摘要

目的 2型糖尿病（Type 2 Diabetes Mellitus, T2DM）作为最常见的代谢性疾病，全球患病人数已超过5亿，并预计在未来数十年内持续增长。随着人口老龄化和生活方式的改变，T2DM和肺癌（Lung Cancer, LC）的共病发病率持续上升。肺癌作为最常见的恶性肿瘤之一，是全球癌症相关死亡的主要原因，其中肺腺癌（Lung Adenocarcinoma, LUAD）为最常见的病理类型。目前对于T2DM与LUAD共病的潜在分子机制尚未明确。

本研究旨在通过生物信息学分析结合细胞实验及动物模型验证，探讨T2DM与LUAD共病现象，研究细胞色素P450家族成员CYP1B1在其中的潜在作用与调控机制，从而为T2DM与LUAD共病研究提供数据支撑与干预靶点。

方法 基于公共数据库GEO和UK Biobank中T2DM与LUAD的转录组与基因组测序数据，采用主成分分析（Principal Component Analysis，PCA）、差异表达分析（Differential Expression Genes Analysis，DEGA）、基于加权网络的基因共表达分析（Weighted Gene Co-expression Network Analysis，WGCNA）、GO富集（Gene Ontology，GO）、KEGG富集分析（Kyoto Encyclopedia of Genes and Genomes Enrichment Analysis，KEGG）及孟德尔随机化（Mendelian Randomization，MR）方法，筛选并验证了T2DM与LUAD共病的关键候选基因CYP1B1。通过Kaplan-Meier Plotter、HPA和TIMER 2.0等在线数据库对CYP1B1的表达及预后意义进行验证。

通过构建CYP1B1敲低的人源肺腺癌细胞A549，利用实时荧光定量（Real-Time Quantitative Polymerase Chain Reaction，RT-qPCR）和蛋白免疫印记（Western Blot，WB）进行敲低效率验证，并在高糖培养条件下进行细胞增殖实验（克隆形成实验）、迁移实验（划痕愈合实验、Transwell迁移实验）和侵袭实验（Transwell侵袭实验）进行细胞层面验证。

通过构建T2DM-LUAD共病裸鼠模型，探究CYP1B1在不同表达水平及不同血糖状态下对皮下移植瘤生长的影响，从而在体内实验水平验证CYP1B1基因在T2DM-LUAD共病中的潜在作用。

结果

PCA结果显示，T2DM患者与健康对照、LUAD患者与非肿瘤肺组织在转录组水平上均表现出显著的表达差异。在T2DM与健康对照之间共筛选出5181个表达基因，其中CYP1B1、IKBKG、MAPK13、PRKCD、MAPK14、PIK3R3等基因在T2DM中显著上调。在LUAD与非肿瘤肺组织之间筛选出2158个差异表达基因，其中CYP1B1、PRKCD、MAPK13、MAPK14、IKBKG、PIK3R3等基因在LUAD中显著上调。  

GO和KEGG富集分析显示，T2DM差异基因显著富集于胰岛素信号通路、NF-κB信号通路、氧化应激信号通路等与T2DM密切相关的生物学过程。通过WGCNA分析，筛选出与T2DM高度相关的基因模块，包含7602个基因，与DEGs结果取交集后得到1466个差异表达基因。

MR分析结果显示，T2DM与LUAD之间存在显著正向因果关系，并筛选出核心基因CYP1B1；在线数据库验证结果表明，CYP1B1高表达导致LUAD患者预后不佳。

CYP1B1在A549细胞中高表达。CYP1B1敲低显著抑制高糖环境下A549细胞的增殖、迁移和侵袭能力。

T2DM裸鼠模型空腹血糖值均大于11.1mmol/L。裸鼠在T2DM状态下，注射A549细胞（shControl）的肿瘤生长速度、体积及重量显著高于注射敲低CYP1B1的A549细胞（shCYP1B1）组。在注射A549细胞的情况下，诱导T2DM的裸鼠组的肿瘤生长速度、体积及重量均高于非T2DM组。

结论 通过转录组数据基因差异表达分析、WGCNA及MR，首次筛选出T2DM与LUAD共病的关键基因CYP1B1，并利用Kaplan-Meier Plotter、HPA、TIMER 2.0等在线数据库验证该基因的关键作用。通过差异基因GO和KEGG富集分析，首次揭示CYP1B1在氧化应激信号通路、胰岛素信号通路、NF-κB信号通路、细胞周期调控等生物学过程中的重要作用。细胞功能实验显示，CYP1B1在A549细胞中高表达，通过shRNA慢病毒成功敲低CYP1B1基因后，显著抑制了高糖环境下A549细胞的增殖、迁移和侵袭能力。动物实验结果进一步证实，在裸鼠体内高糖环境下，CYP1B1表达能够显著促进肺腺癌细胞增殖，而基因敲低后可有效抑制肿瘤生长。本研究首次从转录组学生物信息学分析、细胞与动物实验验证多层次视角，证实了CYP1B1在高糖环境下通过激活氧化应激信号通路，调控肺癌细胞增殖、迁移和侵袭，促进肺腺癌的发生发展。

Abstract

Objective

Type 2 Diabetes Mellitus (T2DM), as the most common metabolic disease, has affected more than 500 million people worldwide, and its prevalence is expected to continue increasing in the coming decades. With population aging and changes in lifestyle, the comorbidity rates of T2DM and Lung Cancer (LC) are rising. Lung cancer, as one of the most common malignant tumors, remains the leading cause of cancer-related deaths globally, with Lung Adenocarcinoma (LUAD) being the most prevalent pathological subtype. However, the underlying molecular mechanisms of T2DM and LUAD comorbidity remain unclear.

This study aims to investigate the comorbidity phenomenon of T2DM and LUAD through bioinformatics analysis combined with cellular and animal model experiments. Specifically, it focuses on exploring the potential role and regulatory mechanisms of the cytochrome P450 family member CYP1B1 in the comorbidity process of these two diseases, providing data support and potential intervention targets for T2DM and LUAD comorbidity research.

Methods

Based on RNA sequencing data related to T2DM and LUAD collected from public databases such as GEO and the UK Biobank, key analyses including Principal Component Analysis (PCA), Differential Expression Genes Analysis (DEGA), Weighted Gene Co-expression Network Analysis (WGCNA), Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and Mendelian Randomization (MR) were conducted to screen and validate the key candidate gene CYP1B1 involved in the comorbidity of T2DM and LUAD. The expression and prognostic significance of CYP1B1 were systematically validated using online databases such as Kaplan-Meier Plotter, the Human Protein Atlas (HPA), and TIMER 2.0.

By constructing LUAD cell models (A549) with CYP1B1 knockdown, the knockdown efficiency was validated using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and Western Blot (WB). Under high-glucose culture conditions, cell function assays—including cell proliferation assays (colony formation assays), migration assays (wound healing and Transwell migration assays), and invasion assays (Transwell invasion assays)—were performed to investigate the functional role of CYP1B1 at the cellular level.

In addition, a T2DM-LUAD comorbidity mouse model was established to compare the growth of subcutaneous xenograft tumors under different CYP1B1 expression levels and varying blood glucose conditions.

Results

PCA results showed that there were significant transcriptomic differences between T2DM patients and healthy controls, as well as between LUAD patients and non-tumor lung tissues. A large number of differentially expressed genes (DEGs) were identified between T2DM patients and healthy controls, among which CYP1B1, IKBKG, MAPK13, PRKCD, MAPK14, and PIK3R3 were significantly upregulated in T2DM. Similarly, many DEGs were found between LUAD and non-tumor lung tissues, with CYP1B1, PRKCD, MAPK13, MAPK14, IKBKG, and PIK3R3 significantly upregulated in LUAD.

GO and KEGG enrichment analyses revealed that DEGs in T2DM were significantly enriched in biological processes closely related to T2DM, including the insulin signaling pathway, NF-κB signaling pathway, and oxidative stress response. Through WGCNA analysis, a gene module highly associated with T2DM was identified, containing 7,602 genes. Following intersection with differentially expressed genes (DEGs), 1,466 overlapping genes were obtained. Intersecting these modules with DEGs resulted in 1,466 differentially expressed genes.

MR analysis demonstrated a significant causal relationship between T2DM and LUAD and identified CYP1B1 as a core gene. Validation using online databases showed that high expression of CYP1B1 was associated with poor prognosis in LUAD patients.

CYP1B1 was highly expressed in A549 cells. Knockdown of CYP1B1 significantly inhibited the proliferation and migration of A549 cells under high-glucose conditions.

In the T2DM nude mouse model, fasting blood glucose levels were all greater than 11.1 mmol/L. When A549 cells were injected, the experimental group exhibited faster tumor growth, larger tumor volume, and heavier tumor weight compared to the control group. Under T2DM conditions, the tumor growth rate, volume, and weight of Experimental Group 1 were significantly higher than those of Experimental Group 2.

Conclusion

Through transcriptomic analysis using DEGA, WGCNA, and MR, CYP1B1 was identified as a core gene associated with the comorbidity of T2DM and LUAD. Its expression and prognostic value were validated using online databases such as Kaplan-Meier Plotter, HPA, and TIMER 2.0. GO and KEGG enrichment analyses of differentially expressed genes revealed that CYP1B1 plays a key role in several biological processes, including oxidative stress signaling, insulin signaling, NF-κB signaling, and cell cycle regulation. CYP1B1 was found to be highly expressed in A549 cells, and its expression was successfully knocked down via shRNA lentiviral transduction. This knockdown significantly suppressed the proliferation, migration, and invasion of A549 cells under high-glucose conditions. The animal experiment results further confirmed that under high-glucose conditions in nude mice, high expression of CYP1B1 significantly promoted the proliferation of lung adenocarcinoma cells, while knockdown of the gene effectively suppressed tumor growth. This study, for the first time, validated from a multi-level perspective: including transcriptomic bioinformatics analysis, cellular experiments, and animal models. CYP1B1 promotes the development and progression of lung adenocarcinoma in high-glucose environments by regulating cell proliferation, migration, and invasion-related signaling pathways.