CAJ | 학술논문

目的筛选影响结直肠癌细胞辐射敏感性的长链非编码RNA(lncRNA)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lncRNA/mRNA芯片筛选在RKO与Lovo、以及2Gy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Gene Ontology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＜0.05).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；Gene Ontology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cyclin D1编码基因CCND1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cyclin D1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cyclin D1表达明显下调(P＜0.05).结论 lncRNA可能通过形成转录复合物与CCND1基因结合,调节cyclin D1蛋白表达进而影响结直肠癌细胞辐射敏感性；且lncRNA对cyclin D1表达的调节不依赖于P53-P21-cyclin D1通路.
목적 사선영향결직장암세포복사민감성적장련비편마RNA(lncRNA)병탐토기작용궤제.방법 선취RKO화Lovo결직장암세포주제도조광후행극륭형성실험,응용단격다파수학모형계산존활분수SF2치병회제제량존활곡선；고통량lncRNA/mRNA심편사선재RKO여Lovo、이급2Gy조광전후RKO세포중표체차이2배이상적lncRNA기인화단백편마기인;채용Gene Ontology연합Pathway종합분석양성표체기인주요작용통로；실시PCR진일보검측험증RKO세포주조광전후P53、P21、세포주기소(cyclin)D1표체변화.결과 극륭형성실험현시,Lovo세포주SF2=0.47,RKO세포주SF2=0.53,Lovo복사민감성현저고우RKO(P＜0.05).고통량lncRNA/mRNA심편사선득도양성표체장련비편마RNA기인268조,단백편마기인270조；Gene Ontology연합Pathway종합분석현시:세포주기상관기인소점비중최대(38.6％),병유다조lncRNA표체수평여cyclin D1편마기인CCND1조간표체차이현저상관.RKO여Lovo량주세포P53화P21상대표체량적차이균무통계학의의(P＞0.05),RKO세포cyclin D1상대표체량명현고우Lovo세포(P＜0.05)；RKO세포주경2Gy제량조사전후,P53화P21상대표체량적차이무통계학의의(P＞0.05),이cyclin D1표체명현하조(P＜0.05).결론 lncRNA가능통과형성전록복합물여CCND1기인결합,조절cyclin D1단백표체진이영향결직장암세포복사민감성；차lncRNA대cyclin D1표체적조절불의뢰우P53-P21-cyclin D1통로.
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 lncRNA/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 lncRNAs 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 Gy 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.