Analyses Of MiRNA-mRNA Network In Radiation Carcinogenesis And Identification Of A Novel Key Molecule MiR-486

BackgroundNuclear energy and nuclear technology have been widely used in industrial,defense and medical fields.However,medical problems related to ionizing radiation damage caused by nuclear accidents and nuclear leaks have always been a public concern,and many aspects have not yet found a good solution.Radiation carcinogenesis is one of them.It is a longterm effect caused by ionizing radiation.The mechanism of occurrence is unknown.Early monitoring,early intervention and specific treatment are very limited.Many problems have not been solved yet.It is urgent to explore radiation-induced carcinogens from new ways.,looking for new targeting molecules,providing new theories and new ideas for early monitoring,early intervention and specific treatment.The role of miRNAs in the development,proliferation,differentiation,apoptosis,immune response,and tumorigenesis of organisms has been reported,but the role in radiation carcinogenesis has been less concerned.So,we have carried out research on the relationship between radiation carcinogenesis and miRNA,and reported the role of several miRNAs,such as miR-100,miR-467 a,miR-200 c and miR-21 in radiation for the first time.And provide a useful reference for carcinogenic research.However,with the deepening of the research,many important problems remain unresolved,including: 1.The key miRNAs in radiation carcinogenesis have not been confirmed;2.It is difficult to fully explain the mechanism of radiation carcinogenesis by single miRNA changes.A new perspective is needed to explain the course of the disease.These problems arise because of the complexity of life activities and the limitations of past research methods and experimental techniques.For miRNAs,it mainly acts by targeting the mRNA of the target gene.Therefore,constructing a network of miRNA-mRNA regulation has become an important hotspot in the area of miRNA research in recent years.Darnell,R.B et al.first reported and proposed the concept of miRNA-mRNA interaction maps in Nature in 2009.Subsequently,many scientists used the miRNA-mRNA network to begin to study the characteristics of tumors from a holistic and "module" perspective,and discovered a number of important new targets,opening up new avenues for cancer research.This method based on miRNA screening to construct miRNA-mRNA network has two important characteristics: 1.Using miRNA and its targeted mRNA as a "module" to describe its influence on disease occurrence and development,in line with modern scientific research.And the "holistic view" is highly scientific;2.From the global perspective,we can more easily see the amount and function of mRNA regulated by each miRNA,so it is also more intuitive and easy to screen out the key miRNAs,thus providing key clues for research on disease control targets.Therefore,we have taken the lead in the international research on radiation-induced carcinogenic "miRNA-mRNA network",and based on this network,miR-486 has been screened as a key miRNA in radiation carcinogenesis for further biological research.To verify the effect of miR-486 on tumor cells and its effect on radiation carcinogenesis from in vivo and in vitro,and we also confirmed its targeted mRNA and binding sites,on the basis we further research on its downstream molecular pathways,which providing new ideas for the research of new targets for radiation carcinogenesisResearch contents and results1.Acquisition of miRNA profile and mRNA profile;Establishment of radiation-induced T cells lymphoma model in mice by exposure to multiple small dose of ? radiation.We identified the difference between lymphoma and normal tissues by Affymetrix miRNA microarray detection and cluster analysis,and we screened out 63 different expression miRNA following confirmed by real-time PCR.Our data showed that there is 15 up-regualted miRNA in radiation-induced T cell lymphoma,miR-762,miR-714,miR-467 a,miR-455,miR-699,miR-712,miR-685,miR-705,miR-494,miR-711,miR-181 d,miR-149,miR-720,miR-744,miR-210;11 down-regulated miRNAs: miR-145,miR-376 b,miR-708,miR-152,miR-143,miR-200 c,miR-193 b,miR-203,miR-100,miR-99 a,miR-486.These data provide hint for further identification of key miRNA.2.Construction of signal pathway of radiation-induced T cells lymphoma285 signal pathway has been identified by GO enrichment analysis,KEGG pathway analysis and miRNA network construction.There are five representative signal pathway: apoptosis pathway,cell cycle pathway,cytokine-cytokines receptor pathway,NF-?B signal pathway and p53 signal pathway.Four prediction methods of TargetScan,miRanda,PicTar and miRBase were applied,1070 target genes were predicted.Then,through the mRNA expression profiling of the radiation carcinogenic model,3160 differential genes were screened,and finally the two results were matched.A total of 567 mRNAs predicted to be consistent with the chip screening results were obtained.Further,we analyze through MAS constructed radiation-induced thymus lymphoma miRNA-mRNA correlation network map.It can provide new theory and experiment evidence for the molecular mechanism.3.miRNA-486 function in radiation induced T cells lymphomaWe analyze the miRNA-mRNA network and found miR-486,a novel miRNA located on the HUB of miRNA-mRNA.We constructed the miR-486 up and down regulation cell model and miR-486 KO mice wih intellectual property.In vitro experiments we found that miR-486 could inhibit lymphoma cell growth and vitality,promoted cell apoptosis.Downregualted miR-486 can increase cell viability,inhibit cell apoptosis.In vivo experiment we found that the subcutaneous tumors of nude mice were constructed by constructing a stable cell line overexpressing miR-486.Overexpression of miR-486 inhibited tumor growth.The radiation-induced carcinogenesis was induced by multiple small-dose ?-irradiation in miR-486 knockout mice: the tumor formation rate of miR-486 knockout mice was significantly increased,promote the occurrence of radiation carcinogenesis.These data indicated that miR-486 played an important role in radiation induced T cells lymphoma.4.Identification the new target gene of miR-486Combined with previous gene expression profiling results,we found that IGF2BP3 has the highest up-regulation in radiation-induced mouse thymic T lymphoma and is consistent with the predicted miR486 target.Therefore,we believe that IGF2BP3 is likely to be an important target for miR-486 to regulate T lymphoma cells.Further detection by the dual luciferase reporter gene revealed that when interacting with the mRNA of IGF2BP3,the luciferase activities of miR-486 and Ad-miR-486 decreased significantly in wild-type group,and the luciferase activity of miR-486 ASO increased.Luciferase activity of mutant group showed no significant change.This indicates that miR-486 target IGF2BP3 in a 3'UTRdependent manner.We found that miR-486 could inhibit IGF2BP3 mRNA and protein.Upregulation of IGFBP3 BP can partly restore the effect of miR-486 on cell viability.5.The mechanism of miR-486 target IGF2BP3miR-486 target IGF2BP3 by binding the 3'UTR of IGF2BP3.There is two miR-486 binding sites in the 3'UTR of IGF2BP3,896-903 site and 928-934 site.The two sites both played roles in this process of target,and the binding with 896-903 contribute more than the binding with 928-934 site.These data showed the theraptuic value in radiation induced carcinogenesis.6.Exploring the mechanism of miR-486 targeting IGF2BP3 to regulate radiation carcinogenesisImmunohistochemistry showed that overexpression of miR-486 inhibited the expression of IGF2BP3 and IGF2,as well as miR-486 knockout promoted IGF2BP3 and IGF2 expression and IGF2.Then Western Blot results suggest that overexpression of miR-486 may act through the signaling pathway of IGF2BP3/IGF2/PI3K/AKT/mTOR,inhibiting proliferation by inhibiting the expression of cell cycle-associated proteins.Such as protein D1,D3,CDK2,CDK4,CDK6,P21,P27.Here is the ideograh of miR-486 regulated the carcinogenesis.miR-486 regulated T cell lymphoma by binding IGF2BP3.The normal level of miR-486 target IGF2BP3.The low level of IGF2BP3 inibited T cell lymphoma.The decrease of miR-486 will decrease the inhibition of IGF2BP3,and the high level of IGF2BP3 promoted cells growth and inhibited apopotosis.Radiation deceased miR-486 level,which will promoted cells growth and inhibited apopotosis.Thus miR-486 played a key role in radiation induced T cells lymphoma???DiscussionAt present,the research on the role of miRNAs in tumors and its mechanism are mainly concentrated in general clinical tumors,but there are relatively few studies in the field of radiation carcinogenesis.The molecular mechanism of radiation carcinogenesis is still unknow,and many important problems are not very clear.According to the literature research and analysis,the construction of molecular network maps for miRNA-mRNA correlation analysis in radiation carcinogenesis has not been reported yet.The role of many miRNAs(such as miR-486)in radiation carcinogenesis has not been reported yet.Based on the demand for medical protection in national defense and national nuclear energy development,this research group has studied the molecular mechanism of radiation carcinogenesis and found several miRNAs closely related to radiation carcinogenesis in the field of miRNA research.The study will clarify the relationship between many miRNAs in radiation carcinogenesis,screen key miRNAs to find new target molecules,clarify their specific functions,and use advanced bioinformatics research techniques such as miRNA chips and gene chips.Large-scale analysis of the difference in expression profiles between tumor tissues and normal tissues,identification of differentially expressed molecules,and then using bioinformatics means to label out the biological significance of these differential expressions,screening out a number of miRNA molecules related to radiation carcinogenesis,and obtaining important molecules,Such as the miR-486 and its targeting site IGF2BP3.Through the analysis of the experimental results,it can be found that the miRNA expression profile and gene expression profile of the radiation-induced carcinogenesis tissue was significantly changed.63 differentially expressed miRNAs were screened,of which 15 were most up-regulated and 11 were most significantly down-regulated.And a map of 283 signal pathways associated with radiation-induced mouse thymic T lymphoma was constructed,showing their importance and their possible role in radiation carcinogenesis.The constructed radiation-induced mouse thymus T lymphoma miRNA-mRNA association network map is helpful to analyze the complex relationship between the miRNAs from the miRNA-mRNA network level,providing new clues and experimental basis for finding key molecules.Fortunately,this study screened a miRNA that is in the HUB status of the miRNA-mRNA association network map and has not been reported in radiation carcinogenesis by analyzing the miRNA-mRNA correlation network map of mouse thymus T lymphoma(ie miR-486),through a series of in-depth studies,found that its expression level is closely related to the proliferation and apoptosis of mouse thymoma cells,and this effect is mainly achieved by 3'UTR-dependent targeting of IGF2BP3.It is strongly suggested that miR-486 plays an important role in radiation carcinogenesis.In conclusion,our study construct animal model,obtained the bioinformatics big data,and identified the novel key molecule.Our data partly elucidated the underlying mechanism of radiation carcinogenesis and provided a potential therapeutic target.