Effect Of Ionizing Radiation On Expression Of Nm23-h1 Gene In Human Non-small Cell Lung Carcinoma Cell Line H446

1 Background and ObjectiveTremdendous benefits have been brought to human being along with the wide and friendly use of radiation sources and nuclear energy,but the ionizing irradiation can cause harmful effects on the human body.It is very important that exposure dose on victims is rapidly estimated.At present,methods on radiation-absorbed dose estimation include the methods of biological and physical dose.Most common and general accepted approach applying to biological close assessment is analyses of chromosomal aberration.Nevertheless,the approach also has obvious defect as not estimating dose with early,quich and high throughput.Therefore,Establishment of the methods on early,quich and high throughput biological dose evaluation using modern biological technology has become the focus of current research.The metastasis suppressor gene family was identified by Steeg PS in 1988.He took a different approach and compared seven cell lines derived from a single murine K-1735 mouse melanoma using differential colony reducing hybridization.Nm23 genes are a conservative gene family that has altitude consanguinity among various living things species including bacterium,yeast cell,Drosophila melanogaster,mouse and human being.At present,that the members have already reached 9 in the human being nm23 clan,are nm23-H1,nm23-H2,DR-nm23,nm23-H4,nm23-H5,nm23-H6, nm23-H7,nm23-H8 and nm23-H9 respectively.Among them,the nm23-H1 gene coding Nucleoside Diphosphate Kinases A(NDPK-A) has been heavily studied,and some scholars have found that NDPK-A was important in tumour occurrence, development and metastasis.There was the negative relevance between the expression of nm23-H1 gene and metastasis with a lot of entity tumour,but displayed positive correlation between the expression of nm23-H1 gene and the prognosis.There were only a few reportes about effect of ionizion radiation on the expression of nm23-H1. For example,there were about the expression of nm23-H1 gene after radiotherapy in tumor,and the prognosis were better with the higher expression of nm23-H1 gene. Meanwhile,it was deficiencily succession studied about the effect of ionizion radiation on extraorgan expression of nm23-H1.Therefor,we studied that expression changes of nm23-H1 gene in human non-small cell lung carcinoma cell line exposed to 0-5 Gy ⁶⁰Coγ-ray by using analyses of RT-PCR and Western blot,and explore feasibility of the gene expression change detection as ionization radiation bio-marker for biological dose assessment.This study might lay the experimental foundation for establishing neotype ionization radiation biodosimetry.2 Methods2.1 Cultivation and treatment of cell.The cells at logarithmic phase were exposed to ⁶⁰Coγ-ray at single irradiation(0,1, 2,3,4 and 5Gy),and then these cells were cultured in the same medium for 0,6,24 and 48 hours after exposure.2.2 Extraction and transcription of total RNAThe totals RNA were extracted from the collecting cells after exposure using Trizol-chloroform-dimethyl carbinol,and then purity and contents of the extracted RNA were measured.The totals RNA were transcripted into cDNA using reverse transcription kit. 2.3 Extraction and treatment of the cell total proteinPMSF was added to the collected cells on ice,and cell lysate was added to the mix. Disrupting the cells for 30 min at 4℃,then the mix was centrifuged with 12000g for 5min.The supernatant was taked from the mix and infused in 0.5ml centrifuge tube. The total proteins were measured atλ= 595nm,and the BSA was used to as standard curve.The surplus proteins were mixed with equal volume 2×SDS,then the mix were boiled in boiling water for 10 minutes and centrifuged using 12000g for 2min.2.4 RT-PCRβ-actin as reference,semi-quantitative RT-PCR was used to test the expression level of nm23-H1 mRNA levels in H446 cell with different doses.Gene System was used to capture pictures and then GeneTools software was used to analyze the bands.2.5 Expression of nm23-H1 by Western blotDifferent proteins with equal quality were respectively subjected to 12%sodium dodecyl sulfate polyacrylamide gel eletrophoresis.The proteins in the gel were transferred to a nitrocellulose membrane,and the membrane was blocked with confining liquid,and incubated with primary antibody and second antibody with turning and stained with DAB.Bioimaging System was used to capture pictures and then GeneTools software was used to analyze the protein bands.2.6 Statistics methodsDataes of the experiment were processed by SPSS software 12.0 with Analysis of Variance of repeated measurement data.The trend of dosages changment was analysised with grade data correlativity analysis.Standard for test wasα=0.05.3 Results3.1 Expression of nm23-H1 mRNA by using RT-PCR after different dose ⁶⁰CoγradiationThe levels of the nm23-H1 mRNA were obviously declined at the range of dose from 0～5Gy otter exposure(P＜0.05),and showed the negative correlation with dosages(r≥0.785,P＜0.05).The levels of mRNA were to increase gradually at the period from 0 to 24 h,and decrease a little at 48 h after the irradiation.3.2 Expression of nm23-H1 protein by Western blot after exposure.Expression of the nm23-H1 protein were obviously declined at the range of dose from 0～5Gy after exposure(P＜0.05),and showed the negative correlation with dosages(r≥0.66,P＜0.05).No significant changes on expression of nm23-H1 protein were observed in the period from 0 to 6h,and the expression reached to peak value at 24h after irradiation and decreased to some extent at 48h.4 Conclusions4.1 Levels of nm23-H1 mRNA and protein expression decreased gradually with increase of the radiation doses,and both between the levels of the expressions and radiation doses all showed a dose-effect response at different time after irradiation.4.2 Nm23-H1 protein maybe not secreting type protein.4.3 The detection of nm23-H1 gene expression changes has a certain extent feasibility as ionization radiation biological marker.