Potential cyclooxygenase-2-independent mechanisms of the anti-proliferation and anti-fibrotic effects of celecoxib on the human intrahepatic biliary epithelial cell line HIBEpiC assessed by bulk-RNA sequencing

This experiment utilized next-generation sequencing (NGS) to identify potential cyclooxygenase-2-independent mechanisms of the anti-proliferation and anti-fibrotic effects of celecoxib on the human intrahepatic biliary epithelial cell line HIBEpiC. HIBEpiC cells were cultured in vitro. Treatments were vehicle, 20 ng/mL of TGF-ß (T20), combination of 10µM of celecoxib plus 20 ng/mL of TGF-ß (C10+T20), or combination of 10µM of 2,5-dimethyl-celecoxib plus 20 ng/mL of TGF-ß (DC10+T20) before NGS. The gene expression were analyzed. Overall, the results suggested that celecoxib had a COX-2-independent inhibitory effect on the proliferation and profibrotic behavior of TGF-ß-stimulated CXCL12+ HIBEpiC cells, probably because of the regulation of the cell cycle and several other signaling pathways. Overall design: HIBEpiC cells were cultured in vitro. Treatments were vehicle, 20 ng/mL of TGF-ß (T20), combination of 10µM of celecoxib plus 20 ng/mL of TGF-ß (C10+T20), or combination of 10µM of 2,5-dimethyl-celecoxib plus 20 ng/mL of TGF-ß (DC10+T20). Vehicle: HIBEpiC cells were cultured in RPMI 1640 medium with 0.5% Bovine Serum Albumin (BSA) and 0.1% DMSO for 28 hours. T20: HIBEpiC cells were cultured in RPMI 1640 medium with 0.5% BSA and 0.1% DMSO for 4 hours, and then the TGF-ß were added to the final concentration of 20 ng/mL for further 24 hours incubation. C10+T20: HIBEpiC cells were cultured in RPMI 1640 medium with 0.5% BSA and 10µM of celecoxib (final concentration of DMSO: 0.1%) for 4 hours, and then the TGF-ß were added to the final concentration of 20 ng/mL for further 24 hours incubation. DC10+T20: HIBEpiC cells were cultured in RPMI 1640 medium with 0.5% BSA and 10µM of 2,5-dimethyl-celecoxib (final concentration of DMSO: 0.1%) for 4 hours, and then the TGF-ß were added to the final concentration of 20 ng/mL for further 24 hours incubation. The cell experiments were performed in triplicate. Then, the cells were harvested for NGS.

本实验采用下一代测序（next-generation sequencing, NGS）技术，旨在探究塞来昔布对人肝内胆管上皮细胞系HIBEpiC发挥抗增殖与抗纤维化作用的非环氧合酶-2（cyclooxygenase-2, COX-2）依赖机制。体外培养HIBEpiC细胞，测序前分别予以四种处理：溶剂对照、20 ng/mL转化生长因子-β（TGF-β，记为T20）、10 μM塞来昔布联合20 ng/mL TGF-β（记为C10+T20），以及10 μM 2,5-二甲基塞来昔布联合20 ng/mL TGF-β（记为DC10+T20），随后对细胞的基因表达谱进行分析。整体实验结果表明，塞来昔布可对TGF-β刺激后的CXCL12阳性HIBEpiC细胞的增殖及促纤维化表型产生COX-2非依赖的抑制效应，其潜在机制可能与调控细胞周期及多条信号通路相关。

总体实验设计：体外培养HIBEpiC细胞，设置四组处理方案如下：
1. 溶剂对照组（Vehicle）：将HIBEpiC细胞置于含0.5%牛血清白蛋白（Bovine Serum Albumin, BSA）与0.1%二甲基亚砜（dimethyl sulfoxide, DMSO）的RPMI 1640培养基中培养28小时。
2. T20组：将HIBEpiC细胞置于含0.5% BSA与0.1% DMSO的RPMI 1640培养基预培养4小时，随后加入TGF-β至终浓度20 ng/mL，继续培养24小时。
3. C10+T20组：将HIBEpiC细胞置于含0.5% BSA与10 μM塞来昔布（DMSO终浓度为0.1%）的RPMI 1640培养基预培养4小时，随后加入TGF-β至终浓度20 ng/mL，继续培养24小时。
4. DC10+T20组：将HIBEpiC细胞置于含0.5% BSA与10 μM 2,5-二甲基塞来昔布（DMSO终浓度为0.1%）的RPMI 1640培养基预培养4小时，随后加入TGF-β至终浓度20 ng/mL，继续培养24小时。
所有细胞实验均设置三次生物学重复，收集细胞后进行下一代测序。