 functional acquisition and loss; and Q RT-PCR was used in the cells with ITGA2 functional acquisition and loss. The expression of HMGA2, FOXL2 and ITGA2 in the primary and metastatic lesions of gastric cancer was detected by immunohistochemistry. The expression levels of HMGA2, FOXL2 and ITGA2 in the primary and metastatic lesions were analyzed statistically. Transwell and tail vein injection metastasis test showed that the invasion and metastasis ability of drug-resistant gastric cancer cells in vivo and in vitro was significantly enhanced compared with parental cells. Q RT-PCR and Western blot detection showed that the expression of E-cadherin was decreased and the expression of Vimentin was increased compared with parental cells. EMT occurred in drug-resistant cells. 423 candidate genes were screened by gene chip. Through bioinformatics analysis, we identified 8 candidate genes related to gene transcription. By checking the Kaplan-Meier Plot website, we found that 4 of them were elevated and the prognosis of gastric cancer was poor. Finally, the expression of HMGA 2 and FOXL2 was found to be negatively correlated with the expression of E-cadherin in gastric cancer tissues by immunohistochemical staining. 2. Downregulation of HMGA 2 and FOXL2 in drug-resistant cells significantly inhibited the invasion and metastasis of gastric cancer cells in vivo and in vitro, promoted the expression of E-cadherin, and inhibited the expression of Vimentin, a mesenchymal marker, respectively. Overexpression of HMGA2 and FOXL2 in parental cells inhibited E-cadherin expression, promoted Vimentin expression and promoted cell invasion and metastasis in vitro and in vivo, suggesting that HMGA2 and FOXL2 regulated the invasion and metastasis of gastric cancer drug-resistant cells by inducing EMT. The expression levels of endogenous HMGA2 and FOXL2 were different in various gastric cancer cell lines, but the co-expression of FOXL2 could be induced by interfering with the expression of HMGA2. At the same time, we found that inhibiting FOXL2 could block the regulation of HMGA2 on the invasion and metastasis of drug-resistant gastric cancer cells and EMT. 4.q RT-PCR results showed that the expression of E2F1 classical downstream target gene was increased in drug-resistant cells, suggesting that the transcriptional activity of E2F1 was enhanced in drug-resistant cells; the co-directional changes of FOXL2 could be induced by interfering with E2F1 expression in drug-resistant and parental cells, suggesting that FOXL2 expression was regulated by E2F1; the results of promoter Luciferase reporter gene The results of immunoprecipitation and Luciferase reporter gene analysis showed that the interaction of HMGA2 and P Rb could promote the transcription of FOXL2 by E2F1. Expression was regulated by FOXL2; down-regulation of ITGA2 significantly inhibited cell invasion and metastasis in vitro and in vivo, resulting in elevated expression of E-cadherin and decreased expression of Vimentin; over-expression of ITGA2 in parental cells promoted cell invasion and metastasis, inhibited E-cadherin expression, and promoted Vimentin expression, suggesting that ITGA2 could also regulate EMT. Immunohistochemical staining showed that the expression of HMGA2, FOXL2 and ITGA2 in lymph nodes and other metastatic lesions of gastric cancer was significantly higher than that in primary lesions, and the expression of HMGA2, FOXL2 and ITGA2 in metastatic cancer was higher than that in non-metastatic cancer. The high expression of HMGA2, FOXL2 and ITGA2 was correlated with the metastasis of gastric cancer. By analyzing the relationship between the expression of HMGA2, FOXL2 and ITGA2 and the survival of gastric cancer patients, it was found that the high expression of any one of the three molecules was correlated with the poor prognosis of gastric cancer patients, and the predictive effect of any two molecular combinations was better than that of single molecule. [Conclusion] This study elucidates a new signaling pathway that promotes EMT and invasion and metastasis of drug-resistant gastric cancer cells. Under the action of chemotherapeutic drugs, the expression of HMGA2 is elevated and the expression of FOXL2 by E2F1 is enhanced through interaction with P Rb, which further leads to EMT and invasion and metastasis of gastric cancer cells by promoting the expression of ITGA2. Overexpression of HMGA2, FOXL2 and ITGA2 was found to be associated with metastasis and poor prognosis in gastric cancer. In conclusion, this study provides a theoretical basis for understanding the intrinsic relationship between drug resistance and metastasis in gastric cancer, and provides a candidate target for developing therapeutic strategies targeting both drug resistance and metastasis in gastric cancer, as well as for evaluating the risk of gastric cancer. The transfer of risk provides new potential markers.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2
，
本文编号：2198564
[Abstract]:[BACKGROUND] Gastric cancer is a serious threat to the health of Chinese people. It has a high morbidity, high mortality and poor prognosis. Chemotherapy is one of the basic treatment methods for gastric cancer and plays an important role in reducing tumor burden. It is noteworthy that gastric cancer after multidrug resistance often progresses rapidly, local invasion and extensive metastasis occur in a short time, suggesting a close relationship between drug resistance and metastasis. Epithelial-mesenchymal transition (EMT) is a malignant progression of cancer cells. Recent studies have shown that EMT, as a key step in initiating metastasis of tumor cells, is closely related to drug resistance of tumor cells. EMT can induce tolerance to a variety of chemotherapeutics, and chemotherapeutically resistant tumor cells often have the characteristics of EMT. The key mechanism of EMT in gastric cancer cells and the target of reversing EMT in gastric cancer cells may provide new ideas for inhibiting the invasion and metastasis of multidrug-resistant cells and improving the prognosis of patients with gastric cancer. 3. The clinical significance of regulating the expression of EMT in gastric cancer tissues was clarified. [Methods] 1. Transwell assay and tail vein lung metastasis assay were used to compare the invasion and metastasis ability of drug-resistant gastric cancer cells and parental cells, and real-time fluorescence quantitative real-time PCR (quantitative Real-time PCR). Q RT-PCR, Western blot and immunofluorescence were used to detect the changes of EMT markers in gastric cancer resistance and parental cells; the key molecules involved in regulating EMT in gastric cancer resistant cells were screened by c-DNA chip combined with bioinformatics analysis; and the candidate molecules and EMT marker E-cadherin were detected by immunohistochemistry in gastric cancer tissues. The expression of HMGA 2 and FOXL 2 was analyzed statistically. 2. Small interfering RNA (si RNA) was synthesized for HMGA 2 and FOXL 2, and the over-expression and sh RNA interference vectors of HMGA 2 and FOXL 2 were constructed. Western blot and Q RT-PCR were used to detect the changes of EMT markers. Western blot was used to detect the changes of HMGA2 and FOXL2 expression after chemotherapy. 3. After intervention of HMGA2 and FOXL2 expression, Western blot and Q RT-PCR were used to detect protein and m RNA levels respectively. Transwell and proto-displaced tumor model were used to detect the invasion and metastasis ability in vitro and in vivo. 4. Functional acquisition and functional deletion cell models were constructed by transient and stable transfection methods. Western blot and Q RT-PCR were used to detect the expression of related molecules at protein and m RNA levels, respectively. Luciferase reporter gene was used to detect the expression of related molecules. E2F1 was used to detect the transcriptional regulation of FOXL2. Immunocoprecipitation and Western blot were used to detect protein interaction. 5. The downstream target gene of FOXL2 was screened by c-DNA chip; the expression of ITGA2 was detected by Western blot and Q RT-PCR in the cells with FOXL2 functional acquisition and loss; and Q RT-PCR was used in the cells with ITGA2 functional acquisition and loss. The expression of HMGA2, FOXL2 and ITGA2 in the primary and metastatic lesions of gastric cancer was detected by immunohistochemistry. The expression levels of HMGA2, FOXL2 and ITGA2 in the primary and metastatic lesions were analyzed statistically. Transwell and tail vein injection metastasis test showed that the invasion and metastasis ability of drug-resistant gastric cancer cells in vivo and in vitro was significantly enhanced compared with parental cells. Q RT-PCR and Western blot detection showed that the expression of E-cadherin was decreased and the expression of Vimentin was increased compared with parental cells. EMT occurred in drug-resistant cells. 423 candidate genes were screened by gene chip. Through bioinformatics analysis, we identified 8 candidate genes related to gene transcription. By checking the Kaplan-Meier Plot website, we found that 4 of them were elevated and the prognosis of gastric cancer was poor. Finally, the expression of HMGA 2 and FOXL2 was found to be negatively correlated with the expression of E-cadherin in gastric cancer tissues by immunohistochemical staining. 2. Downregulation of HMGA 2 and FOXL2 in drug-resistant cells significantly inhibited the invasion and metastasis of gastric cancer cells in vivo and in vitro, promoted the expression of E-cadherin, and inhibited the expression of Vimentin, a mesenchymal marker, respectively. Overexpression of HMGA2 and FOXL2 in parental cells inhibited E-cadherin expression, promoted Vimentin expression and promoted cell invasion and metastasis in vitro and in vivo, suggesting that HMGA2 and FOXL2 regulated the invasion and metastasis of gastric cancer drug-resistant cells by inducing EMT. The expression levels of endogenous HMGA2 and FOXL2 were different in various gastric cancer cell lines, but the co-expression of FOXL2 could be induced by interfering with the expression of HMGA2. At the same time, we found that inhibiting FOXL2 could block the regulation of HMGA2 on the invasion and metastasis of drug-resistant gastric cancer cells and EMT. 4.q RT-PCR results showed that the expression of E2F1 classical downstream target gene was increased in drug-resistant cells, suggesting that the transcriptional activity of E2F1 was enhanced in drug-resistant cells; the co-directional changes of FOXL2 could be induced by interfering with E2F1 expression in drug-resistant and parental cells, suggesting that FOXL2 expression was regulated by E2F1; the results of promoter Luciferase reporter gene The results of immunoprecipitation and Luciferase reporter gene analysis showed that the interaction of HMGA2 and P Rb could promote the transcription of FOXL2 by E2F1. Expression was regulated by FOXL2; down-regulation of ITGA2 significantly inhibited cell invasion and metastasis in vitro and in vivo, resulting in elevated expression of E-cadherin and decreased expression of Vimentin; over-expression of ITGA2 in parental cells promoted cell invasion and metastasis, inhibited E-cadherin expression, and promoted Vimentin expression, suggesting that ITGA2 could also regulate EMT. Immunohistochemical staining showed that the expression of HMGA2, FOXL2 and ITGA2 in lymph nodes and other metastatic lesions of gastric cancer was significantly higher than that in primary lesions, and the expression of HMGA2, FOXL2 and ITGA2 in metastatic cancer was higher than that in non-metastatic cancer. The high expression of HMGA2, FOXL2 and ITGA2 was correlated with the metastasis of gastric cancer. By analyzing the relationship between the expression of HMGA2, FOXL2 and ITGA2 and the survival of gastric cancer patients, it was found that the high expression of any one of the three molecules was correlated with the poor prognosis of gastric cancer patients, and the predictive effect of any two molecular combinations was better than that of single molecule. [Conclusion] This study elucidates a new signaling pathway that promotes EMT and invasion and metastasis of drug-resistant gastric cancer cells. Under the action of chemotherapeutic drugs, the expression of HMGA2 is elevated and the expression of FOXL2 by E2F1 is enhanced through interaction with P Rb, which further leads to EMT and invasion and metastasis of gastric cancer cells by promoting the expression of ITGA2. Overexpression of HMGA2, FOXL2 and ITGA2 was found to be associated with metastasis and poor prognosis in gastric cancer. In conclusion, this study provides a theoretical basis for understanding the intrinsic relationship between drug resistance and metastasis in gastric cancer, and provides a candidate target for developing therapeutic strategies targeting both drug resistance and metastasis in gastric cancer, as well as for evaluating the risk of gastric cancer. The transfer of risk provides new potential markers.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2
，
本文编号：2198564
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