本文選題：HIF-2α　切入點(diǎn)：低氧　出處：《浙江大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：全球結(jié)腸癌的發(fā)病率占所有惡性腫瘤第三位,病死率居第四位。由于結(jié)直腸癌的早期缺乏特異性癥狀,一旦確診大部分已屬中、晚期。結(jié)直腸癌的治療以手術(shù)為主,配合化療、放療和靶向分子治療等綜合治療手段。尋找生物學(xué)靶標(biāo)用于癌變監(jiān)測(cè)和干預(yù)是結(jié)直腸癌防治研究的一個(gè)重要方向。 結(jié)腸癌的發(fā)生發(fā)展與癌基因的突變、抑癌基因的失活密切相關(guān)。c-Myc作為重要原癌基因在多種惡性腫瘤中都呈高表達(dá)。c-Myc基因?qū)?xì)胞具有雙重作用,可刺激細(xì)胞增殖,也可促進(jìn)細(xì)胞凋亡。因此,研究c-Myc在腫瘤中的作用及相關(guān)調(diào)控機(jī)制對(duì)結(jié)腸癌的診治有重要價(jià)值。 結(jié)腸腫瘤組織生長中心區(qū)會(huì)不同程度出現(xiàn)缺氧和壞死。低氧環(huán)境對(duì)腫瘤組織的生長具有重要影響。低氧微環(huán)境參與腫瘤細(xì)胞增殖、凋亡、侵襲轉(zhuǎn)移及血管生成等調(diào)控。低氧誘導(dǎo)因子HIF廣泛表達(dá)于哺乳動(dòng)物的各種組織細(xì)胞中,以促進(jìn)機(jī)體對(duì)低氧的適應(yīng)過程,是細(xì)胞在基因轉(zhuǎn)錄水平協(xié)調(diào)缺氧變化的最主要調(diào)節(jié)因子。本研究將通過系統(tǒng)研究低氧微環(huán)境下結(jié)腸癌細(xì)胞株中HIF-1α/HIF-2α與c-Myc的表達(dá)情況；同時(shí)利用siRNA干擾技術(shù),了解HIF-1α/HIF-2α對(duì)c-Myc的調(diào)控作用；利用蛋白酶體抑制劑MG132預(yù)處理后結(jié)腸癌細(xì)胞低氧培養(yǎng)c-Myc蛋白的表達(dá),以了解泛素化是否參與了c-Myc蛋白的降解過程。 研究方法： 1、不同低氧處理時(shí)間點(diǎn),提取結(jié)腸癌細(xì)胞HCT116和SW480的總蛋白,Western blot檢測(cè)HIF-1α、HIF-2α、c-Myc及p-c-Myc的表達(dá)變化。 2、不同低氧處理時(shí)間點(diǎn),提取結(jié)腸癌細(xì)胞HCT116和SW480的總RNA,qPCR檢測(cè)c-Myc mRNA表達(dá)的變化。低氧處理24h后,雙熒光素酶報(bào)告基因檢測(cè)c-Myc啟動(dòng)子轉(zhuǎn)錄活性的變化。 3、采用siRNA靶向干擾HIF-1α或HIF-2α的表達(dá),低氧培養(yǎng)24h后,Western blot檢測(cè)c-Myc蛋白表達(dá)的變化。 4、采用MG132處理結(jié)腸癌細(xì)胞HCT116和SW480,分別常氧和低氧下培養(yǎng),Western blot檢測(cè)c-Myc蛋白表達(dá)的變化。 研究結(jié)果： 1、低氧微環(huán)境下結(jié)腸癌細(xì)胞株中HIF-1α/HIF-2α與c-Myc蛋白表達(dá)的變化。隨低氧時(shí)間延長,HIF-1α在4h左右達(dá)到高峰,繼續(xù)延長低氧時(shí)間表達(dá)水平逐漸下降；而HIF-2α蛋白在24h表達(dá)保持持續(xù)升高。c-Myc和p-c-Myc蛋白水平隨低氧時(shí)間延長逐步減弱。 2、低氧微環(huán)境下結(jié)腸癌細(xì)胞株中c-Myc mRNA及轉(zhuǎn)錄活性的變化。隨著低氧處理時(shí)間的延長,結(jié)腸癌細(xì)胞株HCT116和SW480中的c-Myc mRNA含量逐漸降低。低氧24h后c-Myc的轉(zhuǎn)錄活性明顯下降。 3、低氧誘導(dǎo)因子對(duì)c-Myc表達(dá)的調(diào)節(jié)。結(jié)腸癌細(xì)胞株HCT116、SW480中,干擾HIF-1α或HIF-2a并低氧處理24h后,c-Myc的表達(dá)增加,且干擾HIF-2a后,c-Myc表達(dá)的增加更為明顯。 4、 MG132對(duì)c-Myc蛋白表達(dá)的影響。結(jié)腸癌細(xì)胞株HCT116、SW480中加入MG132預(yù)處理后c-Myc蛋白的表達(dá)明顯增加,且呈濃度依賴性。 研究結(jié)論： 1、慢性低氧微環(huán)境下,結(jié)腸癌細(xì)胞中HIF-2α為優(yōu)勢(shì)表達(dá)的低氧誘導(dǎo)因子,伴隨著c-Myc啟動(dòng)子轉(zhuǎn)錄活性、mRNA及蛋白質(zhì)表達(dá)的下降。 2、慢性低氧微環(huán)境下,結(jié)腸癌細(xì)胞中c-Myc的表達(dá)受低氧誘導(dǎo)因子和泛素化的影響,其中HIF-2α對(duì)c-Myc的調(diào)控作用比HIF-1α更為明顯。
[Abstract]:The global incidence of colorectal cancer accounted for third of all malignancies, the mortality rate ranks fourth. Due to the lack of early colorectal cancer specific symptoms, once the diagnosis is most advanced. In the treatment of colorectal cancer with surgery, chemotherapy, radiotherapy and targeted molecular therapy and other comprehensive treatment. Looking for biological targets for cancer surveillance and intervention is an important direction of research colorectal cancer prevention.
The occurrence and development of cancer gene mutation and colon cancer, activity is closely related to.C-Myc as an oncogene in a variety of malignant tumors with high expression of.C-Myc gene has a dual effect on cell loss of tumor suppressor that can stimulate cell proliferation, also promote cell apoptosis. Therefore, diagnosis and treatment of colon cancer and related regulation. The c-Myc in tumor mechanism are of great value.
Colon tumor growth center will be different degrees of hypoxia and necrosis. Has an important effect on the growth of tumor tissue hypoxia. Hypoxia is involved in tumor cell proliferation, apoptosis, invasion and angiogenesis. The regulation is widely expressed in mammalian animal factor HIF induced by hypoxia in various tissues and cells, to promote the body to hypoxia the adaptation process is the most cells in the major regulator of gene transcription. The expression of coordination of hypoxia in this study through the systematic study of hypoxia in human colon cell lines HIF-1 alpha /HIF-2 alpha and c-Myc; at the same time using siRNA interference technology, understand the regulatory role of HIF-1 alpha /HIF-2 alpha on c-Myc expression by pretreatment; proteasome inhibitor MG132 after hypoxia in cultured c-Myc colon cancer cell junction proteins, in order to understand the ubiquitination is involved in the degradation process of c-Myc.
Research methods:
1, the total protein of HCT116 and SW480 in colon cancer cells was extracted by different hypoxia treatment time points, and Western blot was used to detect the changes in the expression of HIF-1 alpha, HIF-2 alpha, c-Myc and p-c-Myc.
2, at different hypoxic treatment time points, the total RNA of HCT116 and SW480 from colon cancer cells was extracted, and qPCR was used to detect the change of c-Myc mRNA expression. After hypoxia treatment 24h, dual luciferase reporter gene was used to detect c-Myc promoter transcriptional activity.
3, the expression of HIF-1 alpha or HIF-2 alpha was interfered with siRNA, and 24h was cultured in hypoxia, and the expression of c-Myc protein was detected by Western blot.
4, HCT116 and SW480 of colon cancer cells were treated with MG132, and the expression of c-Myc protein was detected by Western blot.
The results of the study:
1, changes in the colorectal cancer cell line HIF-1 alpha /HIF-2 alpha and c-Myc protein expression in node hypoxia. With prolonged hypoxia, alpha HIF-1 reached a peak at about 4h, continue to extend the time of hypoxia and the expression level decreased; HIF-2 alpha protein in 24h expression increasing.C-Myc and protein levels of p-c-Myc with prolonged hypoxia gradually weakened.
2, the changes of c-Myc mRNA and transcriptional activity in colon cancer cell lines under hypoxic microenvironment. With the prolongation of hypoxia treatment time, the content of c-Myc mRNA in colon cancer cell lines HCT116 and SW480 decreased gradually. C-Myc activity decreased significantly after hypoxia 24h.
3, hypoxia inducible factor regulates the expression of c-Myc. In colon cancer cell line HCT116 and SW480, the expression of c-Myc increases after interfering with HIF-1 alpha or HIF-2a and 24h after hypoxia treatment, and the increase of c-Myc expression is more obvious after interfering HIF-2a.
4, the effect of MG132 on the expression of c-Myc protein. The expression of c-Myc protein in the colon cancer cell line HCT116, MG132 pretreated with MG132 was significantly increased, and was in a concentration dependent manner.
The conclusions are as follows:
1, in chronic hypoxic microenvironment, HIF-2 alpha is a dominant hypoxia inducible factor, which is associated with the transcriptional activity of c-Myc promoter, and the decrease of mRNA and protein expression.
2, in chronic hypoxia microenvironment, the expression of c-Myc in colon cancer cells is influenced by hypoxia inducible factor and ubiquitination. HIF-2 alpha plays a more significant role in regulating c-Myc than HIF-1 alpha.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.35

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