AFB1诱导的B-2A13恶性转化细胞中circRNA-miRNA-mRNA的筛选及其作用的初步研究

发布时间：2018-03-29 08:23

  本文选题：黄曲霉毒素B1　切入点：CYP450　出处：《南京医科大学》2017年硕士论文

【摘要】：目的:筛选与AFB1诱导的P50B-2A13细胞恶性转化相关的circRNAs、miRNAs、mRNAs,探索circRNA-miRNA-mRNA在AFB1诱导B-2A13细胞恶性转化中的靶向调控作用。方法:首先用miRNA芯片、mRNA芯片和cricRNA芯片检测经AFB1诱导的P50B-2A13恶性转化细胞以及同期对照细胞(P50B-Vector细胞)中miRNAs、mRNAs和circRNAs表达谱,比较分析这两种细胞之间差异表达的miRNAs、mRNAs和circRNAs。以miRNA为中心,采用TargetScan软件分析了差异表达的miRNAs靶基因,将这些靶基因与差异表达mRNA相匹配,保留共有的且表达水平相反的miRNA-mRNA调控关系,根据miRNA与circRNA靶向关系,通过筛选出的miRNAs在circRNA-miRNA库中找到对应的差异表达的circRNA,初步构建circRNA-miRNA-mRNA调控关系。结合pathway分析和疾病富集分析筛选出与肺癌可能相关的mRNAs,通过RT-qPCR对关键mRNAs,miRNA及circRNA进行验证。采用western blot验证mRNA在蛋白水平的表达。最后用瞬时转染siRNA干扰方法抑制ID3和FGF5基因的表达,用平板克隆和划痕愈合实验检测它们对P50 B-2A13细胞的增殖与迁移的影响。结果:(1)在P50B-2A13细胞和P50 B-Vector细胞之间,miRNA芯片分析得到差异表达的miRNAs有60个,其中表达上调的有37个,表达下调的有23个,mRNA芯片分析得到差异表达的mRNAs有1014个,其中表达上调的有771个,表达下调的有243个,circRNA芯片分析得到差异表达的circRNAs有4313个,其中表达上调的有1496个,表达下调的有2812个。(2)对差异表达的miRNAs、mRNAs和circRNAs靶向关联分析得到GJA1、ID3和FGF5为候选功能靶基因,miR-23c 等 6 个 miRNAs 为调控中心以及 hsa_circ_0002842 等 85 个 circRNA 为海绵作用的调控关系。(3)RT-qPCR验证表明,miRNAs和mRNAs的表达和芯片结果高度一致,circRNA的表达与芯片结果一致性达到80%。(4)在P50-B-2A13细胞中,敲减ID3可以明显抑制细胞的克隆形成能力,但抑制FGF5对细胞克隆形成能力影响不大;抑制ID3和FGF5对划痕愈合都能起到一定的抑制作用,在划痕24h之后,空白组(P50 B-2A13)、对照组(P50 B-2A13+Negative control)、敲降ID3(P50B-2A13+ID3-siRNA)和敲降 FGF5(P50B-2A13+FGF5-siRNA)划痕愈合度分别为 53.9%、51.1%、38.8%和 30.6%。结论:(1)以差异表达的miRNA为中心的靶向关联分析,结合肿瘤相关的差异表达的mRNA,初步发现以GJA1、ID3和FGF5为功能基础,miR-23c等多个miRNAs为调控中心、hsa_circ_0002842等多个circRNA为海绵作用的调控关系可能与AFB1诱导的B-2A13细胞恶性转化相关。(2)ID3对恶性转化的P50 B-2A13细胞的克隆形成能力和划痕愈合能力有一定的影响;FGF5虽然对细胞克隆形成能力的影响不显著,但可影响细胞的划痕愈合能力。
[Abstract]:Objective: to screen circRNAsmiRNAsmiRNAsmRNAss associated with malignant transformation of P50B-2A13 cells induced by AFB1, and to explore the role of circRNA-miRNA-mRNA in the targeted regulation of B-2A13 cell malignant transformation induced by AFB1. Methods: firstly, AFB1 induced P50B-2A13 malignancy was detected by miRNA microarray and cricRNA chip. The expression profiles of miRNAsmRNAs and circRNAs were observed in the transformed cells and control cells (P50B-Vector cells). The differentially expressed miRN AsmRNAs and circRNAs were compared between the two cells. The differentially expressed miRNAs target genes were analyzed by TargetScan software, and the differentially expressed mRNA was matched with these target genes. The regulatory relationship of miRNA-mRNA with the opposite level of expression was retained, according to the targeting relationship between miRNA and circRNA. The differentially expressed circRNAs were found in the circRNA-miRNA library, and the regulatory relationship of circRNA-miRNA-mRNA was preliminarily constructed. The mRNASs that might be related to lung cancer were screened by pathway analysis and disease enrichment analysis. The key mRNAsmiRNAs and circRNA were verified by RT-qPCR. Western blot was used to verify the expression of mRNA at protein level. The expression of ID3 and FGF5 genes was inhibited by transient transfection of siRNA interference. The effects on proliferation and migration of P50 B-2A13 cells were detected by plate cloning and scratch healing assay. Results: there were 60 miRNAs differentially expressed between P50B-2A13 cells and P50 B-Vector cells, 37 of which were up-regulated. There were 1014 differentially expressed mRNAs in 23 down-regulated mRNAs microarray analysis, of which 771 were up-regulated, and 4313 were differentially expressed in 243 down-regulated circRNAs, among which 1496 were up-regulated. The down-regulated expression of differential expression of miRNAsmRNAs and circRNAs targeting association analysis showed that 6 miRNAs, such as GJA1T ID3 and FGF5 as candidate functional target genes, were the regulatory center and 85 circRNA of hsa_circ_0002842 were sponges. The results showed that the expression of miRNAs and mRNAs was highly consistent with the microarray results. Knockout ID3 could obviously inhibit the ability of cell clone formation, but inhibition of FGF5 had little effect on the ability of cell clone formation, inhibition of ID3 and FGF5 could inhibit the healing of scratches to some extent, and after 24 hours of scratch, inhibition of ID3 and FGF5 could inhibit the ability of cell clone formation. The scratch healing degree of P50 B-2A13, P50 B-2A13 Negative control, ID3(P50B-2A13 ID3-siRNAs and FGF5(P50B-2A13 FGF5-siRNAs in the blank group was 53.8% and 30.8%, respectively. Combined with the differential expression of mRNAs related to tumor, we preliminarily found that the regulatory relationship of multiple miRNAs such as GJA1T ID3 and FGF5 as the regulatory center, such as hsacirc0002842 and circRNA as sponge, may be related to the malignant transformation of B-2A13 cells induced by AFB1. The clone forming ability and scratch healing ability of P50 B-2A13 cells transformed by sex had a certain effect on the ability of cell clone formation although the effect of FGF5 on cell clone formation was not significant. But can affect the cell scratch healing ability.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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1 Salmon SE ,王永红;人类肿瘤克隆形成的检测方法——生长条件及其应用[J];国外医学.遗传学分册;1987年02期

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