Verify The Alternations Of Transcriptional Profile In AHH-1 Co-culture With Same Cells Irradiated By ⁶⁰γ-ray And Analysis The Array Data By Bioinformatics

With more and more widely application of the nuclear energy,the underlying danger of ionization radiation to human beings is increasing.Since now,the complicated effects of low dose radiation especially the random and long-term effects can not figure out.Strongly limit the widely use of the application,assessment of the danger and prevention of the radiation.So it is necessary to study the mechanism of the low dose radiation.Nagasawa H first found in 1992 that recombination across sister achromatizes happened in 30-50%cells though only 1%cells were hitted by particle.this effect were verified by many experiments, they found that larger and more extensive biological effects than the effects estimated by the dose-survival curve.By the appearance of the microbeam,let accurate amount of rays go through single cell or cytoplasm,they found out unirradiate cells besides have similar response to the irradiate cells,and find out many mutation of genes such as CD59,etc.scholars call this effects " bystander effects".RIBBEs become a Hotpoint to study on low dose effects. But still now,the reason,mechanism and endpoints of the bystander effects are still unsure. The microarray technology which compare the great amount of genes' expression,make the complicate research of gene expression more concise and effective,are widely used by researchers.It give us a new convenient,precise way to make sure the pathway of bystander effect.Application of this advanced technology provides a useful tool for the research on radiation-induced bystander effects,we found out large amount of genes express changed in irradiate and bystander cells.Using bioinformatics tools to analysis the large amount of data, aim to find out some new mechanism of radiation induced damage and gives a effective way to study the diversity and complexity of low dose effects.Contents1.Using microarrays to detect the alternations of transcriptional profile in human lymphoblast cells exposure to different dose of⁶⁰Coγrays.2.Verify the RNA expression of the candidate genes,find out more relationship between the irradiate cells and the bystander cells through further study the positive results, explore possible mechanism of the effects.Methods1.Mainly using bioinformatics tools such as software,online analysis,and network database on the microarray data analysis2.Repeat the preparation process of microarray;detect the RNA expression of all the genes by PCR reaction. 3.Irradiate cells and bystander cells were cultured for long term,add into phosphorate inhibitor,detect the change of the protein enzyme activity.4.Statistic method:single sample t-test,single-factor variance analysis.Results1.The transcriptional profile in AHH-1 co-culture with same cells irradiated by 60γ-ray: compare to the normal cells,2.0Gy bystander cells(P2.0) 107 genes up-regulated,6 genes down-regulated,0.5Gy bystander cells(P0.5) 57 genes up-regulated,8 genes down-regulated,2.0Gy irradiate cells(Z2.0) 90 genes up-regulated,349 genes down-regulated,0.5Gy irradiate cells(Z 0.5) bystander 67 genes up-regulated,29 genes down-regulated.Some genes changed similarly both in 0.5 and 2.0 Gy irradiation,17 genes of them expression level increase with the dose.5 genes changed similarly both in irradiate and bystander effects.6 genes changes varied between irradiate and bystander cells.2.Results of the cluster analysis:Find out the differential expression genes in irradiate or co-culture cells are mainly included in the membrane receptor,cell connection, material transfer.Meanwhile there are genes in transcription,translate,cell cycling,apoptosis,ROS,oxidative phosphorylation etc.Researchers consider cells exposure to low dose radiation will not die immediately,but provoke membrane secrete and intercellular communications.The results of cluster happened to have same character.3.BAX,ZNF652,GINS4,FOS,APl Sl changed both in Z0.5 and P0.5,Indicate they may related with bioeffect of low dose radiation.Find ZNF652(zinc protein 652) and GINS4 (have no function classification) have some similarity in sequence,and then APlSl(adaptor-related protein complex 1,sigma 1 subunit) is not far in distance.They may have some relationship in structure and the function.And they both participate in transcription regulator or protein transfer.4.Genes changed similarly both in 0.5 and 2.0 Gy irradiation,Indicate they may not change with the dose.Blast results of these sequences shows PAPLN(papilin,proteoglycan-like sulfated glycoprotein) near to the LOC375010,and away with 5 proteins of BAX, Indicate they may have some relationship in the structure and the function.5.Many zinc protein expression changed in irradiate and bystander cells.ZC3H7A,which participate in TGF-βsignal pathway up-regulated in Z2.0 TGF-β1 also up-regulated in array,ZMAT3(WIGl,p53 target zinc finger protein),ZNF652(have effects in DNA and tumor access) also changed obvious. 6.Bioinformatics analysis of the genes unknown.BLAST results shows there were few genes well known similary to our genes unknown.The sequence of LOC375010 is similar with gene CACS4,which is a cancer susceptibility candidate gene,had been named after our chip finished,CACS4 is up-regulated in irradiate cells as well.Maybe there is some connection between these two genes.7.Functional analysis of genes may closely relate with radiation induced bystander effects. Though foud from web database,many genes participate in important pathway,such as in TP53,BAX and TGF-βrelated pathways.8.In the confirmatory experiment of 15 candidate genes induced by radiation or bystander effects,there are apparently differences of the RNA expression in these genes. 8.1 Verified the RNA expressions of transmembrane related genes:PTPRJ(CD 148, Protein tyrosine phosphatase,receptor type,J),VDAC3(voltage-dependent anion channel 3),PAPLN;VDAC3 have similarly change between PCR experiments and microarray data,But it is down-regulated in low dose while up-regulated in higher dose and bystander cells. 8.2 Verified the RNA expressions of cellular material transduction related genes:APl Sl, PDP2(pyruvate dehydrogenase phosphatase isoenzyme 2);AP1S1 is one of the genes had most obvious difference in RNA expression of bystander cells.PCR verified the changes in bystander effect(P<0.05),but there is no obvious change in irradiate cells. And the up-regulation is growing with the dosage.PDP2 changed same with the array data in our confirmation experiment in Z0.5,P0.5 and P2.0.While change not so obviously in Z2.0. 8.3 Verified the RNA expressions of genes in nuclear regulation of translation,cell cycle and apoptosis:ZNF652,FOS(TPR(translocated promoter region(to activated MET oncogene)),ZMAT3,CFLAR(CASP8 and FADD-like apoptosis regulator);ZNF652 is down-regulated in many kind of tumor cells,while this gene is up-regulated in Z0.5,P0.5 and P2.0 cells both in qPCR experiments and microarray data(P<0.05).ZMAT3 is up-regulated in Z0.5,P0.5,Z2.0 cells in array data,same with PCR results,(P<0.05) up-regulation is more obvious in Z0.5 cells.CFLAR is down-regulated in Z2.0 cells,but up-regulated in Z0.5 and Z2.0 cells(P<0.05). 8.4 Verified the RNA expressions of genes unknown:LOC375010(hypothetical protein),C12orf5(TIGAR,chromosome 12 open reading frame 5),GINS4(GINS complex subunit 4 ),YAF2(YY1 associated factor 2),SSFA2(Sperm specific antigen 2). LOC375010 is up-regulated in Z2.0 cells(P<0.05)there is a up regulate tendency in PCR results of bystander cells,more obvious than array data.These genes changes mostly similary with the array data,indicate that they may play important role in radiation or bystander effects.C12orf5 has been found to be down-regulated in other array data, down-regulated in Z2.0 cells both in array data and RT-PCR verification(P<0.05),while up-regulated obviously in Z0.5 and P0.5,down regulated in P2.0 in RT-PCR experiment.9.We have found that activity of Caspase8 decrease in irradiate and bystander effects,and repeated experiment detect obvious decrease(P<0.05),this pheromones disappear when add into phosphorate inhibitor,the activity of Caspase8 down to the same level.We also found a increase of Caspase8 activity in 30-day cultured of irradiate cells (P<0.05),there is no obvious change in bystander cells.Conclusion:1.There are many genes expression changed after cells exposure to y rays and cells exposure to irradiate cells.The effects are obviously different between different dose of y rays on irradiate and bystander cells.Indicate that bystander effects have something to do with the dose.Some genes changed similarly both in 0.5 and 2.0 Gy irradiation,some genes changed similarly both in irradiate and bystander effects.Pointed out those changes have nothing to do with the dose or the way of exposure.2.In the gene expression level of bystander cells,changes were mostly happened in membrane receptor,cell connection,ion transfer and matter transfer,Researchers consider cells exposure to low dose radiation will not die immediately,but provoke membrane secrete and intercellular communications.The results of cluster happened to have same character3.Muti-sequence cluster shows some genes may have some relationship in structure and the function,their nucleic acid or protein have shorter distance.4.Find many candidate genes in important cell signal passages,such as in TP53,BAX and TGF-βsignal passages.Some of these genes have not been reported,they are E2F,CFLAR,ZC3H7A,ZNF423,etc.Compond with some important zinc protein ZMAT3,ZNF652(have effects in DNA and tumor access);membrane receptor PTPRJ (CD148),CD58,glycosidoprotein PAPLN;Some secrete genes AP1S1,enegy metabolize related genes,may participated in radiation induced bystander effects.study the gene unknown,may help us to find out some new function and effect in low dose radiation.5.Caspase 8 activity detection:there is a decrease of Caspase 8 activity in irradiated an bystander cells(P<0.05),this phenomenon disappear when add into phosphoriate inhibitor, the activity of Caspase8 down to the same level.We also found an increase of Caspase8 activity in long-term cultured of irradiate cells,there is no obvious change in bystander cells.This may because of low dose radiation and bystander effect could induce up-regulation of apoptosis inhibitor(BAX,CFLAR),these protein can combine Caspase 8 down-regulate its activity.Caspase 8 activity decrease after add phosphoriate inhibitor may because of inhibition of large amount of phosphoriate proteins could decrease the activity of Caspase 8.There may have obvious difference between irradiate cells and bystander cells and in their long term effects.Need to further study more related proteins to verify this phenomenon.