DLK1-DIO3 基因體銘印區域基因表現與人類胚胎幹細胞株分化潛能之關聯性

Abstract

選擇優質的人類胚胎幹細胞株 (human embryonic stem cell lines, hESCs lines) 對於 hESC 相關的研究及治療模式能否成功是關鍵的。然而， 在 hESC 的培養中經常能觀察到一些基因或表觀基因修飾的不穩定，包括基因銘印不穩定等現象。經由銘印機制調控的銘印基因當其表現失調時被認為與許多生長缺陷及癌症發生相關。我們利用 bisulfite 定序技術發現 DLK1-DIO3 銘印區域相較於其它受測區域更容易在長期培養的 hESC 中遭受異常的高度甲基化修飾。此外，源自該銘印區域經母源表現之非編碼 RNA (non-coding RNAs) 其表現在長期培養的 hESC 中遭受到抑制也為一相關異常。為了探討該銘印區域是否與 hESC 細胞特性有關，本研究主要依據該區域中名為 MEG3 長片段非編碼 RNA (lncRNA) 與其下游之 lncRNA 及微 RNA (microRNA, miRNA) 之表現，將 hESC 亞細胞株分為 “MEG3-ON” 與 “MEG3-OFF” 兩組。與 MEG3-ON 組別相比，這些 ncRNA 的表現在 MEG3-OFF 組別受到抑制。初步分化 hESC 為類胚體 (Embyoid body, EB) 的實驗結果顯示，由 MEG3-OFF hESC 衍生出的第十二天的 EB 其形態不正常且三胚層相關的基因表現異常。轉錄組分析顯示，與 MEG3-ON 組別相比，許多與發育及癌症相關的基因在 MEG3-OFF 組別中表現失調。特別的是，大部份的失調基因與神經系統有關。我們在數種 hESC 細胞株與誘發性多潛能幹細胞 (hiPSC) 株中驗證了 MEG3 失去表現的確與一些神經系相關的基因受到調降有關，之後利用 siRNA 及 shRNA 方式調降 MEG3 表現後同樣偵測到這些基因的表現降低。藉由神經分化的實驗證明，與 MEG3-ON hESC 所分化的類神經系細胞相比，將 MEG3-OFF hESC 誘導分化成類神經系細胞後，階段性的神經標記表現較低，且神經軸突的形成較少。因此，本研究推論避免使用 DLK1-DIO3 銘印區域失調的 MEG3-OFF hESC 能維持具備較好的神經分化潛能的實驗效率，其對於在基礎研究與進一步的治療應用將有所助益。

Selection of high-quality human embryonic stem cell (hESC) lines is crucial for successful hESC-based research and therapeutic models; however, some genetic and epigenetic instability, including imprinting instability, have been frequently found in cultured hESCs. Imprinted genes should be correctly regulated by Epigenetic machinery. Defective dosages of imprinted gene expression are associated with various growth defects and cancers. When screening the imprinting status in prolonged cultured hESCs by bisulfite sequencing, we observed that the DLK1-DIO3 imprinted locus was more susceptible to abnormal hypermethylation than other examined imprinted loci. Also, repression of maternally expressed non-coding RNAs (ncRNAs) from this locus was one of the first signs of epigenetic deregulation in the prolonged cultured hESCs. To investigate whether this locus correlates with hESC properties, we classified hESC sublines into “MEG3-ON” and “MEG3-OFF” primarily based on the expression levels of a long non-coding RNA from this locus (lncRNA), named MEG3, as well as its down-stream lncRNA and microRNAs (miRNAs). Through embryoid body (EB) formation, we found that the 12-Day-old EBs derived from MEG3-OFF hESCs, where DLK1-DIO3 ncRNAs were repressed, displayed smaller size and aberrant expressions of three germ layer genes. Transcriptome profiling showed that many genes involved in developmental events and different cancer types were deregulated in undifferentiated MEG3-OFF hESCs, compared with MEG3-ON hESCs. Notably, most of these deregulated genes are associated with neural lineage. When validating array results in three hESC lines and two hiPSC lines, we found an association between MEG3 repression and downregulation of several neural lineage-related genes. Direct manipulation of MEG3 via siRNA and shRNA knockdown approach further suggested a potential causative effect that MEG3 reduction leads to a decrease in these neural lineage gene expressions. When performing neural differentiation, lower expression levels of stage-specific neural lineage markers and reduced neurite formation were observed in the neural lineage-like cells derived from MEG3-OFF hESCs compared with those in MEG3-ON groups. Taken together, this study suggested that prevention of using MEG3-OFF hESCs where DLK1-DIO3 locus was deregulated may maintain the experimental efficiency with proper neural differentiation potential, which will benefit basic research and further therapeutic applications in this field.