Study On The Mechanism Of Mir-124-3p Regulating The Physiological Activity Of Pancreatic Cancer Cells Via Affecting Copper Absorption

In the past two decades,pancreatic cancer has become the fourth leading cause of cancer death.The survival rate of patients diagnosed with pancreatic cancer in five years is only 3%-15%.Pancreatic cancer is characterized by high drug resistance,high metastasis,poor prognosis and easy recurrence.MicroRNAs(microRNAs)are highly conserved non-coding microRNAs that regulate gene expression at post-transcriptional level by binding to target genes that complement their seed sequences to degrade target genes or inhibit translation.Clinical studies have found that many of the microRNAs have significant anti-cancer function,and the treatment of cancer can be improved by regulating the level of microRNAs.Many studies have found that the expression of microRNA-124 is low in most cancers,and restoring the level of microRNA-124 has significant anti-cancer effect.Copper is an indispensable transition metal involved in the redox process.As a catalytic cofactor of various enzymes in organisms,copper plays an important role in antioxidant defense,iron homeostasis,electron transport chain and various biochemical processes.However,uncontrolled accumulation of copper may lead to increased oxidative stress leading to cell death.Therefore,the absorption and transport of copper on the cell surface and the transfer of intracellular proteins are the key factors of copper homeostasis under physiological conditions.According to the known mechanism of copper transport,human copper transporter 1(hCTR1)is an important component of copper transport.It locates in the cell membrane and is responsible for intracellular transport of copper.hCTR1 has a high affinity for copper ions.The process of copper transport is an energy-consuming independent process,which is affected by extracellular acidic pH.In this study,we first verified the anti-cancer effect of microRNA-124-3p on pancreatic cancer cells.The inhibition of microRNA-124-3p on pancreatic cancer cells was time-and concentration-dependent,and inhibited cell migration and invasion significantly.Then the target genes of the solute transporter family(SLC)were screened using the NCBI database and experimental methods on the Internet.Two important transporters of the solute transporter family(SLC)were found,namely,the monocarboxylic acid transporter SLC16A1(MCT1)and the copper transporter SLC31A1(CTR1).Through consulting the literature,it was known that MCT1 was the target of the role of microRNA-124,while CTR1 had not been reported yet,and then through double-carboxylic transporter SLC16A1.Luciferase assay confirmed the targeting effect of miR-124 on the 3'UTR region of CTR1,which could significantly inhibit the activity of luciferase.Therefore,we found a new target gene of anti-oncogene micro 124.Later experiments showed that either the over-expression of microRNA-124-3p or the reduction of copper uptake by CTR1 could significantly inhibit cell proliferation in mitochondrial metabolism and oxidative stress.The changes of ROS and ATP in cells were also detected.Reducing copper ion level could increase ROS and decrease ATP in cells,but could not cause cell death.Further experiments showed that inhibiting copper absorption and inhibiting the proliferation of cancer cells could lead to autophagy,which could resist cell death caused by the decrease of copper ion level.The combination of CQ and TM,an autophagy inhibitor,could lead to the death of pancreatic cancer cells.The results of this research reveal the new anticancer mechanism of the tumor suppressor gene mir-124,and discuss the adverse factors of using copper chelator TM to treat cancer in clinical treatment,which provides a new idea for the clinical treatment of pancreatic cancer.