MCU通过LETM1维持线粒体钙稳态调节口腔鳞状细胞癌细胞转移

MCU regulates metastasis of oral squamous cell carcinoma cells by maintaining mitochondrial calcium homeostasis through LETM1

摘要:

目的 探讨线粒体钙离子单向转运蛋白（mitochondrial calcium uniporter，MCU）与亮氨酸拉链/EF跨膜蛋白-1（leucine zipper/EF hand-containing transmembrane-1，LETM1）在调控线粒体钙稳态机制中的作用以及对口腔鳞状细胞癌细胞转移的影响。方法 选用口腔鳞状细胞癌CAL-27细胞株作为研究对象，构建MCU慢病毒干扰载体和过表达质粒，合成LETM1 siRNA干扰序列。采用细胞划痕实验、Transwell细胞侵袭实验检测MCU表达水平对CAL-27细胞侵袭、迁移的影响；细胞内钙离子荧光探针（Fluo-4 AM）以及JC-1线粒体膜电位测定实验检测MCU表达水平对CAL-27细胞内钙离子浓度及线粒体膜电位的影响；免疫荧光和Western blot检测CAL-27细胞内MCU、LETM1以及上皮-间充质转化（epithelial-mesenchymal transition，EMT）相关蛋白表达水平。结果 MCU过表达促进CAL-27细胞迁移和侵袭，MCU沉默组及MCU过表达联合LETM1 siRNA组细胞迁移和侵袭能力均明显受到抑制（F=45.302、1 300.246；均P>0.05）；MCU过表达组细胞内钙荧光强度和线粒体膜电位明显上调，MCU沉默组和MCU过表达联合LETM1 siRNA组细胞内钙荧光强度和线粒体膜电位水平均明显降低（F=75.585、141.767；均P<0.01）；MCU过表达组MCU、LETM1、N-cadherin以及Vimentin蛋白表达水平明显升高，而E-cadherin蛋白表达水平明显降低；MCU沉默组MCU、LETM1、N-cadherin及Vimentin蛋白表达水平较其他处理组明显降低，而E-cadherin蛋白表达水平明显升高（均P<0.01）。结论 MCU可调控LETM1及EMT标志物的表达，干扰LETM1可阻断MCU上调线粒体钙离子浓度和线粒体膜电位水平，降低口腔鳞状细胞癌细胞侵袭和迁移能力。

Abstract:

Objective To investigate the role of mitochondrial calcium uniporter（MCU） and leucine zipper/EF hand-containing transmembrane-1 （LETM1） in regulating mitochondrial calcium homeostasis and its effect on the metastasis of oral squamous cell carcinoma cells.Methods Oral squamous cell carcinoma CAL-27 cell line was selected as the research objects. MCU lentiviral interference vector and overexpression plasmid were constructed. LETM1 siRNA interference sequence was synthesized. Cell scratch test and Transwell cell invasion test were used to detect the effect of MCU expression level on the invasion and migration of CAL-27 cells. Intracellular calcium fluorescent probe（Fluo-4 AM ） and JC-1 mitochondrial membrane potential assay were used to detect the effect of MCU expression level on intracellular calcium concentration and mitochondrial membrane potential of CAL-27 cells. The expression levels of MCU，LETM1 and epithelial-mesenchymal transition （EMT）-related proteins in CAL-27 cells were detected by immunofluorescence and Western blot.Results Migration and invasion of CAL-27 cells were promoted by overexpression of MCU. Migration and invasion of CAL-27 cells were significantly inhibited in MCU silencing group and MCU overexpression combined with LETM1 siRNA interference group（F=45.302，1 300.246； all P>0.05）. The intracellular calcium fluorescence intensity and mitochondrial membrane potential were significantly increased in MCU overexpression group，and significantly decreased in MCU silencing group and MCU overexpression combined with LETM1 siRNA interference group（F=75.585，141.767；all P<0.01）. The protein expression levels of MCU，LETM1，N-cadherin and Vimentin in MCU overexpression group were significantly increased，while the protein expression level of E-cadherin was significantly decreased. The protein expression levels of MCU，LETM1，N-cadherin and Vimentin in MCU silencing group were significantly lower than those in other treatment groups，while the protein expression level of E-cadherin was significantly increased（all P<0.01）.Conclusion MCU can regulate the expression of LETM1 and EMT markers. Interference with LETM1 can block MCU up-regulation of mitochondrial calcium concentration and mitochondrial membrane potential and reduce the invasion and migration of oral squamous cell carcinoma cells.