本文關(guān)鍵詞：miR-483-3P下調(diào)RIOK3蛋白表達并促進神經(jīng)母細胞瘤的增殖侵襲和遷移　出處：《青島大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 神經(jīng)母細胞瘤 mi R-483-3P 細胞增殖 侵襲 靶基因

【摘要】：目的通過實驗研究micro RNA-483-3P(mi R-483-3P)對神經(jīng)母細胞瘤細胞的生物學(xué)行為的影響,諸如增殖能力、侵襲能力及遷移能力。利用生物信息學(xué)軟件預(yù)測mi R-483-3P的靶基因并加以驗證,以及探索mi R-483-3P對所預(yù)測的靶基因的影響。方法mi RNA微陣列芯片(Micro Array)的結(jié)果發(fā)現(xiàn),與原發(fā)瘤相比,mi R-483-3P在神經(jīng)母細胞轉(zhuǎn)移瘤中表達水平明顯上調(diào),差異倍數(shù)顯著,并居于所檢測出的差異表達的mi RNA的第一位。利用陽離子脂質(zhì)體Lipofectamine TM2000將化學(xué)合成的mi R-483-3P inhibitor、mi R-483-3P inhibitor的陰性對照序列(negative control)分別轉(zhuǎn)染入人神經(jīng)母細胞瘤SH-SY-5Y細胞株中,實時熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(RT-q PCR)技術(shù)檢測各組神經(jīng)母細胞瘤經(jīng)處理后mi R-483-3P的表達水平,細胞計數(shù)試劑盒(CCK-8)法檢測各組神經(jīng)母細胞瘤細胞的增殖情況,Transwell小室實驗檢測各組神經(jīng)母細胞瘤細胞的體外侵襲以及遷移能力。利用多個靶基因預(yù)測數(shù)據(jù)庫,綜合預(yù)測mi R-483-3P的靶基因,熒光素酶報告基因檢測實驗、Western blot實驗加以驗證。結(jié)果實驗所取的9對神經(jīng)母細胞瘤組織與癌旁正常組織,RT-QPCR結(jié)果顯示,mi R-483-3P在神經(jīng)母細胞瘤腫瘤組織中表達水平明顯高于癌旁正常組織(P0.01);與轉(zhuǎn)染了mi R-483-3P inhibitor的陰性對照組相比,癌細胞轉(zhuǎn)染mi R-483-3P inhibitor后,mi R-483-3P被抑制表達水平顯著下調(diào)(P0.01),細胞的增殖情況和體外遷移能力均降低(P0.05)。經(jīng)多個靶基因生物信息學(xué)軟件分析,預(yù)測出RIOK3是mi R-483-3P的靶基因之一,當(dāng)神經(jīng)母細胞瘤細胞轉(zhuǎn)染了mi R-483-3P inhibitor后熒光酶的活性明顯升高(P0.01)。與mi R-483-3P inhibitor陰性對照組相比,當(dāng)細胞轉(zhuǎn)染了mi R-483-3P inhibitor后,RIOK3蛋白的表達水平明顯升高(P0.01)。結(jié)論RIOK3是miR-483-3P的靶基因之一,miR-483-3P能下調(diào)RIOK3蛋白的表達,mi R-483-3P可顯著促進神經(jīng)母細胞瘤細胞的增殖及體外遷移能力。
[Abstract]:Objective to investigate the effects of micro RNA-483-3P(mi R-483-3P on the biological behavior of neuroblastoma cells, such as proliferation. Invasive ability and migration ability. The target gene of mi R-483-3P was predicted and verified by bioinformatics software. And to explore the effect of mi R-483-3P on the predicted target gene. Methods the results of mi RNA microarray microarray microarray showed that compared with the primary tumor. The expression of mi R-483-3P was up-regulated in neuroblastoma, and the difference was significant. The chemically synthesized mi R-483-3P was chemically synthesized using cationic liposome Lipofectamine TM2000. Inhibitor. Negative control sequence of mi R-483-3P inhibitor. The cells were transfected into human neuroblastoma SH-SY-5Y cell lines. Real-time quantitative polymerase chain reaction (RT-q PCR) technique was used to detect the expression of mi R-483-3P in neuroblastoma after treatment. The proliferation of neuroblastoma cells in each group was detected by CCK-8 assay. The invasiveness and migration ability of neuroblastoma cells in each group were detected by Transwell chamber assay. The target genes of mi R-483-3P were comprehensively predicted by using multiple target gene prediction databases. Results 9 pairs of neuroblastoma tissues and adjacent normal tissues were detected by RT-QPCR. The expression of miR-483-3P in neuroblastoma was significantly higher than that in adjacent normal tissue (P 0.01). Compared with the negative control group transfected with mi R-483-3P inhibitor, the cancer cells were transfected with mi R-483-3P inhibitor. Mi R-483-3P was significantly down-regulated (P0.01). The cell proliferation and migration ability in vitro were decreased (P 0.05). It was predicted that RIOK3 was one of the target genes of mi R-483-3P by multiple target gene bioinformatics software analysis. The activity of fluorescence enzyme increased significantly after transfection of mi R-483-3P inhibitor into neuroblastoma cells (P 0.01). Compared with mi R-483-3P inhibitor negative control group. When the cells were transfected with mi R-483-3P inhibitor. Conclusion RIOK3 is one of the target genes of miR-483-3P. MiR-483-3P can down-regulate the expression of RIOK3 protein. Mi R-483-3P can significantly promote the proliferation and migration of neuroblastoma cells in vitro.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R739.4

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