發(fā)布時間：2019-07-04 07:16

【摘要】：目的檢測原代海馬神經(jīng)元經(jīng)不同濃度錳染毒后其lncRNA及mRNA的表達情況,通過生物信息學的方法分析、預測并初步驗證由哪些基因、lncRNA以及micro RNA可能形成ceRNA調(diào)控模型通路,為進一步研究lncRNA在錳致神經(jīng)毒性中的可能作用提供線索。方法培養(yǎng)SD大鼠的原代海馬神經(jīng)元隨機分成4組,至第4天分別以0、100、400、800μM濃度的氯化錳溶液染毒24小時,芯片檢測海馬神經(jīng)元中l(wèi)ncRNA以及mRNA的表達情況,使用從聚法分析不同濃度染錳后原代海馬神經(jīng)元中mRNA和lncRNA的總體表達譜;火山圖用于表示篩選出統(tǒng)計學差異大于等于2倍和p小于等于0.05的mRNA和lncRNA差異表達情況;GO分析用于將差異表達的mRNA進行功能歸類注釋;Pathway分析用于研究差異表達mRNA的代謝通路;交叉分析用于找出錳暴露后海馬神經(jīng)元中共同上調(diào)或下調(diào)的mRNA和lncRNA;利用lncRNA與mRNA的共表達網(wǎng)絡并通過target scan、blast比對等工具預測可能的ceRNA通路,并通過Q-PCR檢測相應基因、lncRNA及microRNA在不同濃度染錳后的大鼠神經(jīng)元中的表達水平。結(jié)果(1)芯片測試結(jié)果表明,100μM組與0μM組相比,表達差異的mRNA共1848個,其中972個上調(diào),876個下調(diào);表達差異的lncRNA共566個,其中337個上調(diào),229個下調(diào)(差異均大于2倍且p0.05)。400μM組與0μM組相比,表達差異的mRNA共3328個,其中1435個上調(diào),1793個下調(diào);表達差異的lncRNA共1161個,其中589個上調(diào),572個下調(diào)(差異均大于2倍且p0.05)。800μM組與0μM組相比,表達差異的mRNA共4022個,其中1828個上調(diào),2194個下調(diào);表達差異的lncRNA共1474個,其中839個上調(diào),635個下調(diào)(差異均大于2倍且p0.05)。(2)GO分析結(jié)果表明,差異表達的mRNA參與到生物途徑、細胞定位以及分子功能中;pathway分析結(jié)果表明,100μM組與0μM組相比,最富集的信號通路是胰島素分泌和細胞周期;400μM組與0μM組相比,最富集的信號通路是類風濕性關節(jié)炎和DNA復制;800μM組與0μM組相比,最富集的信號通路是胰島素分泌和DNA復制。(3)交叉分析結(jié)果表明,和0μM組相比,100、400、800μM組海馬神經(jīng)元中共同上調(diào)的有135個lncRNA和373個mRNA,共同下調(diào)的有150個lncRNA和560個mRNA。(4)lncRNA與mRNA共表達分析結(jié)果表明,Pearson相關系數(shù)達98%以上的共表達相關組合共9個,選取基因Arsi并使用target scan檢索到與其一致性分值最高的microRNA-125b,通過blast比對及預測工具發(fā)現(xiàn)基因Arsi,microRNA-125b,lncRNA-BC089928三者可能形成ce RNA調(diào)控通路。(5)Q-PCR驗證結(jié)果表明,隨著染錳濃度的升高,基因Arsi與lncRNA-BC089928的表達顯著下調(diào)(p0.05),而microRNA-125b的表達則顯著上調(diào)(p0.05),三者很可能形成ceRNA調(diào)控模型。結(jié)論不同濃度染錳后的海馬神經(jīng)元中存在不同數(shù)量表達差異的mRNA和lncRNA,并存在一定數(shù)量共同上調(diào)或下調(diào)的mRNA和lncRNA,且lncRNA-BC089928可能通過與microRNA-125b共同調(diào)控基因Arsi形成ceRNA調(diào)控通路,提示lncRNA可能在錳致海馬神經(jīng)元神經(jīng)毒性中發(fā)揮了重要作用。
[Abstract]:Objective to detect the expression of lncRNA and mRNA in primary hippocampal neurons exposed to different concentrations of manganese, and to predict and preliminarily verify which genes, lncRNA and micro RNA may form ceRNA regulatory model pathway by bioinformatics analysis, so as to provide clues for further study on the possible role of lncRNA in manganese-induced neurotoxicity. Methods the primary hippocampal neurons of SD rats were randomly divided into 4 groups and exposed to 0 100400800 渭 M manganese chloride solution for 24 hours. The expression of lncRNA and mRNA in hippocampal neurons was detected by microarray. The overall expression profiles of mRNA and lncRNA in primary hippocampal neurons exposed to different concentrations of manganese were analyzed by polymerase chain reaction. The volcanic map was used to show the differential expression of mRNA and lncRNA with statistical difference greater than or equal to 2 times and p less than or equal to 0.05; GO analysis was used to classify and annotate the differentially expressed mRNA; Pathway analysis was used to study the metabolic pathway of differentially expressed mRNA; and cross analysis was used to identify mRNA and lncRNA; co-upregulated or down-regulated in hippocampal neurons after manganese exposure. The co-expression network of lncRNA and mRNA was used to predict the possible ceRNA pathway by target scan,blast ratio, and the expression levels of lncRNA and microRNA in rat neurons exposed to manganese at different concentrations were detected by Q-PCR. Results (1) the results of chip test showed that there were 1848 differentially expressed mRNA in 100 渭 M group compared with 0 渭 M group, of which 972 were up-regulated and 876 were down-regulated, of which 566 were up-regulated and 229 were down-regulated (the difference was more than 2 times and p0.05). Compared with 0 渭 M group, there were 3328 mRNA expression differences in 400 渭 M group, of which 1435 were up-regulated and 1793 were down-regulated. There were 1161 differentially expressed lncRNA, of which 589 were up-regulated and 572 were down-regulated (the difference was more than 2 times and p0.05). Compared with 0 渭 M group, there were 4022 mRNA expression differences in 800 渭 M group, of which 1828 were up-regulated and 2194 were down-regulated. There were 1474 differentially expressed lncRNA, of which 839 were up-regulated and 635 were down-regulated (the difference was more than 2 times and p0.05). (2). The results of GO analysis showed that the differentially expressed mRNA was involved in biological pathway, cell localization and molecular function, and the results of pathway analysis showed that insulin secretion and cell cycle were the most abundant signal pathways in 100 渭 M group compared with 0 渭 M group. Compared with 0 渭 M group, the most abundant signal pathways in 400 渭 M group were rheumatoid arthritis and DNA replication. Compared with 0 渭 M group, insulin secretion and DNA replication were the most abundant signaling pathways in 800 渭 M group. (3) the results of cross analysis showed that there were lncRNA and 373 mRNA, co-down-regulated mRNA. and mRNA. (4) lncRNA and mRNA co-expression in hippocampal neurons in 100400800 渭 M group compared with 0 渭 M group. The results showed that there were 9 co-expression related combinations with Pearson correlation coefficient above 98%. The gene Arsi was selected and the microRNA-125b, with the highest consistency score was found by blast comparison and prediction tools. (5) the results of Q-PCR verification showed that the expression of gene Arsi and lncRNA-BC089928 was significantly down-regulated (p0.05), while the expression of microRNA-125b was significantly up-regulated (p0.05) with the increase of manganese exposure concentration, and the results of Q-PCR verification showed that the expression of gene Arsi and lncRNA-BC089928 was significantly down-regulated (p0.05), while the expression of microRNA-125b was significantly up-regulated (p0.05). The three are likely to form ceRNA regulation model. Conclusion there are different quantities of mRNA and lncRNA, in hippocampal neurons exposed to different concentrations of manganese, and there are a certain number of mRNA and lncRNA, up-regulated or down-regulated by different concentrations of manganese. LncRNA-BC089928 may form ceRNA regulatory pathway by co-regulating gene Arsi with microRNA-125b, suggesting that lncRNA may play an important role in neurotoxicity of hippocampal neurons induced by manganese.
【學位授予單位】：廣西醫(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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本文編號：2509731