GEMIN6调控NSCLC增殖、迁移及自噬相关机制的初步研究
发布时间：2018-09-16 22:02
【摘要】：[目的]非小细胞肺癌(NSCLC)全球高发,为癌性死亡的首位原因,其驱动机制至今未阐明,给诊治带来诸多挑战。云南省为肺癌高发大省,亟待探索NSCLC特异性的驱动机制。剪接体功能异常可致下游基因差异剪接,进而参与肿瘤的发生。作为剪接体组装的关键复合物,SMN复合物的众多组分逐渐成为癌症的诊治靶点。但是GEMIN6基因编码蛋白作为SMN复合物的重要组分,其在NSCLC中发挥的角色仍是未解之谜。目前,SMN复合物的其他组分被证实参与癌细胞增殖等恶性生物学行为,甚至可调节自噬活性。与此同时,中国传统药物在NSCLC自噬活性的调节中逐渐崭露头角,日渐成为NSCLC用药的研究热点。因此,本研究拟从细胞及组织水平探讨GEMIN6与NSCLC恶性生物学行为的相关性,通过检测细胞增殖能力、迁移活性、周期进程以及自噬标志蛋白,旨在获得GEMIN6参与NSCLC增殖和迁移等恶性生物学行为的实验学证据,进一步探讨黄连素等传统中药单体是否可通过GEMIN6调控NSCLC的恶性进程,为云南地区NSCLC的诊治提供干预新靶点和治疗新思路。[方法]采用Westernblotting技术检测NSCLC细胞系中GEMIN6的基础表达量;利用siRNA敲减A549和H1975细胞系中GEMIN6的表达,Westernblotting技术验证敲减;通过MTS细胞增殖实验、Transwell实验、流式细胞术检测GEMIN6对NSCLC细胞生长的影响;采用Westernblotting技术验证自噬标记蛋白Beclinl和LC3-Ⅰ/Ⅱ的表达水平改变;通过MTS法检测黄连素对A549细胞的半数抑制浓度;结合实验室前期研究结果,采用Westernblotting技术验证4类中药单体作用后A549细胞中GEMIN6及自噬标记蛋白Beclinl的表达水平改变;通过免疫组织化学方法和HE染色方法检测NSCLC的癌组织和良性对照组织中GEMIN6的表达水平,运用SPSS软件分析GENMIN6与NSCLC的临床相关性。[结果]1. Kaplan-Meier plotter生存曲线分析表明高表达GEMIN6的肺癌患者生存期较对照组低2. GEMIN6在NSCLC癌组织中表达高于对照组,但临床相关性不大3. GEMIN6增强NSCLC的增殖和迁移能力4. GEMIN6促进NSCLC发生G1-S期转变5. GEMIN6抑制NSCLC的自噬活性6.在A549细胞中,黄连素和姜黄素可升高GEMIN6以及Beclinl的表达;双氢青蒿素降低GEMIN6的表达并且轻度升高Beclinl的表达;红景天苷升高GEMIN6表达但对Beclinl的表达无影响。双氢青蒿素可能通过GEMIN6发挥其抑癌活性,但另外3种单体均非通过GEMIN6调控A549细胞的生长。[结论]在NSCLC中特异性高表达的GEMIN6通过促进G1/S期过渡,升高A549和H1975细胞的恶性增殖能力和迁移能力,同时抑制A549细胞的自噬活性;双氢青蒿素在下调A549细胞中GMEIN6表达的同时可轻度上调自噬水平,GEMIN6可能为其下游作用靶标;黄连素、姜黄素以及红景天苷可在不同程度上改变A549细胞中GEMIN6的表达,但无具体证据表明它们对NSCLC自噬活性的调控与GEMIN6表达水平改变相关。
[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，
本文编号：2244956
[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，
本文编号：2244956
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