Radiation Induced IER5Expression And The Interaction Of CDC25B In HepG2Cells

Objectives: Primary liver cancer （PHC） in the digestive tract tumormalignant is higher, the incidence of malignant tumors in the fifth in the wholeworld, and the mortality rate is the third in the digestive tract malignant tumor.Cell cycle refers to the cell from the end of the first division until the endof the second division of process, divided into interphase and divide. Atpresent the treatment of primary liver cancer include surgery, chemotherapy,radiation therapy, interventional treatment methods, radiofrequency ablationmethod, and surgical treatment face cannot completely resection,postoperative recurrence rate is high, large trauma problems, in recent years,with the three dimensional suitable shape radiotherapy intensity modulatedradiation therapy and the development of technology, radiation therapy hasbecome an effective method for treatment of liver cancer.Interphase is dividedinto G1phase and S phase and G2phase. cell division phase.（M）, includingthe early, middle, late and last （G0）, and cell cycle phase from G1to S to G2to M is the G1/S and G2/M two inspection point, some emergencies causedby stagnation of cell reaction makes the cell cycle in check point, called thecell cycle arrest.CDC25family is an important factor of cell cycle regulation, the activityof CDKs family proteins by CDC25precise regulation of CDC25familyincludes three homologous isomer （CDC25A and CDC25B, CDC25C）,CDC25family is a group of Sue/bifunctional enzyme tyrosine, which play animportant role in cell cycle regulation. CDC25B genes in humanchromosomes20p13, previous studies have shown that CDC25B expressioncan increase the growth of tumor cells, contrary to inhibit the expression ofCDC25B may inhibit the growth of tumor cells and its mechanism of action isCDC25B may make tumor cell block in s period or earlier, but the specific mechanism is still not very clear, remains to be further research. CDC25B inthe cell cycle plays a monitoring role, its target protein in the pathway and thefinal status, as many pathways, the final effect of protein CDC25B in mice,the CDC25B was found excessive expression in fetal mouse liver, tips may beassociated with embryonic development, its play a role in the growth anddevelopment. Now to the role of CDC25B still have a lot of mechanism areunclear, needs further research.IER5belongs to early slow response gene family, is the short forImmediate early response5, one of the genes located on chromosome1q25.3,Williams etal., confirmed through the experiment IER5is a rich in proline andwith multiple sites for protein, nucleic acid of our previous studies havedemonstrated that elevated levels of radiation can induce the transcription ofIER5rise, play a role in the apoptosis induced by radiation. IER5throughphosphorylation and （or） phosphorylation have rapid adjustment function toexternal stimuli, suggesting that IER5may play an important role in mitosis tothe cell, but the specific mechanism remains unclear. Upfront for AHH1, Helaand HepG2IER5research, studies have found that it can make the cell blocks,inducing apoptosis and inhibition of IER5gene expression can promoteHepG2cells to enter S phase proportion increases; IER5gene expressionincreased HepG2cells to enter S phase proportion reduced. At present, somescholars study found that in chronic myelogenous leukemia cells inducedIER5protein increased, its CDC25B mRNA expression. Therefore, this studywas to investigate radiation induced HepG2IER5and CDC25B mRNAexpress the change of the quantity, and HepG2IER5protein and CDC25Bpromoter DNA interactions.A study shows IER5protein expression increased in chronicmyelogenous leukemia cells, can reduce CDC25B mRNA expression. So wespeculated IER5gene could inhibit CDC25B expression is involved in cellcycle regulation, there are lack of research reports at home and abroad.Given is still lack of at home and abroad studies of IER5geneexpression of liver cancer and CDC25B relations, more not seen IER5protein expression related to radiation induced liver cancer cells and CDC25Brelations research reports. Therefore, this research intends to adopt transienttransfection IER5lower expression cell line, liposome mediated genetransfection technology is adopted to establish the high IER5gene expressioncell line, through, reverse transcription polymerase chain reaction （reversetranscription-polymerase chain reaction, RT-PCR） and immuneco-precipitation of chromatin （CHIP） analysis method study of radiationinduced IER5gene expression, and CDC25B in HepG2cell cycle regulation,whether through a competitive combination of CDC25B promoter DNAbinding sites involved in cell cycle regulation.Methods:1transient transfection technique is adopted to establish the IER5: thelow expression of HepG2cells with liposome as a carrier, the previous buildIER5lower expression plasmid transfection to HepG2cells, cell transfectionempty plasmid as the control group.2liposome mediated gene transfection technology is adopted to establishthe high IER5gene expression of cell line （HepG2/IER5is named for thecells, study has been builded successful）.2gy,4gy⁶⁰Co-γ ray irradiationexpression, expression and control group in HepG2cells and the RT-PCRdetection of IER5mRNA and CDC25B mRNA levels; Through chromatinimmune co-precipitation （CHIP） analysis and CDC25B IER5protein promoterDNA interactions.Results:12gy,4gy after⁶⁰Co-γ ray irradiation, HepG2cells IER5and CDC25BmRNA expression changes: compared with no irradiation group （0gy）,irradiation group of HepG2cells IER5mRNA expression after rising todecline over6hours reach the highest.And CDC25B mRNA levels rise afterfalling first,and6hours reach the lowest.4gy⁶⁰Co-γ rays irradiation than2gy group IER5mRNA expression was increased significantly, CDC25BmRNA levels decreased more significantly. As shown in （table, chart）, promptIER5and CDC25B may exist mutual antagonism effect. 22gy,4gy after6hours to reach the highest ray irradiation, HepG2/IER5IER5cells and CDC25B mRNA expression changes: after⁶⁰Co-γ rayirradiation, compared with HepG2cell HepG2/IER5IER5mRNA trends isconsistent, but the content is more obvious, cell HepG2/IER5CDC25BmRNA levels consistent with HepG2cells change trend, and the contentdecreased more significantly, and further illustrate IER5and CDC25B mayexist mutual antagonism.32gy,4gy, after⁶⁰Co-γ ray irradiation the low expression IER5HepG2cells and CDC25B mRNA expression changes: after⁶⁰Co-γ ray irradiation,compared with HepG2cells,the lower expression HepG2cells IER5mRNAand CDC25B mRNA content has no obvious change, and again IER5andCDC25B may exist mutual antagonism.4chromatin immune co-precipitation analysis showed that radiationinduced IER5mRNA expression increased, the combination of IER5proteincontestability CDC25B promoter DNA locus, thus inhibiting the expression ofCDC25B, involved in cell cycle regulation.Conclusion: these results indicate that radiation induced IER5proteinexpression increased, and IER5protein may be through a competitivecombination of CDC25B promoter DNA binding sites, inhibit the expressionof CDC25B, make HepG2cell block in s period or earlier, resulting inapoptosis of HepG2cells.