Activation Of TMEM16A Ca²⁺-activated Cl^- Channels By ROCK1/moesin Promotes Breast Cancer Migration And Invasion

PartⅠROCK1 promotes TMEM16A channel activities by phosphorylating T558-moesin in breast cancer cellsObjective:To explore the mechanism of Rho A/ROCK participating in gate regulation of TMEM16A channel.Methods:TMEM16A currents activated by calcium stimulation in 1μM was collected from T47D cells before and after the ROCK1 inhibitor Y-27632 was given by whole-cell patch clamp technique.We Used Western blot to detect the protein expression of p-ROCK,ROCK,p-moesin and moesin in T47D cells transfected with Rho A active mutant V14 and inactive mutant 19N plasmids.TMEM16A currents activated by calcium stimulation in 1μM was collected from T47D cells transfected with Rho A active mutant V14 and inactive mutant 19N plasmid,moesin continuously phosphorylates T558D mutant and phosphorylation inhibits T558A mutant plasmid by whole-cell patch clamp technique.And verify the effect of moesin phosphorylation on TMEM16A channel current in HEK293 cells transfected with TMEM16A overexpressing.Results:The addition of Y-27632 inhibited the Ca²⁺activated TMEM16A chloride current in T47D breast cancer cells.Transfection of Rho A active mutant V14 plasmid resulted in increased expression of phosphorylated ROCK1 and phosphorylated moesin protein.Compared with the inactive mutant Rho A-19N,the active mutant Rho A-V14significantly increased the calcium-activated chloride current in T47D cells.Shows that Rho A activates ROCK1 can promote TMEM16A activation.Compared with the moesin T558A mutant that inhibits phosphorylation,the overexpression of the continuous phosphorylated moesin T558D mutant significantly increased the TMEM16A calcium-activated chloride current in T47D cells and HEK293 cells co-transfected with TMEM16A.ROCK1 promotes the activity of TMEM16A channel through the phosphorylation of moesin T558.Conclusion:ROCK1 promotes calcium-activated chloride currents in breast cancer cells T47D.The Rho A/ROCK1 signaling pathway activates calcium-activated chloride currents in breast cancer cells through phosphorylation of moesin T558.PartⅡMoesin regulates the function of TMEM16A channel to promote breast cancer cell invasion and metastasisObjective:To study the mechanism of moesin in promoting breast cancer invasion and metastasis.Methods:The TCGA database was used to analyze the ROC curve,combined survival curve and correlation of TMEM16A and moesin in breast cancer patients.The migration and invasion of breast cancer cells with different TMEM16A and moesin expression levels was verified by wound healing assay and transwell assay.Analyze the effect of different moesin expression on the invasion and migration of TMEM16A overexpression breast cancer cells promoted.The wound healing assay and transwell test were used to detect the invasion and migration of breast cancer MCF-7 cells after transfection of moesin persistent phosphorylation mutant（moesin-T558D）and inhibitory phosphorylation mutant（moesin-T558A）plasmids.Analyze the effect of moesin phosphorylation on the invasion and migration ability of breast cancer cells.These results were also replicated in the T47D breast cancer cell lines.Results:TMEM16A overexpression promotes the invasion and migration of breast cancer cells MCF-7 and T47D.After knocking down moesin,compared with the control group（moesin scramble）,the invasion and migration of breast cancer cells promoted by TMEM16A are inhibited.Compared with the empty plasmid control group,the overexpression of moesin-T558D promoted the invasion and migration of breast cancer cells,and the inhibition of phosphorylated moesin-T558A mutant inhibited the invasion and migration of breast cancer cells compared with the moesin-T558D group.It is proved that moesin promotes the invasion and migration of breast cancer cells through phosphorylation of T558.Conclusion:TCGA database analysis shows that the combined expression of TMEM16A and moesin is associated with poor prognosis of breast cancer.The invasion and migration of breast cancer cells promoted by the high expression of TMEM16A can be inhibited by moesin knockdown.Moesin mediates the invasion and migration of breast cancer cells through threonine 558 phosphorylation.