Objectives: To explore a causal relationship between ferroptosis-related gene heat shock protein A5 (HSPA5) and hepatocellular carcinoma (HCC).
Methods: A two-sample Mendelian randomization (MR) design was employed to evaluate the causal relationships among HSPA5, regulatory T cells (Tregs), and HCC. Single nucleotide polymorphisms (SNPs) associated with HSPA5, Tregs and HCC were selected as instrumental variables through publicly available genome-wide association studies (GWAS) databases. MR analysis was used to assess the direct effect of HSPA5 on HCC, followed by two-step MR to analyze the potential mediating role of Tregs. Reverse MR analysis was conducted with HCC as the exposure and HSPA5 as the outcome. Inverse variance weighting was the primary method for testing causal associations in all MR analyses. Robustness of the results was confirmed through MR-Egger, weighted median, weighted mode, and simple mode methods. Heterogeneity of instrumental variables was evaluated using Cochrane's Q statistic, while pleiotropy was tested by MR-Egger intercept and MR-PRESSO, with leave-one-out sensitivity analysis performed for robustness. Data from The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) were utilized to verify the expression levels of HSPA5 in HCC tissues and its correlation with Tregs to reveal the interaction mechanisms between HSPA5 and Tregs in HCC progression and their relationship with patient prognosis.
Results: MR analysis showed a positive correlation between elevated HSPA5 expression and HCC risk (all P<0.01), while reverse MR analysis found no statistically significant association between HCC and HSPA5 (P>0.05). HSPA5 expression was significantly correlated with Tregs function (all P<0.05), and the enrichment of Tregs in HCC microenvironment was positively associated with HCC progression (all P<0.05). Mediation analysis indicated that Tregs accounted for 5.00% and 7.45% of the mediation effect between HSPA5 and HCC. TCGA and HPA database analysis revealed that both HSPA5 mRNA and protein expression levels were higher in HCC tissues compared to normal tissues, and high HSPA5 expression was significantly associated with poor prognosis. Immune infiltration analysis confirmed a significant positive correlation between HSPA5 and Tregs, with high Tregs infiltration closely related to HCC progression.
Conclusions: Elevated HSPA5 expression is significantly associated with HCC development and poor prognosis. HSPA5 may promote HCC progression by regulating the function of Tregs in the tumor microenvironment.
目的: 探讨铁死亡相关基因热休克蛋白A5（HSPA5）与肝癌发展的因果关系及其作用机制。方法: 采用两步两样本孟德尔随机化（MR）设计评估HSPA5、调节性T细胞及肝癌之间的因果关系。首先通过公开的全基因组关联研究（GWAS）数据库筛选与HSPA5、调节性T细胞、肝癌相关的单核苷酸多态性作为工具变量，采用MR分析HSPA5对肝癌的直接影响，接着通过两步MR分析调节性T细胞在这一过程中可能的中介作用。同时，以肝癌为暴露因素，HSPA5为结局变量进行了反向MR分析。所有MR分析均以逆方差加权法作为主要的因果关联检验方法，通过MR-Egger、加权中值法、加权众数法和简单众数法验证结果的稳健性，并采用Cochrane’s Q统计量评估工具变量的异质性，MR-Egger回归的截距项和MR-PRESSO进行多效性检验，留一法进行敏感性分析。利用癌症基因组图谱（TCGA）数据库和人类蛋白质图谱（HPA）数据库验证HSPA5在肝癌组织中的表达水平及其与调节性T细胞的相关性，揭示HSPA5与调节性T细胞在肝癌进展中的相互作用机制及其与患者预后的关系。结果: MR分析结果显示，HSPA5表达增加与肝癌风险呈正相关（均P<0.01），而反向MR分析未发现肝癌与HSPA5之间存在关联（P>0.05）。HSPA5表达与调节性T细胞功能显著相关（均P<0.05），且调节性T细胞在肝癌微环境中的富集与肝癌进展呈正相关（均P<0.05）。中介效应分析结果显示，调节性T细胞在HSPA5和肝癌间的中介效应分别占5.00%和7.45%。TCGA和HPA数据库分析显示，肝癌组织中HSPA5的mRNA和蛋白质表达水平均高于正常组织，且高表达与患者不良预后显著相关。免疫浸润分析验证了HSPA5与调节性T细胞之间的显著正相关性，且调节性T细胞在肝癌中的高浸润与肝癌进展密切相关。结论: HSPA5的高表达与肝癌的发生及不良预后显著相关，并可能通过调控调节性T细胞在肿瘤微环境中的功能促进肝癌发展。.
Keywords: Ferroptosis; Genome-wide association study; Heat shock protein A5; Hepatocellular carcinoma; Mendelian randomization; Regulatory T cells.