miRNA在结肠癌细胞奥沙利铂耐药中表达及相关调控性分子的研究

发布时间：2018-03-16 13:15

本文选题：miRNA　切入点：奥沙利铂耐药　出处：《郑州大学》2016年博士论文　论文类型：学位论文

【摘要】：背景结直肠癌是第三类常见癌症,也是世界范围内癌症死亡的第三大原因。在过去的10年中,已发现一类非编码微小RNA分子(mi RNA)在癌症发展中具有抑癌基因和癌基因的作用。已证实在结直肠癌中许多mi RNA的表达发生了变化,并且有些变化可能与通过靶向肿瘤相关基因的调控诱导肿瘤发生有关。目的研究mi RNA在人结肠癌细胞(SW480、HCT-15)和对奥沙利铂耐药的结肠癌细胞(SW480/OXA、HCT-15/OXA)中的差异化表达,对差异表达显著的mi RNA在耐药中的调控机制进行初步研究,为进一步探索有效的逆转肿瘤耐药的方法开拓新领域。方法(1)采用药物持续作用和大剂量药物间歇诱导相结合的方法,建立耐奥沙利铂的人结肠癌细胞系SW480/OXA、HCT-15/OXA。(2)利用基因芯片技术检测亲代与耐药结肠癌细胞中mi RNA的表达差异,并采用实时荧光定量PCR技术进行确证,将差异表达mi RNA的反义核苷酸和模拟物转染亲代细胞,测定其对耐药的影响。(3)采用生物信息学对耐药细胞中显著低表达的hsa-mi R-137的靶基因进行预测,利用荧光报告载体实验来验证预测靶基因,Western blot技术检测靶基因蛋白表达水平。Real-time PCR检测亲代细胞转染hsa-mi R-137-ASO后靶基因m RNA转录水平,Western blot检测蛋白水平,验证mi RNA-137对靶基因的调控作用。(4)MTT法测定结肠癌组织原代细胞对奥沙利铂耐药性,并利用实时荧光定量PCR和免疫组化法进一步研究耐药性、has-mi R-137的m RNA水平与靶基因YB-l蛋白表达水平之间的关系。结果(1)成功建立了耐受OXA的SW480/OXA和HCT-15/OXA模型,与亲代细胞相比,两株耐药细胞均产生了中度耐药,对作用机理不同的化疗药物也产生了一定的交叉耐药。(2)基因芯片检测和荧光PCR验证显示,在人结肠癌亲代细胞与耐药细胞之间,hsa-mi R-93、hsa-mi R-21、hsa-mi R-96、hsa-mi R-22、hsa-mi R-191、hsa-mi R-192、hsa-mi R-137存在明显差异性表达。MTT试验结果显示hsa-mi R-93、hsa-mi R-137、hsa-mi R-21和hsa-mi R-22具有调节OXA杀伤结肠癌细胞作用的功能。(3)mi RNA预测软件综合测定YB-l为hsa-mi R-137直接作用的候选靶基因。绿色荧光蛋白报告载体实验和实时荧光定量PCR证实,结肠癌细胞中抑制hsa-mi R-137m RNA的表达后,内源性YB-l的m RNA表达升高。Western Blot实验证实结肠癌细胞中hsa-mi R-137对YB-l蛋白表达具有负性调节作用。(4)real-time PCR显示hsa-mi R-137m RNA表达水平与结肠癌组织对OXA的敏感性密切相关,表达越高,敏感性越强。免疫组化显示,OXA敏感结肠癌组织中YB-l蛋白高表达比例较低,而耐药组织中高表达比例较高。结论(1)人结肠癌细胞OXA耐药与多种mi RNA的异常表达相关。(2)人结肠癌细胞中has-mi R-137的靶基因为YB-l,并具有负性调节作用。(3)has-mi R-137可通过靶基因YB-l调控人结肠癌细胞对OXA的敏感性。(4)结肠癌组织耐药标本has-mi R-137表达降低,YB-l蛋白增加,OXA耐药与has-mi R-137和靶基因YB-l表达密切相关。
[Abstract]:Background Colorectal cancer is the third most common type of cancer and the third leading cause of cancer deaths worldwide. A class of noncoding minute RNA RNAs have been found to play a role in tumor suppressor genes and oncogenes in the development of cancer. It has been proved that the expression of mi RNA has changed in colorectal cancer. Some of the changes may be related to the induction of tumorigenesis by targeting oncogene. Objective to study the differential expression of mi RNA in human colon cancer cell line SW480, HCT-15) and oxaliplatin resistant colon cancer cell line SW480 / OXA- 15 / OXA. A preliminary study on the regulatory mechanism of the differentially expressed mi RNA in drug resistance was carried out. In order to further explore effective methods to reverse drug resistance in cancer and explore new fields. Methods 1) the combination of drug persistence and intermittent induction of large doses of drugs was used. A human colon cancer cell line SW480 / OXAHHCT-15 / OXA.2was established to detect the difference in the expression of mi RNA between parental and drug-resistant colon cancer cells by gene chip technique, and was confirmed by real-time fluorescence quantitative PCR. The antisense nucleotides and analogue of differentially expressed mi RNA were transfected into parental cells to determine their effect on drug resistance. Bioinformatics was used to predict the target gene of hsa-mi R-137, which was significantly underexpressed in drug resistant cells. The expression level of target gene protein was detected by Western blot technique. Real-time PCR was used to detect the transcription level of target gene m RNA after transfection of hsa-mi R-137-ASO. Western blot was used to detect the protein level. To verify the regulatory effect of mi RNA-137 on target gene. MTT assay was used to determine the resistance of primary colon cancer cells to oxaliplatin. The relationship between the expression of target gene YB-l protein and the level of m RNA of drug-resistant OXA R-137 was further studied by real-time fluorescent quantitative PCR and immunohistochemistry. Results 1) the SW480/OXA and HCT-15/OXA models of OXA tolerance were successfully established, compared with those of parental cells. Two strains of drug-resistant cells developed moderate drug resistance, and also produced a certain cross-resistance to chemotherapeutic drugs with different mechanisms. The microarray analysis and fluorescence PCR verification showed that the two cell lines were resistant to drugs. Hsa-mi R-93hsa-mi R-137hsa-mi and hsa-mi R-22 hsa-mi R-192 hsa-mi R-192 hsa-mi R-192 hsa-mi R-192 hsa-mi R-137 showed that hsa-mi R-93hsa-mi R-137hsa-mi R-21 and hsa-mi R-22 had the function of regulating the function of OXA in killing colon cancer cells. YB-l was identified as a candidate target gene for direct action of hsa-mi R-137. Green fluorescent protein report vector experiment and real-time fluorescence quantitative PCR were used. After inhibiting the expression of hsa-mi R-137m RNA in colon cancer cells, The increase of m RNA expression of endogenous YB-l. Western Blot assay confirmed that hsa-mi R-137 negatively regulated the expression of YB-l protein in colon cancer cells. The expression of hsa-mi R-137m RNA was closely related to the sensitivity of colon cancer tissue to OXA, and the higher the expression was, the higher the expression of hsa-mi R-137m RNA was. The higher the sensitivity, the lower the expression of YB-l protein in OXA-sensitive colon cancer tissues. Conclusion the drug resistance of human colon cancer cell line OXA is related to the abnormal expression of various mi RNA.) the target gene of has-mi R-137 in human colon cancer cells is YB-land has a negative regulatory effect. [conclusion] the drug resistance of human colon cancer cell line OXA is related to the abnormal expression of a variety of mi RNA, and the target gene of has-mi R-137 can pass through the target base. Because YB-l regulates the sensitivity of human colon cancer cells to OXA, the expression of has-mi R-137 in drug-resistant colon cancer tissues decreases and the increase of OXA-resistance is closely related to the expression of has-mi R-137 and target gene YB-l.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.35

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