第一临床医学院知识库

目的：莪术醇具有强的肿瘤抑制作用，但其对胆管癌细胞的影响、作用机制及具体靶点尚不明确。本文拟通过莪术醇体外干预人胆管癌细胞，利用蛋白组学技术研究莪术醇对人胆管癌细胞生物学特性（细胞增殖、迁移、周期及凋亡）的影响及作用靶点，通过免疫组化实验验证药物靶点在胆管癌组织中的表达情况，通过慢病毒介导的基因沉默进行功能验证。

方法：以人胆管癌RBE、HCCC-9810细胞株及临床胆管癌组织标本为研究对象，采用CCK8法、细胞划痕实验分别检测不同浓度莪术醇对胆管癌细胞增殖、迁移的影响；流式细胞术检测经不同浓度莪术醇处理后的细胞周期分布及凋亡特征；利用双向电泳技术分析对照组及干预组的差异表达点，并利用MALDI-TOF/TOF质谱对差异蛋白进行分析，筛选出莪术醇抗胆管癌作用的潜在靶标CDKL3，并通过qRT-PCR及Western blot进行验证；通过构建慢病毒介导的CDKL3-shRNA，观察CDKL3下调对RBE细胞生物学行为的影响；通过IHC及qRT-PCR验证CDKL3在胆管癌组织中的表达情况。

结果： 在一定浓度范围内（50-100μg/ml）莪术醇能显著抑制人胆管癌细胞的增殖及迁移，并将胆管癌细胞周期阻滞在G1期，诱导细胞凋亡（P<0.01）。应用蛋白质组学技术筛选出对照组及实验组的差异表达蛋白并通过生物信息学分析筛选出CDKL3进行后续实验。qRT-PCR及Western blot证实莪术醇处理RBE细胞后CDKL3表达降低（P<0.01）。随后经CDKL3-shRNA慢病毒载体转染RBE细胞后，细胞的增殖、迁移能力显著降低，G1期细胞比例增加，凋亡率升高（P<0.01）。最后通过免疫组化及qRT-PCR证实胆管癌组织中CDKL3高表达（P<0.01）。

结论：1. 莪术醇能显著抑制胆管癌细胞的增殖、迁移及侵袭能力，并通过阻滞细胞分裂诱导其凋亡。CDKL3在胆管癌组织中的显著高表达，在胆管癌的发生发展中可能起重要作用，是莪术醇抗胆管癌治疗的潜在靶标。2. CDKL3低表达能显著抑制胆管癌细胞的恶性表型（增殖、迁移及侵袭能力），并通过阻滞细胞周期诱导其凋亡。莪术醇可通过下调CDKL3抑制抗胆管癌细胞发生、发展。

Objective: Curcumol exerts strong anticancer effect but its role and specific target for cholangiocarcinoma is not yet clear. This paper intends to explore the role of curcumol in human cholangiocarcinoma cells, to study the effects of which on the biological characteristics of human cholangiocarcinoma cells (cell proliferation, migration, cell cycle and apoptosis) and to find effective therapeutic targets of curcumol. The potential target was validated by immunohistochemistry (IHC), and the further functional role in cholangiocarcinoma cells was detected by using Lentivirus-mediated vector.
Methods: Human cholangiocarcinoma RBE and HCCC-9810 cell lines and cholangiocarcinoma tissues were used in this study. The inhibitory efficiency of curcumol on cholangiocarcinoma cells was estimated with CCK8 assay. Scratch assay was used to examine the cell migration ability. Alteration of cell cycle and apoptosis rate after curcumol treatments was detected by flow cytometry. The differentially expressed proteins of the curcumol-treated RBE cell proteins were gained based on the two-dimensional gel electrophoresis technology and verified by MALDI-TOF/TOF. CDKL3 was screened as a potential target of curcumol against cholangiocarcinoma and validated by using qRT-PCR and Western blot. We further investigated the role of silencing CDKL3 on the biological behavior of RBE cells by using RNA interference. The expression of CDKL3 in cholangiocarcinoma was validated by IHC and qRT-PCR. 
Result: Curcumol owns remarkably inhibitory effects on the proliferation and migration of cholangiocarcinoma cell lines and induces apoptosis via arrest cell cycle in G1 phase in a certain concentration range (50-100μg/ml) (P<0.01). Based on two-dimensional gel electrophoresis technology and bioinformatics We think of that CDKL3 might be as a potential target and which was used to the last experiments. qRT-PCR and Western blot data showed that the expression of CDKL3 was decreased after RBE cells treated with curcumol (P<0.01). After CDKL3 was down-regulated with lentiviral mediated shRNA, the cell proliferation and migration ability were significantly reduced, and the proportion of G1 phase cells and the apoptosis rate were also increased (P<0.01). IHC and qRT-PCR data showed that CDKL3 was highly expressed in cholangiocarcinoma tissues.
Conclusion: 1. Curcumol had remarkably inhibitory effects on the proliferation and migration of cholangiocarcinoma cell lines and induce apoptosis via arrest cell cycle in G1 phase. CDKL3 was confirmed to be a potential target of curcumol for anti cholangiocarcinoma via t two-dimensional gel electrophoresis technology. The level of CDKL3 in the cholangiocarcinoma tissues is significantly elevated and may play a role in the oncogene.2. Down-regulation of CDKL3 could remarkably inhibit the proliferation and migration ability of cholangiocarcinoma cells and induce apoptosis via arrest cell cycle in G1 phase. Curcumol exerts anticancer effect in cholangiocarcinoma cells via down-regulating CDKL3Objective: Curcumol exerts strong anticancer effect but its role and specific target for cholangiocarcinoma is not yet clear. This paper intends to explore the role of curcumol in human cholangiocarcinoma cells, to study the effects of which on the biological characteristics of human cholangiocarcinoma cells (cell proliferation, migration, cell cycle and apoptosis) and to find effective therapeutic targets of curcumol. The potential target was validated by immunohistochemistry (IHC), and the further functional role in cholangiocarcinoma cells was detected by using Lentivirus-mediated vector.
Methods: Human cholangiocarcinoma RBE and HCCC-9810 cell lines and cholangiocarcinoma tissues were used in this study. The inhibitory efficiency of curcumol on cholangiocarcinoma cells was estimated with CCK8 assay. Scratch assay was used to examine the cell migration ability. Alteration of cell cycle and apoptosis rate after curcumol treatments was detected by flow cytometry. The differentially expressed proteins of the curcumol-treated RBE cell proteins were gained based on the two-dimensional gel electrophoresis technology and verified by MALDI-TOF/TOF. CDKL3 was screened as a potential target of curcumol against cholangiocarcinoma and validated by using qRT-PCR and Western blot. We further investigated the role of silencing CDKL3 on the biological behavior of RBE cells by using RNA interference. The expression of CDKL3 in cholangiocarcinoma was validated by IHC and qRT-PCR. 
Result: Curcumol owns remarkably inhibitory effects on the proliferation and migration of cholangiocarcinoma cell lines and induces apoptosis via arrest cell cycle in G1 phase in a certain concentration range (50-100μg/ml) (P<0.01). Based on two-dimensional gel electrophoresis technology and bioinformatics We think of that CDKL3 might be as a potential target and which was used to the last experiments. qRT-PCR and Western blot data showed that the expression of CDKL3 was decreased after RBE cells treated with curcumol (P<0.01). After CDKL3 was down-regulated with lentiviral mediated shRNA, the cell proliferation and migration ability were significantly reduced, and the proportion of G1 phase cells and the apoptosis rate were also increased (P<0.01). IHC and qRT-PCR data showed that CDKL3 was highly expressed in cholangiocarcinoma tissues.
Conclusion: 1. Curcumol had remarkably inhibitory effects on the proliferation and migration of cholangiocarcinoma cell lines and induce apoptosis via arrest cell cycle in G1 phase. CDKL3 was confirmed to be a potential target of curcumol for anti cholangiocarcinoma via t two-dimensional gel electrophoresis technology. The level of CDKL3 in the cholangiocarcinoma tissues is significantly elevated and may play a role in the oncogene.2. Down-regulation of CDKL3 could remarkably inhibit the proliferation and migration ability of cholangiocarcinoma cells and induce apoptosis via arrest cell cycle in G1 phase. Curcumol exerts anticancer effect in cholangiocarcinoma cells via down-regulating CDKL3.