|THE MECHANISM OF NICKEL CHLORIDE EXPOSURE PROMOTING THE OCCURRENCE, INVASION, AND METASTASIS OF LUNG CANCER BY INFLAMMATORY PATHWAYS
|Place of Conferral
|氯化镍 NiCl2 暴露 exposure 肺癌 lung cancer 炎症通路 inflammatory pathways 发生 occurrence 侵袭转移 invasion and metastasis
方法：（1）利用National Health and Nutrition Examination Survey（NHANES）数据库和重金属职业暴露队列人群分析镍化合物暴露与肺癌患者患病和发病的关系，采用限制性三次样条法（Restricted cubic splines function，RCS）、logistic回归分析镍化合物暴露与肺癌患者患病和发病风险的剂量反应关系。构建体外细胞和体内动物氯化镍（Nickel Chloride，NiCl2）暴露模型，分析NiCl2暴露对肺癌的影响。（2）基于NHANES数据库，通过RCS和logistic回归分析镍化合物暴露与血小板计数/淋巴细胞计数（platelet lymphocyte ratio，PLR）和中性粒细胞/淋巴细胞比值（neutrophil lymphocyte ratio，NLR）的关系；利用GEO数据库分析镍化合物暴露样本中差异表达基因，并筛选出核心炎症因子白细胞介素6（interleukin 6，IL-6）；通过KM-plotter数据库分析IL-6与肺癌患者预后的关系；qRT-PCR验证NiCl2暴露对IL-6表达的影响；（3）通过KM-plotter数据库分析转录激活因子3（Signal transducer and activator of transcription 3，STAT3）与肺癌患者预后的关系；通过构建的体外细胞和体内动物NiCl2暴露模型，分析NiCl2暴露对IL-6和STAT3表达的影响以及对肺癌细胞增殖能力的影响。（4）通过生物信息学预测转录因子STAT3的靶基因三基序蛋白3（tripartite motif-containing protein 31，TRIM31）；通过收集到的队列数据，构建Cox回归模型分析TRIM31表达与肺癌患者预后的关系；利用细胞功能实验分析TRIM31表达对肺癌细胞侵袭和转移能力的影响；染色质免疫沉淀（Chromatin immunoprecipitation，ChIP）qPCR实验确定转录因子STAT3与TRIM31启动子结合位点；蛋白质免疫共沉淀（Co-immunoprecipitation，Co-IP）质谱实验鉴定E3泛素连接酶TRIM31结合的下游靶蛋白肿瘤抑制基因肿瘤蛋白p53（The tumor-suppressor gene tumor protein p53，TP53）；免疫组化和Western blot分析肺癌组织和细胞中TRIM31和TP53蛋白的表达情况，并分析其相关性；通过Co-IP实验、蛋白酶体抑制剂（proteasome inhibitor，MG132）、放线菌酮（Cycloheximide，CHX）、泛素化实验，证实E3泛素连接酶TRIM31对TP53蛋白的影响；通过功能回复实验，分析NiCl2暴露下STAT3、TRIM31、TP53上下游调控网络，以及对肺癌细胞侵袭和转移能力的影响。
（1）基于NHANES人群数据分析发现，镍化合物暴露增加肺癌患者的患病风险，其OR（95% CI）为1.85（0.79-4.34），且镍化合物暴露与肺癌患者的患病风险存在明显非线性的剂量反应关系（Poverall<0.05且Pnon liner<0.05）。（2）重金属职业暴露队列人群数据证实高剂量的镍化合物暴露增加肺癌的发病风险，其OR（95% CI）为1.12（0.73-11.72）。（3）体内动物NiCl2暴露模型结果显示，NiCl2暴露促进肺癌细胞生长，且随着暴露浓度增加，肺癌细胞生长能力越强（P<0.05）。（4）体外细胞NiCl2暴露模型结果显示，NiCl2暴露促进肺癌细胞的增殖、侵袭和转移能力。
（1）基于NHANES人群数据分析发现，镍化合物暴露会导致PLR和NLR等炎症指标异常升高，且他们之间存在剂量反应关系（P<0.05）。（2）基于GEO数据库分析发现，镍化合物暴露样本中炎症因子IL-6属于差异表达的核心基因，且其明显上调表达（LogFC=2.03，P<0.05）。（3）体外细胞NiCl2暴露模型的qRT-PCR结果显示，NiCl2暴露促进炎症因子IL-6上调表达（P<0.05），且随着NiCl2暴露浓度越高，IL-6表达水平越高。（4）KM-plotter数据库分析发现，高表达IL-6降低肺癌患者的总生存期（Overall Survival，OS）和无瘤进展期（Progression Free Survival，PFS），其HR（95% CI）分别为1.32（1.16-1.49）和1.27（1.05-1.54）。
（1）生物信息学显示炎症因子IL-6和STAT3存在相关性（P<0.05），且高表达STAT3降低肺癌患者的OS和PFS，其HR（95% CI）分别为1.11（0.94-1.31）和1.34（1.10-1.62）。（2）体外细胞NiCl2暴露模型的Western Blot结果显示，NiCl2暴露促进IL-6、p-JAK、p-STAT3蛋白上调表达（P<0.05）。（3）体内动物NiCl2暴露模型的Western Blot结果显示，NiCl2暴露同样会促进IL-6、p-JAK、p-STAT3蛋白上调表达。（4）体内动物NiCl2暴露模型的免疫组化实验结果显示，NiCl2暴露促进肺癌细胞的增殖能力。
（1）UCSC和JASPAR数据库分析发现TRIM31作为转录因子STAT3的下游靶基因，存在多个启动子结合位点。（2）TRIM31在肺癌细胞组织中高表达（P<0.05），单因素和多因素Cox回归分析发现高表达的TRIM31降低肺癌患者的OS，其HR（95% CI）分别为2.13（1.28-3.55）和1.75（1.03-2.96），TRIM31可以作为肺癌患者预后的独立危险因素。（3）划痕和Transwell实验结果显示，敲低TRIM31后降低肺癌细胞的侵袭和转移能力。（4）体内细胞和体外动物NiCl2暴露模型的免疫组化和Western Blot结果显示，NiCl2暴露促进TRIM31蛋白上调表达（P<0.05）。（5）ChIP-qPCR实验和体外细胞NiCl2暴露模型结果显示，STAT3与TRIM31启动子存在结合位点，且NiCl2暴露依赖STAT3磷酸化方式介导TRIM31的转录调控，促进TRIM31蛋白上调表达。（6）Co-IP质谱和Ubibrowser数据库分析发现TP53蛋白是E3泛素连接酶TRIM31的下游靶蛋白，存在直接结合；免疫荧光共定位发现TRIM31和TP53在胞质中存在共定位现象，且免疫组化和Western blot分析发现TRIM31和TP53蛋白存在负相关性（P<0.05）。（7）CHX、MG132、泛素化实验发现TRIM31在RING结合域与TP53结合，并泛素化降解TP53蛋白。（8）体内细胞和体外动物NiCl2暴露模型结果显示，NiCl2暴露通过STAT3/TRIM31轴下调TP53蛋白表达，并促进肺癌细胞侵袭和转移能力。
Objectives: As a common malignant tumor, lung cancer has many morbidities and mortality. The occurrence and development of lung cancer are closely related to environmental pollution and genetic changes. In recent years, studies have found that in addition to smoking, environmental pollution and occupational exposure are important risk factors for lung cancer. As a common occupational exposure factor, nickel compound exposure can easily lead to the occurrence of lung cancer, and can affect the body to produce related inflammatory reactions, but its specific mechanism is unknown. To explore the mechanism of lung cancer occurrence and development from the perspective of environmental exposure and molecular level, reveal the new mechanism of lung cancer occurrence and development caused by nickel compound exposure, and find the key genes regulating the occurrence and development of lung cancer, to provide theoretical basis for disease prevention and early intervention of high-risk population exposed to nickel compounds in metal mining and smelting industry.
Methods: (1) The National Health and Nutrition Examination Survey (NHANES) database and the heavy metal occupational exposure cohort were used to analyze the relationship between nickel compound exposure and the prevalence and incidence of lung cancer patients. Restricted cubic spline’s function (RCS) and logistic regression analysis were used to analyze the dose-response relationship between nickel compound exposure and the risk of lung cancer patients. Nickel chloride (NiCl2) exposure models in vitro and in vivo were constructed to analyze the effect of NiCl2 exposure on lung cancer. (2) Based on the NHANES database, the relationship between nickel compound exposure and platelet lymphocyte ratio (PLR) and neutrophil lymphocyte ratio (NLR) were analyzed by RCS and logistic regression. The differentially expressed genes in nickel compound exposed samples were analyzed by GEO database, and the core inflammatory factor interleukin 6 (IL-6) was screened out. The relationship between IL-6 and prognosis of lung cancer patients was analyzed by KM-plotter database. The effect of NiCl2 exposure on IL-6 expression was verified by qRT-PCR. (3) The relationship between signal transducer and activator of transcription 3 (STAT3) and prognosis of lung cancer patients was analyzed by KM-plotter database. The effects of NiCl2 exposure on the expression of IL-6 and STAT3 and the proliferation of lung cancer cells were analyzed by constructing an in vitro cell and in vivo animal NiCl2 exposure model. (4) Tripartite motif-containing protein 31 (TRIM31), the target gene of transcription factor STAT3, was predicted by bioinformatics. Through the collected cohort data, Cox regression model was constructed to analyze the relationship between TRIM31 expression and prognosis of lung cancer patients. The effect of TRIM31 expression on the invasion and metastasis of lung cancer cells was analyzed by cell function assay. Chromatin immunoprecipitation (ChIP) qPCR assay was used to determine the binding site of transcription factor STAT3 to TRIM31 promoter. The tumor-suppressor gene tumor protein p53 (TP53), a downstream target protein of E3 ubiquitin ligase TRIM31, was identified by Co-immunoprecipitation (Co-IP) mass spectrometry. Immunohistochemistry and Western blot were used to analyze the expression of TRIM31 and TP53 protein in lung cancer tissues and cells, and their correlation was analyzed. The effect of E3 ubiquitin ligase TRIM31 on TP53 protein was confirmed by Co-IP assay, proteasome inhibitor (MG132), cycloheximide (CHX) and ubiquitination assay. Through functional recovery experiments, the upstream and downstream regulatory networks of STAT3, TRIM31 and TP53 under NiCl2 exposure were analyzed, as well as their effects on the invasion and metastasis of lung cancer cells.
1. Exposure to nickel compounds promotes the occurrence of lung cancer.
(1) Based on the analysis of NHANES population data, we found that nickel compound exposure increased the risk of lung cancer patients, with an OR (95 % CI) of 1.85 (0.79-4.34), and there was a significant nonlinear dose-response relationship between nickel compound exposure and the risk of lung cancer patients (Poverall<0.05 and Pnon liner<0.05). (2) The data of heavy metal occupational exposure cohort confirmed that exposure to high doses of nickel compounds increased the risk of lung cancer, with an OR (95 % CI) of 1.12 (0.73-11.72). (3) The results of NiCl2 exposure model in vivo showed that NiCl2 exposure could promote the growth of lung cancer cells, and with the increase of exposure concentration, the growth ability of lung cancer cells was stronger (P<0.05). (4) The results of in vitro cell NiCl2 exposure model showed that NiCl2 exposure promoted the proliferation, invasion, and metastasis of lung cancer cells.
2. The abnormal high expression of inflammatory factors in lung cancer caused by nickel chloride exposure.
(1) Based on the analysis of NHANES population data, we found that nickel compound exposure could lead to abnormal elevation of inflammatory indicators such as PLR and NLR, and there was a dose-response relationship between them (P<0.05). (2) Based on GEO database analysis, we found that the inflammatory factor IL-6 in nickel compound exposure samples belonged to the core gene of differential expression, and its expression was significantly up-regulated (LogFC=2.03, P<0.05). (3) After constructing an in vitro cell NiCl2 exposure model, qRT-PCR results showed that NiCl2 exposure promoted the up-regulated expression of inflammatory factor IL-6 (P<0.05), and the higher the concentration of NiCl2 exposure, the higher the expression level of IL-6. (4) KM-plotter database analysis showed that high expression of IL-6 reduced the overall survival (OS) and progression free survival (PFS) of lung cancer patients, with HR (95 % CI) of 1.32 (1.16-1.49) and 1.27 (1.05-1.54), respectively.
3. Nickel chloride exposure promotes the growth of lung cancer cells via IL-6/STAT3 inflammatory pathway.
(1) Bioinformatics showed that there was a correlation between inflammatory factors IL-6 and STAT3 (P<0.05), and high expression of STAT3 reduced OS and PFS in patients with lung cancer, with HR (95 % CI) of 1.11 (0.94-1.31) and 1.34 (1.10-1.62), respectively. (2) After constructing an in vitro cell NiCl2 exposure model, Western Blot results showed that NiCl2 exposure promoted the up-regulated expression of IL-6, p-JAK and p-STAT3 proteins (P<0.05). (3) When the in vivo animal NiCl2 exposure model was constructed, Western Blot results showed that NiCl2 exposure also promoted the up-regulation of IL-6, p-JAK and p-STAT3 proteins. (4) The animal NiCl2 exposure model in vivo was constructed, and the results of immunohistochemistry showed that NiCl2 exposure promoted the proliferation of lung cancer cells.
4. Nickel chloride exposure promotes invasion and metastasis of lung cancer cells through STAT3/TRIM31 axis.
(1) UCSC and JASPAR database analysis showed that TRIM31, as a downstream target gene of transcription factor STAT3, had multiple promoter binding sites. (2) TRIM31 was highly expressed in lung cancer tissues (P<0.05). Univariate and multivariate Cox regression analysis showed that high expression of TRIM31 reduced the OS of lung cancer patients, with HR (95 % CI) of 2.13 (1.28-3.55) and 1.75 (1.03-2.96), respectively. TRIM31 can be used as an independent risk factor for the prognosis of lung cancer patients. (3) The results of wound healing and Transwell assays showed that knockdown of TRIM31 reduced the invasion and metastasis of lung cancer cells. (4) After constructing in vivo cell and in vitro animal NiCl2 exposure models, immunohistochemistry and Western Blot results showed that NiCl2 exposure promoted the up-regulation of TRIM31 protein expression (P<0.05). (5) The results of ChIP-qPCR experiment and in vitro cell NiCl2 exposure model showed that there was a binding site between STAT3 and TRIM31 promoter, and NiCl2 exposure mediated the transcriptional regulation of TRIM31 by STAT3 phosphorylation and promoted the up-regulation of TRIM31 protein. (6) Co-IP mass spectrometry and Ubibrowser database analysis showed that TP53 protein was the downstream target protein of E3 ubiquitin ligase TRIM31 and had direct binding. Immunofluorescence co-localization showed that TRIM31 and TP53 were co-localized in the cytoplasm, and immunohistochemistry and Western blot analysis showed a negative correlation between TRIM31 and TP53 proteins (P<0.05). (7) CHX, MG132 and ubiquitination experiments found that TRIM31 binds to TP53 in the RING binding domain and ubiquitinates and degrades TP53 protein. (8) The results of NiCl2 exposure model in vivo and in vitro showed that NiCl2 exposure down-regulated TP53 protein expression through the STAT3/TRIM31 axis and promoted the invasion and metastasis of lung cancer cells.
(1) Exposure to nickel compounds increased the risk of lung cancer, and there was a significant dose-response relationship between the concentration of nickel compounds and the risk of lung cancer. At the same time, NiCl2 exposure can promote the proliferation, invasion, and metastasis of lung cancer cells.
(2) Nickel compound exposure led to abnormal expression of inflammatory markers such as PLR and NLR, and there was a dose-response relationship between them. NiCl2 exposure increases the up-regulated expression of inflammatory factor IL-6, and the high expression of IL-6 reduces the prognosis of lung cancer patients.
(3) The high expression of STAT3 reduced the prognosis of patients with lung cancer. NiCl2 exposure promoted the up-regulated expression of IL-6, p-JAK and p-STAT3, and NiCl2 exposure promoted the proliferation of lung cancer cells through the IL-6/STAT3 inflammatory pathway.
(4) TRIM31 was highly expressed in lung cancer tissues, and high expression of TRIM31 is an independent risk factor for lung cancer patients. At the same time, high expression of TRIM31 promoted the invasion and metastasis of lung cancer cells. NiCl2 exposure mediated the transcriptional regulation of TRIM31 by phosphorylation of the transcription factor STAT3 and promotes the up-regulation of TRIM31 expression. NiCl2 exposure promoted the ubiquitination and degradation of TP53 protein in a STAT3/TRIM31 axis-dependent manner, and promoted the invasion and metastasis of lung cancer cells.
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|工学 - 环境科学与工程（可授工学、理学、农学学位） - 环境科学
|Yongbin Lu. 氯化镍暴露依赖炎症通路促进肺癌发生及侵袭转移的机制研究[D]. 兰州. 兰州大学,2023.
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