【摘要】：[目的]非小細(xì)胞肺癌(NSCLC)全球高發(fā),為癌性死亡的首位原因,其驅(qū)動機(jī)制至今未闡明,給診治帶來諸多挑戰(zhàn)。云南省為肺癌高發(fā)大省,亟待探索NSCLC特異性的驅(qū)動機(jī)制。剪接體功能異常可致下游基因差異剪接,進(jìn)而參與腫瘤的發(fā)生。作為剪接體組裝的關(guān)鍵復(fù)合物,SMN復(fù)合物的眾多組分逐漸成為癌癥的診治靶點。但是GEMIN6基因編碼蛋白作為SMN復(fù)合物的重要組分,其在NSCLC中發(fā)揮的角色仍是未解之謎。目前,SMN復(fù)合物的其他組分被證實參與癌細(xì)胞增殖等惡性生物學(xué)行為,甚至可調(diào)節(jié)自噬活性。與此同時,中國傳統(tǒng)藥物在NSCLC自噬活性的調(diào)節(jié)中逐漸嶄露頭角,日漸成為NSCLC用藥的研究熱點。因此,本研究擬從細(xì)胞及組織水平探討GEMIN6與NSCLC惡性生物學(xué)行為的相關(guān)性,通過檢測細(xì)胞增殖能力、遷移活性、周期進(jìn)程以及自噬標(biāo)志蛋白,旨在獲得GEMIN6參與NSCLC增殖和遷移等惡性生物學(xué)行為的實驗學(xué)證據(jù),進(jìn)一步探討黃連素等傳統(tǒng)中藥單體是否可通過GEMIN6調(diào)控NSCLC的惡性進(jìn)程,為云南地區(qū)NSCLC的診治提供干預(yù)新靶點和治療新思路。[方法]采用Westernblotting技術(shù)檢測NSCLC細(xì)胞系中GEMIN6的基礎(chǔ)表達(dá)量;利用siRNA敲減A549和H1975細(xì)胞系中GEMIN6的表達(dá),Westernblotting技術(shù)驗證敲減;通過MTS細(xì)胞增殖實驗、Transwell實驗、流式細(xì)胞術(shù)檢測GEMIN6對NSCLC細(xì)胞生長的影響;采用Westernblotting技術(shù)驗證自噬標(biāo)記蛋白Beclinl和LC3-Ⅰ/Ⅱ的表達(dá)水平改變;通過MTS法檢測黃連素對A549細(xì)胞的半數(shù)抑制濃度;結(jié)合實驗室前期研究結(jié)果,采用Westernblotting技術(shù)驗證4類中藥單體作用后A549細(xì)胞中GEMIN6及自噬標(biāo)記蛋白Beclinl的表達(dá)水平改變;通過免疫組織化學(xué)方法和HE染色方法檢測NSCLC的癌組織和良性對照組織中GEMIN6的表達(dá)水平,運用SPSS軟件分析GENMIN6與NSCLC的臨床相關(guān)性。[結(jié)果]1. Kaplan-Meier plotter生存曲線分析表明高表達(dá)GEMIN6的肺癌患者生存期較對照組低2. GEMIN6在NSCLC癌組織中表達(dá)高于對照組,但臨床相關(guān)性不大3. GEMIN6增強(qiáng)NSCLC的增殖和遷移能力4. GEMIN6促進(jìn)NSCLC發(fā)生G1-S期轉(zhuǎn)變5. GEMIN6抑制NSCLC的自噬活性6.在A549細(xì)胞中,黃連素和姜黃素可升高GEMIN6以及Beclinl的表達(dá);雙氫青蒿素降低GEMIN6的表達(dá)并且輕度升高Beclinl的表達(dá);紅景天苷升高GEMIN6表達(dá)但對Beclinl的表達(dá)無影響。雙氫青蒿素可能通過GEMIN6發(fā)揮其抑癌活性,但另外3種單體均非通過GEMIN6調(diào)控A549細(xì)胞的生長。[結(jié)論]在NSCLC中特異性高表達(dá)的GEMIN6通過促進(jìn)G1/S期過渡,升高A549和H1975細(xì)胞的惡性增殖能力和遷移能力,同時抑制A549細(xì)胞的自噬活性;雙氫青蒿素在下調(diào)A549細(xì)胞中GMEIN6表達(dá)的同時可輕度上調(diào)自噬水平,GEMIN6可能為其下游作用靶標(biāo);黃連素、姜黃素以及紅景天苷可在不同程度上改變A549細(xì)胞中GEMIN6的表達(dá),但無具體證據(jù)表明它們對NSCLC自噬活性的調(diào)控與GEMIN6表達(dá)水平改變相關(guān)。
[Abstract]:[objective] Non-small cell lung cancer (NSCLC) has a global high incidence of (NSCLC), which is the leading cause of cancer death. The driving mechanism of NSCLC has not been clarified yet, which brings many challenges to diagnosis and treatment. Yunnan Province is a major province with high incidence of lung cancer. It is urgent to explore the specific driving mechanism of NSCLC. Aberrant splicing can result in differentially splicing downstream genes, and then participate in tumorigenesis. Many components of SMN complex, which is the key component of splice assembly, have gradually become the target of cancer diagnosis and treatment. However, as an important component of SMN complex, the role of GEMIN6 gene encoding protein in NSCLC remains a mystery. At present, other components of SMN complex have been proved to be involved in malignant biological behaviors such as cancer cell proliferation, and even regulate autophagy activity. At the same time, the regulation of NSCLC autophagy activity of traditional Chinese drugs has gradually emerged and become the research hotspot of NSCLC drug. Therefore, the purpose of this study was to explore the relationship between GEMIN6 and malignant biological behavior of NSCLC at cell and tissue levels, and to detect cell proliferation, migration activity, cycle progression and autophagy marker protein. To obtain experimental evidence of GEMIN6 involved in malignant biological behavior such as proliferation and migration of NSCLC, and to further explore whether traditional Chinese medicine monomer, such as berberine, can regulate the malignant process of NSCLC through GEMIN6. To provide a new target of intervention and new ideas for the treatment of NSCLC in Yunnan. [methods] the basic expression of GEMIN6 in NSCLC cell line was detected by Westernblotting technique, the expression of GEMIN6 in A549 and H1975 cell lines was verified by siRNA knockdown and Western blotting technique was used to verify the knockout, and the transwell assay was used to test the proliferation of MTS cells. Flow cytometry was used to detect the effect of GEMIN6 on the growth of NSCLC cells, Westernblotting technique was used to verify the changes of the expression levels of Beclinl and LC3- 鈪，

本文編號：2244956