整合调控网络分析筛选骨髓增生异常综合征ABAT基因相关长链非编码RNA SET2-1及其对细胞增殖和凋亡的影响

Screening of ABAT gene related LncRNA-SET2-1 based on integrated regulatory network analysis and its effect on cell proliferation and apoptosis in myelodysplastic syndrome

目的构建具有骨髓增生异常综合征(myelodysplastic syndromes,MDS)特征的整合调控网络,从中筛选出调控ABAT(4-aminobutyrate aminotransferase)基因的长链非编码RNA(long non-coding RNA,LncRNA),并初步探讨其在MDS中的表达和功能。方法采用甲基化芯片和全基因组表达谱基因芯片对MDS患者和对照者的骨髓标本进行差异基因筛选,将筛选出的差异表达基因(differentially expressed genes,DEGs)、差异甲基化基因(differentially methylated genes,DEMs)、差异表达LncRNA(differentially expressed LncRNAs,DELs)进行整合分析,构建共表达调控网络,从中筛选出与ABAT基因具有相关性的LncRNA-SET2-1。采用qRT-PCR检测MDS患者和对照者骨髓标本中LncRNA-SET2-1和ABAT的表达水平,并在MDS转白细胞系SKM-1、急性白血病细胞系THP-1,以及对照细胞系HEK-293T中检测LncRNA-SET2-1的表达水平。建立稳定过表达LncRNA-SET2-1的SKM-1、THP-1细胞系,qRT-PCR检测ABAT表达变化、CCK-8法检测细胞增殖活力变化、流式细胞术检测细胞凋亡变化。并在ABAT敲减的SKM-1、THP-1细胞中检测LncRNA-SET2-1表达变化。结果通过共表达调控网络分析,筛选得到LncRNA-SET2-1与ABAT的相关性最高。MDS患者及SKM-1、THP-1细胞中LncRNA-SET2-1和ABAT的表达水平均显著下调(P<0.0001)。LncRNA-SET2-1过表达后,ABAT的表达量显著上调(P<0.01),而ABAT敲减后,LncRNA-SET2-1的表达量未发生显著变化。LncRNA-SET2-1过表达后,SKM-1、THP-1细胞的增殖活力显著下降,而凋亡细胞比例显著升高。结论LncRNA-SET2-1可能为ABAT的上游调控分子。LncRNA-SET2-1在MDS患者和MDS转白细胞系中均异常低表达,具有抑制MDS转白细胞增殖活力及促进细胞凋亡的功能。

Objective By constructing an integrated regulatory network with the characteristics of myelodysplastic syndrome(MDS),we tried to screen the long non-coding RNA(Lnc RNA)that regulates the ABAT(4-aminobutyrate aminotransferase)gene,and to preliminarily explore its expression and function in MDS.Methods The differential genes of bone marrow samples collected from MDS patients and controls were screened by methylation microarray and whole genome expression profile microarray.Integrated analysis of the differentially expressed genes(DEGs),differentially methylated genes(DEMs)and differentially expressed Lnc RNAs(DELs)were conducted for constructing a co-expression regulatory network.The Lnc RNA-SET2-1 correlated with ABAT gene was screened from the integrated regulatory network.The expression levels of Lnc RNA-SET2-1 and ABAT in bone marrow samples of MDS patients and controls were detected by q RT-PCR.The expression level of Lnc RNA-SET2-1 was detected in MDS transformed leukemia cell line SKM-1,acute leukemia cell line THP-1 and control cell line HEK-293 T.The SKM-1 and THP-1 cell lines with stable overexpression of Lnc RNA-SET2-1 were established.In these cell lines,the expression of ABAT was detected by q RT-PCR,the cell viability by CCK-8 and apoptosis by flow cytometry.The expression changes of Lnc RNA-SET2-1 were also detected in ABAT knockdown SKM-1 and THP-1 cells.Results Through the analysis of co-expression regulatory network,Lnc RNA-SET2-1 had the highest correlation with ABAT.The expression levels of Lnc RNASET2-1 and ABAT in MDS patients,SKM-1 cells and THP-1 cells were significantly down-regulated(P<0.0001).After overexpression of Lnc RNA-SET2-1,the expression level of ABAT was significantly increased(P<0.01).When ABAT was knocked down,the expression of Lnc RNA-SET2-1 did not change significantly.When Lnc RNA-SET2-1 was overexpressed,the cell viability of SKM-1 and THP-1 cells was reduced significantly,and the cell apoptosis was increased significantly.Conclusion Lnc RNA-SET2-1 may be the upstream regulator of ABAT.Lnc RNA-SET2-1 with functions of cell proliferation inhibition and cell apoptosis induction was aberrantly down-regulated in MDS patients and MDS transformed leukemia cell lines.