抑制USP18表达对人肝癌Hep3B细胞的抑制作用

发布时间：2018-01-20 02:07

本文关键词： USP18 肝癌细胞 siRNA 生物学活性　出处：《南昌大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:检测USP18在肝癌细胞中的表达及对其生物学活性(细胞增殖、细胞克隆、细胞周期、细胞凋亡)的影响。方法:1、通过半定量逆转录聚合酶链反应(RT-PCR)、蛋白印迹(Western Blot)技术从肝癌细胞株HepG2、SMMC7721、Huh7,HepG2.2.15和Hep3B中筛选USP18基因高表达细胞株。2、设计并化学合成针对USP18的3对小干扰RNA(small interfering RNA,siRNA),采用阳离子脂质体法瞬间转染。筛选出高表达细胞株后,采用离体培养该高表达USP18肝癌细胞,并设Nomal Control组、Negative Control组及USP18-siRNA-1、USP18-siRNA-2、USP18-siRNA-3各转染组。3、通过半定量逆转录聚合酶链反应(RT-PCR)和蛋白印迹(Western Blot)法检测转染后各组细胞中USP18在mRNA和蛋白水平表达的变化,明确处理后USP18沉默的效果。4、通过MTT比色法描绘生长曲线和平板克隆形成实验检测转染后各组细胞增殖能力的变化。5、运用Annexin V-FITC/PI染色流式细胞术检测细胞凋亡和流式细胞术检测细胞周期分布。结果:1、RT-PCR及Western blot结果显示:五种肝癌细胞株中,USP18基因均有表达,HBV阳性肝癌细胞中的表达高于HBV阴性肝癌细胞,其中Hep3B细胞表达最高,其次为HepG2.2.15、Huh7、SMMC7721,在HepG2细胞中表达最低。选择USP18表达最高的Hep3B细胞进行后续实验。2、Hep3B细胞转染USP18 siRNA 24 h后,在荧光显微镜下观察绿色荧光蛋白的表达情况,随机取10个低倍视野,计数带有荧光蛋白的细胞占转染细胞总数的百分比来评价转染效率。结果显示转染效率达91±5.67%。3、3个特异性USP18-siRNA转染组转染48小时后,其USP18的mRNA和蛋白表达与另外2组比较受到明显抑制,结果显示转染干扰组USP18-siRNA-1抑制效果最明显(P0.05),而阴性对照组与正常组相比差异无显著性(p0.05)。4、MTT比色法描绘生长曲线及平板克隆形成实验显示,转染干扰组与正常组及阴性对照组相比,细胞增殖速度明显减慢(p0.05),而阴性对照组与正常组相比差异无显著性(p0.05)。5、Annexin V-FITC/PI染色流式细胞术检测细胞凋亡结果显示:转染干扰组与正常组及阴性对照组相比,细胞凋亡率明显增加(p0.05),而阴性对照组与正常组相比差异无显著性(p0.05)。6、流式细胞术检测细胞周期实验显示,转染干扰组与正常组及阴性对照组相比,细胞周期分布差异有统计学差异(p0.05),出现G1期阻滞,S-G2期细胞显著减少。而阴性对照组与正常组相比差异无显著性(p0.05)。结论:1、HepG2、SMMC7721、Huh-7,HepG2.2.15和Hep3B细胞中,Hep3B为USP18基因最高表达细胞株。2、siRNA干扰介导USP18基因沉默后,可抑制人肝癌Hep3B细胞的生长、增殖,促进细胞凋亡,并阻滞细胞周期于G1期。
[Abstract]:Objective: to investigate the expression of USP18 in hepatocellular carcinoma (HCC) cells and its biological activity (cell proliferation, cell clone, cell cycle, apoptosis). Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot technique were used to detect the expression of Huh7 from HepG2 cell line SMMC7721. The high expression cell line of USP18 gene was screened from HepG2.2.15 and Hep3B. Three pairs of small interfering RNA(small interfering siRNAs for USP18 were designed and chemically synthesized. The high expression USP18 hepatoma cell lines were screened by cationic liposome method. The cells were cultured in vitro, and Nomal Control group was set up. Negative Control and USP18-siRNA-1 were transfected with USP18-siRNA-3 and USP18-siRNA-3 respectively. By semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot). After transfection, the expression of USP18 in mRNA and protein was detected. The effect of USP18 silencing was determined. The growth curve was described by MTT colorimetry and the changes of proliferation ability of each group were detected by plate clone formation assay. Apoptosis was detected by Annexin V-FITC / Pi staining and cell cycle distribution was detected by flow cytometry. The results of RT-PCR and Western blot showed that the expression of USP18 gene was higher in HBV-positive hepatoma cells than in HBV negative HCC cells. The expression of Hep3B cells was the highest, followed by HepG2.2.15Huh7H7 SMMC7721. The Hep3B cells with the highest USP18 expression were selected to carry out the following experiment. 2Hep3B cells were transfected with USP18 siRNA for 24 h. The expression of green fluorescent protein (GFP) was observed under fluorescence microscope and 10 low-power visual fields were randomly selected. The transfection efficiency was evaluated by counting the percentage of the cells with fluorescent protein in the total number of transfected cells. The results showed that the transfection efficiency was 91 卤5.67.3. The mRNA and protein expression of USP18 in the three specific USP18-siRNA transfection groups was significantly inhibited compared with the other two groups after 48 hours of transfection. The results showed that the inhibitory effect of USP18-siRNA-1 was the most obvious in the interference group, but there was no significant difference between the negative control group and the normal group. MTT colorimetric method was used to depict the growth curve and the plate clone formation experiment showed that compared with the normal group and the negative control group, the proliferation rate of the transfected interference group was significantly slower than that of the negative control group (P 0.05). However, there was no significant difference between the negative control group and the normal group (P 0.05). The results of flow cytometry with Annexin V-FITC / Pi staining showed that the rate of apoptosis in the transfected interference group was significantly higher than that in the normal group and the negative control group (p0.05). But there was no significant difference between the negative control group and the normal group. Flow cytometry analysis showed that the transfection interference group was compared with the normal group and the negative control group. The difference of cell cycle distribution was statistically significant (p0.05), and G1 phase arrest appeared. The S-G2 phase cells decreased significantly, but there was no significant difference between the negative control group and the normal control group (P 0.05). Conclusion: 1: 1 HepG2 SMMMC7721 Huh-7. In HepG2.2.15 and Hep3B cells, Hep3B is the highest expression cell line of USP18 gene. 2 siRNA interference mediates the silencing of USP18 gene. It can inhibit the growth and proliferation of human hepatoma Hep3B cells, promote cell apoptosis, and block the cell cycle in G1 phase.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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