RESUMO
BACKGROUND: Autophagy is believed to participate in embryonic development, but whether the expression of autophagy-associated genes undergoes changes during the development of human embryonic kidneys remains unknown. METHODS: In this work, we identified 36,151 human renal cells from embryonic kidneys of 9-18 gestational weeks in 16 major clusters by single-cell RNA sequencing (scRNA-seq), and detected 1350 autophagy-related genes in all fetal renal cells. The abundance of each cell cluster in Wilms tumor samples from scRNA-seq and GDC TARGET WT datasets was detected by CIBERSORTx. R package Monocle 3 was used to determine differentiation trajectories. Cyclone tool of R package scran was applied to calculate the cell cycle scores. R package SCENIC was used to investigate the transcriptional regulons. The FindMarkers tool from Seurat was used to calculate DEGs. GSVA was used to perform gene set enrichment analyses. CellphoneDB was utilized to analyze intercellular communication. RESULTS: It was found that cells in the 13th gestational week showed the lowest transcriptional level in each cluster in all stages. Nephron progenitors could be divided into four subgroups with diverse levels of autophagy corresponding to different SIX2 expressions. SSBpod (podocyte precursors) could differentiate into four types of podocytes (Pod), and autophagy-related regulation was involved in this process. Pseudotime analysis showed that interstitial progenitor cells (IPCs) potentially possessed two primitive directions of differentiation to interstitial cells with different expressions of autophagy. It was found that NPCs, pretubular aggregates and interstitial cell clusters had high abundance in Wilms tumor as compared with para-tumor samples with active intercellular communication. CONCLUSIONS: All these findings suggest that autophagy may be involved in the development and cellular heterogeneity of early human fetal kidneys. In addition, part of Wilms tumor cancer cells possess the characteristics of some fetal renal cell clusters.
RESUMO
PURPOSE: Differential gene expression profiles between normal bladder mucosas and bladder transitional cell carcinomas TCC were detected. MATERIALS AND METHODS: cDNA microarrays were prepared by spotting PCR products of 12,800 human genes onto specially treated glass slides. The cDNA probes were prepared by labeling normal bladder mucosa mRNA and TCC tissue mRNA with Cy3-dUTP and Cy5-dUTP respectively through reverse transcription. The arrays were then hybridized against the cDNA probe mixture and the fluorescent signals were scanned. The ratios of Cy5/Cy3 were computed. Northern analysis was used to confirm the results of microarray hybridization. RESULTS: Eighty-three genes (0.65%), whose ratios of Cy5/Cy3 were greater than 4.0 or less than 0.25, were screened out after 10 groups of hybridization. In the cancerous tissues 28 of them showed higher expression and 55 lower. Twenty-three genes are unregistered in GenBank. These differentially expressed genes are always involved in the physiological processes such as signal transduction, apoptosis and cell cycle, etc. CONCLUSIONS: This technique provides a powerful method for quantitative analysis of the expression levels of thousands of genes in parallel, and is used to identify genes involved in TCC carcinogenesis. The data obtained by this means are comparable to those obtained by other methods. Using cDNA microarrays to define alterations in gene expression associated with a specific cancer may be an efficient way to uncover the clues to specific molecular derangements that account for its pathogenesis and thus identify potential targets for therapeutic intervention.
