CAJ | 학술논문

背景与目的:LRP16基因是新发现的白血病相关基因,其生物学功能还远未被阐明.本研究基于生物信息学分析探讨人类LRP16基因的生物学功能.方法:采用生物信息学方法分析LRP16基因启动子及编码蛋白的结构、功能,并对预测结果进行实验验证.建立pGL3-Basic重组载体,并进行荧光素酶活性分析.构建ORF-pcDNA3.1+真核表达载体,转染入HL-60、K562细胞后,采用单细胞凝胶电泳法检测HL-60细胞紫外线损伤,流式细胞术检测K562细胞周期.结果:LRP16基因启动子是一个典型的Ⅱ型真核肩动子,核心调控区位于-600 bp以内,具有7个与细胞周期、造血调控、细胞增殖及DNA损伤修复等有关的顺式作用元件.LRP16基因长型编码蛋白在148-315氨基酸残基之间具有与人类组蛋白H2AlC末端相类似的同源序列hismacro、COG2110和A1pp.紫外线照射HL-60细胞后,LRP16过表达组的彗星细胞数量、慧尾长度明显小于空质粒对照组,且活细胞数量明显多于空质粒对照组.过表达LRP16基因的K562细胞的增殖速度和G_2/M期、S期细胞均明显高于空质粒对照组,且提前达到平台期.结论:基于生物信息学方法分析表明LRP16基因具有促进白血病细胞增殖、调控细胞周期及抗紫外线的DNA损伤作用.
배경여목적:LRP16기인시신발현적백혈병상관기인,기생물학공능환원미피천명.본연구기우생물신식학분석탐토인류LRP16기인적생물학공능.방법:채용생물신식학방법분석LRP16기인계동자급편마단백적결구、공능,병대예측결과진행실험험증.건립pGL3-Basic중조재체,병진행형광소매활성분석.구건ORF-pcDNA3.1+진핵표체재체,전염입HL-60、K562세포후,채용단세포응효전영법검측HL-60세포자외선손상,류식세포술검측K562세포주기.결과:LRP16기인계동자시일개전형적Ⅱ형진핵견동자,핵심조공구위우-600 bp이내,구유7개여세포주기、조혈조공、세포증식급DNA손상수복등유관적순식작용원건.LRP16기인장형편마단백재148-315안기산잔기지간구유여인류조단백H2AlC말단상유사적동원서렬hismacro、COG2110화A1pp.자외선조사HL-60세포후,LRP16과표체조적혜성세포수량、혜미장도명현소우공질립대조조,차활세포수량명현다우공질립대조조.과표체LRP16기인적K562세포적증식속도화G_2/M기、S기세포균명현고우공질립대조조,차제전체도평태기.결론:기우생물신식학방법분석표명LRP16기인구유촉진백혈병세포증식、조공세포주기급항자외선적DNA손상작용.
Background and Objective:LRP16 is a human novel gene linked to leukemia identified recently.However. its biological function is not fully clarified so far.This study was to investigate the biological function of human LRP16 gene by database-aided bioinformatics analysis. Methods:The structures and functions of LRP16 gene promoter and its coding protein were analyzed using bioinformatics prediction,and further experimental testing was performed.The recombinants of pGL3-basic and LRP16 promoter subclones were constructed for luciferase activity analysis.The recombinant of LRP16 open reading frame coding sequence and pcDNA3.1 eukaryotic expression vector was established and transfected into HL-60 and K562 cell lines.DNA damage of HL-60 cells after ultraviolet irradiation was evaluated using single cell gel electrophoresis. Cell cycle of K562 cells was analyzed by flow cytometry. Results:LRP16 promoter was a typical class Ⅱ eukaryotic promoter and its core regulation sequence was located within upstream -600 bp of transcriptional start site.In addition,seven cis-acting elements,which may be implicated in cell cycle,hematopoiesis regulation, cell proliferation and repair of DNA damage,were identified.Long type LRP16 coding protein contained homologous sequences of hismacro, COG2110,and A1PP with human histone H2A1C between 148 and 315 amino acid residue.The number of comet cells and the length of comet tail in HL-60 cells irradiated were significantly decreased and the number of living cell was significantly increased in LRP16-overexpression group compared with empty plasmid control group.The proliferation rate and ratio or quantity of G_2/M and S phases were significantly increased in LRP16-overexpression K562 group compared with empty plasmid control group.LRP16-overexpression in K562 cells promoted the transition of G_1 to S phase and plateau phase of cell proliferation was advanced.Conclusions:Promoter regulation prediction and protein domain analysis based on bioinformatics contribute to the study of gene function.LRP16 may play an important role in leukemia progression by promoting cell proliferation,regulating cell cycle,and antagonizing radiationinduced DNA damage.