【摘要】：目的:PR-Set7/SETD8/KMT5a(SET8)是能夠特異性單甲基化組蛋白H4K20位的賴氨酸甲基轉(zhuǎn)移酶,它在基因的轉(zhuǎn)錄、基因組的穩(wěn)定性、異染色質(zhì)的形成、細(xì)胞周期的進(jìn)展及發(fā)育中均發(fā)揮著重要調(diào)控作用,這意味著它極有可能參與到腫瘤的發(fā)生、發(fā)展過程中。我們前期實驗發(fā)現(xiàn)SET8與肝癌的生存期密切相關(guān)。本研究主要探討SET8基因?qū)Ω伟㏒MMC-7721細(xì)胞增殖、凋亡和侵襲轉(zhuǎn)移的影響。方法:1設(shè)計并體外轉(zhuǎn)錄合成針對人SET8基因4個特異性熒光標(biāo)記的siRNA,并通過脂質(zhì)體(Lipofectamine TM2000)將siRNA轉(zhuǎn)入SMMC-7721細(xì)胞。利用Western blot檢測4個siRNA對SET8蛋白表達(dá)的抑制作用,篩選有效的siRNA。2采用MTS法,檢測SET8基因沉默對SMMC 7721細(xì)胞的增殖活性的影響。3采用Annexin V-PE/7-AAD雙染色法檢測SET8基因沉默誘導(dǎo)SMMC-7721細(xì)胞凋亡的情況。4采用劃痕愈合實驗和Transwell遷移實驗,分別檢測SET8基因沉默對SMMC-7721細(xì)胞遷移能力的影響。5采用Matrigel侵襲實驗檢測SET8基因沉默對SMMC-7721細(xì)胞侵襲能力的影響。結(jié)果:1與正常對照組和陰性對照組相比,SET8 siRNA-2轉(zhuǎn)染組明顯抑制了SET8蛋白表達(dá)水平,SET8蛋白表達(dá)抑制率為63.9%。2 MTS實驗結(jié)果顯示,與陰性對照轉(zhuǎn)染組和正常對照組相比,SET8siRNA-2轉(zhuǎn)染后的24~72小時抑制了SMMC 7721細(xì)胞增殖(P0.01)。MTS實驗說明了SET8基因沉默抑制肝癌細(xì)胞增殖。3流式細(xì)胞技術(shù)檢測細(xì)胞凋亡結(jié)果顯示,與陰性對照轉(zhuǎn)染組和正常對照組相比,SET8 siRNA-2轉(zhuǎn)染后肝癌SMMC-7721細(xì)胞凋亡率差異無統(tǒng)計學(xué)意義(P0.05)。上述實驗表明SET8基因沉默對肝癌細(xì)胞凋亡無影響。4劃痕愈合實驗結(jié)果顯示,陰性對照轉(zhuǎn)染組和正常對照組相比,轉(zhuǎn)染SET8 siRNA-2的SMMC-7721細(xì)胞向劃痕中間遷移的距離明顯縮小,差異有統(tǒng)計學(xué)意義(P0.01)。Transwell遷移實驗結(jié)果顯示,與陰性對照轉(zhuǎn)染組和正常對照組相比,轉(zhuǎn)染SET8 siRNA-2的SMMC-7721細(xì)胞穿過基底膜的數(shù)量顯著降低(P0.01)。上述實驗表明SET8基因沉默抑制肝癌細(xì)胞遷移能力。5 Matrigel侵襲實驗結(jié)果顯示,將SET8 siRNA-2重組質(zhì)粒轉(zhuǎn)染SM MC-7721細(xì)胞后,檢測SET8對細(xì)胞侵襲能力的影響,與對照組相比轉(zhuǎn)染SET8 si RNA-2的SMMC-7721細(xì)胞穿膜細(xì)胞數(shù)明顯減少,差異有統(tǒng)計學(xué)意義(P0.01)。上述實驗結(jié)果表明SET8基因沉默抑制肝癌細(xì)胞侵襲能力。結(jié)論:SET8通過調(diào)控肝癌細(xì)胞的增殖、遷移及侵襲而影響肝細(xì)胞癌的轉(zhuǎn)歸。
[Abstract]:Objective: PR-Set7 / SETD8 / KMT5a (SET8) is a lysine methyltransferase capable of specific monomethylated histone H4K20. It plays an important role in gene transcription, genomic stability, heterochromatin formation, cell cycle progression and development. This means that it is highly likely to be involved in the tumorigenesis and development process. Our previous study found that SET 8 was closely related to the survival time of liver cancer. The aim of this study was to investigate the effects of SET8 gene on the proliferation, apoptosis, invasion and metastasis of SMMC-7721 cells. Methods SMMC-7721 cells were transfected into SMMC-7721 cells by liposome (Lipofectamine TM2000). Western blot was used to detect the inhibitory effect of four siRNAs on the expression of SET8 protein. The effective siRNA.2 was screened by MTS method. To detect the effect of SET8 gene silencing on the proliferation activity of SMMC 7721 cells, the apoptosis of SMMC-7721 cells induced by SET8 gene silencing was detected by Annexin V-PE-7-AAD double staining method. 4. Scratch healing test and Transwell migration test were used to detect the apoptosis of SMMC-7721 cells induced by SET8 gene silencing. The effect of SET8 gene silencing on the migration ability of SMMC-7721 cells was detected by Matrigel invasion assay. The effect of SET8 gene silencing on the invasion ability of SMMC-7721 cells was examined. Results compared with normal control group and negative control group, the expression level of SET8 protein was significantly inhibited by the transfection of T8 siRNA-2. The inhibition rate of SET8 protein expression was 63.9.2 MTS. Compared with the negative control group and the normal control group, SET8 siRNA-2 inhibited the proliferation of SMMC 7721 cells at 2472 hours (P0.01) .MTS experiment showed that SET8 gene silencing inhibited the proliferation of hepatoma cells. 3. The results of flow cytometry showed that SET8 gene silencing inhibited the proliferation of hepatoma cells. Compared with the negative control group and the normal control group, there was no significant difference in apoptosis rate of SMMC-7721 cells after transfection of SMMC-7721 cells with SET8 siRNA-2 (P0.05). The results showed that SET8 gene silencing had no effect on apoptosis of hepatoma cells. The results showed that the distance between SMMC-7721 cells transfected with SET8 siRNA-2 and normal control group was significantly smaller than that in negative control group, and the migration distance between SMMC-7721 cells transfected with SET8 siRNA-2 was significantly smaller than that in normal control group. The difference was statistically significant (P0.01) .Transwell migration assay showed that the number of SMMC-7721 cells transfected with SET8 siRNA-2 through the basement membrane was significantly lower than that in the negative control group and the normal control group (P0.01). The results indicated that SET8 gene silencing inhibited the migration ability of hepatoma cells. 5 Matrigel invasion assay showed that the effect of SET8 siRNA-2 recombinant plasmid on the invasion ability of SMMC-7721 cells was detected after transfection of SET8 siRNA-2 recombinant plasmid into SMMC-7721 cells. Compared with the control group, the number of SMMC-7721 cells transfected with SET8si RNA-2 was significantly decreased (P0.01). These results suggest that SET 8 gene silencing inhibits the invasion of hepatoma cells. ConclusionSet 8 affects the prognosis of hepatocellular carcinoma by regulating the proliferation, migration and invasion of HCC cells.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.7

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