直肠癌炎症蛋白因子的遗传驱动：孟德尔随机化方法在临床预后研究中的应用

doi:10.12122/j.issn.1673-4254.2024.07.16

摘要/Abstract

摘要：

目的利用孟德尔随机化（MR）方法，探索炎症蛋白因子与直肠癌风险之间的因果关系，为直肠癌的预防和治疗提供新的策略。方法从全基因组关联分析（GWAS）数据集中选取与直肠癌相关的信息，使用91个炎症蛋白因子作为暴露变量。采用双样本MR分析模型评估炎症蛋白因子与直肠癌的因果联系，并通过异质性、多效性和敏感性分析评估结果的稳健性。使用5种MR分析模型，包括逆方差加权法（IVW）、加权中位数法（Weighted median）、模型选择法（MR-Egger）、简单模型法和加权模型法。选择2021年12月~2023年12月南方医科大学南方医院收治的86 例未经治疗的直肠腺癌患者开展临床研究，使用RT-qPCR技术定量分析程序性死亡配体1（PD-L1）、轴突诱导蛋白1（AXIN1）和β-神经生长因子（β-NGF）基因表达，使用Spearman秩相关系数分析基因表达与临床特征（包括吸烟情况、肿瘤最大直径、是否有转移、肿瘤-淋巴结-转移分期、分化程度和病理分型）之间的相关性。结果 MR分析显示AXIN1（OR=0.866，95% CI：0.754-0.994，P=0.040；IVW模型）、β-NGF（OR=0.914，95% CI：0.843-0.990，P=0.028；IVW模型）（OR=0.884，95% CI：0.784-0.998，P=0.047，Weighted median模型）和PD-L1（OR=0.903，95% CI：0.824-0.989，P=0.028，IVW模型）风险降低有与直肠癌显著的因果关系。研究中不存在异质性（IVW法和MR Egger法检测的P>0.05），不存在多效性（P>0.05），模型稳定。敏感性分析显示，AXIN1、β-NGF和PD-L1的剩余单核苷酸多态性（SNP）效应值的均值分别为-0.1425、-0.0973和-0.1011，均与各自的主效应值-0.144、-0.1和-0.1接近，验证了结果的可靠性。临床研究显示，PD-L1基因的表达量与直肠癌的TNM分期显著相关，尤其是在TNM分期IV期患者中，PD-L1的表达量显著高于I、II、III期（P=0.007）。AXIN1和β-NGF基因的表达量与肿瘤的分化程度显著相关；低分化的直肠癌样本中，AXIN1和β-NGF的表达量显著高于中分化和高分化样本（P<0.001）。结论炎症蛋白因子AXIN1、β-NGF和PD-L1的水平降低与直肠癌风险降低有显著的因果关系，且这些因子的表达量与直肠癌的TNM分期和肿瘤分化程度相关，可能是直肠癌治疗和预防的新靶点。

关键词: 直肠癌, 炎症蛋白因子, 孟德尔随机化, 临床研究

Abstract:

Objective To explore the causal relationship between inflammatory protein markers and the risk of colorectal cancer using a Mendelian randomization (MR) approach. Methods We obtained data pertaining to colorectal cancer from Genome-Wide Association Study (GWAS) datasets and used 91 inflammatory protein markers as the exposure variables. A two-sample MR analysis model was used to assess the causal link between the inflammatory markers and colorectal cancer risk. The robustness of the results was evaluated through heterogeneity, pleiotropy, and sensitivity analyses using 5 MR models: Inverse Variance Weighted (IVW), Weighted Median, MR Egger, Simple Mode, and Weighted Mode. We examined the mRNA expressions of PD-L1, AXIN1, and β-NGF using RT-qPCR in 86 untreated patients with colorectal adenocarcinoma admitted in Nanfang Hospital between December, 2021 and December 2023, and analyzed their correlation with the clinical characteristics of the patients. Results Using the IVW model, MR analysis revealed significant causal associations between a reduced risk of colorectal cancer and lowered expressions of AXIN1 (OR=0.866, 95% CI: 0.754-0.994, P=0.040), β-NGF (OR=0.914, 95% CI: 0.843-0.990, P=0.028; OR=0.884, 95% CI: 0.784-0.998, P=0.047 using Weighted Median model), and PD-L1 (OR=0.903, 95% CI: 0.824-0.989, P=0.028). No significant heterogeneity or pleiotropy was observed, indicating good stability of the results. Sensitivity analysis confirmed the reliability of the findings. The clinical study demonstrated a significant correlation between PD-L1 expression and TNM staging, particularly in stage IV patients (P=0.007). AXIN1 and β-NGF expression levels were significantly correlated with the degree of tumor differentiation, and their expressions were higher in poorly differentiated samples (P<0.001). Conclusion Lowered expressions of inflammatory protein markers AXIN1, β-NGF, and PD-L1 are causally correlated with a reduced risk of colorectal cancer and their expression levels are associated with TNM staging and tumor differentiation. These markers may thus serve as potential targets for colorectal cancer treatment and prevention.

Key words: colorectal cancer, inflammatory protein markers, Mendelian randomization, clinical study

李和平, 李高桦, 张学华, 王亚楠. 直肠癌炎症蛋白因子的遗传驱动：孟德尔随机化方法在临床预后研究中的应用[J]. 南方医科大学学报, 2024, 44(7): 1361-1370.

Heping LI, Gaohua LI, Xuehua ZHANG, Yanan WANG. Genetic drivers for inflammatory protein markers in colorectal cancer: a Mendelian randomization approach to clinical prognosis study[J]. Journal of Southern Medical University, 2024, 44(7): 1361-1370.

图/表 10

参考文献 30

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Gene	Forward primer (5'-3')	Reverse primer (5'-3')
PD-L1	5'-TGGCATTTGCTGAACGCAT-3'	5'-TGCTTGTCCAGATGACTTCG-3'
AXIN1	5'-AGCCTGCTGTACTGCTGCTA-3'	5'-TGCAGAGTGAGCGTGTACTC-3'
β-NGF	5'-GCTACATCGAGGAGGCTGTT-3'	5'-CACAGTGTCCTCAGGTTTGG-3'
GAPDH	5'-ACCACAGTCCATGCCATCAC-3'	5'-TCCACCACCCTGTTGCTGTA-3'

Gene	Forward primer (5'-3')	Reverse primer (5'-3')
PD-L1	5'-TGGCATTTGCTGAACGCAT-3'	5'-TGCTTGTCCAGATGACTTCG-3'
AXIN1	5'-AGCCTGCTGTACTGCTGCTA-3'	5'-TGCAGAGTGAGCGTGTACTC-3'
β-NGF	5'-GCTACATCGAGGAGGCTGTT-3'	5'-CACAGTGTCCTCAGGTTTGG-3'
GAPDH	5'-ACCACAGTCCATGCCATCAC-3'	5'-TCCACCACCCTGTTGCTGTA-3'

3 inflammatory protein factors	MR model	P	OR	95% CI
AXIN1	Inverse variance weighted	0.040	0.866	0.754-0.994
	Weighted median	0.155	0.873	0.724-1.053
	MR Egger	0.126	0.720	0.502-1.035
	Simple mode	0.254	0.846	0.649-1.102
	Weighted mode	0.273	0.873	0.697-1.092
β-NGF	Inverse variance weighted	0.028	0.914	0.843-0.990
	Weighted median	0.047	0.884	0.784-0.998
	MR Egger	0.718	1.035	0.862-1.242
	Simple mode	0.126	0.840	0.677-1.041
	Weighted mode	0.080	0.857	0.726-1.010
PD-L1	Inverse variance weighted	0.028	0.903	0.824-0.989
	Weighted median	0.060	0.905	0.816-1.004
	MR Egger	0.706	0.957	0.765-1.198
	Simple mode	0.341	0.923	0.787-1.083
	Weighted mode	0.080	0.898	0.801-1.006

3 inflammatory protein factors	MR model	P	OR	95% CI
AXIN1	Inverse variance weighted	0.040	0.866	0.754-0.994
	Weighted median	0.155	0.873	0.724-1.053
	MR Egger	0.126	0.720	0.502-1.035
	Simple mode	0.254	0.846	0.649-1.102
	Weighted mode	0.273	0.873	0.697-1.092
β-NGF	Inverse variance weighted	0.028	0.914	0.843-0.990
	Weighted median	0.047	0.884	0.784-0.998
	MR Egger	0.718	1.035	0.862-1.242
	Simple mode	0.126	0.840	0.677-1.041
	Weighted mode	0.080	0.857	0.726-1.010
PD-L1	Inverse variance weighted	0.028	0.903	0.824-0.989
	Weighted median	0.060	0.905	0.816-1.004
	MR Egger	0.706	0.957	0.765-1.198
	Simple mode	0.341	0.923	0.787-1.083
	Weighted mode	0.080	0.898	0.801-1.006

88 inflammatory protein factors	P	OR	95% CI
Level of eukaryotic translation initiation factor 4e-binding protein 1 in blood plasma	0.330693955	1.05701024	0.945~1.182
Adenosine deaminase measurement	0.400744522	0.971110383	0.907~1.040
Artemin measurement	0.577886958	1.028293723	0.932~1.134
Caspase-8 measurement	0.893225803	1.01109646	0.861~1.188
Eotaxin measurement	0.418629293	1.049686291	0.933~1.181
C-c motif chemokine 19 measurement	0.138858991	0.953521049	0.895~1.016
C-c motif chemokine 20 measurement	0.58707287	1.027424201	0.932~1.133
C-c motif chemokine 23 measurement	0.193446913	1.0380569	0.981~1.098
C-c motif chemokine 25 measurement	0.276211279	1.024595882	0.981~1.070
C-c motif chemokine 28 measurement	0.155521941	1.077788608	0.972~1.195
C-c motif chemokine 4-like measurement	0.104239836	0.949281216	0.892~1.011
Natural killer cell receptor 2b4 measurement	0.883114482	1.006982684	0.918~1.105
Cd40 measurement	0.228753652	1.032385457	0.980~1.087
T-cell surface glycoprotein cd5 measurement	0.365387247	0.943265519	0.831~1.070
Level of t-cell differentiation antigen cd6 in blood plasma	0.100531596	0.951163531	0.896~1.010
Cub domain-containing protein 1 measurement	0.832570494	0.988924375	0.892~1.096
Macrophage colony-stimulating factor 1 measurement	0.506667178	0.959861227	0.851~1.083
Cystatin-d measurement	0.72428164	1.008161078	0.964~1.055
Fractalkine measurement	0.462560826	1.040793854	0.935~1.158
Cxcl1 measurement	0.894677494	1.00525697	0.930~1.086
C-x-c motif chemokine 10 measurement	0.771709442	0.982307712	0.871~1.108
C-x-c motif chemokine 11 measurement	0.575284323	1.033187503	0.922~1.158
C-x-c motif chemokine 5 measurement	0.799499229	0.988392831	0.903~1.082
C-x-c motif chemokine 6 measurement	0.911547109	0.989014351	0.814~1.202
C-x-c motif chemokine 9 measurement	0.228872998	0.930704757	0.828~1.046
Delta and notch-like epidermal growth factor-related receptor measurement	0.940378359	0.996546078	0.910~1.091
Protein s100-a12 measurement	0.408502837	1.040250552	0.947~1.142
Fibroblast growth factor 19 measurement	0.188440681	0.917624127	0.807~1.043
Fibroblast growth factor 21 measurement	0.090121312	0.912044138	0.820~1.015
Fibroblast growth factor 23 measurement	0.100383968	1.100299985	0.982~1.233
Fibroblast growth factor 5 measurement	0.996902105	1.00008661	0.957~1.045
Fms-related tyrosine kinase 3 ligand measurement	0.533761185	1.029291159	0.940~1.127
Glial cell line-derived neurotrophic factor measurement	0.407072647	1.044508504	0.942~1.158
Hepatocyte growth factor measurement	0.262585949	0.944119121	0.854~1.044
Interferon gamma measurement	0.237368214	1.110258305	0.933~1.321
Interleukin-10 measurement	0.116811526	0.911618611	0.812~1.023
Interleukin-10 receptor subunit alpha measurement	0.748459744	1.014930285	0.927~1.111
Interleukin-10 receptor subunit beta measurement	0.636886216	1.017501244	0.947~1.094
Interleukin-12 subunit b measurement	0.985645686	0.999389385	0.935~1.068
Interleukin-13 measurement	0.459701443	1.057809342	0.911~1.228
Interleukin-15 receptor subunit alpha measurement	0.640753925	1.021046126	0.936~1.114
Interleukin-17a measurement	0.849139422	0.989209252	0.885~1.106
Interleukin-17c measurement	0.881546189	0.991402959	0.885~1.111
Interleukin 18 measurement	0.558552042	0.97218849	0.885~1.069
Interleukin-18 receptor 1 measurement	0.090188729	0.95511571	0.906~1.007
Interleukin-1 alpha measurement	0.677978683	0.984846788	0.916~1.058
Interleukin-2 measurement	0.07740399	0.917987666	0.835~1.009
Interleukin-20 measurement	0.291150659	1.093808053	0.926~1.292
Interleukin-20 receptor subunit alpha measurement	0.597326832	1.034418796	0.912~1.173
Interleukin-22 receptor subunit alpha-1 measurement	0.286716746	0.953002592	0.872~1.041
Interleukin-24 measurement	0.521908621	0.938998028	0.774~1.138
Interleukin-2 receptor subunit beta measurement	0.905772546	1.010513096	0.850~1.202
Level of interleukin-33 in blood plasma	0.867584002	0.987423578	0.851~1.146
Interleukin-4 measurement	0.747455619	0.978873314	0.860~1.115
Interleukin-5 measurement	0.711859457	1.024422635	0.901~1.164
Interleukin-6 measurement	0.775696404	1.015280674	0.915~1.127
Interleukin-7 measurement	0.716962035	1.025544025	0.895~1.175
Interleukin-8 measurement	0.752783452	1.015097112	0.925~1.114
Transforming growth factor beta-1 measurement	0.274065715	1.058249902	0.956~1.171
Leukemia inhibitory factor measurement	0.206989335	1.076015903	0.960~1.206
Leukemia inhibitory factor receptor measurement	0.332957247	1.045419336	0.956~1.144
Ccl2 measurement	0.202036076	1.044503498	0.977~1.117
Monocyte chemotactic protein-2 measurement	0.265061104	1.021836151	0.984~1.061
Monocyte chemotactic protein 3 measurement	0.237594093	1.065347928	0.959~1.183
Monocyte chemotactic protein-4 measurement	0.503878948	1.019953512	0.963~1.081
Macrophage inflammatory protein 1a measurement	0.914789496	0.994719076	0.903~1.096
Matrix metalloproteinase 1 measurement	0.691649178	0.981202618	0.893~1.078
Matrix metalloproteinase 10 measurement	0.279953505	1.051622418	0.960~1.152
Level of neurturin in blood plasma	0.304288769	0.936474867	0.826~1.061
Neurotrophin-3 measurement	0.457455683	1.042340882	0.934~1.163
Osteoprotegerin measurement	0.077956745	1.07367806	0.992~1.162
Oncostatin-m measurement	0.382798408	0.940220439	0.819~1.080
Stem cell factor measurement	0.774417755	0.988552924	0.914~1.070
Sir2-like protein 2 measurement	0.628422372	0.96738532	0.846~1.106
Signaling lymphocytic activation molecule measurement	0.991033477	1.000583468	0.904~1.108
Sulfotrasferase 1a1 measurement	0.214016095	0.958306538	0.896~1.025
Stam binding protein measurement	0.746256217	1.020153145	0.904~1.151
Transforming growth factor-alpha measurement	0.605271992	0.95352472	0.796~1.142
Tumor necrosis factor measurement	0.179961864	1.078953805	0.966~1.206
Lymphotoxin-alpha measurement	0.244034995	1.026859418	0.982~1.074
Tumor necrosis factor receptor superfamily member 9 measurement	0.962790331	1.002600075	0.899~1.118
Tumor necrosis factor ligand superfamily member 14 measurement	0.850366904	0.994105748	0.935~1.057
Tnf-related apoptosis-inducing ligand measurement	0.148528335	0.931451642	0.846~1.026
Tnf-related activation-induced cytokine measurement	0.575971297	1.020575767	0.950~1.096
Thymic stromal lymphopoietin measurement	0.845461662	0.985914611	0.855~1.137
Tumor necrosis factor ligand superfamily member 12 measurement	0.597249576	1.019992959	0.948~1.098
Urokinase-type plasminogen activator measurement	0.559931482	0.974045442	0.892~1.064
Vascular endothelial growth factor a measurement	0.346474973	0.97745845	0.932~1.025