TRIM59和COMMD9促进非小细胞肺癌增殖和迁移的分子机制研究

发布时间：2018-05-04 21:49

本文选题：TRIM59 + p53　；参考：《南昌大学》2016年博士论文

【摘要】：第一部分TRIM59促进非小细胞肺癌增殖和迁移研究目的:TRIM家族是一类在炎症、免疫、抗病毒和癌症发生发展过程中有重要作用超家族蛋白。作为TRIM家族的一员,之前有文献报道TRIM59通过泛素化p53促进胃癌的发生发展,但是TRIM59在非小细胞肺癌(NSCLC)中的生理学功能和作用机制并不清楚。本课题通过QPCR方法,得到TRIM蛋白家族成员在非小细胞肺癌中的m RNA表达情况。进一步探讨TRIM59对非小细胞肺癌细胞生长、迁移和细胞周期的影响及其作用机制。研究方法:1、用QPCR方法筛选4株非小细胞肺癌细胞(H1299,A549,SPC-A1和H292)相对于正常人类支气管上皮细胞(HBE)中差异表达的TRIM基因。2、在蛋白水平上验证各株细胞中TRIM59表达情况,并且检测其在14种人类正常组织中的表达情况。3、在转染TRIM59特异性si RNA情况下,通过饱和密度实验、低血清实验、克隆集落形成实验、划痕实验、Transwell实验、流式细胞术和免疫荧光实验,检测干扰TRIM59表达对非小细胞肺癌细胞生长、迁移和细胞周期的影响。4、降低TRIM59表达水平,然后进行QPCR实验,免疫蛋白印迹实验,检测细胞周期蛋白和p53蛋白表达情况。研究结果:1、相比于HBE细胞,在4株NSCLC细胞中TRIM3、TRIM7、TRIM14、TRIM16、TRIM21、TRIM22、TRIM29、TRIM59、TRIM66和TRIM70高表达,TRIM4、TRIM9、TRIM36、TRIM46、TRIM54、TRIM67和TRIM76低表达。而TRIM17、TRIM40、TRIM42、TRIM43、TRIM48、TRIM49、TRIM50、TRIM51、TRIM60、TRIM63、TRIM64和TRIM77在NSCLC细胞和HBE细胞中未检测到。2、相比于HBE细胞,TRIM59蛋白在4株NSCLC细胞中高表达。TRIM59在骨髓和脾脏中高表达,在肺和肌肉中低表达,而在其它正常组织(脂肪、血液、直肠、心脏、肾脏、肝脏、淋巴结、皮肤、胃和睾丸)中未检测到。3、干扰TRIM59蛋白表达明显抑制NSCLC细胞生长、克隆集落形成、迁移,并且G2/M期细胞增多,PCNA蛋白表达降低。4、干扰TRIM59蛋白表达,细胞周期蛋白CDC2、CCNA2、CCNB1、CCNB2和CDC25C的m RNA水平降低,其中CDC2和CDC25C蛋白水平也降低,而CCNB1的蛋白水平没有变化。在4株NSCLC细胞和HBE细胞中干扰TRIM59表达,发现H1299细胞中未检测到p53表达,HBE、H292和A549细胞中p53表达没有改变,而在SPC-A1细胞中p53蛋白水平有所降低。研究结论:1、TRIM59在NSCLC细胞中高表达,并且促进NSCLC细胞生长、克隆集落形成和迁移。2、TRIM59促进NSCLC细胞从DNA合成后期进入细胞分裂期,并且上调细胞周期蛋白的表达。3、TRIM59并非通过p53信号通路来影响NSCLC细胞的生理学行为。第二部分COMMD9在非小细胞肺癌中与TFDP1结合促进TFDP1/E2F1转录活性研究目的:COMMD家族是在炎症、铜离子代谢、钠离子稳态、胞内分选和癌症中发挥重要作用的一类蛋白家族。文献报道COMMD9可能与TFDP1有相互作用,但是还未验证,并且COMMD9在癌症中的作用还未见报道。本课题的研究目的是探讨COMMD9对非小细胞肺癌生长、迁移、细胞周期和自噬的影响,检测COMMD9和TFDP1是否有相互作用及作用形式,并且是否对TFDP1/E2F1转录活性有影响。研究方法:1、用QPCR方法筛选4株非小细胞肺癌细胞(H1299,A549,SPC-A1和H292)相对于正常人类支气管上皮细胞(HBE)中COMMD家族差异表达基因。用免疫蛋白印迹(Western blot)实验检测COMMD9蛋白在NSCLC细胞和组织中的表达情况。2、用si RNA干扰COMMD9表达,通过结晶紫染色实验、克隆集落形成实验、软琼脂实验、划痕实验、Transwell实验、流式细胞术和Western blot实验,检测降低COMMD9表达对非小细胞肺癌细胞生长、迁移、细胞周期和自噬的影响。3、构建相关质粒,通过免疫共沉淀实验研究COMMD9和TFDP1相互作用及作用结构域。4、干扰COMMD9表达,采用荧光素酶报告基因、QPCR和Western blot方法,检测TFDP1/E2F1转录活性变化,以及p53信号通路的改变。研究结果:1、相比于HBE细胞,在4株NSCLC细胞中COMMD3、COMMD4、COMMD5和COMMD8的m RNA水平低表达,而COMMD9的m RNA和蛋白水平都高表达。NSCLC组织中COMMD9蛋白表达高于对应的癌旁组织。2、干扰COMMD9表达明显抑制NSCLC细胞生长、克隆集落形成、迁移,阻滞细胞G1期到S期的进程,并且促进自噬的发生。3、成功构建COMMD9和TFDP1全长以及截短的表达质粒,COMMD9可以和TFDP1发生相互作用。COMMD9的COMM结构域和TFDP1的DNA结合结构域是这两个蛋白相互作用的主要结合区域。4、干扰COMMD9表达抑制TFDP1/E2F1转录活性及下游基因的表达,并且p53蛋白水平上调,p53下游基因的表达也受到影响。研究结论:1、COMMD9在NSCLC中高表达,并且促进NSCLC细胞生长、克隆集落形成、迁移,促进细胞周期从DNA合成前期进入DNA合成期,并且抑制细胞自噬。2、COMMD9通过COMM结构域与TFDP1结合,并且TFDP1中DNA结合结构域对于这两个蛋白的相互作用是必需的。3、COMMD9促进TFDP1/E2F1转录活性,并且抑制p53信号通路。
[Abstract]:The first part TRIM59 promotes the proliferation and migration of non-small cell lung cancer: the TRIM family is an important class of superfamily proteins in the process of inflammation, immunity, antiviral and cancer development. As a member of the TRIM family, it has been reported that TRIM59 has promoted the development of gastric cancer by ubiquitination of p53, but TRIM59 is not small. The physiological function and mechanism of cell lung cancer (NSCLC) is not clear. This subject has obtained the m RNA expression of TRIM protein family in non-small cell lung cancer by means of QPCR method. The effect of TRIM59 on the growth, migration and cell cycle of non-small cell lung cancer and its mechanism of action are further discussed. 1, TRIM59 screening with QPCR method TRIM gene.2 differentially expressed in 4 non small cell lung cancer cells (H1299, A549, SPC-A1 and H292) compared with normal human bronchial epithelial cells (HBE). The expression of TRIM59 in each cell was verified at the protein level, and the expression of the TRIM was detected in the 14 normal human tissues, and the expression of TRIM59 was detected in the specific Si RNA of TRIM59. The supersaturation density experiment, the low serum experiment, the clone colony formation experiment, the scratch test, the Transwell experiment, the flow cytometry and the immunofluorescence test, detect the effect of interference TRIM59 expression on the growth, migration and cell cycle of non small cell lung cancer cells.4, reduce the expression level of TRIM59, and then carry out QPCR test, immunoblotting experiment and detection. Cell cycle protein and p53 protein expression. 1, compared to HBE cells, TRIM3, TRIM7, TRIM14, TRIM16, TRIM21, TRIM22, TRIM29, TRIM59, in the 4 NSCLC cells 0, TRIM63, TRIM64 and TRIM77 were not detected in NSCLC cells and HBE cells. Compared to HBE cells, TRIM59 protein expressed high expression in 4 strains of NSCLC cells in bone marrow and spleen, low expression in the lungs and muscles, but not in other normal tissues (fat, blood, straight intestine, heart, kidney, liver, lymph node, skin, stomach, and testicle) The expression of TRIM59 protein inhibited the growth of NSCLC cells, cloned colony formation, migration, and G2/M cell growth, PCNA protein expression decreased.4, interfered the expression of TRIM59 protein, cyclin CDC2, CCNA2, CCNB1, CCNB2 and CDC25C. The expression of TRIM59 was disturbed in 4 NSCLC cells and HBE cells, and the expression of p53 was not detected in the H1299 cells. The p53 expression in HBE, H292 and A549 cells was not changed, but the p53 protein level in the SPC-A1 cells decreased. The conclusion: 1, TRIM59 was highly expressed in the cells and promoted the colony formation. The formation and migration of.2, TRIM59 promotes NSCLC cells to enter the cell division phase from the late stage of DNA synthesis, and up regulation of the expression of cyclin.3, TRIM59 does not affect the physiological behavior of NSCLC cells through p53 signaling. Second part COMMD9 in non-small cell lung cancer and TFDP1 binding to promote TFDP1/E2F1 transcriptional activity research: COMMD home The family is a family of proteins that play an important role in inflammation, copper ion metabolism, sodium ion homeostasis, intracellular separation and cancer. The literature reports that COMMD9 may interact with TFDP1 but has not been verified, and the role of COMMD9 in cancer has not yet been reported. The purpose of this study is to explore the growth and migration of COMMD9 for non-small cell lung cancer. Migration, cell cycle and autophagy, whether COMMD9 and TFDP1 have interaction and action form, and whether it affects TFDP1/E2F1 transcriptional activity. 1, screening 4 non small cell lung cancer cells (H1299, A549, SPC-A1 and H292) with the QPCR method for the COMMD family difference table in normal human bronchial epithelial cells (HBE). The expression of COMMD9 protein in NSCLC cells and tissues was detected by immunoblotting (Western blot),.2 expression in NSCLC cells and tissues was detected by Si RNA, by crystal violet staining, cloned colony formation experiment, soft agar test, scratch test, Transwell experiment, flow cytometry and Western blot experiment to detect reduced COMMD9 table. The effect of.3 on the growth, migration, cell cycle and autophagy of non-small cell lung cancer cells, the construction of related plasmids, the interaction of COMMD9 and TFDP1 and the function domain.4, interfering COMMD9 expression by immunoprecipitation experiment, using luciferase reporter gene, QPCR and Western blot method to detect the change of TFDP1/E2F1 transcriptional activity and p53 letter. 1, compared to HBE cells, the levels of M RNA, COMMD4, COMMD5 and COMMD8 in 4 NSCLC cells were low, while COMMD9 m RNA and protein levels were highly expressed in.NSCLC tissues. Migrate, block the process of G1 phase to S phase, and promote the occurrence of autophagy by.3, successfully construct COMMD9 and TFDP1 full length and a truncated expression plasmid, COMMD9 can interact with TFDP1 in.COMMD9's COMM domain and TFDP1 DNA binding domain is the main binding region of the two proteins interacting with.4, interference expression Inhibition of TFDP1/E2F1 transcriptional activity and expression of downstream genes, and up regulation of p53 protein levels, and the expression of the downstream p53 genes are also affected. Conclusions: 1, COMMD9 is highly expressed in NSCLC, and promotes the growth of NSCLC cells, colony formation and migration, promotes cell cycle from the early stage of DNA synthesis into DNA synthesis, and inhibits autophagy. 2, COMMD9 is combined with TFDP1 through the COMM domain, and the interaction of DNA in TFDP1 domain for these two proteins is essential.3, COMMD9 promotes TFDP1/E2F1 transcriptional activity and inhibits p53 signaling pathways.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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