SET8基因調控肝細胞肝癌轉歸的研究
發(fā)布時間：2018-07-22 13:58
【摘要】：目的:PR-Set7/SETD8/KMT5a(SET8)是能夠特異性單甲基化組蛋白H4K20位的賴氨酸甲基轉移酶,它在基因的轉錄、基因組的穩(wěn)定性、異染色質的形成、細胞周期的進展及發(fā)育中均發(fā)揮著重要調控作用,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。本研究主要探討SET8基因對肝癌SMMC-7721細胞增殖、凋亡和侵襲轉移的影響。方法:1設計并體外轉錄合成針對人SET8基因4個特異性熒光標記的siRNA,并通過脂質體(Lipofectamine TM2000)將siRNA轉入SMMC-7721細胞。利用Western blot檢測4個siRNA對SET8蛋白表達的抑制作用,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SMMC-7721細胞凋亡的情況。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細胞侵襲能力的影響。結果:1與正常對照組和陰性對照組相比,SET8 siRNA-2轉染組明顯抑制了SET8蛋白表達水平,SET8蛋白表達抑制率為63.9%。2 MTS實驗結果顯示,與陰性對照轉染組和正常對照組相比,SET8siRNA-2轉染后的24~72小時抑制了SMMC 7721細胞增殖(P0.01)。MTS實驗說明了SET8基因沉默抑制肝癌細胞增殖。3流式細胞技術檢測細胞凋亡結果顯示,與陰性對照轉染組和正常對照組相比,SET8 siRNA-2轉染后肝癌SMMC-7721細胞凋亡率差異無統(tǒng)計學意義(P0.05)。上述實驗表明SET8基因沉默對肝癌細胞凋亡無影響。4劃痕愈合實驗結果顯示,陰性對照轉染組和正常對照組相比,轉染SET8 siRNA-2的SMMC-7721細胞向劃痕中間遷移的距離明顯縮小,差異有統(tǒng)計學意義(P0.01)。Transwell遷移實驗結果顯示,與陰性對照轉染組和正常對照組相比,轉染SET8 siRNA-2的SMMC-7721細胞穿過基底膜的數(shù)量顯著降低(P0.01)。上述實驗表明SET8基因沉默抑制肝癌細胞遷移能力。5 Matrigel侵襲實驗結果顯示,將SET8 siRNA-2重組質粒轉染SM MC-7721細胞后,檢測SET8對細胞侵襲能力的影響,與對照組相比轉染SET8 si RNA-2的SMMC-7721細胞穿膜細胞數(shù)明顯減少,差異有統(tǒng)計學意義(P0.01)。上述實驗結果表明SET8基因沉默抑制肝癌細胞侵襲能力。結論:SET8通過調控肝癌細胞的增殖、遷移及侵襲而影響肝細胞癌的轉歸。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.7
本文編號：2137686
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.7
【參考文獻】
相關期刊論文 前2條
1 Roberto Iezzi;Maurizio Pompili;Alessandro Posa;Giuseppe Coppola;Antonio Gasbarrini;Lorenzo Bonomo;;Combined locoregional treatment of patients with hepatocellular carcinoma: state of the art[J];World Journal of Gastroenterology;2016年06期
2 María L Bacigalupo;Malena Manzi;Gabriel A Rabinovich;María F Troncoso;;Hierarchical and selective roles of galectins in hepatocarcinogenesis, liver fibrosis and inflammation of hepatocellular carcinoma[J];World Journal of Gastroenterology;2013年47期
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