Study Of The Effects And Related Mechanisms Of Millimeter Wave Irradiation On Human Melanoma Cells

Melanoma is a kind of skin malignant tumor with high drug resistant and metastasis rate.Current traditional methods such as surgery,radiotherapy and chemotherapy can not cure it and result in poor prognosis.Therefore,it is necessary to explore new therapeutic methods.Millimeter wave is a kind of electromagnetic field with frequency band of 30-300 GHz and wavelength of 1-10 mm.Research have showen that millimeter wave has a variety of biological effects on organisms.At present,it has two main applications on clinical treatment:analgesia,immune and inflammatory diseases treatment.In particular,some investigations have shown that millimeter wave has ability to inhibit the growth of certain tumor cells.Based on this finding,this study intends to investigate the influence of biological effects induced by 35.2 GHz which is often used in the clinic)millimeter wave on human melanoma A375 cells and explore the mechanism of this if exists.PURPOSETo explore the effects of 35.2 GHz millimeter wave irradiation on the cell viability and apoptosis of A375 cells,and further explore from the perspective of transcriptome level through the RNA-seq technology.Finally,it was preliminarily verified in tumor-bearing mice whether 35.2 GHz millimeter wave could inhibit tumor growth.CONTENTS AND METHODSThis research is divided into three parts:electromagnetic calculation experiment,in vitro experiment and in vivo verification experiment.The electromagnetic calculation experiment uses SEMCAD software to calculate the millimeter wave irradiation energy absorbed by the cells,which is expressed by the specific absorption rate(SAR).By setting up four sets of experiments,the effects of different culture materials and antenna placement positions on SAR were explored.The uniformity and intensity of SAR were compared and analyzed to obtain the optimal irradiation parameters in in vitro experiment.In in vitro experiment,A375 cells were divided into irradiation and non-irradiation group.The irradiation group received millimeter wave irradiation with frequency of 35.2 GHz,power density of 0.1 mW/cm2(transmited into the bottom of dish),and the irradiation time were 15,30,60,90 min;The environmental conditions of the non-irradiation group were consistent with those of the irradiation group except for millimeter wave irradiation.After irradiation,CCK-8 method was used to detect the change of A375 cell activity.Annexin-V/PI double staining method was used to detect apoptosis.Western blot was used to detect the expression of apoptosis-related protein.RNA-seq technology was used to detect the effect of irradiation on transcriptome of A375 cells.In in vivo verification experiment,the tumor-bearing Balb/c mice were divided into two groups(8 mice in each group),and one group was irradiation group:the mouse nose was exposed to the millimeter wave,15 min per day for 5 days.The environmental conditions of the non-irradiation group were consistent with those of the irradiation group except for millimeter wave irradiation.On the first day of irradiation,the long and short radius of the tumor were measured using a vernier caliper,and the tumor volume was calculated according to the volume calculation formula.The effect of millimeter wave on tumor growth was expressed in terms of tumor volume growth rate.RESULTSElectromagnetic calculation experiment:The optimal irradiation parameters were obtained by electromagnetic calculation.The antenna was placed below the plastic petri dish which formed the upward irradiation.In in vitro experiments:A375 cells were irradiated for 15,30,60,90 min.The CCK-8 results showed a time-dependent inhibition of the cell viability of A375 cells,and when irradiated for 90 min the cell viability was lowest.The results of Annexin-V/PI double staining showed that the apoptosis rate of A375 increased with the increase of irradiation time.The results of Western blot showed that the expression of cleaved caspase-3 and cleaved caspase-8 protein were up-regulated by millimeter wave irradiation.A total of 453 genes differential expressed were selected by RNA-seq,of which 140 were differentially up-regulated and 313 were down-regulated.Functional analysis of the differential expressed genes revealed that the major enrichment pathways of these genes were MicroRNAs in cancer,TGF-beta signaling pathway,and FoxO cell signaling pathway.In in vivo experiment:After the 5 days millimeter wave irradiation on the tumor-bearing mice in the irradiation group,the tumor volume growth rate was significantly slower than that of the non-irradiation group,but the difference was not statistically significant(P>0.05).CONCLUSIONIn summary,35.2 GHz millimeter wave irradiation induced apoptosis of human melanoma cells in vitro.Furthermore,its biological effects also influence many pathways including cell proliferation and angiogenesis;and the results in in vivo study showed a slower trend of tumor growth rate in irradiation group.Though there was no significant difference,this work provides a reference for subsequent living experiments.