Clear cell renal cell carcinoma (ccRCC) frequently features a high level of tumor heterogeneity. Elucidating the chromatin landscape of ccRCC at the single-cell level could provide a deeper understanding of the functional states and regulatory dynamics underlying the disease. Here, we performed single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) on 19 ccRCC samples, and whole-exome sequencing was used to understand the heterogeneity between individuals. Single-cell transcriptome and chromatin accessibility maps of ccRCC were constructed to reveal the regulatory characteristics of different tumor cell subtypes in ccRCC. Two long noncoding RNAs (RP11-661C8.2 and CTB-164N12.1) were identified that promoted the invasion and migration of ccRCC, which was validated with in vitro experiments. Taken together, this study comprehensively characterized the gene expression and DNA regulation landscape of ccRCC, which could provide new insights into the biology and treatment of ccRCC. Significance: A comprehensive analysis of gene expression and DNA regulation in ccRCC using scATAC-seq and scRNA-seq reveals the DNA regulatory programs of ccRCC at the single-cell level.

Clear cell renal cell carcinoma (ccRCC) arising in the setting of von Hippel-Lindau (VHL) disease is a rare type of kidney cancer and features VHL germline mutation. This type of ccRCC is rarely characterised at the single-cell level. In this work, whole-exome sequencing and single-cell RNA sequencing (scRNA-seq) were conducted on one ccRCC sample with VHL disease. Integrating scRNA-seq and whole-exome sequencing data by the Seurat package, we determined the relationship between single-cell transcriptome features and gene mutations. Immunohistochemistry and immunofluorescence were performed on one VHL germline mutation ccRCC and six non-VHL germline mutation ccRCC samples. We revealed the gene expression characteristics of ccRCC with VHL germline mutation. The frameshift mutations in OBP2A and BCR1, and the elevated expression of COX7A1 were most specific characteristics of ccRCC tumor cells with VHL mutation. And the extensive infiltration of exhausted T cells was the characteristic of tumor microenvironment. In addition, we discovered the relationship between genetic mutations and immune checkpoints. This work highlights the single-cell transcriptome and DNA-level information of this rare ccRCC and will provide more genetic insights and references into this rare disease.

Regulating the occurrence and progression of tumor cells at the epigenetic level is a new insight of clear cell renal cell carcinoma (ccRCC). Chromatin accessibility is an important pathway of epigenetic regulation, which may explain the mystery of tumor occurrence. Assay for transposase-accessible chromatin sequencing (ATAC-seq) provides insight into the epigenetic regulatory features of ccRCC, especially at the single-cell level. In this study, we performed scATAC-seq of 3 ccRCC samples and captured a total of 18,703 high-quality cell nuclei and 104,818 unique peaks. Our protocol for nuclear extraction was reliable and stable, which can be used to deal with fresh and frozen single-cell suspensions. We presented basic methods for scATAC-seq data analysis, such as cell clustering, gene activity scoring, cell subtype specific peaks, transcription factors, motif and motif footprinting analysis. Taken together, our data indicated the valuable epigenetic features of ccRCC, which will provide more references for the study of ccRCC.

Clear cell renal cell carcinoma (ccRCC) with sarcomatoid differentiation is a rare histological type of ccRCC, characterized by aggressive clinical behavior and poor prognosis. However, the malignant characteristics of this disease have yet to be fully elucidated. Here, we performed single‐cell RNA sequencing (scRNA‐seq), single‐cell assay for transposase‐accessible chromatin using sequencing (scATAC‐seq), and WES analysis on a single patient with sarcomatoid ccRCC. By integrating scRNA‐seq and scATAC‐seq data from 19 previous classical ccRCC samples (19,819 tumor cells and 21,684 nuclei), we firstly identified the transcriptional regulatory features of sarcomatoid differentiated tumor cells by scATAC‐seq, primarily evidenced by active binding with FOS / JUND , FOSL1 / JUN , and FOSL2 . In addition, we identified DST , FRMD4A , and PREX2 as tumor markers for ccRCC with sarcomatoid differentiation, which were validated in a cohort study. Moreover, we determined that PREX2 played a malignant role in ccRCC with sarcomatoid differentiation in vitro and in vivo, facilitating tumor progression by inhibiting PTEN and activating the PI3K/AKT pathway. This study demonstrated the comprehensive gene expression and DNA regulation information of ccRCC with sarcomatoid differentiation, highlighting its malignant characteristics, thereby offering novel insights for the diagnosis and treatment of sarcomatoid ccRCC.

QPCR results after ASO (5717 and 5608) treatment of 2 lncRNAs in caki-2 and 786-O cell lines.Three or six PCR by-wells were prepared for each sample. NC, negative control.

QPCR results of the expression of 4 deferent lncRNA in cytoplasm and nucleus. Three PCR by-wells were prepared for each sample.