Objective: To investigate the differences in the expression profiles of circular RNA (circRNA) between luminal breast cancer cells and normal breast cells.
Methods: Total RNA extracted from luminal breast cancer cells MCF7 and normal breast cells MCF10A was digested with Rnase R to remove linear RNAs and enrich circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNAs using a random priming method, and were hybridized onto the circRNA hybridization array. The circRNA expression profiles of MCF7 and MCF10A cells were analyzed using Agilent Feature Extraction software. Quantile normalization and subsequent data processing were performed, and volcano plot filtering and hierarchical clustering were utilized to analyze the circRNA expression patterns. The expressions of 3 circRNAs with significant log fold changes were validated using qPCR.
Results: The hybridization array data revealed significant differences in the circRNA expression profiles between MCF7 and MCF10A cells. Compared with those of MCF10A cells, the 12910 circRNAs expressed in MCF7 cells showed 5964 up-regulated, 81 consistently regulated, and 6865 down-regulated circRNAs; 343 circRNAs showed a log fold change by more than 2 folds, among which 213 circRNAs were up-regulated and 130 were down-regulated. Nine circRNAs showed differential expressions by more than 2 folds, including 8 up-regulated ones, namely hsa_circRNA_061260 (6.02 folds), hsa_circRNA_103933 (5.96 folds), hsa_circRNA_005239 (5.84 folds), hsa_circRNA_100689 (5.69 folds), hsa_circRNA_004087 (5.60 folds), hsa_circRNA_104420 (5.25 folds), hsa_circRNA_104421 (5.13 folds) and hsa_circRNA_101222 (5.03 folds); only one circRNA was down-regulated, namely hsa_circRNA_104864 (5.09 folds). The expressions of hsa_circRNA_100689, hsa_circRNA_005239 and hsa_circRNA_104864 were further validated by qPCR, which yielded consistent results with the microarray data.
Conclusions: The circRNA expression profiles differ significantly between luminal breast cancer cells and normal breast cells. These differentially expressed circRNAs may serve as potential novel targets for the diagnosis of luminal breast cancer.
目的: 探讨环状RNA(circRNA)在Luminal亚型乳腺癌细胞与正常乳腺细胞中表达谱的差异。
方法: 分别提取Luminal亚型细胞MCF7和正常乳腺细胞MCF10A的总RNA；使用NanoDrop ND-1000对total RNA质量进行检测，用核糖核酸酶R分解total RNA以除去线性RNA并富集circRNA；利用随机引物法扩增富集的circRNA并转录成荧光cRNA；将荧光标记cRNA杂交到circRNA微阵列芯片上，扫描得到两种细胞的circRNA表达谱；在Agilent Feature Extraction软件中进行原始数据提取，并对采集到的阵列图像进行分位数归一化和后续数据处理，进行火山图和聚类热图分析；最后，选取差异表达倍数较高的3条circRNA进行实时荧光定量PCR(qPCR)鉴定。
结果: 提取的MCF7和MCF10A细胞总RNA纯度高、完整度较好；扫描微阵列芯片上标记的荧光信号，发现两种细胞的circRNA表达谱明显不同；与MCF10A细胞相比，在MCF7细胞的全部12 910条circRNA表达归一化结果中，有5964条上调，81条表达水平一致，6865条下调；表达差异(Log fold change)在2倍以上的有343条，其中213条上调，130条下调；表达差异在5倍以上的有9条，其中8条上调，分别为：hsa_circRNA_061260(6.02倍)、hsa_circRNA_103933(5.96倍)、hsa_circRNA_005239(5.84倍)、hsa_circRNA_100689(5.69倍)、hsa_circRNA_004087(5.60倍)、hsa_circRNA_104420(5.25倍)、hsa_circRNA_104421(5.13倍)和hsa_circRNA_101222(5.03倍)，1条下调：hsa_circRNA_ 104864(5.09倍)；经qPCR鉴定hsa_circRNA_100689、hsa_circRNA_005239和hsa_circRNA_104864的差异表达趋势与芯片结果相符。
结论: Luminal亚型乳腺癌细胞与正常乳腺细胞的circRNA表达差异较大，其中表达上调或下调的circRNA有望成为Luminal亚型乳腺癌诊断的新靶标。
Keywords: breast cancer; circRNA; expression profiles; luminal type.