RNA-seq analysis identifies an Lgr6 isoform and novel markers characterizing adult skin stem cells. Transcription profiling of Lgr6+ and Lgr6- basal keratinocytes in the epidermis

Adult stem cells are a focus of research in developmental skin biology and skin carcinogenesis attributable tobecause of their life-long pivotalcritical role in epidermal renewal. This study aimed at a thorough transcriptional characterization of stem cells carrying the Lgr6 receptor (cofactor of Frizzled in Wnt signaling). We used quantitative RNA deep seq and gene set enrichment analysis on α6+/Lgr6- and α6+/Lgr6+ epidermal basal (α6+) cells. These basal cells were isolated from epidermal sheets of transgenic hairless mice containing the Lgr6-EGFP-Ires-CreERT2/R26R-LacZ cassettes (Tg/Tg and Tg/wt) by flow cytometric sorting on EGFP fluorescence.Epidermal cell suspensions were prepared from transgenic hairless mouse strains containing the Lgr6-EGFP-Ires-CreERT2/R26R-LacZ cassettes (Tg/Tg and Tg/wt). Basal cells (integrin α6+) were sorted by EGFP expression (Lgr6+ vs -). Quantitative RNA deep seq and gene set enrichment analysis was performed on α6+/Lgr6- and α6+/Lgr6+ cells.We demonstrated that this approach permits isolation of cells expressing the cassette with a high purity in isolating cells that transcribe the cassette, and identified a collection of protein encoding genes as well as non-coding RNAs differentially expressed between Lgr6+ and Lgr6-these basal cells (Tg/wt) types. In addition, we discovered that in presumed Lgr6-null mice (Tg/Tg), a Lgr6 isoform (delta-Lgr6) is transcribed encoding a protein isoform lacking 75 N-terminal amino acids (delta-Lgr6). Evidence for the existence of a human orthologue (delta-LGR6) was found by screening a proteome database. Thus, we have established in Lgr6+ cells a RNA expression profile characteristic of cancer-related pathways and stem cells, and identified a set of genes co-expressed with Lgr6 which may serve further (functional) characterization and identification of these cells, also in human skin.

成体干细胞因其在表皮更新中终身发挥关键核心作用，成为皮肤发育生物学与皮肤癌变研究领域的核心关注对象。本研究旨在对表达Lgr6受体（Wnt信号通路中Frizzled的辅助因子）的干细胞开展全面的转录组学表征。我们针对α6+/Lgr6-与α6+/Lgr6+表皮基底细胞（整合素α6阳性细胞）开展了定量深度RNA测序（RNA-seq）与基因集富集分析。
此类基底细胞通过增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）荧光流式细胞分选，从携带Lgr6-EGFP-Ires-CreERT2/R26R-LacZ基因盒（Tg/Tg与Tg/wt基因型）的转基因无毛小鼠表皮片中分离得到。
研究人员从携带上述基因盒（Tg/Tg与Tg/wt基因型）的转基因无毛小鼠品系中制备表皮细胞悬液，通过EGFP荧光表达水平将整合素α6阳性的基底细胞分为Lgr6+与Lgr6-两组，并对两组细胞开展定量深度RNA测序与基因集富集分析。
本研究证实，该分选方法可高纯度分离出转录该基因盒的细胞，并鉴定出Tg/wt基因型小鼠的Lgr6+与Lgr6-基底细胞之间差异表达的一系列蛋白编码基因与非编码RNA。
此外，本研究发现，在预设的Lgr6敲除小鼠（Tg/Tg基因型）中，存在一种Lgr6剪接变体（delta-Lgr6），其转录产物编码的蛋白缺失75个N端氨基酸。通过筛选蛋白质组数据库，本研究找到了人类直系同源变体delta-LGR6存在的证据。
综上，本研究明确了Lgr6+细胞的RNA表达谱特征，该特征兼具癌症相关通路与干细胞特性，并鉴定出一组与Lgr6共表达的基因，这些基因可用于后续（功能）表征与这类细胞的识别，在人类皮肤样本中亦适用。