Sema3C在神經膠質瘤中的功能及分子調控機制研究
發(fā)布時間：2019-04-27 14:35
【摘要】：目的:探討在膠質瘤中調控Semaphorin3c表達調控機制。方法:1、生物信息學軟件分析和預測可能調控Sema3C表達的microRNA:(1)GeneBank中輸入Sema3C基因,找出該基因mRNA的3'UTR序列。(2)采用TargetScan和miRanDa軟件,將Sema3C mRNA的3'UTR序列輸入其中,評分選擇可能調控Sema3C表達的5-10個候選microRNA。(3)通過PubMed軟件進行文獻檢索,在2中選擇的5-10個候選microRNA中進一步縮小范圍,將待檢測目的microRNA縮小至2-3個。2、檢測microRNA對Sema3C表達的調控:(1)訂制針對人microRNA的模擬物(mimics)和抑制劑。(2)體外常規(guī)培養(yǎng)人膠質瘤細胞系(A172和U251),采用Lipotamine2000轉染法將目的microRNA的模擬物(mimics)和抑制劑,及其相應的對照(模擬物對照和抑制劑對照)分別轉染入以上細胞系,提取總蛋白和總RNA,Westernblot和熒光定量PCR法檢測Sema3C的表達。3、檢測待選microRNA是否可影響Sema3C mRNA的3'UTR的活性:(1)提取人膠質瘤細胞系A172的總RNA,進行cDNA的反轉錄,擴增軟件中預測出可能有microRNA結合的序列,將其連接到pmirGLo質粒上。(2)將microRNA的模擬物和抑制劑及其對照、含有Sema3C mRNA的3'UTR的pmirGLo載體等分別轉染入HEK293T細胞中,對細胞樣品進行裂解后檢測各組熒光信號值。4、收集膠質瘤組織(約30例)和周邊正常腦組織(約30例)后,進行如下實驗:(1)提取組織總RNA和總蛋白,Western blot和實時熒光定量PCR法檢測Sema3C和microRNA的表達。(2)統計學分析Sema3C和microRNA的表達在膠質瘤和周邊癌組織中是否存在相關性。結果:1、Sema3C的組織學表達特點免疫組織化學法檢測發(fā)現Sema3C在膠質瘤中的過表達的頻率(78.2%)明顯高于周邊正常組織(20.0%);低表達Sema3C的膠質瘤患者預后較好(p=0.017);臨床指標分析發(fā)現,其表達量與組織學分型(p=0.008)和病理分級(p=0.002)相關;在Sema3C表達量較高的組織中IDH1(可溶性異檸檬酸脫氫酶1)突變的發(fā)生率低(p=0.0001);此外,還與Ki67指數相關(p=0.02)。2、Sema3C的細胞學功能沉默Sema3C表達后,細胞的增殖和侵襲能力明顯降低;對增殖能力的調控是通過影響細胞周期實現的;Sema3C可通過改變細胞的上皮-間質轉換影響細胞的侵襲能力等。3、Sema3C致病機制的研究Sema3C在膠質瘤細胞中受mi R-142-5p的表達調控,miR-142-5p是通過影響Sema3C m RNA的翻譯水平實現的;miR-142-5p也可調控膠質瘤細胞的增殖和侵襲能力,并且該作用的發(fā)揮也可能通過影響細胞的上皮-間質細胞轉化而發(fā)生。4、Sema3C和miR-142-5p組織表達相關性miR-142-5p在膠質瘤組織的表達隨著級別的升高而降低;Sema3C和miR-142-5p在膠質瘤組織中的表達成負相關。結論:1、Sema3C在膠質瘤中可作為不良預后指標用于臨床診斷方面的繼續(xù)研究;2、Sema3C在膠質瘤中發(fā)揮癌基因的作用,該作用的發(fā)揮可能通過調控上皮-間質細胞轉化而實現;3、miR-142-5p在膠質瘤中負向調控Sema3C的表達,提示在膠質瘤病變過程中可能存在miR-142-5p/Sema3C致病途徑。
[Abstract]:Objective: to investigate the regulation mechanism of Semaphorin3c expression in glioma. Methods: 1. Bioinformatics software was used to analyze and predict the input of Sema3C gene into microRNA: (1) GeneBank, which might regulate the expression of Sema3C. (2) the 3'UTR sequence of Sema3C mRNA was inputted into it by TargetScan and miRanDa software. The 5 / 10 candidate microRNA. (3), which may regulate the expression of Sema3C, were retrieved by PubMed software and further narrowed in the 5 / 10 candidate microRNA selected in 2, and the target microRNA to be detected was reduced to 2. 2. To detect the regulation of Sema3C expression by microRNA: (1) (mimics) and inhibitors targeting human microRNA were made. (2) Human glioma cell lines (A172 and U251) were cultured routinely in vitro. The target microRNA mimicants (mimics) and inhibitors were transfected by Lipotamine2000. And the corresponding control (mock control and inhibitor control) were transfected into the above cell lines respectively. The total protein and total RNA,Westernblot were extracted and the expression of Sema3C was detected by fluorescence quantitative PCR. To detect whether microRNA could affect the activity of 3'UTR of Sema3C mRNA: (1) the total RNA, of human glioma cell line A172 was extracted for reverse transcription of cDNA, and the sequence of microRNA binding was predicted in the amplification software. (2) the mimics and inhibitors of microRNA and their control, pmirGLo vector containing Sema3C mRNA 3'UTR and so on were transfected into HEK293T cells respectively, and the fluorescent signal values of each group were detected after the cell samples were lysed. After collecting glioma tissues (about 30 cases) and peripheral normal brain tissues (about 30 cases), the following experiments were carried out: (1) Total RNA and total protein were extracted from the tissues. Western blot and real-time fluorescence quantitative PCR were used to detect the expression of Sema3C and microRNA. (2) the correlation between the expression of Sema3C and microRNA in glioma and peripheral carcinoma was analyzed statistically. Results: (1) Immunohistochemistry showed that the frequency of Sema3C overexpression in glioma (78.2%) was significantly higher than that in peripheral normal tissues (20.0%). The prognosis of glioma patients with low expression of Sema3C was better (p < 0. 017), and the expression level was correlated with histological type (p < 0. 008) and pathological grade (p < 0. 002), and the expression level was correlated with histological type (p < 0. 008). The incidence of IDH1 (soluble isocitrate dehydrogenase 1) mutation was lower in the tissues with higher expression of Sema3C (p < 0.0001). In addition, it was also correlated with Ki67 index (p = 0.02). 2. After silencing Sema3C expression by cytological function of Sema3C, the ability of cell proliferation and invasion was significantly decreased, and the regulation of proliferation ability was realized by affecting the cell cycle. Sema3C can affect the invasiveness of glioma cells by changing the epithelial-interstitial transformation. 3. The study of the pathogenesis of Sema3C to investigate the regulation of Sema3C expression in glioma cells by the expression of mi R _ 2 ~ 2 ~ 2 ~ (5) p, P < 0.01, P < 0.05, P < 0.05. MiR-142-5p is realized by influencing the translation level of Sema3C m RNA; MiR-142-5p can also regulate the proliferation and invasion of glioma cells, and this effect may also occur by affecting epithelial-stromal cell transformation. 4, The expression of Sema3C and miR-142-5p-related miR-142-5p in glioma tissues decreased with the increase of grade. There was a negative correlation between the expression of Sema3C and miR-142-5p in glioma. Conclusion: (1) Sema3C can be used as an indicator of poor prognosis for further study in clinical diagnosis, and Sema3C may play an oncogene role in gliomas, which may be achieved by regulating epithelial-stromal cell transformation. 3. The negative regulation of the expression of Sema3C in gliomas suggests that miR-142-5p/Sema3C may be involved in the pathogenesis of glioma.
【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.4
本文編號：2467058
[Abstract]:Objective: to investigate the regulation mechanism of Semaphorin3c expression in glioma. Methods: 1. Bioinformatics software was used to analyze and predict the input of Sema3C gene into microRNA: (1) GeneBank, which might regulate the expression of Sema3C. (2) the 3'UTR sequence of Sema3C mRNA was inputted into it by TargetScan and miRanDa software. The 5 / 10 candidate microRNA. (3), which may regulate the expression of Sema3C, were retrieved by PubMed software and further narrowed in the 5 / 10 candidate microRNA selected in 2, and the target microRNA to be detected was reduced to 2. 2. To detect the regulation of Sema3C expression by microRNA: (1) (mimics) and inhibitors targeting human microRNA were made. (2) Human glioma cell lines (A172 and U251) were cultured routinely in vitro. The target microRNA mimicants (mimics) and inhibitors were transfected by Lipotamine2000. And the corresponding control (mock control and inhibitor control) were transfected into the above cell lines respectively. The total protein and total RNA,Westernblot were extracted and the expression of Sema3C was detected by fluorescence quantitative PCR. To detect whether microRNA could affect the activity of 3'UTR of Sema3C mRNA: (1) the total RNA, of human glioma cell line A172 was extracted for reverse transcription of cDNA, and the sequence of microRNA binding was predicted in the amplification software. (2) the mimics and inhibitors of microRNA and their control, pmirGLo vector containing Sema3C mRNA 3'UTR and so on were transfected into HEK293T cells respectively, and the fluorescent signal values of each group were detected after the cell samples were lysed. After collecting glioma tissues (about 30 cases) and peripheral normal brain tissues (about 30 cases), the following experiments were carried out: (1) Total RNA and total protein were extracted from the tissues. Western blot and real-time fluorescence quantitative PCR were used to detect the expression of Sema3C and microRNA. (2) the correlation between the expression of Sema3C and microRNA in glioma and peripheral carcinoma was analyzed statistically. Results: (1) Immunohistochemistry showed that the frequency of Sema3C overexpression in glioma (78.2%) was significantly higher than that in peripheral normal tissues (20.0%). The prognosis of glioma patients with low expression of Sema3C was better (p < 0. 017), and the expression level was correlated with histological type (p < 0. 008) and pathological grade (p < 0. 002), and the expression level was correlated with histological type (p < 0. 008). The incidence of IDH1 (soluble isocitrate dehydrogenase 1) mutation was lower in the tissues with higher expression of Sema3C (p < 0.0001). In addition, it was also correlated with Ki67 index (p = 0.02). 2. After silencing Sema3C expression by cytological function of Sema3C, the ability of cell proliferation and invasion was significantly decreased, and the regulation of proliferation ability was realized by affecting the cell cycle. Sema3C can affect the invasiveness of glioma cells by changing the epithelial-interstitial transformation. 3. The study of the pathogenesis of Sema3C to investigate the regulation of Sema3C expression in glioma cells by the expression of mi R _ 2 ~ 2 ~ 2 ~ (5) p, P < 0.01, P < 0.05, P < 0.05. MiR-142-5p is realized by influencing the translation level of Sema3C m RNA; MiR-142-5p can also regulate the proliferation and invasion of glioma cells, and this effect may also occur by affecting epithelial-stromal cell transformation. 4, The expression of Sema3C and miR-142-5p-related miR-142-5p in glioma tissues decreased with the increase of grade. There was a negative correlation between the expression of Sema3C and miR-142-5p in glioma. Conclusion: (1) Sema3C can be used as an indicator of poor prognosis for further study in clinical diagnosis, and Sema3C may play an oncogene role in gliomas, which may be achieved by regulating epithelial-stromal cell transformation. 3. The negative regulation of the expression of Sema3C in gliomas suggests that miR-142-5p/Sema3C may be involved in the pathogenesis of glioma.
【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.4
【參考文獻】
相關期刊論文 前1條
1 曾冉;況建國;;膠質瘤發(fā)病機制的研究進展[J];廣東醫(yī)學;2013年06期
，本文編號：2467058
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