本文選題：miR-147a + PDPK1　； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2017年碩士論文

【摘要】：背景:隨著經(jīng)濟(jì)技術(shù)水平的不斷提高,電離輻射已經(jīng)完全充滿了人們的日常生活。微小RNA(micro RNA,mi RNA)已經(jīng)被發(fā)現(xiàn)在不同類型的輻射損傷中呈差異表達(dá)狀態(tài)。有研究發(fā)現(xiàn)mi R-147a是放射損傷后的一個(gè)重要差異表達(dá)基因,它可以促使正常細(xì)胞發(fā)生凋亡導(dǎo)致輻射損傷的敏感性。PDPK1介導(dǎo)AKT的磷酸化,PI3K/PDPK1/AKT信號通路在輻射損傷的防護(hù)中起著重要作用,輻射后mi R-147a與PDPK1有相反關(guān)系。目的:研究mi R-147a與PDPK1之間的靶標(biāo)關(guān)系,為下一步探討mi R-147a和PDPK1基因在輻射致癌發(fā)展過程提供實(shí)驗(yàn)基礎(chǔ)。方法:利用生物信息學(xué)軟件Target Scan,mi Randa數(shù)據(jù)庫,預(yù)測mi R-147a調(diào)控PDPK1基因結(jié)合序列。設(shè)計(jì)合成PDPK1 3'-UTR目的基因片段,將目的基因片段克隆到GV272熒光素酶報(bào)告基因載體,通過T4連接酶反應(yīng)構(gòu)建PDPK1 3'-UTR雙熒光素酶報(bào)告基因野生型載體(GV272-PDPK1 3'-UTR)以及突變型載體(GV272-PDPK13'-UTR-mut);另外將mi R-147a PCR擴(kuò)增片段和GV268熒光素酶報(bào)告基因載體相連,通過交換被交換反應(yīng)構(gòu)建GV268-mi R-147a重組載體�；驕y序鑒定重組載體,驗(yàn)證重組載體構(gòu)建成功。使用轉(zhuǎn)染試劑Lipofectamine TM 2000 Reagent將這兩種重組載體質(zhì)粒共轉(zhuǎn)染到293T細(xì)胞中,然后利用雙熒光素酶基因檢測系統(tǒng)檢測其活性,以及利用western blot和熒光定量PCR驗(yàn)證二者相互關(guān)系,進(jìn)而確定mi R-147a與PDPK1是否具有直接調(diào)控作用。結(jié)果:PCR基因測序結(jié)果表明,PCR擴(kuò)增后含有PDPK1 3'-UTR和PDPK1 3'-UTR序列的片段的大小和序列與Gene Bank的報(bào)道序列一致。成功構(gòu)建了兩個(gè)含有mi R-147a結(jié)合位點(diǎn)的重組質(zhì)粒及其突變重組質(zhì)粒。雙熒光素酶報(bào)告基因測定結(jié)果表明,與用PDPK1 3'-UTR突變載體質(zhì)粒轉(zhuǎn)染的mi R-147a組相比,熒光素酶活性與陰性對照組相比沒有顯著差異。通過轉(zhuǎn)染PDPK1 3'-UTR載體,mi R-147a組熒光素酶的活性被明顯抑制。熒光素酶活性與陰性對照組相比有顯著性差異(P0.05)。在western、PCR、熒光顯微鏡驗(yàn)證PDPK1與mi R-147a的表達(dá)的實(shí)驗(yàn)中,mi R-147a組與陰性對照組相比表達(dá)量減少,具有顯著性差異(P0.05);而加過抑制劑后,mi R-147a組表達(dá)量增多,具有顯著性差異(P0.05)。結(jié)論:成功構(gòu)建兩組重組載體;PDPK1基因是mi R-147a的靶基因,mi R-147a結(jié)合到PDPK1基因的3'-UTR區(qū)域,mi R-147a和PDPK1具有負(fù)性調(diào)控表達(dá),mi R-147a對轉(zhuǎn)錄后水平的PDPK1基因具有直接抑制作用。
[Abstract]:Background: with the development of economy and technology, ionizing radiation is full of people's daily life.Minute RNA(micro RNAi RNAs have been found to be differentially expressed in different types of radiation damage.It has been found that mi R-147a is an important differentially expressed gene after radiation injury. It can induce apoptosis of normal cells. PDPK1 mediates phosphorylation of AKT. PI3K / PDPK1 / AKT signaling pathway plays an important role in the protection of radiation injury.After radiation, mi R-147a has the opposite relationship with PDPK1.Aim: to study the target relationship between miR-147a and PDPK1, and to provide the experimental basis for the further study of the gene of mi R-147a and PDPK1 in the process of radiation carcinogenesis.Methods: Target Scanmi Randa database was used to predict the binding sequence of PDPK1 gene regulated by miR-147a.The target gene fragment of PDPK1 3'-UTR was designed and synthesized and cloned into GV272 luciferase reporter gene vector.Through T4 ligase reaction, PDPK1 3'-UTR double luciferase reporter gene wild-type vector, GV272-PDPK13, UTR-UTRand mutant vector, GV272-PDPK13-UTR-mutin, were constructed, and the PCR amplified fragment of mi R-147a was linked to GV268 luciferase reporter gene vector.GV268-mi R-147a recombinant vector was constructed by exchange exchange reaction.The recombinant vector was identified by gene sequencing, and the recombinant vector was successfully constructed.The recombinant plasmids were co-transfected into 293T cells with Lipofectamine TM 2000 Reagent, and their activity was detected by double luciferase gene detection system, and the relationship between them was verified by western blot and fluorescence quantitative PCR.Furthermore, it is determined whether mi R-147a and PDPK1 have direct regulation.Results the results of DNA sequencing showed that the size and sequence of the fragments containing PDPK1 3'-UTR and PDPK1 3'-UTR were consistent with the reported sequence of Gene Bank.Two recombinant plasmids containing mi R-147a binding site and their mutated recombinant plasmids were successfully constructed.The results of double luciferase reporter gene analysis showed that there was no significant difference in luciferase activity compared with the negative control group compared with the miR-147a group transfected with PDPK1 3'-UTR mutant plasmid.Luciferase activity was significantly inhibited by transfection of PDPK1 3'-UTR vector Mi R-147a.Luciferase activity was significantly different from that of negative control group (P 0.05).In the experiment of PDPK1 and mi R-147a expression verified by fluorescence microscope, the expression of PDPK1 and mi R-147a group was significantly lower than that of negative control group (P 0.05), but after adding inhibitor, the expression of miR-147a group was increased with significant difference (P 0.05).Conclusion: two groups of recombinant vector pPDPK1 gene were successfully constructed. The target gene of mi R-147a was mil R-147a binding to the 3'-UTR region of PDPK1 gene, and the expression of miR-147a and PDPK1 had direct inhibitory effect on PDPK1 gene at post-transcriptional level.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R818

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本文編號：1734976