circ_0000619通过调控miR-595/GLS轴对食管鳞状细胞癌细胞增殖及谷氨酰胺代谢的影响

目的:探讨circ_0000619/miR-595/GLS轴对人食管鳞状细胞癌（esophageal squamous cell carcinoma,ESCC）KYSE150细胞增殖、迁移、侵袭和谷氨酰胺代谢的影响及其可能的机制。方法:采用qPCR法检测KYSE150细胞中circ_0000619、miR-595、谷氨酰胺酶（glutaminase,GLS）mRNA等表达水平，Western blot检测KYSE150细胞中GLS蛋白的表达情况，使用细胞计数试剂盒-8（cell counting kit-8，CCK-8）试剂盒、Transwell法分别检测KYSE150细胞增殖、迁移及侵袭能力，使用相应的检测试剂盒检测KYSE150细胞谷氨酰胺消耗、谷氨酸产生及ATP产生水平，利用生物信息学分析技术及双荧光素酶报告基因实验分析并检测circ_0000619、miR-595与GLS mRNA之间的相互作用关系。结果:circ_0000619在ESCC细胞系中呈现高表达，其亲本基因DENND4A mRNA在食管癌组织中呈高表达（P<0.01）；敲减circ_0000619显著抑制KYSE150细胞的增殖、侵袭和迁移能力（P<0.01），并显著抑制KYSE150细胞的谷氨酰胺消耗、谷氨酸及ATP产生水平（P<0.01）；敲减circ_0000619显著上调miR-595表达（P<0.01），抑制GLS mRNA（P<0.01）及蛋白的表达；双荧光素酶报告基因实验显示在KYSE150细胞中，circ_0000619与miR-595存在靶向结合、miR-595与GLS存在靶向结合（P<0.01）。circ_0000619敲低显著降低了KYSE150细胞谷氨酰胺代谢和细胞增殖，并且这些作用被miR-595抑制或GLS过表达部分逆转。结论:circ_0000619能通过靶向调控miR-595/GLS轴增强ESCC细胞谷氨酰胺代谢及细胞增殖，并可能成为潜在的ESCC治疗靶点。

The effect of circ_ 0000619 on the proliferation and glutamine metabolism of esophageal squamous cell carcinoma cells by regulating miR-595/GLS axis

Objective:To investigate the effect and its possible mechanism of circ_0000619/miR-595/GLS axis on cell proliferation,migration,invasion and glutamine metabolism of human esophageal squamous cell carcinoma (ESCC) KYSE150 cells.Methods:qPCR was used to detect circ_0000619,miR-595 and glutaminase (GLS) mRNA expression levels in KYSE150 cells.Western blot was used to detect GLS protein levels in KYSE150 cells.Cell counting kit-8 (CCK-8)and Transwell assays were used to investigate KYSE150 cell proliferation,invasion and migration,respectively.Glutamine consumption,glutamate production and ATP production levels were detected by indicated assay kits.Bioinformatics analysis technology and dual luciferase reporter gene assay were used to analyze and detect the interaction among circ_0000619,miR-595 and GLS mRNA.Results:circ_0000619 was highly expressed in ESCC cell lines,and its parental gene DENND4A mRNA was highly expressed in esophageal cancer tissues (P<0.01).Knockdown of circ_0000619 significantly inhibited the cell proliferation,invasion and migration of KYSE150 cells (P<0.01),and significantly inhibited the glutamine consumption level,glutamate and ATP production level of KYSE150 cells (P<0.01).Knockdown of circ_0000619 significantly increased the expression of miR-595 (P<0.01) and inhibited the mRNA (P<0.01) and protein expression of GLS.Dual luciferase reporter gene assay demonstrated that circ_0000619 had targeted binding with miR-595,and miR-595 had targeted binding with GLS in KYSE150 cells(all P<0.01).Glutamine metabolism and cell proliferation,were significantly decreased by circ_0000619 knockdown in KYSE150 cells,and these effects were partly reversed by miR-595 inhibition or GLS over-expression.Conclusion:circ_ 0000619 can enhance glutamine metabolism and cell proliferation of ESCC cells through targeted regulation of miR-595/GLS axis,and may become a potential target for ESCC therapy.