LARG通过Rac1依赖途径限制饥饿诱导的肺腺癌自噬
发布时间：2019-06-06 13:02
【摘要】：背景:Rho家族蛋白特异性鸟苷酸交换因子LARG是调节Rho家族蛋白GDP结合形式向GTP结合形式转化的调节因子。作为Rho GEFs的一种,LARG被报道主要通过调节Rho家族蛋白活性来参与细胞移动,收缩以及有丝分裂,但是目前很少有LARG参与自噬调控的研究。LARG在血癌和实体肿瘤当中所扮演的角色是依赖于不同信号通路的。在急性髓样细胞白血病中作为原癌基因的LARG在乳腺癌和大肠癌中扮演的却是抑癌基因的角色。因此,LARG在实体肿瘤当中需要更深入的研究。在本文中,我们将就LARG在肺腺癌中的表达以及它与营养缺乏诱导的自噬是否存在调控关系进行研究。方法:首先,通过体内和体外实验研究LARG在肺腺癌中的表达。我们选用了6组肺癌细胞系和HBEC细胞系来检测LARG在体外细胞系中蛋白水平和转录水平的表达。同时,我们还通过免疫组化检测了30例肺腺癌临床样本中LARG的表达。接着,通过营养缺乏诱导细胞自噬研究LARG与自噬之间是否存在调控关系。我们利用HBSS处理细胞一定时间来模拟肿瘤细胞在发生发展过程中遇到的营养缺乏条件并配合sh RNA干扰技术和超表达技术,通过检测自噬标志物来判断自噬水平的变化。利用GST Pull Down技术找出受LARG调控的Rho家族小分子GTPase蛋白,验证LARG与自噬的关联是否由该蛋白介导。最后,检测营养缺乏条件下癌细胞的增殖能力和细胞凋亡来判断癌细胞的生存能力是否受LARG的影响。结果:首先,LARG在肺腺癌细胞系A549、H1299、H446和H1993中蛋白水平和转录水平的表达均低于HBEC,其中在来源于三期肺腺癌的H1993细胞系中表达量最少。在临床样本方面,LARG在一期、二期肺腺癌中的表达量要明显高于三期、四期。其次,通过HBSS处理后,LARG表达量低的细胞比表达量高的细胞表现出更明显的自噬表型,包括p62/SQSTM-1积累的减少、自噬溶酶体的增加。GST pull down的结果显示在A549和H1993细胞中LARG调控的是Rac1的活性而不是Rho A,并且LARG对自噬的影响就是通过Rac1活性的变化来实现的。最后,LARG通过自噬调控着p53和p21CDKN1A的蛋白水平表达量,进而影响了营养缺乏状态下肺腺癌细胞的凋亡和增殖能力。结论:LARG在肺腺癌中伴随着癌症的发展是逐渐缺失表达的,这种缺失表达通过降低Rac1的活性增强了营养缺乏状态下肺腺癌细胞的自噬。增强的自噬通过降解p53的积累抑制凋亡的同时通过上调p21CDKN1A抑制增殖,最终促进了肺腺癌细胞在营养缺乏状态下的生存。全文结果告诉我们,在肺腺癌中LARG很可能通过对自噬的限制作用起到了抑癌基因的角色,并且它不是调控Rho A活性的分子靶点。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
本文编号：2494382
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
【参考文献】
相关期刊论文 前1条
1 李慧;;肺癌的治疗现状及研究新进展[J];实用中西医结合临床;2015年05期
，本文编号：2494382
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