Coding and Non-coding mRNA expression after exposure of Breast Primary Epithelial Cells to different doses of X-ray

Low and high doses of X-rays are used in medicine as diagnostic and therapeutic tools, respectively. While response to high doses of radiation is well known, contradictions exist about effects of low-dose irradiation. Therefore, improving the knowledge on the consequences of low-dose irradiation could help to address this controversy. Moreover, describing new insights into high-dose irradiation would improve new cancer therapies combining radiation and gene therapy. As long non-coding RNAs (lncRNAs) seems to be engaged to almost all biological functions, including response to DNA damage, we aimed to describe the participation of lncRNAs in the response to different doses of X-ray exposure. We observed that, in human breast epithelial cells, different sets of coding and non-coding transcripts are differentially regulated at moderate and high doses compared to low doses. The validation of expression of five lncRNAs only regulated at high and moderate X-ray doses supports our results. Altogether, we could conclude that response to moderate and high dose irradiation versus response to low-doses also differs in terms of lncRNA expression. Therefore, further studies on the participation of lncRNAs in this response to radiation would help to address controversies regarding low-dose irradiation response and to improve therapies using high-dose irradiation. Breast primary epithelial cells were exposed to 0.02Gy, 0.1Gy and 2Gy of X-rays using medical devices and sham-irradiated samples were used as a control. To irradiate cells at low doses of X-ray, cells growing in 25cm2 flasks were irradiated with two (0.02 Gy) and ten automatic shots (0.1 Gy) under a mammography X-ray diagnostic device (SENO DMR plur, General electric). A Therapax STX 150 machine (Pantak) was used to irradiate BPECs at high doses (2Gy). RNA collection of the samples was done at three different days in order to have biological triplicates per each condition. Afterwards, total RNA was hybridized to Sureprint G3 Human Gene Expression v2 Microarray (Agilent) that includes probes to Broad Institute Human lincRNA catalog (Khalil et al. 2009; Guttman et al. 2009) among other ncRNAs. Differential expression analysis was carried out on non-control probes with an empirical Bayes approach on linear models (limma) (Smyth 2004). Results were corrected for multiple testing according to the False Discovery Rate (FDR) method (Benjamini & Hochberg 1995). Genes were selected as significant using a B-statistic cut-off B>0 and Fold Change (FC) > 1.2 or >-1.2.

医学领域中，低剂量与高剂量X射线分别被用作诊断与治疗工具。尽管人们对高剂量辐射的生物学应答机制已有充分认知，但低剂量辐射的效应仍存在诸多学术争议。因此，深化对低剂量辐射所致后果的认知，有助于解决这一争端。此外，挖掘高剂量辐射的全新研究视角，也可为放射治疗与基因治疗联合的新型癌症疗法提供助力。
鉴于长链非编码RNA（long non-coding RNAs, lncRNAs）几乎参与了包括DNA损伤应答在内的所有生物学过程，本研究旨在探究lncRNAs在不同剂量X射线暴露应答中的作用。我们观察到，在人乳腺上皮细胞中，相较于低剂量照射组，中剂量与高剂量照射组的编码及非编码转录本呈现出显著的差异化调控模式。针对仅在中、高剂量X射线照射下出现表达差异的5种lncRNAs开展的验证实验，进一步佐证了上述研究结果。综合来看，相较于低剂量辐射应答，中、高剂量辐射的应答在lncRNA表达层面同样存在显著差异。因此，后续针对lncRNAs在辐射应答中作用的研究，将有助于解决低剂量辐射应答相关的学术争议，并优化高剂量辐射的临床治疗方案。
本研究使用医疗设备对乳腺原代上皮细胞分别施以0.02Gy、0.1Gy及2Gy剂量的X射线照射，以假照射样本作为对照组。针对低剂量X射线照射组：将培养于25cm²培养瓶中的细胞，使用乳腺X线诊断设备（SENO DMR plur，通用电气（General Electric））分别进行2次（0.02Gy）与10次（0.1Gy）自动照射。对于高剂量（2Gy）照射组，则采用Therapax STX 150设备（Pantak）对乳腺原代上皮细胞（Breast Primary Epithelial Cells, BPECs）进行照射。为保证每个实验条件均设置生物学重复，我们于三个不同日期分别完成样本的RNA收集。随后，将总RNA与Sureprint G3人类基因表达v2微阵列芯片（安捷伦（Agilent））进行杂交，该芯片包含针对布罗德研究所人类基因间区长链非编码RNA目录（Khalil等，2009；Guttman等，2009）以及其他非编码RNA的探针。差异表达分析采用针对线性模型的经验贝叶斯分析工具limma（Smyth，2004），仅针对非对照探针进行分析。结果通过错误发现率（False Discovery Rate, FDR）方法进行多重检验校正（Benjamini & Hochberg，1995）。以B统计量阈值B>0，且折叠变化（Fold Change, FC）>1.2或<-1.2作为基因显著性筛选标准。