本文選題：黃曲霉毒素B1　切入點：CYP450　出處：《南京醫(yī)科大學》2017年碩士論文

【摘要】：目的:篩選與AFB1誘導的P50B-2A13細胞惡性轉化相關的circRNAs、miRNAs、mRNAs,探索circRNA-miRNA-mRNA在AFB1誘導B-2A13細胞惡性轉化中的靶向調控作用。方法:首先用miRNA芯片、mRNA芯片和cricRNA芯片檢測經AFB1誘導的P50B-2A13惡性轉化細胞以及同期對照細胞(P50B-Vector細胞)中miRNAs、mRNAs和circRNAs表達譜,比較分析這兩種細胞之間差異表達的miRNAs、mRNAs和circRNAs。以miRNA為中心,采用TargetScan軟件分析了差異表達的miRNAs靶基因,將這些靶基因與差異表達mRNA相匹配,保留共有的且表達水平相反的miRNA-mRNA調控關系,根據miRNA與circRNA靶向關系,通過篩選出的miRNAs在circRNA-miRNA庫中找到對應的差異表達的circRNA,初步構建circRNA-miRNA-mRNA調控關系。結合pathway分析和疾病富集分析篩選出與肺癌可能相關的mRNAs,通過RT-qPCR對關鍵mRNAs,miRNA及circRNA進行驗證。采用western blot驗證mRNA在蛋白水平的表達。最后用瞬時轉染siRNA干擾方法抑制ID3和FGF5基因的表達,用平板克隆和劃痕愈合實驗檢測它們對P50 B-2A13細胞的增殖與遷移的影響。結果:(1)在P50B-2A13細胞和P50 B-Vector細胞之間,miRNA芯片分析得到差異表達的miRNAs有60個,其中表達上調的有37個,表達下調的有23個,mRNA芯片分析得到差異表達的mRNAs有1014個,其中表達上調的有771個,表達下調的有243個,circRNA芯片分析得到差異表達的circRNAs有4313個,其中表達上調的有1496個,表達下調的有2812個。(2)對差異表達的miRNAs、mRNAs和circRNAs靶向關聯(lián)分析得到GJA1、ID3和FGF5為候選功能靶基因,miR-23c 等 6 個 miRNAs 為調控中心以及 hsa_circ_0002842 等 85 個 circRNA 為海綿作用的調控關系。(3)RT-qPCR驗證表明,miRNAs和mRNAs的表達和芯片結果高度一致,circRNA的表達與芯片結果一致性達到80%。(4)在P50-B-2A13細胞中,敲減ID3可以明顯抑制細胞的克隆形成能力,但抑制FGF5對細胞克隆形成能力影響不大;抑制ID3和FGF5對劃痕愈合都能起到一定的抑制作用,在劃痕24h之后,空白組(P50 B-2A13)、對照組(P50 B-2A13+Negative control)、敲降ID3(P50B-2A13+ID3-siRNA)和敲降 FGF5(P50B-2A13+FGF5-siRNA)劃痕愈合度分別為 53.9%、51.1%、38.8%和 30.6%。結論:(1)以差異表達的miRNA為中心的靶向關聯(lián)分析,結合腫瘤相關的差異表達的mRNA,初步發(fā)現(xiàn)以GJA1、ID3和FGF5為功能基礎,miR-23c等多個miRNAs為調控中心、hsa_circ_0002842等多個circRNA為海綿作用的調控關系可能與AFB1誘導的B-2A13細胞惡性轉化相關。(2)ID3對惡性轉化的P50 B-2A13細胞的克隆形成能力和劃痕愈合能力有一定的影響;FGF5雖然對細胞克隆形成能力的影響不顯著,但可影響細胞的劃痕愈合能力。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114

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1 Salmon SE ,王永紅;人類腫瘤克隆形成的檢測方法——生長條件及其應用[J];國外醫(yī)學.遺傳學分冊;1987年02期

2 黃明,吳e，

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