Establishment Of A Specific Cu (Ⅰ) Reporting System For Living Cells Based On Metallothionein Screening

Copper is one of the essential trace elements in life activities.It exists in organisms as Cu(I)and Cu(II).Due to its redox potential,copper ions can be used as a cofactor for much of enzymes and participate in some important life activities after combining with enzymes.The formation of connective tissue,anti-oxidation effect,cellular respiration,conduction of neural signals,and maturation of extracellular matrix are all related to copper ion.The abnormal level of intracellular copper ion is closely related to the occurrence and development of Wilson’s disease,Menks disease,senile dementia and other diseases.The level of copper ions is higher in tumor cells than in normal cells,and that the proliferation and spread of tumor cells are closely related to the intracellular copper ions.There are many tools available to detect intracellular copper ions,such as isotope labeling.ICP-MS,and chemical fluorescent probes.Although the use of chemo fluorescence probes to track the intracellular storage of metal ions is well advanced,they still have many limitations that need to be addressed,such as the small range of copper ion concentrations that can be detected by copper probes or the inability to detect the dynamics of intracellular copper ion changes in real time.Therefore,we sought to establish a living cell reporting system for copper ions.First,we find copper-sensitive genes that reflect intracellular copper ion changes specifically.For example,Metallothionein(MT),a cysteine-rich,small molecular weight protein,is a typical copper ion binding protein and plays an important role in the sudden increase of copper ion concentration.MT is divided into four subtypes,MT1,MT2,MT3,MT4,which are localized in cytoplasm and are involved in the uptake,transport,and regulation of copper in biological systems.It has also been proved that MTs protect cells from oxidative stress and are involved in differentiation,proliferation,or apoptosis of normal and cancer cells.Different MTs have different metal-binding preferences,which are related to functional specificity.Here,we screened the copper-response MTs in pancreatic cancer cells through data-mining analysis,RNA-seq and qPCR analysis.We found that MT1E,MT1F and MT1X mRNA were significantly upregulated after exogenous copper ions induction.By constructing the stable cell lines with MT1E,MT1F,or MT1X promoter-driven EGFP as reporters,we found that only PMT1F-EGFP could specifically and stably report the intracellular Cu(Ⅰ)changes in multiple cell lines,including Panc-1,8988T,293T,HepG2.and normal hepatic cells HL,indicating that PMT1F-EGFP is an ideal candidate for the in vivo Cu(Ⅰ)reporter.Using PMTF1-EGFP reporter,we found that MEK inhibitors(U0126)and Astragaloside Ⅳ could significantly increase intracellular copper ions.According to these results,we conclude that PMTF1-EGFP reporter effectively senses intracellular copper alteration,and can be used as a tool to screen copper-target drugs and to study the mechanism in copper-related cellular physiology and pathology.