PB3A干预SW480大肠癌细胞中miRNAs及其靶基因表达的意义

发布时间：2018-08-31 17:25

【摘要】：PB3A是从蜂胶中分离出的黄酮单体,它对包括大肠癌细胞、宫颈癌细胞、白血病细胞、肝癌细胞、肺癌细胞等多种癌细胞的增殖抑制和诱导凋亡作用已被证明,但其在癌症发生和发展中对miRNA表达量的影响,从而发挥的抗肿瘤机制尚未清楚。本研究调查了蜂胶黄酮PB3A对大肠癌细胞中miRNA表达谱的影响,寻求与大肠癌发生、发展密切相关的差异表达miRNAs,从而为大肠癌诊断治疗的药物靶点筛选提供新的方法和思路。本研究中首先用MTT比色法检测不同浓度的(25、50、75和100μg/m L)PB3A,在不同时间(24h、48h、72h))对SW480大肠癌细胞增殖的影响,结果显示,随着PB3A浓度增加细胞存活率降低,随着作用时间延长药物增殖抑制作用也增高,PB3A对SW480大肠癌细胞以剂量和时间依赖性起增殖抑制作用。然后以miRNA芯片同样条件(100μg/m L PB3A,24h)处理SW480细胞,提总RNA进行实时荧光RT-PCR实验验证miRNA表达谱芯片的真实性和可靠性,结果显示进行验证的12个miRNA中mi R-22-5p、mi R-211-5p、mi R-198、mi R-769-3P、miRNA-4321、miRNA-204-5P、miRNA-4326、miRNA-219a-2-3P、mi R-4697-3p、mi R-142-3p等10条miRNA的表达情况与micro RNA芯片的检测结果一致,表达差异有统计学差异(P0.05);而mi R-761的表达差异无统计学意义(P0.05),mi R-642a-5P在SW480细胞中差异表达量与miRNA芯片结果相反。下一步根据miRNA qRT-PCR验证结果挑选mi R-4326,合成mi R-4326模拟物和无关序列,对SW480细胞进行转染,结果显示mi R-4326成功地转染到大肠癌SW480细胞,与无关序列转染的对照组相比mi R-4326模拟物转染组mi R-4326的表达量显著提高,差异有统计学意义(P0.05)。进一步对PB3A干预后miRNA芯片结果和qRT-PCR结果中显示同样趋势的差异表达miRNAs进行靶基因预测并对预测出靶基因进行功能富集分析。利用mi RWalk,Microt4,mi Randa等12个数据库预测差异表达miRNAs的靶基因结果显示,mi R-22-5p有575个靶基因、mi R-802有896个靶基因、mi R-296-5p有906个靶基因、mi R-18a-5p有762个靶基因、mi R-3064-5p有209个靶基因、mi R-211-5p有1730个靶基因、mi R-769-3P有728个靶基因、miRNA-4321有7个靶基因、miRNA-204-5P有1693个靶基因、miRNA-4326有299个靶基因、miRNA-219a-2-3P有626个靶基因、mi R-4697-3p有115个靶基因、mi R-142-3p有534个靶基因、mi R-198有859个靶基因。差异表达miRNAs靶基因GO富集分析结果显示,这14条miRNA通过调控其靶基因参与多种细胞组分形成、分子功能和生物过程,其中获得注释信息的靶基因数最多的是生物过程,因此这些miRNA可能通过调控其参与多种生物过程的靶基因而发挥抑癌或致癌作用。miRNAs靶基因基因功能主要富集于转录因子和拷贝数变异相关的基因、蛋白激酶基因、组蛋白以及致癌基因等基因功能上;蛋白功能与Ras通路、PDGF信号通路、整合素信号通路、EGF受体信号通路、血管生成、FGF信号通路、Wnt信号通路、p53通路、凋亡信号通路等肿瘤发生和发展中起重要作用的信号通路有关。KEGG信号通路分析发现这14条miRNAs靶基因涉及肿瘤通路、Wnt信号通路、胞吞通路、轴突导向、慢性骨髓性白血病、MAPK信号通路、前列腺癌、胰岛素信号通路、胶质瘤、黑素生成、肌动蛋白细胞骨架调控、结肠癌发生等信号通路。其中肿瘤通路、Wnt信号通路、MAPK信号通路、轴突导向通路、胞吞通路都是细胞癌变、癌症发生、发展中重要的通路。本研究qRT-PCR实验结果提示SW480细胞中PB3A干预后差异表达的mi R-22-5p、mi R-211-5p、mi R-198、mi R-769-3P、miRNA-4321、miRNA-204-5P、miRNA-4326、miRNA-219a-2-3P、mi R-4697-3p、mi R-142-3p等10条miRNA可能在PB3A抗癌作用中起重要作用。生物信息学分析结果提示这些PB3A作用下差异表达miRNAs可能通过调控涉及多种细胞组分形成、分子功能和生物过程的靶基因,参与肿瘤通路、Wnt信号通路、MAPK信号通路、轴突导向通路、胞吞通路等重要的信号通路,从而抑制大肠癌细胞的增殖、侵袭、迁移并诱导其凋亡。
[Abstract]:PB3A is a flavonoid monomer isolated from propolis. It has been shown to inhibit the proliferation and induce apoptosis of colorectal cancer cells, cervical cancer cells, leukemia cells, liver cancer cells, lung cancer cells and other cancer cells. However, the effect of PB3A on the expression of microRNAs in cancer occurrence and development has not yet been clarified. In this study, we investigated the effect of propolis flavonoid PB3A on the expression profile of microRNAs in colorectal cancer cells, and sought to differentially express microRNAs closely related to the occurrence and development of colorectal cancer, thus providing new methods and ideas for screening drug targets for the diagnosis and treatment of colorectal cancer. The proliferation of SW480 colorectal cancer cells was inhibited by PB3A at different time (24h, 48h, 72h). The results showed that the survival rate of SW480 colorectal cancer cells decreased with the increase of PB3A concentration and the inhibitory effect of PB3A on the proliferation of SW480 colorectal cancer cells increased with the prolongation of action time. PB3A inhibited the proliferation of SW480 colorectal cancer cells in a dose-and time-dependent manner. 00ug/m L PB3A, 24h) treated SW480 cells, extracted total RNA for real-time fluorescence RT-PCR assay to verify the authenticity and reliability of the microarray expression profile. The results showed that of the 12 verified microRNAs, 10 microRNAs including mi R-22-5p, mi R-211-5p, mi R-198, mi R-769-3P, microNA-4321, microNA-204-5P, microNA-4326, microNA-219a-2-3P, mi R-4697-3p, and MI R-142-3p were detected. There was no significant difference in the expression of MI R-761 (P 0.05). The differential expression of MI R-642a-5P in SW480 cells was contrary to that of micro RNA chip. The next step was to select mi R-4326 and synthesize mir-4326 mimic and mir-4326 mimic according to the results of micro RNA qRT-PCR. MiR-4326 was successfully transfected into SW480 cells with unrelated sequence. The expression of MI R-4326 in mimic transfection group was significantly higher than that in unrelated sequence transfection group (P 0.05). The results of microarray and qRT-PCR showed that the expression of MI R-4326 in mimic transfection group was significantly higher than that in unrelated sequence transfection group (P 0.05). The target genes of differentially expressed microRNAs with the same trend were predicted and analyzed by functional enrichment. Twelve databases including MIRWalk, Microt4, and MIRanda were used to predict the target genes of differentially expressed microRNAs. The results showed that there were 575 target genes in MIR-22-5p, 896 target genes in MIR-802, 906 target genes in MIR-296-5p, and 906 target genes in MIR-18a-5p. There are 762 target genes, 209 target genes for MI R-3064-5p, 1730 target genes for MI R-211-5p, 728 target genes for MI R-769-3P, 7 target genes for miNA-4321, 1693 target genes for miNA-204-5P, 299 target genes for miNA-4326, 626 target genes for MI R-219a-2-3P, 115 target genes for MI R-4697-3p, 534 target genes for MI R-142-3p, and R-434 target genes for MI R-432-5P. There were 859 target genes in 198. Differentially expressed microRNAs target gene GO enrichment analysis showed that these 14 microRNAs were involved in many cellular components formation, molecular functions and biological processes by regulating their target genes. Among them, the most annotated target genes were biological processes, so these microRNAs may be involved in a variety of biological processes by regulating them. MicroRNAs target genes are mainly enriched in transcription factors and copy number variation-related genes, protein kinase genes, histones and oncogenes; protein functions are associated with Ras pathway, PDGF signaling pathway, integrin signaling pathway, EGF receptor signaling pathway, angiogenesis, and FGF KEGG signaling pathway analysis revealed that these 14 microRNAs target genes were involved in tumor pathway, Wnt signaling pathway, endocytosis pathway, axonal guidance, chronic myeloid leukemia, MAPK signaling pathway, prostate cancer, insulin and so on. Signal pathways, such as glioma, melanogenesis, actin cytoskeleton regulation, colon carcinogenesis, and so on. Tumor pathway, Wnt signaling pathway, MAPK signaling pathway, axon-directed pathway, and endocytosis pathway are all important pathways in cell carcinogenesis, cancer occurrence and development. The results of qRT-PCR in this study suggest that PB3A in SW480 cells is an intervention. Differentially expressed 10 microRNAs, including mi R-22-5p, mi R-211-5p, mi R-198, mi R-769-3P, microNA-4321, microNA-204-5P, microNA-4326, microNA-219a-2-3P, mi R-4697-3p, and MI R-142-3p, may play an important role in the anticancer effect of PB3A. Bioinformatics analysis suggests that the differentially expressed microNAs under the action of PB3A may involve a variety of cells. Component formation, molecular function and target genes involved in biological processes, tumor pathway, Wnt signaling pathway, MAPK signaling pathway, axon-directed pathway, endocytosis pathway and other important signaling pathways, thereby inhibiting the proliferation, invasion, migration and inducing apoptosis of colorectal cancer cells.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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