白血病抑制因子通过调控CD44的表达增强结直肠癌细胞干性特征

Leukemia inhibitory factor promotes stemness features of colorectal can⁃cer cells by modulating CD44 expression

目的:探讨白血病抑制因子(leukemia inhibitory factor,LIF)通过调控肿瘤干细胞(cancer stem cells,CSCs)分子标志物CD44的表达增强结直肠癌(colorectal cancer,CRC)细胞干性特征的分子机制。方法:利用TCGA公共数据库和RNAscope原位杂交方法分析LIF基因在CRC组织中的表达情况;应用慢病毒感染系统构建稳定敲减LIF的CRC细胞系(HCT116和Caco2细胞);实验分CRC细胞对照组、CRC细胞添加LIF组、CRC细胞敲减LIF对照组和CRC细胞敲减LIF组;应用干细胞成球实验、MTT、RTCA、平板集落及迁移实验检测LIF对CRC细胞的影响;应用RT-qPCR和Western blot检测LIF对CRC肿瘤球细胞干性相关标志物CD44和转录因子ELF3表达的影响;应用RT-qPCR检测敲减LIF后CD44剪接变异体的表达变化。结果:CRC组织中LIF的表达水平高于癌旁组织(P<0.01),且LIF高表达的CRC患者无病生存期缩短(P<0.05),外源性LIF可增强CRC细胞的成球、增殖和迁移能力(P<0.05),敲减LIF可抑制CRC细胞的增殖和迁移(P<0.05)。外源性LIF可上调CRC肿瘤球细胞CD44的表达(P<0.05),而敲减LIF可抑制CD44的表达(P<0.01),同时CD44剪接变异体的转录水平降低。外源性添加LIF和敲减LIF可分别增强和降低转录因子ELF3的表达水平(P<0.05)。结论:LIF通过上调转录因子ELF3,增强CRC细胞CD44的表达,进而增强CRC细胞的干性特征,促进CRC细胞的恶性进展。

AIM:To explore the molecular mechanism by which leukemia inhibitory factor(LIF)enhances the stemness characteristics of colorectal cancer(CRC)cells by regulating the expression of the molecular marker CD44 in cancer stem cells(CSCs).METHODS:The expression of LIF in CRC tissues was analyzed using the TCGA public database and RNAscope in situ hybridization method.Stable knockdown of LIF was constructed in CRC cell lines(HCT116 and Caco2 cells)using a lentiviral infection system.The experiment was divided into 4 groups:CRC cells control group,CRC cells plus LIF group,CRC cells knockdown control group,and CRC cells knockdown LIF group.The impact of exogenous LIF on CRC cells´capacity for spheroidization was assessed through stem cell spheroidization assays.The ability of CRC cells to proliferate and migrate was evaluated using MTT,RTCA,plate cloning,and migration tests following the addition or removal of LIF.The impact of LIF on the expression of the transcription factor ELF3 and CD44 in CRC tumor cells was determined using RT-qPCR,a fully automated protein expression analysis system,and Western blot.RT-qPCR was used to identify changes in CD44 splicing variant expression following LIF knockdown.RESULTS:The results demonstrated that the expression level of LIF in CRC tissue was higher than that in neighboring cancer tissue(P<0.01),and that patients with CRC who had high expression of LIF had a shorter disease-free survival time(P<0.05).While LIF knockdown can decrease CRC cells´capability to proliferate and migrate(P<0.05),exogenous LIF can boost CRC cells´sphericity,proliferation,and migration capacity(P<0.05).Exogenous LIF can raise(P<0.05)CD44 expression in CRC tumor cells,however LIF reduction can lower(P<0.01)CD44 expression and lower the transcription level of CD44 splicing variants.Exogenous injection of LIF or LIF knockdown can cause a significant change(P<0.05)in the expression level of the transcription factor ELF3.CONCLUSION:LIF improves the stemness characteristics of colorectal cancer cells by upregulating the transcription factor ELF3,which also promotes the development of malignancy by increasing CD44 expression in CRC cells.