【摘要】：【背景】胃癌嚴(yán)重危害國(guó)人健康,其發(fā)病率高,死亡率高,預(yù)后較差�；熓俏赴┲委煹幕A(chǔ)手段之一,在降低腫瘤負(fù)荷方面發(fā)揮著重要作用。在治療的初始階段多數(shù)患者體內(nèi)的胃癌細(xì)胞對(duì)化療藥物反應(yīng)敏感。然而,隨著治療的進(jìn)展,腫瘤細(xì)胞逐漸發(fā)生多藥耐藥。值得注意的是,發(fā)生多藥耐藥后的胃癌往往進(jìn)展迅速,在短時(shí)間內(nèi)就出現(xiàn)局部侵犯和全身廣泛轉(zhuǎn)移,提示胃癌耐藥與轉(zhuǎn)移之間存在密切關(guān)系。上皮間質(zhì)轉(zhuǎn)分化(Epithelial-mesenchymal transition,EMT)是腫瘤細(xì)胞惡性進(jìn)展的重要驅(qū)動(dòng)因素。近年來(lái)的研究表明,EMT作為啟動(dòng)腫瘤細(xì)胞轉(zhuǎn)移的關(guān)鍵步驟與腫瘤細(xì)胞的耐藥有著十分密切的關(guān)系,表現(xiàn)為:腫瘤細(xì)胞發(fā)生EMT后可對(duì)多種化療藥物產(chǎn)生耐受,而化療耐藥的腫瘤細(xì)胞也常常具有EMT的特征。因此,深入探索耐藥細(xì)胞發(fā)生EMT的關(guān)鍵機(jī)制,尋找可以逆轉(zhuǎn)胃癌耐藥細(xì)胞EMT的靶點(diǎn)可為抑制胃癌多藥耐藥細(xì)胞的侵襲轉(zhuǎn)移和改善患者預(yù)后提供新的思路�！灸康摹�1.明確EMT在胃癌耐藥細(xì)胞侵襲轉(zhuǎn)移中的作用。2.篩選并驗(yàn)證調(diào)控胃癌耐藥細(xì)胞EMT的關(guān)鍵分子并明確其作用機(jī)制。3.明確調(diào)控胃癌細(xì)胞EMT的分子在胃癌組織中表達(dá)的臨床意義�！痉椒ā�1.利用Transwell實(shí)驗(yàn)和裸鼠尾靜脈注射肺轉(zhuǎn)移實(shí)驗(yàn)比較胃癌耐藥細(xì)胞和親本細(xì)胞侵襲轉(zhuǎn)移能力的差別;利用實(shí)時(shí)熒光定量PCR(quantitative Real-time PCR,q RT-PCR)技術(shù)、western blot技術(shù)和免疫熒光技術(shù)檢測(cè)胃癌耐藥和親本細(xì)胞中EMT標(biāo)志物的變化;采用c DNA芯片結(jié)合生物信息學(xué)分析的方法篩選參與調(diào)控胃癌耐藥細(xì)胞EMT的關(guān)鍵分子;采用免疫組化檢測(cè)候選分子與EMT標(biāo)志物E-cadherin在胃癌組織中的表達(dá),并統(tǒng)計(jì)分析其相關(guān)性。2.合成針對(duì)HMGA2和FOXL2的小干擾RNA(small interfering RNA,si RNA),構(gòu)建HMGA2和FOXL2的過表達(dá)和sh RNA干擾載體,分別通過瞬時(shí)和穩(wěn)定轉(zhuǎn)染構(gòu)建HMGA2和FOXL2的功能缺失和功能獲得細(xì)胞模型。分別采用Transwell和原位移植瘤轉(zhuǎn)移模型實(shí)驗(yàn)評(píng)價(jià)細(xì)胞的體內(nèi)外侵襲轉(zhuǎn)移能力,采用western blot和q RT-PCR技術(shù)檢測(cè)EMT標(biāo)志物的變化。采用western blot技術(shù)檢測(cè)化療藥物處理后HMGA2和FOXL2表達(dá)的變化。3.干預(yù)HMGA2和FOXL2表達(dá)后,采用western blot和q RT-PCR分別從蛋白和m RNA水平檢測(cè)相關(guān)分子表達(dá);采用Transwell和原位移植瘤模型檢測(cè)細(xì)胞體內(nèi)外侵襲轉(zhuǎn)移能力。4.采用瞬時(shí)和穩(wěn)定轉(zhuǎn)染的方法構(gòu)建功能獲得和功能缺失細(xì)胞模型;采用western blot和q RT-PCR分別從蛋白和m RNA水平檢測(cè)相關(guān)分子表達(dá);采用Luciferase報(bào)告基因檢測(cè)E2F1對(duì)FOXL2的轉(zhuǎn)錄調(diào)控;采用免疫共沉淀技術(shù)聯(lián)合western blot檢測(cè)蛋白相互作用。5.采用c DNA芯片篩選FOXL2下游靶基因;在FOXL2功能獲得和功能缺失的細(xì)胞里采用western blot和q RT-PCR檢測(cè)ITGA2表達(dá);在ITGA2功能獲得和功能缺失的細(xì)胞里采用q RT-PCR、western blot和免疫熒光檢測(cè)EMT標(biāo)志物的變化,采用Transwell和裸鼠原位移植瘤模型檢測(cè)細(xì)胞侵襲轉(zhuǎn)移能力變化。6.采用免疫組化在胃癌原發(fā)灶和轉(zhuǎn)移灶組織中檢測(cè)HMGA2、FOXL2和ITGA2的表達(dá),并統(tǒng)計(jì)分析其表達(dá)水平在原發(fā)灶、轉(zhuǎn)移灶中差別及其表達(dá)水平與患者預(yù)后的關(guān)系�！窘Y(jié)果】1.Transwell和尾靜脈注射轉(zhuǎn)移實(shí)驗(yàn)結(jié)果表明胃癌耐藥細(xì)胞的體內(nèi)外侵襲轉(zhuǎn)移能力較親本細(xì)胞顯著增強(qiáng)。q RT-PCR和western blot檢測(cè)發(fā)現(xiàn)耐藥細(xì)胞與親本細(xì)胞相比E-cadherin表達(dá)下降,Vimentin表達(dá)升高,表明耐藥細(xì)胞發(fā)生了EMT。通過基因芯片篩查我們獲得了423個(gè)在耐藥細(xì)胞中表達(dá)升高2倍以上的候選基因,通過生物信息學(xué)分析我們鎖定了8個(gè)與基因轉(zhuǎn)錄相關(guān)的候選分子,通過查閱Kaplan-Meier Plot網(wǎng)站發(fā)現(xiàn)其中4個(gè)基因表達(dá)升高與胃癌預(yù)后差相關(guān)。最后,通過對(duì)胃癌組織進(jìn)行免疫組化染色發(fā)現(xiàn)HMGA2和FOXL2表達(dá)與上皮標(biāo)志物E-cadherin表達(dá)呈負(fù)相關(guān)。2.在耐藥細(xì)胞中分別下調(diào)HMGA2和FOXL2顯著抑制細(xì)胞的體內(nèi)外侵襲轉(zhuǎn)移能力,并促進(jìn)上皮標(biāo)志物E-cadherin的表達(dá),抑制間質(zhì)標(biāo)志物Vimentin的表達(dá);在親本細(xì)胞中分別過表達(dá)HMGA2和FOXL2則抑制E-cadherin表達(dá),促進(jìn)Vimentin表達(dá)并促進(jìn)細(xì)胞的體內(nèi)外侵襲轉(zhuǎn)移,表明HMGA2和FOXL2通過誘導(dǎo)EMT調(diào)控了胃癌耐藥細(xì)胞的侵襲轉(zhuǎn)移。同時(shí),我們發(fā)現(xiàn)短時(shí)間化療藥物處理能使胃癌細(xì)胞中HMGA2和FOXL2表達(dá)升高,表明兩者可被化療藥物誘導(dǎo)表達(dá)。3.內(nèi)源性HMGA2和FOXL2在多種胃癌細(xì)胞系中表達(dá)水平不同,但干預(yù)HMGA2的表達(dá)均可引起FOXL2發(fā)生同向表達(dá)變化。同時(shí),我們發(fā)現(xiàn)抑制FOXL2可阻斷HMGA2對(duì)胃癌耐藥細(xì)胞侵襲轉(zhuǎn)移和EMT的調(diào)控作用,提示HMGA2可能通過影響FOXL2發(fā)揮功能。4.q RT-PCR結(jié)果表明E2F1經(jīng)典下游靶基因在耐藥細(xì)胞中表達(dá)升高,提示耐藥細(xì)胞中E2F1轉(zhuǎn)錄活性增強(qiáng);在耐藥和親本細(xì)胞中干預(yù)E2F1表達(dá)可以引起FOXL2同向變化,表明FOXL2表達(dá)受E2F1調(diào)控;啟動(dòng)子Luciferase報(bào)告基因結(jié)果顯示FOXL2啟動(dòng)子區(qū)域存在E2F1的結(jié)合位點(diǎn);免疫共沉淀及Luciferase報(bào)告基因結(jié)果表明HMGA2與p Rb相互作用可以促進(jìn)E2F1對(duì)FOXL2的轉(zhuǎn)錄。5.基因芯片篩選結(jié)果表明ITGA2是FOXL2潛在的下游基因;在耐藥細(xì)胞下調(diào)FOXL2可以導(dǎo)致ITGA2表達(dá)下降,提示ITGA2的表達(dá)受FOXL2的調(diào)控;在耐藥細(xì)胞中下調(diào)ITGA2顯著抑制細(xì)胞的體內(nèi)外侵襲轉(zhuǎn)移能力,并導(dǎo)致E-cadherin表達(dá)升高,Vimentin表達(dá)降低;在親本細(xì)胞中過表達(dá)ITGA2則促進(jìn)細(xì)胞的侵襲轉(zhuǎn)移、抑制E-cadherin表達(dá)、促進(jìn)Vimentin表達(dá),表明ITGA2同可以通過調(diào)控EMT促進(jìn)胃癌耐藥細(xì)胞的侵襲轉(zhuǎn)移。同時(shí),我們發(fā)現(xiàn)ITGA2可以拮抗FOXL2的促EMT作用。6.免疫組化染色結(jié)果表明HMGA2、FOXL2和ITGA2在胃癌淋巴結(jié)和其他轉(zhuǎn)移灶中的表達(dá)水平顯著高于原發(fā)灶的水平,而且轉(zhuǎn)移癌中HMGA2、FOXL2和ITGA2的表達(dá)高于非轉(zhuǎn)移癌,提示HMGA2、FOXL2和ITGA2高表達(dá)與胃癌轉(zhuǎn)移相關(guān)。通過對(duì)HMGA2、FOXL2和ITGA2的表達(dá)與胃癌患者生存情況進(jìn)行分析,發(fā)現(xiàn)三個(gè)分子中任意一個(gè)分子高表達(dá)都與胃癌患者預(yù)后不良相關(guān),且其中任意兩個(gè)分子組合的預(yù)測(cè)效果優(yōu)于單一分子預(yù)測(cè)效果�！窘Y(jié)論】本課題闡明了一條新的促進(jìn)胃癌耐藥細(xì)胞EMT和侵襲轉(zhuǎn)移的信號(hào)通路。在化療藥物的作用下,HMGA2表達(dá)升高并通過與p Rb相互作用促進(jìn)E2F1對(duì)FOXL2的表達(dá),后者進(jìn)一步通過促進(jìn)ITGA2的表達(dá)從而導(dǎo)致胃癌細(xì)胞發(fā)生EMT和侵襲轉(zhuǎn)移能力的增強(qiáng)。通過臨床標(biāo)本驗(yàn)證,我們發(fā)現(xiàn)HMGA2、FOXL2和ITGA2高表達(dá)與胃癌轉(zhuǎn)移和預(yù)后不良相關(guān)。綜上所述,本課題為認(rèn)識(shí)胃癌耐藥和轉(zhuǎn)移的內(nèi)在聯(lián)系提供了理論基礎(chǔ),為開發(fā)同時(shí)靶向胃癌耐藥和轉(zhuǎn)移的治療策略提供了候選靶點(diǎn),也為評(píng)估胃癌患者轉(zhuǎn)移風(fēng)險(xiǎn)提供了新的潛在標(biāo)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2
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本文編號(hào)：2198564