Mechanism Of ELF-MF Induced Autophagic Cell Death In NSCLC By Mir-486

lung cancer today is one of the most common and high mortality cancers which seriously harm to human’s health.The cancers even get worse prognosis which treated with advanced surgery and a variety of new chemotherapy drugs.According to the statistics,the efficiency of combination with chemotherapy in lung cancer is only 14%to 40%.Extremely low frequency magnetic field(ELF-MF),a magnetic field with frequencies less than 300.Our previous study optimized the intensity and duration of ELF-MF which inhibit the proliferation of tumor cells.We screened the anti-tumor spectrum of ELF-MF,and confirmed the ELF-MF had a function in inhibiting animal models of hepatocellular and melanoma tumors.But,the mechanism of ELF-MF expose to treating tumors is unclear.Latest studies reported that the electromagnetic fields also increase autophagy activity in Neuroblastoma cells via regulating microRNA(miRNA)which inhibited the progress of Neurodegenerative diseases.miRNAs is a kind of important regulatory factor involved in a wide range of biology.The aberrant expression of miRNA has been proved to be linked to the molecular pathogenesis of non-small cell lung cancer.Autophagy is not only a conserved defense cellular mechanism but also a programmed mechanisms of cell death,The vitality of autophagy in tumor cells is lower than the normal cells.Autophagic cell death can be activated under certain conditions and play an essential role in the development of tumor cell death.We guess that ELF-MF might affect tumor autophagy vitality by miRNA which inhibiting the development and progression in tumor.To reveal the molecular of how the ELF-MF physical factors changed into biological effects,the lines of lung cells exposing to ELF-MF maked use of analyzing the activity of autophagy and apoptosis genes.1.ELF-MF induced autophagic cell death in lung cancerThe in vivo therapeutic examinations were carried out on C57/B6 mice bearing LLC tumor cells.Lewis lung cancer cell were injected subcutaneously into the right armpits of the C57/B6 animals,animals were randomly divided into three groups:sham MF group(tumor mice were not exposed to MF),ELF-MF group(tumor mice were exposed to MF)and cisplatin-group tumor mice were administrated with 5mg/kg of cisplatin once every three days).On day 30,tumor challenged and then sacrificed.In vitro,A549 and LLC cells were treated with true and false magnetic field respectively and cells were obtained in 2days,4 days and 6 days.The results show that compared with sham MF group,ELF-MF could reduce the tumors obviously.Moreover,immunehistochemical analysis showed that Ki-67 was lower in lung tissues of mice treated with ELF-MF than sham MF treatment.ELF-MF promotes LC3B,Beclinl and ATG5,autophagic related important proteins and enhanced protein level of apoptosis protein PARP and Caspase3 in tumor tissue.Immunehistochemical analysis showed that LC3B and Cleaved caspase3 was highter in ELF-MF tumor tissues.ELF-MF increased protein levels of LC3B,Beclinl and ATG5 in A549 and LLC cells after treatment of 2D,4D and 6D.It is the same result for activity of caspase-3 and PARP cleavage with a manner of time dependent.Laser scanning confocal microscope was used to observe the number of Sham treated cells was significantly more than that of ELF-MF group.These data confirmed that ELF-MF could induce the autophagic cell death in vivo and vitro.2.ELF-MF induced autophagic cell death in lung cancer through upregulating theexpression of miR-486Twelve lung related miRNAs were found to study.MiR-223,miR-92a and miR-486 were differentially between the 2 groups.Especially,miR-486 expression was frequently up-regulated in the ELF-MF tissues.Then,we also found that the ELF-MF can raise expression of miR-486 in A549 and LLC cells and the up regulation effect was time dependent.High expression of miR-486 in A549 and LLC cells can enhance the level of autophagy and apoptosis.We interfere with autophagy protein ATG5 by siRNA which makes the loss of autophagy function,it was found that the overexpression of miR-486 attenuated the effect on cell growth.Further use of siRNA to inhibit A549 and LLC cells in miR-486,we found that ELF-MF decreased induced-autophagy significantly on tumor cells after inhibition of miR-486 expression levels.Dates show that miR-486 is significantly regulated by ELF-MF and play an important role in inhibiting tumor cells.3.MiR-486 regulate autophagy through targeting BCAP and inhibiting AKT/mTOR signaling pathwayWe predicted target genes of miR-486 using the website and founded the region of BCAP 3’UTR.ELF-MF treatment could decrease expression level of creatine phosphate 3 kinase adaptor protein 1(phosphoinositide-3-kinase adaptor proteinl,BCAP)in the cells.Furthermore,luciferase report assay identify that BCAP is a direct target of miR-486.High expression of miR-486 in A549 cells can reduce the expression of BCAP from gene and protein levels.In addition,we found that overexpression of miR-486 or siRNA-BCAP can inhibit the expression of p-AKT and p-mTOR in A549 cellsIn summary,this study found that ELF-MF can induced autophagic cell death in animal models of lung cancer and lung cancer cell death;ELF-MF treatment in mouse tumor tissues and in lung cancer cells with high expression of mir-486.Over expression of mir-486 can inhibit Akt/mTOR pathway activation by targeting BCAP which contributing to the occurrence of autophagy in tumor cells.The results confirmed the function and molecular mechanism of miRNA in the biological effect of magnetic field,and put forward a new theoretical basis for the scientific explanation of the effect of magnetic field on tumor.