Dataset Information

RNA-seq analysis in Cornea epithelial cells (CECs), skin epithelial cells (SECs), LSCs after knocking down PAX6 (3-D shPAX6 LSCs) and SESCs transduced with PAX6(3-D PAX6+ SESCs) upon 3-D differentiation

ABSTRACT: Purpose: We find that Wnt7a-PAX6 axis determine corneal epithelial cell fate. To obtain global evidence for successful cell fate conversion, we performed gene expression profiling by RNA-seq on CECs, SECs, and LSCs after knocking down PAX6 and on SESCs transduced with PAX6 upon 3-D differentiation. Methods: Under 3-D culture condition, limbal stem cell (LSCs) can be differentiated to Cornea epithelial cells (CECs), and skin epithelial stem cells (SESCs) can be differentiated to skin epithelial cells (SECs). Total RNA was isolated from CECs, SECs, and LSCs after knocking down PAX6 (3-D shPAX6 LSCs) and on SESCs transduced with PAX6 (3-D PAX6+ SESCs) upon 3-D differentiation. Libraries were prepared following published standard protocol (Fox-Walsh K et al., 2011, genomics, 266-71). mRNA profiles were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. Results: Following optimized decoding and mapping workfollow, we mapped about 5 million sequence reads to the human genome and identified more than 23659 transcripts per sample. Conclusions: Hierarchical clustering analysis of differentially expressed gene signatures revealed that the gene expression pattern of SESCs with PAX6 transduction was strikingly similar to that of CECs, whereas the profile of LSCs with PAX6 knockdown was highly related to that in SECs upon differentiation. These data therefore provided global evidence for a decisive role of the WNT7A/PAX6 axis in cell fate conversion from SESCs to CECs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE54322 | GEO | 2014/07/11

SECONDARY ACCESSION(S): PRJNA236350

REPOSITORIES: GEO

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Project description:We previously demonstrated that Alox5 deficiency impairs the function of LSCs and prevents the initiation of BCR-ABL-induced CML. To identify the pathways in which Alox5 gene regulates function of LSCs, we performed a comparative DNA microarray analysis using total RNA isolated from non-BCR-ABL-expressing Lin-Sca-1+c-Kit+, BCR-ABL-expressing wild type LSCs and BCR-ABL-expressing Alox5-/- LSCs. The result was validated by quantitative real-time PCR analysis of non-BCR-ABL-expressing Lin-Sca-1+c-Kit+, BCR-ABL-expressing wild type LSCs and BCR-ABL-expressing Alox5-/- LSCs. We have shown that Alox5 is a critical regulator of leukemia stem cells (LSCs) in a BCR-ABL-induced chronic myeloid leukemia (CML) mouse model, and we hypothesize that the Alox5 pathway represents a major molecular network that regulates LSC function. Therefore, we sought to further dissect this pathway by comparing the gene expression profiles of wild type and Alox5-/- LSCs derived from our mouse model for BCR-ABL-induced CML. DNA microarray analysis revealed a small group of candidate genes that exhibited changes in the levels of transcription in the absence of Alox5 expression. In particular, we noted that the expression of the Msr1 gene was up-regulated in Alox5-/- LSCs, suggesting that Msr1 might suppress the proliferation of LSCs. Using our CML mouse model, we show that Msr1 is down-regulated by BCR-ABL and this down-regulation is partially restored by Alox5 deletion, and that Msr1 deletion causes acceleration of CML development. Moreover, Msr1 deletion markedly increases LSC function through its effects on cell cycle progression and apoptosis. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and ?-Catenin. Together, these results demonstrate that Msr1 suppresses LSCs and CML development. The enhancement of Msr1 function may be of significance in the development of novel therapeutic strategies targeting CML. To identify genes that are regulated by BCR-ABL in LSCs and LSCs without Alox5 gene, we compared the gene profile between wild type(WT) LSCs or Alox5-/- LSCs.

Dataset Information

RNA-seq analysis in Cornea epithelial cells (CECs), skin epithelial cells (SECs), LSCs after knocking down PAX6 (3-D shPAX6 LSCs) and SESCs transduced with PAX6(3-D PAX6+ SESCs) upon 3-D differentiation

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