POFUT1影响食管鳞癌细胞生物学行为及其机制的初步研究

Preliminary study on the biological behavior and mechanism of POFUT1 affecting esophageal squamous cell carcinoma cells

目的探讨蛋白O-岩藻糖基转移酶1(POFUT1)对食管鳞癌细胞增殖、迁移、周期和凋亡的影响及其潜在机制。方法利用siRNA转染人食管鳞癌细胞KYSE30和EC109细胞株,按转染情况分为POFUT1敲低组(转染POFUT1 siRNA)和阴性对照组(转染NC siRNA)。应用实时荧光定量聚合酶链式反应(qRT-PCR)和蛋白质印迹法验证敲低效果。通过CCK-8法、Transwell实验检测不同的POFUT1表达对细胞增殖、迁移能力的影响;采用流式细胞术检测敲低POFUT1后食管鳞癌细胞周期和凋亡的变化。通过转录组测序分析POFUT1敲低组与阴性对照组KYSE30细胞中的差异表达基因,利用KEGG数据库分析潜在的信号通路。结果与阴性对照组相比,POFUT1敲低组细胞增殖能力及迁移细胞数均明显降低,差异均有统计学意义(P<0.05)。与阴性对照组相比,POFUT1敲低组凋亡细胞比例增加,同时,细胞周期G2/M期细胞比例增加,差异均有统计学意义(P<0.05)。转录组测序分析及qRT-PCR结果显示,敲低POFUT1后,多条癌症相关信号通路分子表达发生改变,其中鞘脂信号通路相关基因S1PR5与MAPK8,以及NOTCH信号通路相关基因NOTCH1、NOTCH2、DLL1表达均降低。结论POFUT1体外敲低导致食管鳞癌细胞增殖和迁移减少、细胞周期阻滞以及凋亡水平升高,POFUT1可能通过鞘脂信号通路、NOTCH信号通路等影响食管鳞癌细胞生物学行为。

Objective To investigate the effects of protein O-fucosyltransferase 1(POFUT1)on the cell proliferation,migration,cell cycle,and apoptosis of esophageal squamous cell carcinoma(ESCC)cells and its potential mechanisms.Methods Human esophageal squamous carcinoma cells KYSE30 and EC109 were transfected with siRNA and divided into POFUT1 knockdown group(transfected with POFUT1 siRNA)and negative control group(transfected with NC siRNA).The effect of knockdown was confirmed using quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blotting.CCK-8 and Transwell assays were utilized to assess the impact of different POFUT1 expression levels on cell proliferation and migration capabilities,and flow cytometry was employed to detect changes in cell cycle and apoptosis following POFUT1 knockdown.Genes that differed in expression between KYSE30 cells in the POFUT1 knockdown group and the negative control group were examined using transcriptome sequencing,and the KEGG database was utilized to analyze potential signaling pathways.Results Compared to the negative control group,both the quantity of migrating cells and the capacity for cell proliferation in the POFUT1 knockdown group were significantly decreased,the differences were statistically significant(P<0.05).Furthermore,compared to the negative control group,the POFUT1 knockdown group had a larger percentage of apoptotic cells as well as a higher percentage of cells in the G2/M phase of the cell cycle,the differences were statistically significant(P<0.05).The results of transcriptome sequencing and qRT-PCR analyses showed that the downregulation of POFUT1 led to altered expression of several molecules involved in cancer-related signaling pathways,among which,the expression of genes S1PR5 and MAPK8 related to sphingolipid signaling pathway as well as those involved in the NOTCH signaling pathway,NOTCH1,NOTCH2,and DLL1,were significantly reduced.Conclusion Knockdown of POFUT1 in vitro leads to reduced proliferation and migration,cell cycle arrest,and increased apoptosis in esophageal squamous cell carcinoma cells.POFUT1 may affect the biological behavior of esophageal squamous cell carcinoma cells through the sphingolipid signaling pathway and the NOTCH signaling pathway.