發(fā)布時間：2019-06-06 13:02

【摘要】：背景:Rho家族蛋白特異性鳥苷酸交換因子LARG是調(diào)節(jié)Rho家族蛋白GDP結(jié)合形式向GTP結(jié)合形式轉(zhuǎn)化的調(diào)節(jié)因子。作為Rho GEFs的一種,LARG被報道主要通過調(diào)節(jié)Rho家族蛋白活性來參與細(xì)胞移動,收縮以及有絲分裂,但是目前很少有LARG參與自噬調(diào)控的研究。LARG在血癌和實體腫瘤當(dāng)中所扮演的角色是依賴于不同信號通路的。在急性髓樣細(xì)胞白血病中作為原癌基因的LARG在乳腺癌和大腸癌中扮演的卻是抑癌基因的角色。因此,LARG在實體腫瘤當(dāng)中需要更深入的研究。在本文中,我們將就LARG在肺腺癌中的表達(dá)以及它與營養(yǎng)缺乏誘導(dǎo)的自噬是否存在調(diào)控關(guān)系進(jìn)行研究。方法:首先,通過體內(nèi)和體外實驗研究LARG在肺腺癌中的表達(dá)。我們選用了6組肺癌細(xì)胞系和HBEC細(xì)胞系來檢測LARG在體外細(xì)胞系中蛋白水平和轉(zhuǎn)錄水平的表達(dá)。同時,我們還通過免疫組化檢測了30例肺腺癌臨床樣本中LARG的表達(dá)。接著,通過營養(yǎng)缺乏誘導(dǎo)細(xì)胞自噬研究LARG與自噬之間是否存在調(diào)控關(guān)系。我們利用HBSS處理細(xì)胞一定時間來模擬腫瘤細(xì)胞在發(fā)生發(fā)展過程中遇到的營養(yǎng)缺乏條件并配合sh RNA干擾技術(shù)和超表達(dá)技術(shù),通過檢測自噬標(biāo)志物來判斷自噬水平的變化。利用GST Pull Down技術(shù)找出受LARG調(diào)控的Rho家族小分子GTPase蛋白,驗證LARG與自噬的關(guān)聯(lián)是否由該蛋白介導(dǎo)。最后,檢測營養(yǎng)缺乏條件下癌細(xì)胞的增殖能力和細(xì)胞凋亡來判斷癌細(xì)胞的生存能力是否受LARG的影響。結(jié)果:首先,LARG在肺腺癌細(xì)胞系A(chǔ)549、H1299、H446和H1993中蛋白水平和轉(zhuǎn)錄水平的表達(dá)均低于HBEC,其中在來源于三期肺腺癌的H1993細(xì)胞系中表達(dá)量最少。在臨床樣本方面,LARG在一期、二期肺腺癌中的表達(dá)量要明顯高于三期、四期。其次,通過HBSS處理后,LARG表達(dá)量低的細(xì)胞比表達(dá)量高的細(xì)胞表現(xiàn)出更明顯的自噬表型,包括p62/SQSTM-1積累的減少、自噬溶酶體的增加。GST pull down的結(jié)果顯示在A549和H1993細(xì)胞中LARG調(diào)控的是Rac1的活性而不是Rho A,并且LARG對自噬的影響就是通過Rac1活性的變化來實現(xiàn)的。最后,LARG通過自噬調(diào)控著p53和p21CDKN1A的蛋白水平表達(dá)量,進(jìn)而影響了營養(yǎng)缺乏狀態(tài)下肺腺癌細(xì)胞的凋亡和增殖能力。結(jié)論:LARG在肺腺癌中伴隨著癌癥的發(fā)展是逐漸缺失表達(dá)的,這種缺失表達(dá)通過降低Rac1的活性增強(qiáng)了營養(yǎng)缺乏狀態(tài)下肺腺癌細(xì)胞的自噬。增強(qiáng)的自噬通過降解p53的積累抑制凋亡的同時通過上調(diào)p21CDKN1A抑制增殖,最終促進(jìn)了肺腺癌細(xì)胞在營養(yǎng)缺乏狀態(tài)下的生存。全文結(jié)果告訴我們,在肺腺癌中LARG很可能通過對自噬的限制作用起到了抑癌基因的角色,并且它不是調(diào)控Rho A活性的分子靶點。
[Abstract]:Background: Rho family protein specific guanylate exchange factor LARG is a regulatory factor regulating the transformation of Rho family protein GDP binding form to GTP binding form. As a kind of Rho GEFs, LARG is reported to be involved in cell migration, contraction and mitosis by regulating the activity of Rho family proteins. However, at present, LARG is rarely involved in autophagy regulation. The role of lag in blood cancer and solid tumor depends on different signaling pathways. LARG, as a proto-Oncogene in acute myeloid leukemia, plays the role of tumor inhibitor in breast cancer and colorectal cancer. Therefore, LARG needs more in-depth study in solid tumors. In this paper, we will study the expression of LARG in lung cancer and its regulatory relationship with autophagy induced by nutritional deficiency. Methods: firstly, the expression of LARG in lung carcinoma was studied in vivo and in vitro. Six groups of lung cancer cell lines and HBEC cell lines were selected to detect the expression of LARG protein and transcription in vitro. At the same time, we also detected the expression of LARG in 30 clinical samples of lung cancer by immunohistochemistry. Then, the regulatory relationship between LARG and autophagy was studied by inducing autophagy by nutritional deficiency. We used HBSS to simulate the nutritional deficiency of tumor cells during the occurrence and development of tumor cells and combined with sh RNA interference technique and overexpression technique to judge the change of autophagy level by detecting autophagy markers. The small molecule GTPase protein of Rho family regulated by LARG was identified by GST Pull Down technique, and the relationship between LARG and autophagy was verified to be mediated by this protein. Finally, the proliferation and apoptosis of cancer cells under the condition of nutritional deficiency were detected to determine whether the survival ability of cancer cells was affected by LARG. Results: first of all, the expression of LARG in lung adenocarcinoma cell lines A549, H1299, H446 and H1993 was lower than that of HBEC, and the expression of H1993 was the least in H1993 cell lines from stage III lung carcinoma. In clinical samples, the expression of LARG in stage I and stage II lung cancer was significantly higher than that in stage III and stage IV. Secondly, after HBSS treatment, the cells with low expression of LARG showed more obvious autophagy phenotype than those with high expression, including the decrease of p62/SQSTM-1 accumulation. The increase of autophagy lysosomes. GST pull down results showed that LARG regulated the activity of Rac1 rather than Rho A in A549 and H1993 cells, and the effect of LARG on autophagy was realized by the change of Rac1 activity. Finally, LARG regulated the protein expression of p53 and p21CDKN1A through autophagy, which affected the apoptosis and proliferation of lung cancer cells under nutritional deficiency. Conclusion: the expression of LARG is gradually absent in lung cancer with the development of cancer. This deletion expression enhances the autophagy of lung adenocarcinoma cells under nutritional deficiency by reducing the activity of Rac1. Enhanced autophagy can inhibit apoptosis by degradation of p53 accumulation and up-regulation of p21CDKN1A to inhibit proliferation, and finally promote the survival of lung cancer cells under nutritional deficiency. The results show that LARG may play the role of tumor inhibitor gene through the restriction of autophagy in lung cancer, and it is not a molecular target for regulating Rho A activity.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R734.2

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本文編號：2494382