cDNA微阵列技术分析NAG7基因对鼻咽癌细胞基因表达谱的影响

The Effects of NAG7 on Gene Expressional Profile of HNE1 Cells Using cDNA Microarray Analysis

运用cDNA微阵列技术分析NAG7基因重表达对HNE1细胞基因表达谱的影响 .抽提HNE1细胞和pcDNA3 1(+) /NAG7/HNE1细胞总RNA ,分离 polyAmRNA ,将mRNA逆转录为cDNA ,并在逆转录过程中用33P dATP进行标记 ,与含有 16 15 0个基因和表达序列标签 (EST)的cDNA表达阵列膜杂交 ,获得基因表达图谱 .ArrayGauge软件分析NAG7基因的重表达所导致的鼻咽癌细胞HNE1基因表达谱改变 ,并用RNA印迹对微阵列杂交结果进行验证 .结果分析表明 ,2倍以上的差异表达基因或EST 179个 ,其中表达上调的 91个 ,表达下调的 88个 ;已明确基因表达产物的上调基因 2 9个 ,下调基因 37个 .在差异表达基因中 ,涉及基因转录调控、信号转导、细胞生长、细胞代谢和细胞凋亡等基因 .RNA印迹证实生长阻滞特异蛋白 1(gas 1)基因表达上调 .特别值得关注的是 ,先前的蛋白质组研究结果亦发现NAG7基因可导致生长阻滞特异蛋白 1表达上调 ,说明gas 1基因在NAG7重表达的HNE1细胞中具有重要作用 ,这为深入研究NAG7基因的作用环节和机理提供了重要的线索 .

To observe whether the re-expression of NAG7 gene could affect the gene expression of HNE1 cells, the cDNA microarray technique was employed to analyze the changes of gene expressions. 250 mug total RNA was extracted and 1 mug polyA mRNA was isolated. Reverse transcription was performed and cDNA probe was random-prime labeled with P-33-dATP and hybridized with the cDNA microarray membrane containing 16 150 genes and ESTs. The hybridized result was-confirmed by Northern blot analysis. FLA-3000A Plate scanner and Array Gauge software were used to screen and analyze the expressions of each gene. The background was eliminated and the differences of signal intensity of matched spots over 2 times were set as a marker to identify the differential expression genes. The results suggested that 179 genes were differential expressed, in which 91 genes were up-regulated and 88 genes were down-regulated in NAG7 re-expressed cells. These genes were involved in gene transcription, regulation, proliferation, metabolism, apoptosis, and so on. Northern blot result testified that growth arrest specific protein 1 (gas 1) expressed up-regulated. Especially, the previous results of proteomic research also found that the protein of gas 1 expressed up-regulated. Therefore, the data suggested that gas I gene plays a critical role in NAG7 re-expressed HNE1 cells, and provides an important clue to elucidate the mechanism of NAG7 gene.