Sema3C在神經(jīng)膠質(zhì)瘤中的功能及分子調(diào)控機(jī)制研究
發(fā)布時(shí)間：2019-04-27 14:35
【摘要】：目的:探討在膠質(zhì)瘤中調(diào)控Semaphorin3c表達(dá)調(diào)控機(jī)制。方法:1、生物信息學(xué)軟件分析和預(yù)測(cè)可能調(diào)控Sema3C表達(dá)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(píng)分選擇可能調(diào)控Sema3C表達(dá)的5-10個(gè)候選microRNA。(3)通過(guò)PubMed軟件進(jìn)行文獻(xiàn)檢索,在2中選擇的5-10個(gè)候選microRNA中進(jìn)一步縮小范圍,將待檢測(cè)目的microRNA縮小至2-3個(gè)。2、檢測(cè)microRNA對(duì)Sema3C表達(dá)的調(diào)控:(1)訂制針對(duì)人microRNA的模擬物(mimics)和抑制劑。(2)體外常規(guī)培養(yǎng)人膠質(zhì)瘤細(xì)胞系(A172和U251),采用Lipotamine2000轉(zhuǎn)染法將目的microRNA的模擬物(mimics)和抑制劑,及其相應(yīng)的對(duì)照(模擬物對(duì)照和抑制劑對(duì)照)分別轉(zhuǎn)染入以上細(xì)胞系,提取總蛋白和總RNA,Westernblot和熒光定量PCR法檢測(cè)Sema3C的表達(dá)。3、檢測(cè)待選microRNA是否可影響Sema3C mRNA的3'UTR的活性:(1)提取人膠質(zhì)瘤細(xì)胞系A(chǔ)172的總RNA,進(jìn)行cDNA的反轉(zhuǎn)錄,擴(kuò)增軟件中預(yù)測(cè)出可能有microRNA結(jié)合的序列,將其連接到pmirGLo質(zhì)粒上。(2)將microRNA的模擬物和抑制劑及其對(duì)照、含有Sema3C mRNA的3'UTR的pmirGLo載體等分別轉(zhuǎn)染入HEK293T細(xì)胞中,對(duì)細(xì)胞樣品進(jìn)行裂解后檢測(cè)各組熒光信號(hào)值。4、收集膠質(zhì)瘤組織(約30例)和周邊正常腦組織(約30例)后,進(jìn)行如下實(shí)驗(yàn):(1)提取組織總RNA和總蛋白,Western blot和實(shí)時(shí)熒光定量PCR法檢測(cè)Sema3C和microRNA的表達(dá)。(2)統(tǒng)計(jì)學(xué)分析Sema3C和microRNA的表達(dá)在膠質(zhì)瘤和周邊癌組織中是否存在相關(guān)性。結(jié)果:1、Sema3C的組織學(xué)表達(dá)特點(diǎn)免疫組織化學(xué)法檢測(cè)發(fā)現(xiàn)Sema3C在膠質(zhì)瘤中的過(guò)表達(dá)的頻率(78.2%)明顯高于周邊正常組織(20.0%);低表達(dá)Sema3C的膠質(zhì)瘤患者預(yù)后較好(p=0.017);臨床指標(biāo)分析發(fā)現(xiàn),其表達(dá)量與組織學(xué)分型(p=0.008)和病理分級(jí)(p=0.002)相關(guān);在Sema3C表達(dá)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(shù)相關(guān)(p=0.02)。2、Sema3C的細(xì)胞學(xué)功能沉默Sema3C表達(dá)后,細(xì)胞的增殖和侵襲能力明顯降低;對(duì)增殖能力的調(diào)控是通過(guò)影響細(xì)胞周期實(shí)現(xiàn)的;Sema3C可通過(guò)改變細(xì)胞的上皮-間質(zhì)轉(zhuǎn)換影響細(xì)胞的侵襲能力等。3、Sema3C致病機(jī)制的研究Sema3C在膠質(zhì)瘤細(xì)胞中受mi R-142-5p的表達(dá)調(diào)控,miR-142-5p是通過(guò)影響Sema3C m RNA的翻譯水平實(shí)現(xiàn)的;miR-142-5p也可調(diào)控膠質(zhì)瘤細(xì)胞的增殖和侵襲能力,并且該作用的發(fā)揮也可能通過(guò)影響細(xì)胞的上皮-間質(zhì)細(xì)胞轉(zhuǎn)化而發(fā)生。4、Sema3C和miR-142-5p組織表達(dá)相關(guān)性miR-142-5p在膠質(zhì)瘤組織的表達(dá)隨著級(jí)別的升高而降低;Sema3C和miR-142-5p在膠質(zhì)瘤組織中的表達(dá)成負(fù)相關(guān)。結(jié)論:1、Sema3C在膠質(zhì)瘤中可作為不良預(yù)后指標(biāo)用于臨床診斷方面的繼續(xù)研究;2、Sema3C在膠質(zhì)瘤中發(fā)揮癌基因的作用,該作用的發(fā)揮可能通過(guò)調(diào)控上皮-間質(zhì)細(xì)胞轉(zhuǎn)化而實(shí)現(xiàn);3、miR-142-5p在膠質(zhì)瘤中負(fù)向調(diào)控Sema3C的表達(dá),提示在膠質(zhì)瘤病變過(guò)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(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.4
本文編號(hào)：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(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.4
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 曾冉;況建國(guó);;膠質(zhì)瘤發(fā)病機(jī)制的研究進(jìn)展[J];廣東醫(yī)學(xué);2013年06期
，本文編號(hào)：2467058
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