Identification of Differentially Expressed Genes and Associated Immune Cell Types In South African Gallbladder Cancer Patients

Introduction: Gallbladder cancer (GBC) is a highly aggressive malignancy with limited therapeutic options, particularly in underrepresented populations, including South Africa. Understanding the molecular landscape of GBC may provide novel insights into its pathogenesis and potential therapeutic targets. Molecular changes are known to be associated with GBC, however, there is a paucity of this information especially in African populations. Furthermore, within the tumour microenvironment, different immune cells contribute to GBC progression. We investigated gene expression patterns in GBC tumours and their association with different immune cells in a cohort of South African patients. Methods: RNA sequencing was conducted on 2 normal and 8 gallbladder cancer tissues from South African patients (Ethics: M160640) to identify differentially expressed genes. Bioinformatics tools were used for pathway analysis, while immune cell quantification was performed using the quanTIseq software and presented as median [IQR]. Verification studies were further carried out using real-time PCR on an independent cohort comprising 7 gallstone samples and 26 gallbladder tumour samples. Results: A total of 65 genes were found to be significantly differentially expressed between the gallbladder tumours and gallstone controls. We also identified 37 upregulated and 28 downregulated genes in this cohort. Among the most upregulated genes, MUC16 was confirmed to be significantly overexpressed in tumours. Normal tissues exhibited a significantly higher proportion of dysregulated genes associated with B cells (17.132 [14.866–18.483], p<0.0001) and M1 macrophages (18.943 [1.097–36.790], p<0.0001) compared to tumours. In contrast, tumours showed a greater association with dysregulated genes linked to regulatory T cells (Tregs) (14.373 [9.696–20.162]) relative to normal tissues. Pathway analysis further revealed the upregulation of defective GALNT12, defective GALNT3, defective C1GALT1C1 and termination of O-glycan biosynthesis, highlighting key mechanisms potentially involved in tumour progression. Conclusion: The study has shown the dysregulation of key genes in South African gallbladder cancer patients. Specifically, MUC16 was verified to be significantly elevated in tumour samples. Furthermore, the association of these dysregulated genes with key immune cells in this patient group may further highlight their roles in dysfunctional immune processes linked with tumourigenesis. Overall design: RNA-seq profiling of Gallbladder tumours (8) and normal gallbladder tissues (2)

## 引言
胆囊癌（Gallbladder Cancer, GBC）是一种侵袭性极强的恶性肿瘤，治疗手段有限，在包括南非在内的代表性不足人群中尤为如此。解析GBC的分子特征图谱，可为其发病机制及潜在治疗靶点提供全新见解。已知分子改变与GBC相关，但此类研究数据较为匮乏，在非洲人群中尤其如此。此外，肿瘤微环境中的不同免疫细胞会促进GBC进展。本研究针对南非患者队列，探究了胆囊癌肿瘤组织的基因表达模式及其与不同免疫细胞的关联。

## 方法
本研究对来自南非患者的2份正常胆囊组织与8份胆囊癌组织进行RNA测序（伦理审批号：M160640），以筛选差异表达基因。采用生物信息学工具进行通路分析，同时使用quanTIseq软件进行免疫细胞定量分析，结果以中位数[四分位距（IQR）]形式呈现。后续采用实时荧光定量PCR（real-time PCR），针对包含7份胆囊结石样本与26份胆囊癌组织样本的独立队列进行验证实验。

## 结果
本研究共筛选出65个在胆囊癌组织与胆囊结石对照样本间存在显著差异表达的基因。该队列中共鉴定出37个上调基因与28个下调基因。在上调幅度最高的基因中，MUC16被证实于肿瘤组织中显著高表达。与肿瘤组织相比，正常组织中与B细胞、M1巨噬细胞相关的失调基因占比显著更高（B细胞：17.132 [14.866–18.483]，p<0.0001；M1巨噬细胞：18.943 [1.097–36.790]，p<0.0001）。与之相反，相较于正常组织，肿瘤组织与调节性T细胞（Tregs）相关的失调基因关联度更高（14.373 [9.696–20.162]）。通路分析进一步显示，GALNT12缺陷、GALNT3缺陷、C1GALT1C1缺陷以及O-糖基化生物合成终止通路出现上调，揭示了可能参与肿瘤进展的关键机制。

## 结论
本研究证实南非胆囊癌患者体内存在关键基因的表达失调。具体而言，MUC16在肿瘤样本中被证实显著高表达。此外，此类失调基因与该患者队列中关键免疫细胞的关联，可进一步阐明其在肿瘤发生相关的免疫功能异常过程中的作用。

## 整体实验设计
对8份胆囊癌组织与2份正常胆囊组织开展RNA测序（RNA-seq）表达谱分析。