高密度Oligo基因芯片筛选人永生化成骨细胞恶性转化中印迹基因的研究

Screening imprinted genes during malignant transformation of immortalized human osteoblastic cells by large-scale oligomicroarray technique

目的探索骨肉瘤发病相关印迹基因,研究印迹基因表达水平对于骨肉瘤发病的重要意义。方法建立人成骨细胞恶性转化模型,收集转化过程中4个不同时间点(40、55、70、90 d)细胞,以未处理第90天传代细胞为对照,提取总RNA,合成生物素标记的cDNA与博奥生物CAP-F0017芯片杂交。用实时荧光定量PCR对随机选取的2个基因在3组样本(对照,40 d,90 d)中的表达情况进行验证。结果以表达差异≥2.0或≤0.5为限,共筛选出10个差异表达印迹基因;CD81、GRB10、NDN、MEST在转化过程中表达下调,H19、MKRN3、SLC22A1L、SLC22A3、TSSC3、CDKN1C在转化过程中表达上调,功能分类显示差异表达基因主要与细胞生长/维持和细胞信号转导相关,随机挑选2个差异表达基因的实时RT-PCR结果与芯片结果相符。结论在人成骨细胞恶性转化过程中发现10个差异表达印迹基因,通过对10个差异表达基因的分析有助于揭示骨肉瘤发生过程中印迹基因的参与机制。

Objective To explore the pathogenesis-related imprinted genes in osteosarcoma and investigate the role of imprinted genes expression in osteosarcoma pathogenesis. Methods The model of malignant transformed immortalized human osteoblastic cells was established and the malignant transformed cells were collected on day 40, 55, 70, 90. The untreated cells passaged from the normal cells on day 90 served as control. After the total RNA extraction, the finally synthesized biotinylated cDNAs were hybridized to CapitalBio Genechip CAP-F0017.2 of the differentially expressed genes. Two genes were chosen at random to further confirm the array results using the SYBR Green real-time PCR in samples of control, day 40 and day 90. Results By an entrance limit of ≥2.0 or ≤0.5, ten imprinted genes which biological functions are mainly related with cell growth/maintenance and signaling transduction were detectable for notablely differential expression, including down-regulated genes of CDS1, GRB10, NDN, MEST and up-regulated genes of H19, MKRN3, SLC22A1L, TSSC3, CDKN1C in the malignant transformation of immortalized human osteoblastic cells. The array results were further confirmed by the real-time RT-PCR. Conclusion Ten imprinted genes were detectable for notablely differential expression. Our results will promote our understanding of the molecular mechanisms of imprinted genes in the course of osteosarcoma pathogenesis.