ALK融合基因在腎透明細(xì)胞癌中的表達(dá)及功能研究
發(fā)布時(shí)間：2018-10-16 20:43
【摘要】：腎透明細(xì)胞癌是腎癌最常見的病理類型，約占腎癌的80%～85%。分子靶向藥物如貝伐單抗，舒尼替尼，索拉非尼等已經(jīng)成為晚期轉(zhuǎn)移性腎癌治療的一二線藥物，雖然在治療上帶來令人鼓舞的成績(jī)，但是達(dá)到完全緩解無病生存的患者還是很少。ALK重排和突變?cè)诙喾N腫瘤的發(fā)生發(fā)展中起著至關(guān)重要的作用，針對(duì)ALK的靶向治療成為腫瘤治療研究的熱點(diǎn)。臨床上已經(jīng)批準(zhǔn)ALK抑制劑用于NSCLC、ACLC、IMT的治療。目前在腎髓質(zhì)癌、腎未分類癌和乳頭狀腎細(xì)胞癌也發(fā)現(xiàn)ALK基因重排形成的融合基因，使得ALK有望成為腎癌新的治療靶點(diǎn)。由于腎透明細(xì)胞癌是最常見腎癌，因此，，本研究目的是探討腎透明細(xì)胞癌患者中是否存在ALK基因重排的現(xiàn)象，探索ALK在腎透明細(xì)胞癌發(fā)生、發(fā)展中的作用。首先應(yīng)用新型的ALK抗體篩查87例腎透明細(xì)胞癌中ALK表達(dá)情況，4例為ALK蛋白表達(dá)陽性，陽性率為4.59%。ALK蛋白主要表達(dá)于細(xì)胞質(zhì)，部分胞核也有表達(dá)。通過5'-RACE技術(shù)和DNA測(cè)序結(jié)果表明，其中2例標(biāo)本有ALK重排，形成EML4-ALK融合基因，都是1型變異體基因（variant1）。FISH結(jié)果同樣證實(shí)這兩例腎透明細(xì)胞癌中存在EML4-ALK融合基因。利用分子克隆方法構(gòu)建EML4-ALKvariant1融合基因慢病毒表達(dá)系統(tǒng)，感染人腎近曲小管上皮細(xì)胞HK-2，探討EML4-ALK在腎癌中的作用。功能學(xué)研究表明，EML4-ALK variant1融合基因轉(zhuǎn)染到HK-2中，能增強(qiáng)HK-2細(xì)胞的生長(zhǎng)和增殖的能力。ALK特異性小分子抑制劑TAE684能選擇性抑制ALK激酶活性，從而抑制穩(wěn)定表達(dá)EML4-ALKvariant1基因的HK-2細(xì)胞的增殖。體內(nèi)實(shí)驗(yàn)結(jié)果顯示，穩(wěn)定轉(zhuǎn)染表達(dá)EML4-ALK variant1基因能使非致瘤性的HK-2細(xì)胞在體內(nèi)成瘤，證明EML4-ALK variant1融合基因在體內(nèi)具有致瘤活性。更重要是，ALK抑制劑Crizotinib能夠顯著抑制ALK驅(qū)動(dòng)的HK-2腫瘤的生長(zhǎng)。本研究首次報(bào)道在人腎透明細(xì)胞癌中存在EML4-ALK融合基因突變，體內(nèi)外功能學(xué)實(shí)驗(yàn)證明，Crizotinib有望成為腎透明細(xì)胞癌治療的新靶向藥物。
[Abstract]:Renal clear cell carcinoma (RCC) is the most common pathological type of RCC, accounting for 80% of RCC. Molecular targeted drugs such as bevacizumab, sunitinib, sorafenil have become first-line and second-line drugs in the treatment of advanced metastatic renal cell carcinoma, although there have been encouraging results in the treatment. ALK rearrangement and mutation play an important role in the occurrence and development of many kinds of tumors. Targeted therapy for ALK has become a hot topic in tumor therapy. ALK inhibitors have been approved clinically for the treatment of NSCLC,ACLC,IMT. At present, fusion gene of ALK gene rearrangement has been found in renal medullary carcinoma, renal unclassified carcinoma and papillary renal cell carcinoma, which makes ALK a new therapeutic target for renal cell carcinoma. Since renal clear cell carcinoma is the most common renal cell carcinoma, the aim of this study was to investigate whether ALK gene rearrangement exists in patients with renal clear cell carcinoma, and to explore the role of ALK in the occurrence and development of renal clear cell carcinoma. The new ALK antibody was used to screen the expression of ALK in 87 cases of renal clear cell carcinoma, 4 cases were positive for ALK protein expression, the positive rate was mainly expressed in the cytoplasm, and some of the nuclei were also expressed. The results of 5'-RACE and DNA sequencing showed that two of them had ALK rearrangement and formed EML4-ALK fusion gene. Both of them were variant 1 genes (variant1). FISH results also confirmed the existence of EML4-ALK fusion gene in these two cases of renal clear cell carcinoma). The expression system of EML4-ALKvariant1 fusion gene lentivirus was constructed by molecular cloning to investigate the role of EML4-ALK in renal cell carcinoma (RCC) by infecting human proximal tubule epithelial cells (HK-2,). Functional studies showed that transfection of EML4-ALK variant1 fusion gene into HK-2 enhanced the ability of HK-2 cells to grow and proliferate. TAE684, a small molecular inhibitor of ALK, selectively inhibited the activity of ALK kinase and thus inhibited the proliferation of HK-2 cells stably expressing EML4-ALKvariant1 gene. The results of in vivo experiments showed that stable transfection of EML4-ALK variant1 gene could induce tumorigenesis of non-tumorigenic HK-2 cells in vivo, which proved that EML4-ALK variant1 fusion gene had tumorigenic activity in vivo. More importantly, Crizotinib, a ALK inhibitor, significantly inhibited the growth of HK-2 tumors driven by ALK. In this study, we first reported the existence of EML4-ALK fusion gene mutation in human renal clear cell carcinoma. Functional experiments in vitro and in vivo demonstrated that Crizotinib may be a new target drug for the treatment of renal clear cell carcinoma.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.11
本文編號(hào)：2275582
[Abstract]:Renal clear cell carcinoma (RCC) is the most common pathological type of RCC, accounting for 80% of RCC. Molecular targeted drugs such as bevacizumab, sunitinib, sorafenil have become first-line and second-line drugs in the treatment of advanced metastatic renal cell carcinoma, although there have been encouraging results in the treatment. ALK rearrangement and mutation play an important role in the occurrence and development of many kinds of tumors. Targeted therapy for ALK has become a hot topic in tumor therapy. ALK inhibitors have been approved clinically for the treatment of NSCLC,ACLC,IMT. At present, fusion gene of ALK gene rearrangement has been found in renal medullary carcinoma, renal unclassified carcinoma and papillary renal cell carcinoma, which makes ALK a new therapeutic target for renal cell carcinoma. Since renal clear cell carcinoma is the most common renal cell carcinoma, the aim of this study was to investigate whether ALK gene rearrangement exists in patients with renal clear cell carcinoma, and to explore the role of ALK in the occurrence and development of renal clear cell carcinoma. The new ALK antibody was used to screen the expression of ALK in 87 cases of renal clear cell carcinoma, 4 cases were positive for ALK protein expression, the positive rate was mainly expressed in the cytoplasm, and some of the nuclei were also expressed. The results of 5'-RACE and DNA sequencing showed that two of them had ALK rearrangement and formed EML4-ALK fusion gene. Both of them were variant 1 genes (variant1). FISH results also confirmed the existence of EML4-ALK fusion gene in these two cases of renal clear cell carcinoma). The expression system of EML4-ALKvariant1 fusion gene lentivirus was constructed by molecular cloning to investigate the role of EML4-ALK in renal cell carcinoma (RCC) by infecting human proximal tubule epithelial cells (HK-2,). Functional studies showed that transfection of EML4-ALK variant1 fusion gene into HK-2 enhanced the ability of HK-2 cells to grow and proliferate. TAE684, a small molecular inhibitor of ALK, selectively inhibited the activity of ALK kinase and thus inhibited the proliferation of HK-2 cells stably expressing EML4-ALKvariant1 gene. The results of in vivo experiments showed that stable transfection of EML4-ALK variant1 gene could induce tumorigenesis of non-tumorigenic HK-2 cells in vivo, which proved that EML4-ALK variant1 fusion gene had tumorigenic activity in vivo. More importantly, Crizotinib, a ALK inhibitor, significantly inhibited the growth of HK-2 tumors driven by ALK. In this study, we first reported the existence of EML4-ALK fusion gene mutation in human renal clear cell carcinoma. Functional experiments in vitro and in vivo demonstrated that Crizotinib may be a new target drug for the treatment of renal clear cell carcinoma.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.11
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