SET8基因调控肝细胞肝癌转归的研究
发布时间：2018-07-22 13:58
【摘要】：目的:PR-Set7/SETD8/KMT5a(SET8)是能够特异性单甲基化组蛋白H4K20位的赖氨酸甲基转移酶,它在基因的转录、基因组的稳定性、异染色质的形成、细胞周期的进展及发育中均发挥着重要调控作用,这意味着它极有可能参与到肿瘤的发生、发展过程中。我们前期实验发现SET8与肝癌的生存期密切相关。本研究主要探讨SET8基因对肝癌SMMC-7721细胞增殖、凋亡和侵袭转移的影响。方法:1设计并体外转录合成针对人SET8基因4个特异性荧光标记的siRNA,并通过脂质体(Lipofectamine TM2000)将siRNA转入SMMC-7721细胞。利用Western blot检测4个siRNA对SET8蛋白表达的抑制作用,筛选有效的siRNA。2采用MTS法,检测SET8基因沉默对SMMC 7721细胞的增殖活性的影响。3采用Annexin V-PE/7-AAD双染色法检测SET8基因沉默诱导SMMC-7721细胞凋亡的情况。4采用划痕愈合实验和Transwell迁移实验,分别检测SET8基因沉默对SMMC-7721细胞迁移能力的影响。5采用Matrigel侵袭实验检测SET8基因沉默对SMMC-7721细胞侵袭能力的影响。结果:1与正常对照组和阴性对照组相比,SET8 siRNA-2转染组明显抑制了SET8蛋白表达水平,SET8蛋白表达抑制率为63.9%。2 MTS实验结果显示,与阴性对照转染组和正常对照组相比,SET8siRNA-2转染后的24~72小时抑制了SMMC 7721细胞增殖(P0.01)。MTS实验说明了SET8基因沉默抑制肝癌细胞增殖。3流式细胞技术检测细胞凋亡结果显示,与阴性对照转染组和正常对照组相比,SET8 siRNA-2转染后肝癌SMMC-7721细胞凋亡率差异无统计学意义(P0.05)。上述实验表明SET8基因沉默对肝癌细胞凋亡无影响。4划痕愈合实验结果显示,阴性对照转染组和正常对照组相比,转染SET8 siRNA-2的SMMC-7721细胞向划痕中间迁移的距离明显缩小,差异有统计学意义(P0.01)。Transwell迁移实验结果显示,与阴性对照转染组和正常对照组相比,转染SET8 siRNA-2的SMMC-7721细胞穿过基底膜的数量显著降低(P0.01)。上述实验表明SET8基因沉默抑制肝癌细胞迁移能力。5 Matrigel侵袭实验结果显示,将SET8 siRNA-2重组质粒转染SM MC-7721细胞后,检测SET8对细胞侵袭能力的影响,与对照组相比转染SET8 si RNA-2的SMMC-7721细胞穿膜细胞数明显减少,差异有统计学意义(P0.01)。上述实验结果表明SET8基因沉默抑制肝癌细胞侵袭能力。结论:SET8通过调控肝癌细胞的增殖、迁移及侵袭而影响肝细胞癌的转归。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
本文编号：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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
【参考文献】
相关期刊论文 前2条
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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