本文選題：RNA激活 + RNA干擾　； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的原發(fā)性肝癌(hepato cellular carcinoma,HCC)是世界上最為常見的惡性腫瘤之一,其發(fā)病率和致死率都位居世界前列。肝癌具有發(fā)病隱匿,潛伏期長,發(fā)展速度快,惡性程度高等特點,手術(shù)治療是目前治療肝癌最好的方法。但由于大多數(shù)患者直至病程中晚期才得以確診,因而錯過了手術(shù)治療的最佳時期。因此,尋找新的治療工具和藥物靶點對肝癌的治療至關(guān)重要。本研究旨在探討小激活RNA(small activating RNA,sa RNA)上調(diào)p21基因表達和小干擾RNA(small interfering RNA,si RNA)沉默MTH1基因表達后,對人肝癌Hep G2細胞增殖、遷移、侵襲和凋亡的影響。方法設(shè)計并合成靶向p21和MTH1基因的sa RNA和si RNA序列,并將其轉(zhuǎn)染到體外培養(yǎng)的人肝癌Hep G2細胞中。實驗分為5組:空白對照組(CT)、陰性對照組(NC)、sa RNA-p21轉(zhuǎn)染組(p21)、si RNA-MTH1轉(zhuǎn)染組(MTH1)和聯(lián)合轉(zhuǎn)染組(p21+MTH1)。q PCR檢測各組p21和MTH1基因m RNA表達情況;Western blot檢測各組p21,MTH1,以及凋亡相關(guān)蛋白表達水平;CCK8法檢測各組Hep G2細胞增殖情況;劃痕實驗檢測細胞遷移能力;Transwell侵襲實驗檢測細胞侵襲能力;流式細胞術(shù)檢測細胞凋亡情況。結(jié)果與空白對照組和陰性對照組相比,sa RNA-p21轉(zhuǎn)染組和聯(lián)合轉(zhuǎn)染組p21基因m RNA和蛋白表達水平明顯上調(diào)(P0.01)。與空白對照組和陰性對照組相比,si RNA-MTH1轉(zhuǎn)染組和聯(lián)合轉(zhuǎn)染組MTH1基因m RNA和蛋白表達水平明顯下調(diào)(P0.01)。與空白對照組和陰性對照相比sa RNA-p21轉(zhuǎn)染組和si RNA-MTH1轉(zhuǎn)染組細胞存活率和劃痕愈合率明顯降低(P0.05),聯(lián)合轉(zhuǎn)染組的細胞存活率和劃痕愈合率與對照組和單轉(zhuǎn)組相比明顯降低(P0.05)。流式細胞術(shù)實驗結(jié)果顯示與空白對照組和陰性對照相比sa RNA-p21轉(zhuǎn)染組和si RNA-MTH1轉(zhuǎn)染組細胞凋亡率明顯升高(P0.01),聯(lián)合轉(zhuǎn)染組細胞凋亡率較對照組和單轉(zhuǎn)組明顯升高(P0.01)。Western blot結(jié)果顯示與空白對照組和陰性對照相比sa RNA-p21轉(zhuǎn)染組和si RNA-MTH1轉(zhuǎn)染組caspase-3蛋白表達量明顯上調(diào)(P0.05),聯(lián)合轉(zhuǎn)染組比單轉(zhuǎn)組效果明顯(P0.05);聯(lián)合轉(zhuǎn)染組Bak蛋白表達量與對照組相比明顯上調(diào)(P0.01),單轉(zhuǎn)組與對照組相比無差異(P0.05)。與空白對照組和陰性對照相比sa RNA-p21轉(zhuǎn)染組和si RNA-MTH1轉(zhuǎn)染組穿過基底膜的細胞數(shù)明顯減少(P0.01),聯(lián)合轉(zhuǎn)染組穿過基底膜的細胞數(shù)較對照組和單轉(zhuǎn)組明顯升高(P0.01)結(jié)論sa RNA-p21可以有效的激活p21基因的表達,si RNA-MTH1可以有效的沉默MTH1基因的表達。通過RNA激活(RNAa)技術(shù)上調(diào)p21基因表達和RNA干擾(RNAi)技術(shù)下調(diào)MTH1基因表達可以抑制人肝癌Hep G2細胞的增殖、遷移和侵襲,并能誘導(dǎo)其凋亡。sa RNA-p21和si RNA-MTH1聯(lián)合組的效果要比單轉(zhuǎn)組好。
[Abstract]:Objective Primary liver cancer (HCC) is one of the most common malignant tumors in the world, and its morbidity and mortality are among the highest in the world. Liver cancer is characterized by occult onset, long incubation period, rapid development and high malignancy. Surgical treatment is the best method for the treatment of liver cancer at present. However, most patients were not diagnosed until the middle and late stage of the disease, thus missing the optimal period of surgical treatment. Therefore, the search for new therapeutic tools and drug targets is essential for the treatment of liver cancer. The aim of this study was to investigate the effects of up-regulation of p21 gene expression and silencing of MTH1 gene expression by small activated RNA(small activating RNA-siRNAs on proliferation, migration, invasion and apoptosis of human hepatoma Hep G2 cells. Methods the sa RNA and si RNA sequences targeting p21 and MTH1 genes were designed and synthesized and transfected into human hepatoma Hep G2 cells in vitro. The experiment was divided into five groups: the blank control group (CTL), the negative control group (RNA-p21) and the co-transfection group (p21 MTH1).q PCR) were used to detect the expression of p21 and MTH1 gene m RNA in each group. Western blot was used to detect the expression of p21 RNA-p21 and apoptosis-related protein (Apoptosis-related protein) in each group. The proliferation of Hep G2 cells was detected by CCK8 assay. The cell migration ability was detected by scratch assay and the cell invasion ability was detected by Transwell invasion assay, and apoptosis was detected by flow cytometry. Results compared with the blank control group and the negative control group, the expression of p21 gene m RNA and protein in the p21 RNA-p21 transfection group and co-transfection group were significantly up-regulated. Compared with the blank control group and the negative control group, the expression level of m RNA and protein of MTH1 gene in the RNA-MTH1 transfection group and co-transfection group were significantly down-regulated. Compared with the control group and the negative control group, the cell survival rate and scratch healing rate of the sa RNA-p21 transfection group and si RNA-MTH1 transfection group were significantly lower than that of the control group and the si RNA-MTH1 transfection group. The cell survival rate and scratch healing rate of the co-transfection group were significantly lower than those of the control group and the single transfer group. The results of flow cytometry showed that the apoptosis rate of sa RNA-p21 transfection group and si RNA-MTH1 transfection group was significantly higher than that of control group and negative control group. The apoptosis rate of co-transfection group was significantly higher than that of control group and single transfer group. The expression of caspase-3 protein in sa RNA-p21 transfection group and si RNA-MTH1 transfection group was significantly up-regulated than that in blank control group and negative control group, the effect of co-transfection group was significantly higher than that in single transfer group, and the expression of Bak protein in co-transfection group was significantly higher than that in control group. Compared with the control group, there was no difference between the single transfer group and the control group. Compared with the blank control group and the negative control group, the number of cells passing through the basement membrane in the sa RNA-p21 transfection group and the si RNA-MTH1 transfection group was significantly reduced, and the number of cells passing through the basement membrane in the co-transfection group was significantly higher than that in the control group and the single transfer group. The expression of MTH1 gene could be effectively silenced by activating p21 gene expression by sisi RNA-MTH1. Upregulation of p21 gene expression and down-regulation of MTH1 gene expression by RNA activator RNAa technique could inhibit the proliferation, migration and invasion of Hep G2 cells, and induce apoptosis of Hep G2 cells in combination with RNA-p21 and si RNA-MTH1.
【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】：2017
【分類號】：R735.7

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相關(guān)期刊論文 前2條

1 Jalil Pirayesh Islamian;Mohsen Mohammadi;Behzad Baradaran;;Inhibition of human esophageal squamous cell carcinomas by targeted silencing of tumor enhancer genes: an overview[J];Cancer Biology & Medicine;2014年02期

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本文編號：1939353