长链非编码RNA调节细胞周期素D1表达对结直肠癌细胞辐射敏感性的影响

Long non-coding RNA influences radiosensitivity of colorectal carcinoma cell lines by regulating cyclin D1 expression

目的筛选影响结直肠癌细胞辐射敏感性的长链非编码RNA（1ncRNA）并探讨其作用机制。方法选取RKO和Lovo结直肠癌细胞株梯度照光后行克隆形成实验。应用单击多靶数学模型计算存活分数SF2值并绘制剂量存活曲线：高通量IncRNA／mRNA芯片筛选在RKO与Lovo、以及2Gv照光前后RKO细胞中表达差异2倍以上的lncRNA基因和蛋白编码基因：采用GeneOntology联合Pathway综合分析阳性表达基因主要作用通路：实时PCR进一步检测验证RKO细胞株照光前后P53、P21、细胞周期素（cyclin）D1表达变化。结果克隆形成实验显示，Lovo细胞株SF2=0．47．RKO细胞株SF2=0．53，Lovo辐射敏感性显著高于RKO（P〈O．05）。高通量lncRNA／mRNA芯片筛选得到阳性表达长链非编码RNA基因268条，蛋白编码基因270条；GeneOntologv联合Pathway综合分析显示：细胞周期相关基因所占比重最大（38．6％），并有多条lncRNA表达水平与cvclinD1编码基因CCNDl组间表达差异显著相关。RKO与Lovo两株细胞P53和P21相对表达量的差异均无统计学意义（P〉0．05），RKO细胞cyclinD1相对表达量明显高于Lovo细胞（P〈0．05）；RKO细胞株经2Gy剂量照射前后，P53和P21相对表达量的差异无统计学意义（P〉0．05），而cvclinD1表达明显下调（P〈0．05）。结论incRNA可能通过形成转录复合物与CCNDl基因结合，调节cvclinD1蛋白表达进而影响结直肠癌细胞辐射敏感性：且lncRNA对cvclinD1表达的调节不依赖于P53-P21-cyclinD1通路。

Objective To screen long non-coding RNA which influences radiosensitivity of colorectal carcinoma cell lines and investigate the mechanism. Methods Under different doses of radiation, colony formation assay and single-hit multi-target model were conducted to draw dose-survival curve and SF2 value of colorectal carcinoma cell lines （RKO, Lovo） was calculated. High-throughput lncRNA/mRNA chips were used to screen lncRNA genes and protein coding genes with expression differences more than 2 folds between RKO, Lovo cell lines and RKO cell line receiving 2Gy radiation. The main action pathway was computed by Gene Ontology analysis combined with Pathway analysis in order to explore the mechanism which induces the effect of lncRNA on radiosensitivity of colorectal carcinoma cell lines. Further experiment on P53, P21, cyclin D1 expression contents of RKO cell line was confirmed by real-time RT-PCR. Results Lovo （SF2=0.47） was more sensitiv to radiation than RKO （SF2=0.53） according to the outcome of colony formation assay. High-throughput IncRNA/mRNA chips identified a total of 268 lncRNA genes and 270 protein coding genes. Gene Ontology analysis showed that the expression of genes associated with cell cycle process were significantly different （38.6%）. There was a significant relationship between expression of several lneRNAs and CCND1 gene.Real-time RT-PCR showed no significant differences of P53 and P21 expression in RKO and Lovo cell lines（P〉0.05）, while cyclin D1 expression of RKO cell line was higher than that of Lovo cell lines（P〈 0.05）. After exposed to 2 Gv doses of radiation, there was an obvious decrease of cyclin D1 expression in RKO cell lines（P〈0.05）, while P53 and P21 expressions were not different（P〉0.05）. Conclusion The possible mechanism is that lncRNAs compose transcription compound to combine with CCND1 gene and influence radiosensitivity of colorectal carcinoma cell lines by regulating expression of cyclin D1, which is independent of P53-P21-cyclin D1 pathway.