RNA-seq of in vitro differentiated human abdominal and gluteal adipocyte cell lines following doxycycline induced RSPO3-knockdown

GWAS studies and our own work have identified RSPO3 as a gene modulating human body fat distribution. The GWAS signal at RSPO3 is coincident with an eQTL in mature adipocytes. To assess the effects of RSPO3 on abdominal and gluteal adipocyte biology, we undertook inducible RSPO3-knockdown in in vitro differentiated immortalized human abdominal and gluteal adipocyte cell lines (DFAT cells). Abdominal and gluteal DFAT preadipocyte cells stably transduced with the tet-pLKO-puro-shRSPO3 (tet-shRSPO3) or tet-pLKO-puro-shCON (tet-shCON) lentiviral vectors were differentiated in standard adipogenic media. On day 13 of differentiation, cells were treated with doxycycline (final concentration of 0.05 µg ml-1), or vehicle, in hormone-free basal media for ~48 hours. On day 15, cells were harvested for RNA. Total RNA purification and on-column DNase I-treatment were performed using the RNeasy Mini kit (Qiagen). RNA-seq was performed on samples from three independent experiments. In addition to tet-shCON cells, vehicle-treated cells were used as controls.

全基因组关联分析（Genome-Wide Association Study, GWAS）及本团队前期研究均已证实，RSPO3是调控人体脂肪分布的关键基因。RSPO3位点的GWAS信号与成熟脂肪细胞中的表达数量性状基因座（expression Quantitative Trait Locus, eQTL）相重合。为探究RSPO3对腹部及臀部脂肪细胞生物学特性的影响，我们在体外分化的永生化人类腹部与臀部脂肪细胞系（DFAT细胞）中构建了可诱导型RSPO3基因敲减模型。将经tet-pLKO-puro-shRSPO3（tet-shRSPO3）或tet-pLKO-puro-shCON（tet-shCON）慢病毒载体稳定转导的腹部及臀部DFAT前体脂肪细胞，置于标准成脂诱导培养基中进行分化培养。于分化第13天，将细胞转移至无激素基础培养基，以终浓度为0.05 μg·mL⁻¹的多西环素或赋形剂处理约48小时。于分化第15天收集细胞并提取RNA。总RNA纯化及柱上DNase I消化均采用RNeasy Mini试剂盒（Qiagen公司）完成。本次RNA测序（RNA Sequencing, RNA-seq）的样本来自三次独立重复实验，对照组除tet-shCON细胞外，同时设置赋形剂处理组作为额外对照。