發(fā)布時間：2018-10-29 11:45

【摘要】：目的異染色質(zhì)蛋白1γ(CBX3)連接組蛋白甲基化從而發(fā)揮轉(zhuǎn)錄沉默、DNA修復(fù)、RNA剪切等功能。研究CBX3在結(jié)腸癌中的生物學(xué)作用及其相關(guān)機制有利于為腫瘤診斷、治療提供新的靶點。方法運用IHC和Western Blot方法分別檢測人結(jié)腸癌組織及其癌旁組織CBX3的表達(dá);利用CRISPR/Cas9基因編輯技術(shù)敲出HCT116細(xì)胞中的CBX3并構(gòu)建穩(wěn)定細(xì)胞系;通過MTS、EDU、克隆形成實驗及流式細(xì)胞儀檢測CBX3對結(jié)腸癌細(xì)胞增殖、周期、凋亡的影響;qRT PCR篩選CBX3影響結(jié)腸癌細(xì)胞周期G1-S期相關(guān)靶點,Western Blot和免疫熒光進一步在蛋白水平上驗證;運用慢病毒質(zhì)粒干擾及過表達(dá)實驗、激酶抑制實驗分析CBX3、CDK6、p21三者之間的關(guān)系;通過裸鼠成瘤實驗在體內(nèi)驗證CBX3對結(jié)腸癌發(fā)生發(fā)展中的作用;IHC和Western Blot進一步分析CDK6與p21在人結(jié)腸癌中的表達(dá)關(guān)系。結(jié)果CBX3在結(jié)腸癌組織中顯著高表達(dá);敲出CBX3明顯抑制結(jié)腸癌細(xì)胞增殖及細(xì)胞周期G1-S期進展,促進下游靶點CDK6和p21上調(diào);CDK6在缺乏CBX3時能通過獨立于激酶的活性調(diào)控p21表達(dá)限制細(xì)胞過度增殖;CBX3在裸鼠體內(nèi)能夠影響結(jié)腸癌細(xì)胞發(fā)生發(fā)展。結(jié)論CBX3通過抑制CDK6/p21的表達(dá)促進結(jié)腸癌發(fā)生發(fā)展,從而破壞了CDK6的負(fù)反饋調(diào)節(jié)機制。
[Abstract]:Objective to methylate heterochromatin 1 緯 (CBX3) binding histone to play a role in transcription silencing, DNA repair and RNA shearing. The study of the biological role of CBX3 in colon cancer and its related mechanisms may provide a new target for tumor diagnosis and treatment. Methods IHC and Western Blot were used to detect the expression of CBX3 in human colon cancer tissues and adjacent tissues respectively, and CRISPR/Cas9 gene editing technique was used to knock out CBX3 in HCT116 cells and construct stable cell lines. The effects of CBX3 on the proliferation, cycle and apoptosis of colon cancer cells were detected by MTS,EDU, clone forming assay and flow cytometry. QRT PCR screening of CBX3 related targets for G1-S phase of colon cancer cell cycle, Western Blot and immunofluorescence were further verified at protein level. The relationship among CBX3,CDK6,p21 was analyzed by plasmid interference and overexpression of lentivirus and kinase inhibition assay, and the role of CBX3 in the development of colon cancer in vivo was verified by tumorigenesis test in nude mice. IHC and Western Blot were used to analyze the expression of CDK6 and p21 in human colon cancer. Results CBX3 was significantly overexpressed in colon cancer tissues, knockout CBX3 significantly inhibited the proliferation of colon cancer cells and progression of cell cycle G1-S phase, and promoted downstream target CDK6 and p21 upregulation. In the absence of CBX3, CDK6 could restrict the proliferation of human colon cancer cells by regulating the expression of p21 independent of kinase activity, and CBX3 could influence the development of colon cancer cells in nude mice. Conclusion CBX3 can promote the development of colon cancer by inhibiting the expression of CDK6/p21, thus destroying the negative feedback regulation mechanism of CDK6.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.35

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