SUMO蛋白在肝細胞癌中差異性調節(jié)p65的細胞內定位和穩(wěn)定性以及影響p65與MANF的相互作用
發(fā)布時間：2018-07-26 20:04
【摘要】：SUMO (small ubiquitin-related modifier)蛋白是一類與泛素非常類似的小分子蛋白,參與重要的蛋白質翻譯后的修飾——SUMO化修飾(SUMOylation)。SUMO化修飾調節(jié)機體的一系列生理學與病理學的過程,與很多疾病的發(fā)生發(fā)展都存在密切的聯(lián)系,例如炎癥性疾病和炎癥相關性疾病,包括肝細胞癌(hepatocellular carcinoma, HCC)。p65是NF-κB家族最重要亞基之一,p65蛋白入核后激活NF-κB信號通路在肝細胞癌的發(fā)生發(fā)展過程中起重要作用。近年來已有文獻證實,在人胚胎腎293T細胞和小鼠3T3纖維細胞中p65蛋白能被外源性的SUMO3蛋白進行SUMO化修飾,但是目前未見p65蛋白與SUMO1、SUMO2蛋白之間相互作用,以及p65蛋白發(fā)生SUMO化修飾之后對HCC影響的研究報道。本課題組前期證實MANF (Mesencephalic Astrocyte-derived Neurotrophic factor,中腦新型膠質細胞源性神經營養(yǎng)因子)能在細胞核內與p65存在相互作用從而抑制NF-κB的轉錄活性,但是SUMO蛋白是否影響p65與MANF的相互作用,未見報道。目的：研究SUMO蛋白在HCC中如何差異性地調節(jié)p65細胞內的水平和定位及是否影響p65與MANF的相互作用。方法：選取乙肝患者及HCC患者的肝組織固定石蠟包埋切片,采用免疫組化的方法在人的肝炎組織、HCC患者的癌組織和癌旁組織中檢測p65、SUMO1及SUMO2/3蛋白的表達情況；在人肝癌組織及肝癌細胞株中采用免疫熒光雙標檢測p65與SUMO1或SUMO2/3是否存在細胞內的共定位現(xiàn)象；在人肝癌組織及肝癌細胞株中采用免疫共沉淀的方法驗證p65與SUMO1或SUMO2/3之間是否存在相互作用以及p65蛋白是否能發(fā)生SUMO化修飾；Western blot在肝癌細胞株中觀察腫瘤微環(huán)境(如：缺氧及炎癥刺激)對p65蛋白的細胞內亞定位以及SUMO1和SUMO2/3蛋白水平的表達是否有影響；免疫共沉淀實驗觀察肝癌細胞株行缺氧及炎癥刺激處理后p65的SUMO化修飾水平是否產生變化；在肝癌細胞株中轉染外源性的SUMO1或SUMO2/3,采用Western blot及核漿分離實驗觀察p65蛋白水平及細胞內亞定位的變化；通過雙熒光素酶報告基因實驗檢測過表達及沉默SUMO1對NF-κB轉錄活性的影響；在肝癌細胞株中,采用MTT、克隆形成實驗、transwell小室實驗觀察SUMO1及SUMO2/3對肝癌細胞增殖及侵襲能力的影響,以及在人肝癌組織中行免疫熒光雙標檢測SUMO1與ki67蛋白之間的相關性,評價SUMO1蛋白是否與肝癌細胞的增殖有關；在肝癌細胞株中分別敲低內源性SUMO1或SUMO2/3,免疫共沉淀實驗檢測p65與MANF蛋白之間的相互作用是否受SUMO蛋白的影響。結果：1.在人肝組織中觀察p65、SUMO1以及SUMO2/3蛋白的表達情況免疫組化結果顯示,在肝炎組織中有少量SUMO1陽性表達細胞；肝癌組織中SUMO1表達量明顯高于癌旁組織,且定位于細胞核中。SUMO2/3的表達與SUMO1不同,SUMO2/3在肝炎組織中有少量表達,細胞質和細胞核中均有分布；癌組織中SUMO2/3的表達明顯低于癌旁組織,主要定位于細胞質中。p65蛋白的表達與SUMO2/3非常類似,肝炎組織中少量表達,癌組織低表達,癌旁組織高表達,但是在肝癌組織的胞核中有p65存在。2. SUMO1與p65的相互作用免疫熒光雙標顯示p65蛋白與SUMO1在HCC患者的癌組織以及肝癌細胞株中存在共定位的現(xiàn)象,主要共定位于細胞核中。在人肝癌組織及肝癌細胞株中,免疫共沉淀實驗也發(fā)現(xiàn)SUMO1與p65的相互作用,并發(fā)現(xiàn)SUMO1可使p65蛋白發(fā)生SUMO化修飾。3. SUMO2/3與p65的相互作用人肝組織連續(xù)切片的DAB染色顯示,p65蛋白與SUMO2/3的表達高度一致。進一步免疫熒光雙標實驗發(fā)現(xiàn),p65與SUMO2/3共定位于細胞質中；同時SUMO2/3表達高的細胞中p65表達多,SUMO2/3不表達的細胞中,p65表達很少。在HCC患者的肝組織及肝癌細胞株中,免疫共沉淀實驗進一步證實SUMO2/3與p65的相互作用,并發(fā)現(xiàn)SUMO2/3可使p65蛋白發(fā)生SUMO化修飾。4.缺氧及炎癥刺激促進SUMO1和SUMO2/3蛋白的表達及p65的SUMO化肝癌細胞株經過缺糖缺氧處理150 min后可發(fā)現(xiàn)有部分p65蛋白入核,SUMO1及SUMO2/3蛋白表達量增加。肝癌細胞株經過TNF-a (10ng/ml)處理30 min,部分p65蛋白入核,細胞質中SUMO2/3蛋白水平明顯增多,但SUMO1蛋白的水平沒有明顯變化。當TNF-a(10ng/ml)處理肝癌細胞株8 hrs時,細胞質中SUMO2/3蛋白的水平與TNF處理30 min時無明顯變化,而細胞核中SUMO2/3蛋白水平明顯增多,細胞質和細胞核中SUMO1蛋白的水平均明顯增高。免疫共沉淀實驗結果顯示：細胞在轉染SUMO化修飾E2酶(Ubc9)及外源性的SUMO質粒后24 hrs,缺糖缺氧處理150min能增加SUMO1蛋白相關的p65的SUMO化修飾水平,但并不影響SUMO2/3相關的p65的SUMO化修飾；細胞轉染24 hrs后采用TNF-α(10ng/ml)刺激30 min,能同時增加SUMO1及SUMO2/3蛋白相關的p65蛋白SUMO化修飾水平。5. SUMO1蛋白參與調節(jié)p65蛋白的入核肝癌細胞株中過表達SUMO1并不影響p65蛋白水平。核漿分離實驗發(fā)現(xiàn),過表達SUMO1能增加缺糖缺氧誘導的p65蛋白入核,相反沉默內源性的SUMO1能抑制p65蛋白入核；同時沉默SUMO1能抑制TNF-α刺激誘導的p65蛋白入核,而過表達SUMO1對TNF-a誘導的p65蛋白入核影響不大。6.敲低內源性SUMO1蛋白抑制NF-κB的轉錄活性在肝癌細胞株中采用雙熒光素酶報告基因實驗方法,結果顯示：正常培養(yǎng)的細胞以及細胞經過TNF-α刺激或缺糖缺氧處理,過表達SUMO1都能抑制NF-κB的轉錄活性；而敲低內源性的SUMO1也能抑制NF-κB的轉錄活性。7.敲低內源性SUMO1抑制肝癌細胞株的增殖與轉移侵襲能力MTT、克隆形成實驗證實：敲除內源性SUMO1抑制了細胞的增殖能力。于是我們進一步在人肝細胞癌的癌組織中采用免疫熒光雙標觀察ki67和SUMO1的相關性,發(fā)現(xiàn)：所有ki67陽性的細胞,SUMO1都是表達陽性。于是我們推測：SUMO1蛋白的表達可能與肝癌細胞的增殖能力有關。transwell實驗顯示,過表達SUMO1能增加肝癌細胞株的轉移侵襲能力,沉默SUMO1抑制了細胞的轉移侵襲能力。8. SUMO2/3穩(wěn)定胞質中p65蛋白的水平與其拮抗p65的泛素化有關肝癌細胞株中過表達SUMO2/3發(fā)現(xiàn),p65蛋白水平隨轉染SUMO2/3的DNA增多而增高。進一步核漿分離實驗發(fā)現(xiàn),SUMO2/3蛋白主要是維持p65蛋白在細胞質中的穩(wěn)定性。在肝癌細胞株中過表達SUMO2/3檢測p65的mRNA水平,發(fā)現(xiàn)SUMO2/3并不影響p65的轉錄水平。同時免疫共沉淀實驗發(fā)現(xiàn),當SUMO2/3蛋白相關的p65的SUMO化修飾增多時,p65蛋白的泛素化減少。該結果提示SUMO2/3蛋白可能是通過拮抗p65的泛素化,減少p65蛋白降解來穩(wěn)定胞質中的p65蛋白水平。9.過表達SUMO2/3蛋白抑制肝癌細胞株的增殖MTT實驗發(fā)現(xiàn),在肝癌細胞株中過表達SUMO2/3抑制了肝癌細胞株的增殖能力,SUMO2/3并不影響肝癌細胞株的轉移與侵襲能力。10. SUMO蛋白影響p65與MANF之間的相互作用免疫共沉淀實驗發(fā)現(xiàn),在肝癌細胞株中敲低內源性SUMO1或SUMO2/3, p65與MANF蛋白之間的相互作用減少。結論：1. SUMO1和SUMO2/3能與p65相互作用并對其進行SUMO化修飾；2.缺糖缺氧和TNF-α能增強SUMO蛋白與p65的相互作用；3.敲低SUMO1能抑制缺糖缺氧和TNF-α誘導的p65蛋白入核,并能抑制NF-κB的轉錄活性；4. SUMO2/3能穩(wěn)定胞質中的p65蛋白水平；5.p65蛋白的泛素修飾拮抗它的SUMO化；6. SUMO1能促進肝癌細胞的增殖與轉移侵襲能力；7.過表達SUMO2/3能抑制肝癌細胞的增殖；8.敲低SUMO1或SUMO2/3影響p65與MANF之間的相互作用。
[Abstract]:SUMO (small ubiquitin-related modifier) protein is a kind of small molecular protein that is very similar to ubiquitin, and participates in a series of physiological and pathological processes of SUMO modification (SUMOylation) modification (SUMOylation).SUMO modification, which is closely related to the occurrence and development of many diseases, for example. Hepatocellular carcinoma (HCC).P65 is one of the most important subunits of the NF- kappa B family, and the activation of NF- kappa B signaling pathway after p65 protein nucleation plays an important role in the development of hepatocellular carcinoma. In recent years, Yi Youwen has demonstrated that in human embryonic kidney 293T cells and mouse 3T3 fibers P65 protein in cells can be modified by exogenous SUMO3 protein by SUMO, but there is no interaction between p65 protein and SUMO1, SUMO2 protein and the effect of SUMO modification on p65 protein. This research group confirmed MANF (Mesencephalic Astrocyte-derived), new medium brain glue. The cytokine derived neurotrophic factor (PNF) can interact with p65 in the nucleus to inhibit the transcriptional activity of NF- kappa B, but it is not reported whether SUMO protein affects the interaction between p65 and MANF. The purpose of this study is to investigate how the SUMO protein can regulate the level and localization of p65 cells in HCC and whether p65 and MANF are influenced by the SUMO protein. Methods: the liver tissues of patients with hepatitis B and HCC were fixed in paraffin embedded section, and the expression of p65, SUMO1 and SUMO2/3 protein were detected by immunohistochemical method in human hepatitis tissue, cancer tissue and para cancerous tissue of HCC patients; p65 and SUMO were detected by double immunofluorescence in human liver cancer tissue and liver cancer cell lines. Whether there is a co localization phenomenon within 1 or SUMO2/3 cells; the interaction between p65 and SUMO1 or SUMO2/3 by immunoprecipitation in human hepatocellular carcinoma and hepatoma cell lines and whether the p65 protein can be modified by SUMO; Western blot is used to observe the microenvironment (such as hypoxia and inflammation) in the hepatocellular carcinoma cell lines. Whether the subcellular localization of p65 protein and the expression of SUMO1 and SUMO2/3 protein have an effect on the expression of SUMO1 and SUMO2/3 protein, and the changes in the SUMO modification level of the liver cancer cell lines after the treatment of hypoxia and inflammatory stimulation were observed. The transfection of exogenous SUMO1 or SUMO2/3 in the hepatocellular carcinoma cell lines was used to use Western blot. The changes of p65 protein level and subcellular localization were observed and the effects of SUMO1 on NF- kappa B transcriptional activity were detected by double luciferase reporter gene experiment. In the hepatocellular carcinoma cell lines, MTT, clone formation experiment, and Transwell chamber experiment were used to observe the proliferation and invasion of liver cancer cells by SUMO1 and SUMO2/3. The effect of the ability and the correlation between SUMO1 and Ki67 protein in the human liver cancer tissue, to evaluate whether the SUMO1 protein is related to the proliferation of hepatoma cells; to knock down the endogenous SUMO1 or SUMO2/3 in the hepatocellular carcinoma cell lines, and to detect the interaction between p65 and MANF protein by the immunoprecipitation test. Results: 1. in human liver tissue, the expression of p65, SUMO1 and SUMO2/3 protein was observed. The immunohistochemical results showed that there was a small number of SUMO1 positive cells in the hepatitis tissues, and the expression of SUMO1 in the liver tissues was significantly higher than that in the paracancerous tissues, and the expression of.SUMO2/3 in the nucleus was different from that of SUMO1. SUMO2/3 in hepatitis was in hepatitis. There were a small amount of expression in the tissue, cytoplasm and nucleus in the nucleus. The expression of SUMO2/3 in the cancer tissues was obviously lower than that in the paracancerous tissues. The expression of.P65 protein in the cytoplasm was very similar to that of SUMO2/3. A small amount of expression in the hepatitis tissue, the low expression of the cancer tissue and high expression of the para cancer tissues were found, but there were p65 in the nucleus of the liver cancer. The interaction of immunofluorescence between.2. SUMO1 and p65 showed that p65 protein was Co located with SUMO1 in the cancer tissue of HCC patients and in the hepatocellular carcinoma cell lines, and was mainly located in the nucleus. The interaction between SUMO1 and p65 was also found in the immunoprecipitation experiment in human liver cancer tissue and liver cancer cell lines, and found SUMO1 can make p65. The DAB staining of the interaction between.3. SUMO2/3 and p65 showed that the p65 protein was highly consistent with the expression of SUMO2/3. Further immunofluorescence double labeling experiment found that p65 and SUMO2/3 were Co located in the cytoplasm, and the p65 expression in the cells with high expression of SUMO2/3 was more than that in the cells that were not expressed in SUMO2/3. 5 there are few expressions. In the liver and liver cancer cell lines of HCC patients, the immunoprecipitation experiment further confirms the interaction between SUMO2/3 and p65, and it is found that SUMO2/3 can make p65 protein SUMO modified.4. anoxia and inflammatory stimulation to promote the expression of SUMO1 and SUMO2/3 protein and p65 SUMO hepatoma cell line through glucose deficiency anoxia treatment 150 min. After that, some p65 protein was found to be nucleed and the expression of SUMO1 and SUMO2/3 protein increased. The hepatocellular carcinoma cell line was treated with TNF-a (10ng/ml) 30 min, some of the p65 protein was nucleed, the level of SUMO2/3 protein in the cytoplasm increased obviously, but the level of SUMO1 protein was not obviously changed. When TNF-a (10ng/ml) treated the hepatocellular carcinoma cell line 8 hrs, the cytoplasm The level of protein and TNF treatment 30 min did not change obviously, but the level of SUMO2/3 protein in the nucleus increased obviously, and the level of SUMO1 protein in cytoplasm and nucleus increased obviously. The result of immunoprecipitation experiment showed that the cells were 24 hrs after transfection of SUMO modified E2 enzyme (Ubc9) and exogenous SUMO plasmid, and 150min could be increased by oxygen deficiency anoxic treatment 150min. The SUMO modification level of p65 associated with SUMO1 protein does not affect the SUMO modification of p65 related to SUMO2/3; cells transfected with TNF- a (10ng/ml) stimulates 30 min after transfection of 24 hrs, and can simultaneously increase the level of SUMO1 and SUMO2/3 protein related protein SUMO1 does not affect the level of p65 protein. The overexpression of SUMO1 can increase the nucleation of p65 protein induced by oxygen deficiency and hypoxia, but the silent endogenous SUMO1 can inhibit the nucleation of p65 protein, while silent SUMO1 can inhibit the nucleation of p65 protein induced by TNF- alpha, and the overexpression of SUMO1 has no effect on TNF-a induced nucleation of the p65 protein. The transcriptional activity of.6. knockout low endogenous SUMO1 protein inhibited NF- kappa B in the hepatocellular carcinoma cell lines using the double luciferase reporter gene test method. The results showed that the transcriptional activity of NF- kappa B could be inhibited by normal cultured cells and cells after TNF- alpha stimulation or glucose deficiency anoxia treatment, and the low endogenous SUMO1 could also be suppressed. NF- kappa B transcriptional activity.7. knocks low endogenous SUMO1 to inhibit the proliferation and metastasis of hepatocellular carcinoma cell line MTT. Cloning experiments confirmed that knockout endogenous SUMO1 inhibited cell proliferation. We further observed the correlation of Ki67 and SUMO1 by immunofluorescence in human hepatocellular carcinoma tissue. All Ki67 positive cells were positive for SUMO1, so we speculated that the expression of SUMO1 protein may be related to the proliferation ability of hepatoma cells in.Transwell experiments. Overexpression of SUMO1 can increase the metastasis and invasion ability of liver cancer cell lines, and silence SUMO1 inhibits the transformation and invasion of.8. SUMO2/3 stable cytoplasm in.8. SUMO2/3 The level of the overexpression of SUMO2/3 in the hepatoma cell line related to its antagonism to the ubiquitination of p65 found that the level of p65 protein increased with the increase of DNA in the transfected SUMO2/3. Further nuclear plasma separation experiments found that SUMO2/3 protein mainly maintained the stability of the p65 protein in the cytoplasm. In the liver cancer cell lines, the level of mRNA was expressed by SUMO2/3 to detect p65. SUMO2/3 does not affect the transcriptional level of p65. At the same time, the immunoprecipitation experiment shows that the ubiquitination of p65 protein decreases when the SUMO modification of SUMO2/3 protein related p65 increases. The results suggest that the SUMO2/3 protein may be by antagonizing the ubiquitination of p65, reducing the p65 protein degradation to stabilize the p65 protein level in the cytoplasm and.9. over expression SUMO2/3. The inhibitory effect of protein inhibition on the proliferation of hepatocellular carcinoma cell strain MTT showed that overexpression of SUMO2/3 in hepatoma cells inhibited the proliferation of hepatocellular carcinoma cell lines, SUMO2/3 did not affect the metastasis and invasion of hepatoma cell lines,.10. SUMO protein affected the interaction between p65 and MANF, and the immune co precipitation experiment was found in the liver cancer cell lines. The interaction between the source SUMO1 or SUMO2/3, p65 and MANF protein decreased. Conclusion: 1. SUMO1 and SUMO2/3 can interact with p65 and modify them with SUMO; 2. the glucose deficiency anoxia and TNF- alpha can enhance the interaction between SUMO protein and p65; 3. knock low SUMO1 can inhibit the lack of glucose and hypoxia and the nucleation of the protein induced by alpha. The transcriptional activity of 4. SUMO2/3 can stabilize the level of p65 protein in the cytoplasm; the ubiquitin modification of 5.p65 protein antagonists its SUMO; 6. SUMO1 can promote the proliferation and metastasis of hepatoma cells; 7. overexpression of SUMO2/3 can inhibit the proliferation of hepatoma cells; 8. knock low SUMO1 or SUMO2/3 to respond to the interaction between p65 and MANF.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7
[Abstract]:SUMO (small ubiquitin-related modifier) protein is a kind of small molecular protein that is very similar to ubiquitin, and participates in a series of physiological and pathological processes of SUMO modification (SUMOylation) modification (SUMOylation).SUMO modification, which is closely related to the occurrence and development of many diseases, for example. Hepatocellular carcinoma (HCC).P65 is one of the most important subunits of the NF- kappa B family, and the activation of NF- kappa B signaling pathway after p65 protein nucleation plays an important role in the development of hepatocellular carcinoma. In recent years, Yi Youwen has demonstrated that in human embryonic kidney 293T cells and mouse 3T3 fibers P65 protein in cells can be modified by exogenous SUMO3 protein by SUMO, but there is no interaction between p65 protein and SUMO1, SUMO2 protein and the effect of SUMO modification on p65 protein. This research group confirmed MANF (Mesencephalic Astrocyte-derived), new medium brain glue. The cytokine derived neurotrophic factor (PNF) can interact with p65 in the nucleus to inhibit the transcriptional activity of NF- kappa B, but it is not reported whether SUMO protein affects the interaction between p65 and MANF. The purpose of this study is to investigate how the SUMO protein can regulate the level and localization of p65 cells in HCC and whether p65 and MANF are influenced by the SUMO protein. Methods: the liver tissues of patients with hepatitis B and HCC were fixed in paraffin embedded section, and the expression of p65, SUMO1 and SUMO2/3 protein were detected by immunohistochemical method in human hepatitis tissue, cancer tissue and para cancerous tissue of HCC patients; p65 and SUMO were detected by double immunofluorescence in human liver cancer tissue and liver cancer cell lines. Whether there is a co localization phenomenon within 1 or SUMO2/3 cells; the interaction between p65 and SUMO1 or SUMO2/3 by immunoprecipitation in human hepatocellular carcinoma and hepatoma cell lines and whether the p65 protein can be modified by SUMO; Western blot is used to observe the microenvironment (such as hypoxia and inflammation) in the hepatocellular carcinoma cell lines. Whether the subcellular localization of p65 protein and the expression of SUMO1 and SUMO2/3 protein have an effect on the expression of SUMO1 and SUMO2/3 protein, and the changes in the SUMO modification level of the liver cancer cell lines after the treatment of hypoxia and inflammatory stimulation were observed. The transfection of exogenous SUMO1 or SUMO2/3 in the hepatocellular carcinoma cell lines was used to use Western blot. The changes of p65 protein level and subcellular localization were observed and the effects of SUMO1 on NF- kappa B transcriptional activity were detected by double luciferase reporter gene experiment. In the hepatocellular carcinoma cell lines, MTT, clone formation experiment, and Transwell chamber experiment were used to observe the proliferation and invasion of liver cancer cells by SUMO1 and SUMO2/3. The effect of the ability and the correlation between SUMO1 and Ki67 protein in the human liver cancer tissue, to evaluate whether the SUMO1 protein is related to the proliferation of hepatoma cells; to knock down the endogenous SUMO1 or SUMO2/3 in the hepatocellular carcinoma cell lines, and to detect the interaction between p65 and MANF protein by the immunoprecipitation test. Results: 1. in human liver tissue, the expression of p65, SUMO1 and SUMO2/3 protein was observed. The immunohistochemical results showed that there was a small number of SUMO1 positive cells in the hepatitis tissues, and the expression of SUMO1 in the liver tissues was significantly higher than that in the paracancerous tissues, and the expression of.SUMO2/3 in the nucleus was different from that of SUMO1. SUMO2/3 in hepatitis was in hepatitis. There were a small amount of expression in the tissue, cytoplasm and nucleus in the nucleus. The expression of SUMO2/3 in the cancer tissues was obviously lower than that in the paracancerous tissues. The expression of.P65 protein in the cytoplasm was very similar to that of SUMO2/3. A small amount of expression in the hepatitis tissue, the low expression of the cancer tissue and high expression of the para cancer tissues were found, but there were p65 in the nucleus of the liver cancer. The interaction of immunofluorescence between.2. SUMO1 and p65 showed that p65 protein was Co located with SUMO1 in the cancer tissue of HCC patients and in the hepatocellular carcinoma cell lines, and was mainly located in the nucleus. The interaction between SUMO1 and p65 was also found in the immunoprecipitation experiment in human liver cancer tissue and liver cancer cell lines, and found SUMO1 can make p65. The DAB staining of the interaction between.3. SUMO2/3 and p65 showed that the p65 protein was highly consistent with the expression of SUMO2/3. Further immunofluorescence double labeling experiment found that p65 and SUMO2/3 were Co located in the cytoplasm, and the p65 expression in the cells with high expression of SUMO2/3 was more than that in the cells that were not expressed in SUMO2/3. 5 there are few expressions. In the liver and liver cancer cell lines of HCC patients, the immunoprecipitation experiment further confirms the interaction between SUMO2/3 and p65, and it is found that SUMO2/3 can make p65 protein SUMO modified.4. anoxia and inflammatory stimulation to promote the expression of SUMO1 and SUMO2/3 protein and p65 SUMO hepatoma cell line through glucose deficiency anoxia treatment 150 min. After that, some p65 protein was found to be nucleed and the expression of SUMO1 and SUMO2/3 protein increased. The hepatocellular carcinoma cell line was treated with TNF-a (10ng/ml) 30 min, some of the p65 protein was nucleed, the level of SUMO2/3 protein in the cytoplasm increased obviously, but the level of SUMO1 protein was not obviously changed. When TNF-a (10ng/ml) treated the hepatocellular carcinoma cell line 8 hrs, the cytoplasm The level of protein and TNF treatment 30 min did not change obviously, but the level of SUMO2/3 protein in the nucleus increased obviously, and the level of SUMO1 protein in cytoplasm and nucleus increased obviously. The result of immunoprecipitation experiment showed that the cells were 24 hrs after transfection of SUMO modified E2 enzyme (Ubc9) and exogenous SUMO plasmid, and 150min could be increased by oxygen deficiency anoxic treatment 150min. The SUMO modification level of p65 associated with SUMO1 protein does not affect the SUMO modification of p65 related to SUMO2/3; cells transfected with TNF- a (10ng/ml) stimulates 30 min after transfection of 24 hrs, and can simultaneously increase the level of SUMO1 and SUMO2/3 protein related protein SUMO1 does not affect the level of p65 protein. The overexpression of SUMO1 can increase the nucleation of p65 protein induced by oxygen deficiency and hypoxia, but the silent endogenous SUMO1 can inhibit the nucleation of p65 protein, while silent SUMO1 can inhibit the nucleation of p65 protein induced by TNF- alpha, and the overexpression of SUMO1 has no effect on TNF-a induced nucleation of the p65 protein. The transcriptional activity of.6. knockout low endogenous SUMO1 protein inhibited NF- kappa B in the hepatocellular carcinoma cell lines using the double luciferase reporter gene test method. The results showed that the transcriptional activity of NF- kappa B could be inhibited by normal cultured cells and cells after TNF- alpha stimulation or glucose deficiency anoxia treatment, and the low endogenous SUMO1 could also be suppressed. NF- kappa B transcriptional activity.7. knocks low endogenous SUMO1 to inhibit the proliferation and metastasis of hepatocellular carcinoma cell line MTT. Cloning experiments confirmed that knockout endogenous SUMO1 inhibited cell proliferation. We further observed the correlation of Ki67 and SUMO1 by immunofluorescence in human hepatocellular carcinoma tissue. All Ki67 positive cells were positive for SUMO1, so we speculated that the expression of SUMO1 protein may be related to the proliferation ability of hepatoma cells in.Transwell experiments. Overexpression of SUMO1 can increase the metastasis and invasion ability of liver cancer cell lines, and silence SUMO1 inhibits the transformation and invasion of.8. SUMO2/3 stable cytoplasm in.8. SUMO2/3 The level of the overexpression of SUMO2/3 in the hepatoma cell line related to its antagonism to the ubiquitination of p65 found that the level of p65 protein increased with the increase of DNA in the transfected SUMO2/3. Further nuclear plasma separation experiments found that SUMO2/3 protein mainly maintained the stability of the p65 protein in the cytoplasm. In the liver cancer cell lines, the level of mRNA was expressed by SUMO2/3 to detect p65. SUMO2/3 does not affect the transcriptional level of p65. At the same time, the immunoprecipitation experiment shows that the ubiquitination of p65 protein decreases when the SUMO modification of SUMO2/3 protein related p65 increases. The results suggest that the SUMO2/3 protein may be by antagonizing the ubiquitination of p65, reducing the p65 protein degradation to stabilize the p65 protein level in the cytoplasm and.9. over expression SUMO2/3. The inhibitory effect of protein inhibition on the proliferation of hepatocellular carcinoma cell strain MTT showed that overexpression of SUMO2/3 in hepatoma cells inhibited the proliferation of hepatocellular carcinoma cell lines, SUMO2/3 did not affect the metastasis and invasion of hepatoma cell lines,.10. SUMO protein affected the interaction between p65 and MANF, and the immune co precipitation experiment was found in the liver cancer cell lines. The interaction between the source SUMO1 or SUMO2/3, p65 and MANF protein decreased. Conclusion: 1. SUMO1 and SUMO2/3 can interact with p65 and modify them with SUMO; 2. the glucose deficiency anoxia and TNF- alpha can enhance the interaction between SUMO protein and p65; 3. knock low SUMO1 can inhibit the lack of glucose and hypoxia and the nucleation of the protein induced by alpha. The transcriptional activity of 4. SUMO2/3 can stabilize the level of p65 protein in the cytoplasm; the ubiquitin modification of 5.p65 protein antagonists its SUMO; 6. SUMO1 can promote the proliferation and metastasis of hepatoma cells; 7. overexpression of SUMO2/3 can inhibit the proliferation of hepatoma cells; 8. knock low SUMO1 or SUMO2/3 to respond to the interaction between p65 and MANF.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7
【相似文獻】
相關期刊論文 前10條
1 周晶,沈志祥;過氧化物酶體增殖物激活受體與肝癌[J];腫瘤防治研究;2004年11期
2 段紀成;楊家和;劉凱;吳孟超;;單用絲裂霉素或表柔比星對肝癌細胞株的抑制作用[J];第二軍醫(yī)大學學報;2007年04期
3 楊盛力;彭靜;陳孝平;;中藥逆轉肝癌多藥耐藥的研究進展[J];肝膽外科雜志;2008年06期
4 魏莉;李愛民;羅榮城;;肝癌多藥耐藥及逆轉的研究進展[J];中國藥房;2009年07期
5 王智;;硒-甲基硒代半胱氨酸誘導肝癌SMMC-7721細胞株的凋亡及機制探討[J];江蘇醫(yī)藥;2012年11期
6 李培坤;耿小平;;肝癌耐藥的基因學研究進展[J];中華腫瘤防治雜志;2012年12期
7 龔民族;;大白鼠肝癌細胞株FSK 7901,FSK 7902的建立和特點[J];解剖學報;1981年03期
8 曹韻貞,湯釗猷;人體肝癌細胞株研究進展[J];腫瘤;1985年03期
9 ;視黃酸逆轉入肝癌細胞株的研究[J];上海醫(yī)科大學學報;1987年05期
10 偶曉明,王俊明,黃辰,關蓮璞,楊培金,林琳;肝癌細胞的融合方法探討[J];西安交通大學學報(醫(yī)學版);1992年01期
相關會議論文 前10條
1 魏緒勇;王建國;馮曉文;王雁;曹國強;陳海;徐驍;鄭樹森;;肝癌組織中氨基�；�-1表達及作用[A];2012中國器官移植大會論文匯編[C];2012年
2 魏緒勇;王建國;馮曉文;王雁;曹國強;陳海;徐驍;鄭樹森;;肝癌組織中氨基�；�-1表達及作用[A];2012年浙江省外科學學術年會論文集[C];2012年
3 唐瑞峰;馬艷鵬;吳建飛;孫建;張文華;黃亮;;白細胞介素2和白細胞介素6對肝癌細胞表達血管內皮生長因子B的調節(jié)[A];第6屆全國疑難及重癥肝病大會論文集[C];2011年
4 唐瑞峰;孫超;馬艷鵬;吳建飛;孫建;張文華;;白細胞介素2和白細胞介素6對肝癌細胞表達血管內皮生長因子D的調節(jié)[A];第6屆全國疑難及重癥肝病大會論文集[C];2011年
5 任軍;;肝癌的生物治療[A];第三屆中國腫瘤學術大會教育論文集[C];2004年
6 陳啟楨;林雅慧;;樟芝菌種于中藥草固態(tài)發(fā)酵萃取液對抗人類肝癌動物試驗功效研究[A];海峽兩岸第十屆菌物學暨第三屆食藥用菌學術研討會論文摘要集[C];2011年
7 楊建林;樊曉暉;;一株新發(fā)現(xiàn)的新城疫病毒高效殺傷肝癌細胞及其作用機制研究[A];2009醫(yī)學前沿論壇暨第十一屆全國腫瘤藥理與化療學術會議論文集[C];2009年
8 孫文潔;廖正凱;周福祥;張紅艷;黃成虎;熊杰;周云峰;;放射線對肝癌細胞中端粒酶啟動子活性的影響[A];2007第六屆全國放射腫瘤學學術年會論文集[C];2007年
9 王魯;湯釗猷;薛瓊;孫惠川;陳軍;高冬梅;趙燕;陳潔;;β干擾素抑制肝癌腫瘤生長和復發(fā)轉移的實驗研究[A];2000全國腫瘤學術大會論文集[C];2000年
10 葉依霞;周永健;李瑜元;張宗勝;;廣州市人群非酒精性脂肪性肝病的自然病程及危險因素[A];中華醫(yī)學會第十六次全國病毒性肝炎及肝病學術會議論文匯編[C];2013年
相關重要報紙文章 前8條
1 李運紅邋胡顏;科學家從壁虎中分離出抗肝癌成分[N];新華每日電訊;2007年
2 張中橋;AFP可作為治療肝癌的新靶標[N];中國醫(yī)藥報;2006年
3 印高樂;臺灣研究發(fā)現(xiàn)黃體酯酮可降低肝癌復發(fā)率[N];醫(yī)藥經濟報;2004年
4 李運紅邋胡顏;我國科學家首次從壁虎中分離出抗腫瘤活性成分[N];醫(yī)藥經濟報;2007年
5 記者 陳建強邋通訊員 李運紅 胡顏;國內首次從壁虎中分離出抗腫瘤活性成分[N];光明日報;2007年
6 李運紅邋胡顏;國內首次從壁虎中分離出抗腫瘤活性成分[N];科技日報;2007年
7 劉道安邋運紅 胡顏;我國學者從壁虎中分離出抗腫瘤活性成分[N];中國醫(yī)藥報;2007年
8 劉道安邋通訊員 運紅 胡顏;壁虎中分離出抗腫瘤活性成分[N];健康報;2007年
相關博士學位論文 前10條
1 張余琴;MiR-20a激活PTEN/PI3K/AKT通路誘導肝癌輻射抵抗[D];南方醫(yī)科大學;2015年
2 葉真龍;Argonaute-2蛋白在肝癌血管生成和擬態(tài)中的功能研究[D];第二軍醫(yī)大學;2015年
3 劉s，
本文編號：2147140
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2147140.html
最近更新
教材專著
