【摘要】：PB3A是從蜂膠中分離出的黃酮單體,它對(duì)包括大腸癌細(xì)胞、宮頸癌細(xì)胞、白血病細(xì)胞、肝癌細(xì)胞、肺癌細(xì)胞等多種癌細(xì)胞的增殖抑制和誘導(dǎo)凋亡作用已被證明,但其在癌癥發(fā)生和發(fā)展中對(duì)miRNA表達(dá)量的影響,從而發(fā)揮的抗腫瘤機(jī)制尚未清楚。本研究調(diào)查了蜂膠黃酮PB3A對(duì)大腸癌細(xì)胞中miRNA表達(dá)譜的影響,尋求與大腸癌發(fā)生、發(fā)展密切相關(guān)的差異表達(dá)miRNAs,從而為大腸癌診斷治療的藥物靶點(diǎn)篩選提供新的方法和思路。本研究中首先用MTT比色法檢測(cè)不同濃度的(25、50、75和100μg/m L)PB3A,在不同時(shí)間(24h、48h、72h))對(duì)SW480大腸癌細(xì)胞增殖的影響,結(jié)果顯示,隨著PB3A濃度增加細(xì)胞存活率降低,隨著作用時(shí)間延長(zhǎng)藥物增殖抑制作用也增高,PB3A對(duì)SW480大腸癌細(xì)胞以劑量和時(shí)間依賴性起增殖抑制作用。然后以miRNA芯片同樣條件(100μg/m L PB3A,24h)處理SW480細(xì)胞,提總RNA進(jìn)行實(shí)時(shí)熒光RT-PCR實(shí)驗(yàn)驗(yàn)證miRNA表達(dá)譜芯片的真實(shí)性和可靠性,結(jié)果顯示進(jìn)行驗(yàn)證的12個(gè)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條miRNA的表達(dá)情況與micro RNA芯片的檢測(cè)結(jié)果一致,表達(dá)差異有統(tǒng)計(jì)學(xué)差異(P0.05);而mi R-761的表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),mi R-642a-5P在SW480細(xì)胞中差異表達(dá)量與miRNA芯片結(jié)果相反。下一步根據(jù)miRNA qRT-PCR驗(yàn)證結(jié)果挑選mi R-4326,合成mi R-4326模擬物和無(wú)關(guān)序列,對(duì)SW480細(xì)胞進(jìn)行轉(zhuǎn)染,結(jié)果顯示mi R-4326成功地轉(zhuǎn)染到大腸癌SW480細(xì)胞,與無(wú)關(guān)序列轉(zhuǎn)染的對(duì)照組相比mi R-4326模擬物轉(zhuǎn)染組mi R-4326的表達(dá)量顯著提高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。進(jìn)一步對(duì)PB3A干預(yù)后miRNA芯片結(jié)果和qRT-PCR結(jié)果中顯示同樣趨勢(shì)的差異表達(dá)miRNAs進(jìn)行靶基因預(yù)測(cè)并對(duì)預(yù)測(cè)出靶基因進(jìn)行功能富集分析。利用mi RWalk,Microt4,mi Randa等12個(gè)數(shù)據(jù)庫(kù)預(yù)測(cè)差異表達(dá)miRNAs的靶基因結(jié)果顯示,mi R-22-5p有575個(gè)靶基因、mi R-802有896個(gè)靶基因、mi R-296-5p有906個(gè)靶基因、mi R-18a-5p有762個(gè)靶基因、mi R-3064-5p有209個(gè)靶基因、mi R-211-5p有1730個(gè)靶基因、mi R-769-3P有728個(gè)靶基因、miRNA-4321有7個(gè)靶基因、miRNA-204-5P有1693個(gè)靶基因、miRNA-4326有299個(gè)靶基因、miRNA-219a-2-3P有626個(gè)靶基因、mi R-4697-3p有115個(gè)靶基因、mi R-142-3p有534個(gè)靶基因、mi R-198有859個(gè)靶基因。差異表達(dá)miRNAs靶基因GO富集分析結(jié)果顯示,這14條miRNA通過調(diào)控其靶基因參與多種細(xì)胞組分形成、分子功能和生物過程,其中獲得注釋信息的靶基因數(shù)最多的是生物過程,因此這些miRNA可能通過調(diào)控其參與多種生物過程的靶基因而發(fā)揮抑癌或致癌作用。miRNAs靶基因基因功能主要富集于轉(zhuǎn)錄因子和拷貝數(shù)變異相關(guān)的基因、蛋白激酶基因、組蛋白以及致癌基因等基因功能上;蛋白功能與Ras通路、PDGF信號(hào)通路、整合素信號(hào)通路、EGF受體信號(hào)通路、血管生成、FGF信號(hào)通路、Wnt信號(hào)通路、p53通路、凋亡信號(hào)通路等腫瘤發(fā)生和發(fā)展中起重要作用的信號(hào)通路有關(guān)。KEGG信號(hào)通路分析發(fā)現(xiàn)這14條miRNAs靶基因涉及腫瘤通路、Wnt信號(hào)通路、胞吞通路、軸突導(dǎo)向、慢性骨髓性白血病、MAPK信號(hào)通路、前列腺癌、胰島素信號(hào)通路、膠質(zhì)瘤、黑素生成、肌動(dòng)蛋白細(xì)胞骨架調(diào)控、結(jié)腸癌發(fā)生等信號(hào)通路。其中腫瘤通路、Wnt信號(hào)通路、MAPK信號(hào)通路、軸突導(dǎo)向通路、胞吞通路都是細(xì)胞癌變、癌癥發(fā)生、發(fā)展中重要的通路。本研究qRT-PCR實(shí)驗(yàn)結(jié)果提示SW480細(xì)胞中PB3A干預(yù)后差異表達(dá)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條miRNA可能在PB3A抗癌作用中起重要作用。生物信息學(xué)分析結(jié)果提示這些PB3A作用下差異表達(dá)miRNAs可能通過調(diào)控涉及多種細(xì)胞組分形成、分子功能和生物過程的靶基因,參與腫瘤通路、Wnt信號(hào)通路、MAPK信號(hào)通路、軸突導(dǎo)向通路、胞吞通路等重要的信號(hào)通路,從而抑制大腸癌細(xì)胞的增殖、侵襲、遷移并誘導(dǎo)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.
【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34

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