结肠癌细胞VCR耐药株的建立及相关miRNA筛选

发布时间：2018-08-31 17:04

【摘要】：背景肿瘤细胞耐药是导致肿瘤化学治疗失败的重要原因,长春新碱(vincristine, VCR)是一类广泛用于肿瘤治疗的化学药物。但是,长期使用可使肿瘤细胞对VCR产生耐药性,肿瘤耐药机制仍不清楚。本研究探讨与结肠癌耐药的微小RNA(microRNA, miRNA),旨在揭示肿瘤耐药机制及为肿瘤细胞耐药逆转提供基础。目的建立长春新碱耐药结肠癌细胞模型,筛选与结肠癌细胞VCR耐药相关的miRNA。方法1.培养结肠癌HCT-8细胞,采用VCR梯度诱导,建立HCT-8/VCR耐药细胞株。2.采用全基因组筛选的研究策略,运用HiSeq 2500测序技术和生物信息学的方法,检测和分析耐药HCT-8/VCR细胞的和非耐药的HCT-8细胞差异性表达的miRNAs,构建其miRNA的差异表达谱。3.针对鉴定的差异miRNA,通过靶基因预测软件进行靶位点确认,通过靶基因所在基因的GO进行功能注释。结果1.利用浓度梯度的VCR建立了VCR耐药的结肠癌细胞HCT-8/VCR, HCT-8/VCR细胞在2×103ng/mLVCR中生长良好。2.共进行了1651种miRNA测序,其中有差异的有48种miRNA,与非耐药的结肠癌HCT-8细胞相比,24种miRNA差异具有统计学意义(P0.05),其中有17种miRNA表达上调(P0.05)。hsa-miR-675-3p表达量上调最高,高达7.497681倍,其次是hsa-miR-100-3p, hsa-miR-125b-1-3p, hsa-miR-582-5p, hsa-miR-3141, chr15_10055, hsa-miR-582-5p, hsa-miR-582-3p, hsa-miR-3687。7种miRNA表达下调(P0.05),下调最高的是hsa-miR-146a-5p,下调7.75199倍,其次是hsa-miR-1247-3p, hsa-miR-181 a-3p, hsa-miR-1247-5p。结论]miRNA的异常表达与结肠癌细胞VCR耐药相关。miRNA表达量的差异可能在结肠癌细胞VCR耐药中起关键作用。
[Abstract]:Background Drug resistance of tumor cells is an important factor leading to the failure of tumor chemotherapy. Vincristine (vincristine, VCR) is a kind of chemotherapeutic drugs widely used in tumor therapy. However, long-term use can result in drug resistance of tumor cells to VCR, and the mechanism of drug-resistance remains unclear. The purpose of this study was to explore the mechanism of drug resistance and to provide a basis for reversal of drug resistance in cancer cells. Objective to establish vincristine resistant colon cancer cell model and screen miRNA. associated with VCR resistance of colon cancer cells. Method 1. Colon cancer HCT-8 cells were cultured and induced by VCR gradient to establish HCT-8/VCR resistant cell line. 2. By using HiSeq 2500 sequencing technique and bioinformatics method, the differentially expressed miRNAs, of drug-resistant HCT-8/VCR cells and non-drug-resistant HCT-8 cells were detected and analyzed by using the strategy of genome screening. The differential expression profile of miRNA was constructed. The target site was identified by target gene prediction software and the functional annotation was performed by GO of the gene in which the target gene was located. Result 1. The VCR resistant colon cancer cell HCT-8/VCR, HCT-8/VCR cells were established by using concentration gradient VCR. The cells grew well in 2 脳 103ng/mLVCR. A total of 1651 miRNA sequences were performed. Among them, 48 miRNA, were significantly different from non-drug-resistant colon cancer HCT-8 cells in 24 miRNA (P0.05), 17 of which were miRNA up-regulated (P0.05), the highest (7.497681 times) in hsa-miR-675-3p expression, and the highest in Hsa-miR-675-3p expression (P0.05). The miRNA expression of hsa-miR-100-3p, hsa-miR-125b-1-3p, hsa-miR-582-5p, hsa-miR-3141, chr15_10055, hsa-miR-582-5p, hsa-miR-582-3p, hsa-miR-3687.7 species was down-regulated (P0.05), and the highest down-regulation of hsa-miR-146a-5p, was 7.75199 times, followed by hsa-miR-1247-3p, hsa-miR-181 a-3p, hsa-miR-1247-5p.. Conclusion: the difference of the expression of miRNA and VCR related to drug resistance in colon cancer cells may play a key role in the drug resistance of colon cancer cell line VCR.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.35

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