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| Status |
Public on Jan 08, 2024 |
| Title |
Bulk transcriptomics analysis of SIX2+CITED1+ cells from Wilms Tumor |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Purpose: Bulk transcriptomics analysis of Wilms tumor SIX2+CITED1+ cells to compare and identify unique nephron progenitor transcriptome profiling (RNA-seq) signature between unfavorable and favorable Wilms Tumors and against human fetal kidney
Methods: Wilms tumor samples were collected and transported on ice at 4°C in RPMI-1640 and a single cell suspensions were prepared following mechanical and enzymatic dissociation as previously publication. Enzymatic dissociation was performed with 125 U/ml collagenase I in RPMI-1640 at 37 °C for 35 min. The digested cells were then passed through a 100-μm cell strainer and a 40-μm cell strainer with washes of 1x PBS. The cell suspension was than centrifuged at 1500 rpm for 5 min and erythrocytes were eliminated using a red blood cell lysis kit. WT SIX2+CITED1+ cells were isolated using SIX2-Cy5 and CITED1-Cy3 Smartflare RNA probes following manufacturer’s instructions and previous publication. Briefly, cells were incubated over night at 37 °C with both RNA probes diluted at 1:20 in PBS and 25ul/ml in RPMI-1640 supplemented with 5% FBS, and 0.2% antimicrobial agent Primocin. After 16-18 h, cells were dissociation using TrypLE 1x for 5 min, cells were centrifuged at 1500 rpm for 5 min and prepared for FACS. RNA extraction was performed immediately after FACS using the RNeasy Micro Kit following manufacturer’s recommendations. After cDNA production and construction of DNA libraries, the samples were run on an Illumina NextSep500 (Illumina). Differential gene expression was analyzed using ERCC ExFold probes with the Remove Unwanted Variation R/Bioconductor software package combined with edgeR
Results: Transcriptomics analysis of Wilms Tumor SIX2+CITED1+ cells in comparison to different tumor types and human fetal kidneys confirmed the nephrogenic signature of hFK-SIX2+CITED1+ and WT-SIX2+CITED1+ cells but highlighted differences in expression of pluripotency and self renewal-related genes like OCT4, FOXO1, SALL, NANOG along with a lower expression of β-catenin, TCF, and other growth factors known to promote differentiation (BMPs, FGFs). Hierarchical clustering of gene expression showed shared similarities between Wilms tumor samples against human fetal kidney samples however, with major differences in gene expression between tumor-to-tumor type was also present.
Conclusion: Our study represents the first transcriptomic characterization of Wilms Tumor cancer stem cells (SIX2+CITED1+) against human fetal kidney SIX2+CITED1+ cells. Identifying specify gene expression and signaling pathway profiles across different subtypes of Wilms Tumor and against human fetal kidney SIX2+CITED1+ cells.
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| Overall design |
Wilms Tumor mRNA profiles of anaplastic, non-anaplastic, and non-anaplastic chemotreated SIX2+CITED1+ cells.
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| Contributor(s) |
Perin L, Da Sacco S, Petrosyan A, Thornton ME, Grubbs BH |
| Citation(s) |
37114795 |
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| Submission date |
Jun 07, 2021 |
| Last update date |
Jan 08, 2024 |
| Contact name |
Brendan H Grubbs |
| Organization name |
University of Southern California
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| Department |
Obstetrics and Gynecology
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| Lab |
Grubbs
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| Street address |
4661 W. Sunset Blvd.
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90027 |
| Country |
USA |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (3) |
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| Relations |
| BioProject |
PRJNA735794 |
| SRA |
SRP323113 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE176342_20Dec17_LP_WT_gencode_h28_RawData_RPKM_Annotated.csv.gz |
1.4 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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