FOXM1通过调控ABCC10促进结直肠癌5-氟尿嘧啶耐药的研究

发布时间：2018-07-08 14:21

本文选题：结直肠癌 + 5-氟尿嘧啶　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景:结直肠癌是最常见的消化道肿瘤之一,其发病率和癌症相关死亡率在各类肿瘤中居于前列。结直肠癌的治疗手段包括手术、放疗、化疗,但其生存预后仍不乐观。5-氟尿嘧啶作为临床结直肠癌化疗的一线用药,其作用机制是通过抑制胸苷酸合酶的活性,影响DNA的合成。一部分患者由于原发性和继发性对5-氟尿嘧啶治疗形成耐药,导致化疗失败,肿瘤复发转移。FOXM1隶属于叉头框转录因子家族,在多种肿瘤化疗药物的耐受中发挥重要作用,但FOXM1对结直肠癌与5-FU耐药的作用和机制尚未明确。方法:1、基因芯片分析在5-FU治疗敏感和不敏感的临床标本中的差异基因。2、分析FOXM1在结直肠癌细胞系中的表达含量和IC50之间的相关性。3、选取细胞系中FOXM1表达含量高的两株细胞和HCT-8/5-FU耐药株,转染慢病毒包装的sh-FOXMl;FOXM1表达低的两株细胞,转染慢病毒包装的FOXM1过表达质粒。验证干扰和过表达效率,筛选稳定转染的细胞,检测5-FU的IC50值,平板克隆形成率,细胞凋亡水平。4、选取FOXM1过表达的细胞系,裸鼠皮下种植,待肿瘤长到一定体积后予以裸鼠腹腔内注射5-FU和相关药物,测量肿瘤体积大小,绘制肿瘤生长曲线,移植瘤做免疫组化检测相关蛋白的表达水平。5、利用生物信息学方法筛选参与5-FU代谢相关的并且启动子区域具有FOXM1结合位点的基因,ChIP实验验证FOXM1对靶基因ABCCI0的调控。6、干扰下游靶基因,检测IC50值和凋亡相关变化,功能拯救实验验证FOXM1通过调控该靶基因参与5-FU的化疗耐受。7、临床样本。结果:经基因芯片筛选和验证,FOXM1在5-FU不敏感的临床样本中表达含量高;结直肠癌细胞系中FOXM1的表达含量与5-FU的IC50值呈正相关;干扰FOXM1能增强结直肠癌细胞对5-FU的敏感性,抑制5-FU作用下克隆形成率,增加5-FU引起的细胞凋亡,反之过表达FOXM1能抑制结直肠癌细胞对5-FU的敏感性;体内实验进一步证实过表达FOXM1能增强结直肠癌移植瘤对5-FU的耐受性;机制研究表明FOXM1通过转录调控ABCC10的表达增强结直肠细胞对5-FU的耐受性,ABCC10抑制剂Vardenafil协同5-FU腹腔注射,能恢复5-FU耐药移植瘤的化疗反应性。讨论:近年来研究发现FOXM1参与吉非替尼、顺铂、多西他赛、表柔霉素等多种化疗药物的耐受,本课题第一次提出FOXM1在5-FU的化疗耐药中发挥重要作用。FOXM1通过转录调控ABC家族成员ABCC10,增加肿瘤细胞内药物的外排,阻断5-FU对细胞的毒性作用,从而影响细胞对药物的敏感性。我们的研究证实FOXM1/ABCC10信号轴在结直肠癌5-FU耐药中的作用,为解决5-FU耐药提供了新方向。
[Abstract]:Background: colorectal cancer is one of the most common digestive tract tumors. The incidence of cancer and cancer related mortality are in the forefront of all kinds of tumors. The treatment methods of colorectal cancer include surgery, radiotherapy, chemotherapy, but the survival prognosis is still not optimistic..5- fluorouracil is the first line of chemotherapy for colorectal cancer, its mechanism is through Inhibition of the activity of thymidylate synthase affects the synthesis of DNA. Some patients are resistant to 5- fluorouracil due to primary and secondary treatment, resulting in chemotherapy failure. Tumor recurrence and metastasis of.FOXM1 is subordinate to the forked frame transcription factor family, which plays an important role in the tolerance of various tumor chemotherapeutic drugs, but FOXM1 is resistant to colorectal cancer and 5-FU. The effect and mechanism of the drug have not been clearly defined. Methods: 1, gene chip analysis was used to analyze the differential gene.2 in the sensitive and insensitive clinical specimens treated by 5-FU. The expression of FOXM1 in the colorectal cancer cell lines and the correlation between IC50 and.3 were analyzed, and two cells and HCT-8/5-FU resistant strains with high FOXM1 expression in the cell lines were selected to transfect the slow disease. Toxic package of sh-FOXMl; FOXM1 expressed low two cells, transfected with FOXM1 overexpressed plasmids packed with lentivirus, verified interference and overexpression efficiency, screened stable transfected cells, detected the IC50 value of 5-FU, the formation rate of flat clones, the level of apoptosis.4, selected FOXM1 overexpressed cell lines, nude mice planted subcutaneously, and the tumor grew to a certain volume. 5-FU and related drugs were injected intraperitoneally in the nude mice. The tumor size was measured, the tumor growth curve was plotted, and the expression level of the related protein was detected by immunohistochemistry. The bioinformatics method was used to screen the genes involved in the metabolism of 5-FU and the FOXM1 binding site in the promoter region. The ChIP experiment verified the target of FOXM1 to the target. Gene ABCCI0 regulating.6, interfering downstream target gene, detecting IC50 value and apoptosis related changes. Functional rescue experiment verifies that FOXM1 is involved in 5-FU chemotherapy tolerance.7, clinical samples. Results: FOXM1 is highly expressed in 5-FU insensitive clinical samples by gene chip screening and verification; FOXM1 in colorectal cancer cell lines. The expression content is positively correlated with the IC50 value of 5-FU; interfering FOXM1 can enhance the sensitivity of colorectal cancer cells to 5-FU, inhibit the clone formation rate under the action of 5-FU and increase the apoptosis induced by 5-FU. Conversely, overexpression of FOXM1 can inhibit the sensitivity of colorectal cancer cells to 5-FU, and in vivo experiments further confirmed that overexpression FOXM1 can enhance colorectal cancer. The tolerance of transplanted tumor to 5-FU showed that FOXM1 enhanced the tolerance of colorectal cells to 5-FU by transcriptional regulation of ABCC10. ABCC10 inhibitor Vardenafil combined with 5-FU intraperitoneal injection to restore the chemotherapeutic responsiveness of 5-FU resistant xenografts. Discussion: in recent years, a study has been made to develop FOXM1 involved in gefitinib, cisplatin, docetaxel, and epirubicin. The tolerance of mycophenin and a variety of chemotherapeutic drugs, the first time that FOXM1 plays an important role in the chemotherapeutic resistance of 5-FU,.FOXM1 is regulated by the transcription of ABC family members ABCC10, increasing the efflux of the drug in the tumor cells, blocking the toxic effects of 5-FU on the cells, and thus affecting the cell sensitivity to the drug. Our study confirmed that FOXM1/ABCC1 0 the role of signal axis in 5-FU resistance of colorectal cancer provides a new direction for resolving 5-FU resistance.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.3

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