RNF2抑制肝癌细胞的恶性生物学行为及其与MANF相互作用的研究

发布时间：2018-05-08 03:22

本文选题：RNF2 + MANF　；参考：《安徽医科大学》2015年硕士论文

【摘要】：环指蛋白2(ring finger protein 2,Ring2又称RNF2或Ring1b)是一个具有环指(ring finger)结构的泛素连接酶(E3),属于多梳蛋白家族(polycomb group,Pc G)中多梳抑制子复合物1(polycomb repressor complex 1,PRC1)中的一员,可介导组蛋白H2A的第119位赖氨酸的泛素化,从而影响染色质的结构,具有基因转录抑制的作用。Pc G家族中的很多蛋白都与肿瘤的发生和发展具有密切联系,而关于RNF2在肝细胞癌(hepatocellular carcinoma,HCC)中所起的作用及其具体机制仍不明确。RNF2作为一个通过酵母双杂交筛选出的与中脑星形胶质细胞源性神经营养因子(mesencephalic astrocyte-derived neurotrophic factor,MANF)具有相互作用的蛋白,在HCC细胞和组织中也与MANF存在密切的相关性。MANF是一种可被内质网应激(Endoplasmic reticulum stress,ERS)所诱导的分泌性蛋白,我们的研究发现其对HCC也有抑制作用。而RNF2可剂量依赖性的调节MANF的蛋白水平,因此我们推测RNF2对HCC的抑制作用可能是通过与MANF的相互作用而实现。目的:1.观测RNF2在人肝细胞癌及非癌组织中的表达及与病人预后的关系;2.研究RNF2对肝癌细胞的影响;3.证明RNF2和MANF的相互作用;方法:在SMMC-7721细胞和TEL-7402细胞中,采用MTT实验、流式细胞术、免疫印迹法检测caspase-3等方法证明RNF2对肝癌细胞的增殖和凋亡的影响;用平板实验、软克隆琼脂实验、划痕实验、Transwell实验等研究RNF2对肝癌细胞的抑制作用;采用细胞免疫荧光、组织免疫荧光、组织免疫共沉淀验证RNF2和MANF在HCC中的相互作用;分别用tunicamycin(TM)和oxygen glucose deprivation(OGD)处理SMMC-7721和TEL-7402细胞,模拟HCC病人体内的应激和缺糖缺氧状态,在沉默和过表达RNF2之后,用RT-PCR和Western blot方法检测MANF的基因和蛋白表达情况;在SMMC-7721细胞中分别转染RNF2-myc质粒(0μg,0.25μg,0.5μg,1.0μg),并分别用TM和OGD处理之后,用免疫印迹实验检测MANF的蛋白表达情况。结果:1.RNF2在人肝细胞癌和非癌肝脏组织中的表达用免疫组织化学技术检测RNF2在150例的肝细胞癌病人和136例的非肿瘤对照肝脏组织中的表达情况。结果发现,RNF2在所有的病例中都有表达,且与非癌对照比较,肝细胞癌病人的肝脏组织中RNF2高表达的比率显著下降(p0.01)。2.RNF2可以增加糖氧剥夺(OGD)处理后肝癌细胞的死亡在SMMC-7721细胞中瞬时转染RNF2的过表达质粒以及si RNA,经OGD处理2.5 h之后,用PI对其进行染色,对视野下红色的PI阳性细胞进行计数,并进行统计分析。结果发现过表达RNF2之后,死亡细胞增加;沉默了RNF2之后,死亡细胞减少,提示RNF2可能会促进在缺糖缺氧情况下肝癌细胞的死亡。3.RNF2促进SMMC-7721细胞的凋亡SMMC-7721细胞中转染RNF2-si RNA质粒并经过OGD处理,然后用流式细胞仪检测细胞的凋亡情况,用免疫印迹技术检测caspase-3的表达情况。结果RNF2可以促进OGD诱导的细胞凋亡及caspase-3的表达。4.RNF2对HCC细胞恶性行为学的影响4.1 RNF2抑制肝癌细胞的增殖在SMMC-7721和TEL-7402细胞中分别转染RNF2-siRNA和RNF-myc质粒,用MTT法及克隆形成实验,分别检测各组细胞的OD值以及单个细胞克隆形成能力。结果发现在沉默RNF2之后,细胞的增殖能力增加,过表达RNF2使细胞的增殖能力减弱。用流式细胞仪检测细胞的活力也发现在沉默RNF2之后,细胞的增殖能力增加。4.2 RNF2抑制肝癌细胞的迁移能力用划痕实验观测了分别转染RNF2-si RNA和RNF2-myc质粒的肝癌细胞的迁移能力。结果发现沉默了RNF2之后,细胞的迁移能力增强,而过表达RNF2使细胞的迁移能力减弱,该结果提示RNF2对肝癌细胞的迁移具有抑制作用。4.3 RNF2对肝癌细胞的侵袭能力具有抑制作用在分别沉默和过表达RNF2的SMMC-7721和TEL-7402细胞中,用transwell实验方法检测细胞的侵袭能力,结果表明在沉默了RNF2的细胞组中,穿过T4小孔的细胞数比转染载体对照的细胞增多;相反,过表达RNF2可抑制细胞的迁移,提示RNF2可以抑制肝癌细胞的侵袭能力。5.RNF2和MANF在SMMC-7721细胞核中共定位SMMC-7721细胞经TM处理6 h或者经OGD处理2.5 h之后,用免疫荧光双标观察RNF2和MANF的表达情况。结果发现,肝癌细胞经上述处理之后,MANF和RNF2在细胞核中共定位。6.RNF2和MANF在HCC病人的肝脏组织中共定位肝细胞癌病人的肝脏组织经包埋切片之后,用免疫荧光双标方法检测RNF2和MANF在组织中的表达情况。结果表明两者在肝癌组织中也存在共定位现象。7.RNF2和MANF在肝组织中的相互作用为观察RNF2和MANF在肝癌组织中是否存在相互作用,我们将肝癌组织研磨提取蛋白之后进行免疫共沉淀实验。用RNF2抗体沉淀组织中的RNF2蛋白,用IB检测免疫沉淀物中是否有MANF;同时用抗MANF的抗体共沉淀RNF2。结果提示,RNF2可以与MANF在肝癌组织中相互作用。8.RNF2不影响MANF基因的转录在SMMC-7721细胞中沉默RNF2或者转染过表达RNF2的质粒,用RT-PCR的方法检测MANF基因的表达水平。结果发现无论是在沉默RNF2还是过表达RNF2的细胞中,与对照组相比MANF的m RNA水平几乎保持不变。9.RNF2影响MANF的蛋白水平在SMMC-7721细胞中,瞬时转染RNF2-si RNA或RNF2-myc质粒,采用免疫印迹方法检测MANF的蛋白表达水平。结果表明在沉默RNF2之后MANF的表达下调;而在过表达RNF2之后MANF的表达上调;用细胞免疫荧光方法检测MANF的表达水平,发现在RNF2被沉默的细胞中,MANF的表达也低,而在RNF2被过表达的细胞中,MANF的表达量也上调。10.RNF2剂量依赖性的调节MANF的蛋白水平在SMMC-7721细胞中分别转染RNF2-myc质粒0μg、0.25μg、0.5μg、1.0μg,并分为对照组、TM刺激组(6 h)、OGD刺激组(2.5 h),用免疫印迹方法来检测MANF的蛋白表达情况。结果发现,随着RNF2-myc转染剂量的逐渐增加MANF的蛋白水平也逐渐增加。提示MANF在蛋白水平上对RNF2存在剂量依赖性。结论:1.RNF2促进肝癌细胞的凋亡;2.RNF2对HCC起抑制作用;3.RNF2和MANF在HCC中有相互作用;4.RNF2不影响MANF基因的转录,但可剂量依赖性的上调MANF的蛋白水平。
[Abstract]:The ring finger protein 2 (ring finger protein 2, Ring2 also known as RNF2 or Ring1b) is a ubiquitin ligase (E3) with the structure of the ring finger (ring finger), belonging to a member of the multi comb protein family (Polycomb group, Pc) 1, which mediates the ubiquitination of the 119th bit lysine of the histone As a result, the structure of chromatin is influenced by the inhibition of gene transcription, many of the proteins in the.Pc G family are closely related to the occurrence and development of tumor, and the role of RNF2 in hepatocellular carcinoma (HCC) and its specific mechanism are still not clearly defined by.RNF2 as a yeast two hybrid. The proteins interacting with mesencephalic astrocyte-derived neurotrophic factor (MANF), and the close correlation with MANF in HCC cells and tissues,.MANF is a secretory protein that can be induced by endoplasmic reticulum stress (Endoplasmic reticulum stress, ERS). The study found that it has a inhibitory effect on HCC, and RNF2 can be dose-dependent to regulate the protein level of MANF. Therefore, we speculate that the inhibitory effect of RNF2 on HCC may be achieved through the interaction with MANF. Objective: 1. to observe the expression of RNF2 in human hepatocellular carcinoma and noncancerous tissues and the relationship with the prognosis of the patients; 2. the study of RNF2 against the liver The effect of cancer cells; 3. the interaction between RNF2 and MANF was demonstrated. Methods: in SMMC-7721 and TEL-7402 cells, the effects of RNF2 on the proliferation and apoptosis of hepatoma cells were proved by MTT experiment, flow cytometry and Western blot detection of Caspase-3, and the study was conducted by flat test, soft cloned agar experiment, scratch test, Transwell experiment and so on. The inhibitory effect of RNF2 on hepatoma cells; using cell immunofluorescence, tissue immunofluorescence, and tissue immunoprecipitation to verify the interaction of RNF2 and MANF in HCC; tunicamycin (TM) and oxygen glucose deprivation (OGD) were used to treat SMMC-7721 and TEL-7402 cells respectively. After overexpression of RNF2, RT-PCR and Western blot were used to detect the gene and protein expression of MANF; RNF2-myc plasmids were transfected in SMMC-7721 cells (0 mu g, 0.25 micron, 0.5 mu g, 1 mu g). The expression of egg white was detected by immunoblotting with TM and immunoblotting. The expression of RNF2 in 150 cases of hepatocellular carcinoma and 136 cases of non tumor control liver tissue was detected by immunohistochemistry. The results showed that RNF2 was expressed in all cases, and compared with non cancer control, the ratio of high expression of RNF2 in the liver tissue of hepatocellular carcinoma patients decreased significantly (P0.01).2. RNF2 could increase the death of hepatoma cells after OGD treatment and transfect RNF2 over expression plasmid and Si RNA in SMMC-7721 cells. After OGD treatment of 2.5 h, it was stained with PI to count the red PI positive cells in the visual field, and the statistical analysis was carried out. The result was that after the expression RNF2, the death cells were increased. After the silence of RNF2, the death cell decreased, suggesting that RNF2 may promote the death of hepatoma cells in the absence of glucose deprivation.3.RNF2 and promote the transfection of RNF2-si RNA plasmid in SMMC-7721 cell apoptosis SMMC-7721 cells and undergo OGD treatment. Then the cell apoptosis is detected by flow cytometry, and caspase-3 is detected by immunoblotting technique. Results RNF2 could promote the apoptosis of OGD induced cells and the expression of Caspase-3. The effect of.4.RNF2 on the malignant behavior of HCC cells 4.1 RNF2 inhibited the proliferation of liver cancer cells in SMMC-7721 and TEL-7402 cells, and the RNF2-siRNA and RNF-myc plasmids were transfected respectively in SMMC-7721 and TEL-7402 cells, and the OD values of each group were detected by MTT method and clone formation test. The ability of single cell clone formation was found. The proliferation ability of cells increased after RNF2 silencing, and overexpression of RNF2 made the proliferation ability of cells weakened. The cell viability was also detected by flow cytometry, and the proliferation ability of cells increased by.4.2 RNF2 to inhibit the migration of liver cancer cells by scratch test. The migration ability of RNF2-si RNA and RNF2-myc plasmids was not transfected. The results showed that after the silence of RNF2, the migration ability of the cells was enhanced and the overexpression of RNF2 reduced the migration ability of the cells. The results suggested that RNF2 had inhibitory effect on the migration of hepatoma cells by the inhibition of.4.3 RNF2 on the invasion ability of hepatoma cells. In the SMMC-7721 and TEL-7402 cells expressing RNF2, the cell invasiveness was detected by Transwell assay. The results showed that the number of cells passing through the T4 cell in the silent RNF2 cell group was more than that of the transfected vector control; on the contrary, the overexpression of RNF2 could inhibit the migration of cells, suggesting that RNF2 could inhibit the liver cancer cells. The expression of RNF2 and MANF was observed by immunofluorescence double labeled.5.RNF2 and MANF in the cell nuclei of the cell nuclei of the SMMC-7721 nuclei after TM treatment 6 h or OGD treated with 2.5 h. The expression of RNF2 and MANF in the tissues was detected by double immunofluorescence method after the liver tissue was embedded in the liver cancer patients. The results showed that the co localization of both.7.RNF2 and MANF in liver tissues was also found to be the interaction of RNF2 and MANF in the liver tissues. We used the liver cancer tissue to lapping and extracting the protein in the immunoprecipitation experiment. The RNF2 protein in the tissue was precipitated with RNF2 antibody and the MANF in the immune precipitates was detected by IB. Meanwhile, the MANF antibody co precipitated RNF2. results suggested that RNF2 can interact with MANF in the liver cancer tissues without affecting the transcription of the MANF gene. SMMC-7721 cells were silent RNF2 or transfected with RNF2 plasmids, and the expression level of MANF gene was detected by RT-PCR method. The results showed that the m RNA level of MANF was almost unchanged in the cells of the silent RNF2 or over the expression of RNF2, and the level of the MANF protein was influenced by the instantaneous transfection. RNF2-si RNA or RNF2-myc plasmids were used to detect the protein expression level of MANF by immunoblotting. The results showed that the expression of MANF was downregulated after the silence of RNF2, and the expression of MANF was up-regulated after overexpressing RNF2, and the expression level of MANF was detected by cell immunofluorescence, and the MANF expression was found in the RNF2 that was silenced, while RNF2 was in RNF2. In the overexpressed cells, the expression of MANF also up-regulated the protein level of.10.RNF2 dose-dependent regulating MANF in SMMC-7721 cells transfected to RNF2-myc plasmid 0 mu g, 0.25 g, 0.5 mu g, 1 u g, and divided into control group, TM stimulation group (6 h), OGD stimulus group (2.5), and detected the protein expression by immunoblotting method. The results found that the protein expression was detected by immunoblotting. The protein level of MANF increased gradually with the gradual increase of RNF2-myc transfection dose. It suggests that MANF has a dose dependence on RNF2 at the protein level. Conclusion: 1.RNF2 promotes the apoptosis of hepatoma cells; 2.RNF2 inhibits HCC; 3.RNF2 and MANF have interaction in HCC; 4.RNF2 does not affect the transcription of the MANF genes, but can be dose-dependent. The protein level of MANF was up-regulated.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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