C. Dampier, M. Devall, J. Bryant, S. Eaton, J. Huyghe, Andre E Kim, Virginia Díez-Obrero, F. V. Duijnhoven, Duncan C. Thomas, S. Powell, W. Gauderman, U. Peters, G. Casey
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引用次数: 0
Abstract
Background: Observational studies indicate that calcium, a micronutrient involved in numerous cell-signaling cascades, may protect against colon cancer. Stratified analyses suggest the protective effect of calcium may depend on sex and anatomic subsite, but these hypotheses have been difficult to test experimentally. Here, we employ human-derived normal colon 3D organoids as a model of the healthy colon crypt to interrogate the effect of calcium exposure on gene expression and the effect of donor sex and biopsy anatomic subsite on the response to calcium. Methods: A total of 39 3D colon organoid lines derived from colon biopsies (22 proximal, 17 distal) obtained from unrelated subjects (21 female, 18 male) during screening colonoscopy were grown under low (1.7 mM) calcium conditions, and a matching set of the same lines were grown under high (5.0 mM) calcium conditions. After 72 hours, total RNA was extracted from organoid lines, libraries were prepared with poly-A selection, and bulk RNA-sequencing was used to measure gene expression. DNA was extracted from the same lines and genotyped using the OncoArray 500K beadchip. Missing genotypes were imputed to the TOPMed reference panel using the TOPMed Imputation Server. Polygenic risk scores for colorectal cancer were calculated for each organoid line using Plink and 135 of 141 genome-wide significant SNPs associated with colorectal cancer in recent genome-wide association studies. Relative expression between low and high calcium conditions was tested using generalized linear models as implemented in DESeq2. Functional enrichment of gene ontologies among differentially expressed genes was performed with ToppFun. The effects of donor sex, biopsy subsite, and organoid polygenic risk score on response to calcium were tested using single and multiple linear regression as implemented in the base R lm function. Results: Exposure to calcium induced differential expression of 767 genes with a false discovery rate of 5%. Differentially expressed genes were enriched for biological processes including extracellular structure organization (Bonferroni p=1.6E-3) and positive regulation of apoptosis (Bonferroni p=2.2E-2). Although stratified analyses demonstrated more differentially expressed genes in 3D colon organoids derived from male subjects, no statistically significant effect of sex, subsite, or polygenic risk score on response to calcium was observed. Conclusion: We tested the effect of calcium exposure on gene expression in human-derived normal colon 3D organoids and found that calcium modulated processes related to differentiation and apoptosis, but the response to calcium was not affected by sex, anatomic subsite, or polygenic risk score. Citation Format: Christopher H. Dampier, Matthew Devall, Jennifer Bryant, Stephen D. Eaton, Jeroen R. Huyghe, Andre E. Kim, Virginia Diez-Obrero, Franzel J. van Duijnhoven, Duncan C. Thomas, Steven M. Powell, W James Gauderman, Ulrike Peters, Graham Casey. Calcium modulates apoptosis and differentiation in human normal colon 3D organoids irrespective of donor sex and biopsy site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2578.
背景:观察性研究表明,钙,一种参与许多细胞信号级联反应的微量营养素,可能预防结肠癌。分层分析表明,钙的保护作用可能取决于性别和解剖亚位,但这些假设很难通过实验来验证。在这里,我们采用人类来源的正常结肠3D类器官作为健康结肠隐窝的模型,来探究钙暴露对基因表达的影响,以及供体性别和活检解剖亚位点对钙反应的影响。方法:在结肠镜筛查过程中,从无亲缘关系的受试者(21名女性,18名男性)获得结肠活检标本(22名近端,17名远端),在低钙(1.7 mM)条件下培养39个3D结肠类器官细胞系,在高钙(5.0 mM)条件下培养一组相同的细胞系。72h后,提取类器官细胞系的总RNA,采用poly-A选择建立文库,采用大体积RNA测序法测定基因表达。从相同的细胞系中提取DNA,使用OncoArray 500K头芯片进行基因分型。使用TOPMed Imputation Server将缺失的基因型输入到TOPMed参考面板。在最近的全基因组关联研究中,使用Plink和141个与结直肠癌相关的全基因组显著snp中的135个计算了每个类器官系的结直肠癌多基因风险评分。采用DESeq2中实现的广义线性模型检验低钙和高钙条件之间的相对表达。利用ToppFun对差异表达基因的基因本体进行功能富集。供体性别、活检亚部位和类器官多基因风险评分对钙反应的影响采用基础R - lm函数中实施的单线性和多元线性回归进行测试。结果:钙暴露诱导767个基因差异表达,错误发现率为5%。差异表达基因富集于细胞外结构组织(Bonferroni p=1.6E-3)和细胞凋亡的正向调控(Bonferroni p= 2.22 e -2)等生物过程。虽然分层分析显示,来自男性受试者的三维结肠类器官中有更多的差异表达基因,但没有观察到性别、亚位点或多基因风险评分对钙反应的统计学显著影响。结论:我们测试了钙暴露对人源性正常结肠3D类器官基因表达的影响,发现钙调节了与分化和凋亡相关的过程,但对钙的反应不受性别、解剖亚位点或多基因风险评分的影响。引用格式:Christopher H. Dampier, Matthew Devall, Jennifer Bryant, Stephen D. Eaton, Jeroen R. Huyghe, Andre E. Kim, Virginia Diez-Obrero, Franzel J. van Duijnhoven, Duncan C. Thomas, Steven M. Powell, W James Gauderman, Ulrike Peters, Graham Casey。钙调节人类正常结肠3D类器官的凋亡和分化,而不受供体性别和活检部位的影响[摘要]。见:美国癌症研究协会2021年年会论文集;2021年4月10日至15日和5月17日至21日。费城(PA): AACR;癌症杂志,2021;81(13 -增刊):2578。