Revealing Cellular Heterogeneity and Key Regulatory Factors of Triple-Negative Breast Cancer through Single-Cell RNA Sequencing.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). TNBC has a poor prognosis due to high intratumoral heterogeneity and metastasis, pointing to the need to explore distinct molecular subtypes and gene regulatory networks. METHODS: The scRNA-seq data of five primary BC samples were downloaded from the Gene Expression Omnibus (GEO) database. Clustering was performed based on filtered and normalized data using the Seurat R package to identify marker genes, which were subsequently annotated to each subset using the CellMarker database. AUCell R package was applied to calculate the hallmark score for each epithelial cell. Marker genes of each subset were screened with FindAllMarkers and their biological functions were analyzed using the Database for Annotation Visualization and Integrated Discovery (DAVID) database. Next, cell-cell communication was performed with the CellChat R package. To identify the key regulatory genes, single-cell regulatory network inference and clustering (SCENIC) analysis was conducted. Finally, the expression and potential biological functions of the key regulatory factors were verified through cellular experiments. RESULTS: A total of 29,101 cells were classified into nine cell subsets, namely, Fibroblasts, Fibroepithelial cells, Epithelial cells 1, Epithelial cells 2, Epithelial cells 3, Endothelial cells, T cells, Plasma B cells and Macrophages. Particularly, the epithelial cells had a higher proportion and higher transforming growth factor-ß (TGF-ß) activity in the TNBC pathotype as compared to the non-TNBC pathotype. Furthermore, four epithelial cell subsets (marked as Stearoyl-CoA Desaturase (SCD1), marker of proliferation Ki67 (MKI67), Annexin A3 (ANXA3) and aquaporin 5 (AQP5)) were identified as having the greatest impact on the TNBC pathotype. Cell-cell interaction analysis revealed that ANXA3-epithelial cell subset suppressed the T cell function through different mechanisms. C-fos gene (FOS) and X-box binding protein 1 (XBP1) were considered critical regulons involved in TNBC progression. Notably, cellular experiments demonstrated that silencing XBP1 and overexpressing FOS inhibited cancer cell invasion. CONCLUSION: The four epithelial cell subsets and two critical regulons identified based on the scRNA-seq data could help explore the underlying intratumoral heterogeneity molecular mechanism and develop effective therapies for TNBC.

The role of C5aR1-mediated hepatic macrophage efferocytosis in NASH.

Non-alcoholic fatty liver disease (NAFLD) has become the first major chronic liver disease in developed countries. 10-20% of NAFLD patients will progress to non-alcoholic steatohepatitis (NASH), and up to 25% of NASH patients may develop cirrhosis within 10 years. Therefore, it is critical to find key targets that may treat this disease. Here, we identified C5aR1 as a highly-expressed gene in NASH mouse model through analyzing Gene Expression Omnibus (GEO) database and confirmed its higher expression in livers of NASH patients than that of NAFL patients. Meanwhile, we verified its positive correlation with patients' serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. In vivo and in vitro experiments revealed that knocking down C5aR1 in liver significantly reduced liver weight ratio and serum ALT and AST levels and attenuated inflammatory cell infiltration and cell apoptosis in the liver of NASH mice as well as enhanced the efferocytotic ability of liver macrophages, suggesting that C5aR1 may play a crucial role in the efferocytosis of liver macrophages. Furthermore, we also found that the expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3), caspase-1, IL-1ß and other inflammation-related factors in the liver were significantly reduced. Our work demonstrates a potential mechanism of how C5aR1 deficiency protects against diet-induced NASH by coordinating the regulation of inflammatory factors and affecting hepatic macrophage efferocytosis.

Integrated Analysis of C16orf54 as a Potential Prognostic, Diagnostic, and Immune Marker across Pan-Cancer.

Chromosome 16 open reading frame 54 (C16orf54) is a protein coding gene, showing a biased expression in the bone marrow, lymph node, and 11 other tissues. Reports on the function of C16orf54 in the onset and development of tumours remain scarce. Clinical information and tumour expression profile data from The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), and Genotype-Tissue Expression (GTEx) were utilized to determine the relationship between C16orf54 expression and prognosis, diagnosis, immune microenvironment, heterogeneity, and stemness across pan-cancer. The findings ascertained that C16orf54 was expressed at a low level in most cancers. Furthermore, C16orf54 could distinguish between cancer and normal tissues with high accuracy in most cancers, and the prognostic significance of low C16orf54 mRNA levels differs across cancers. C16orf54 expression was positively linked to the stromal, immune, and ESTIMATE scores. On the other hand, C16orf54 was reported to be negatively correlated with tumour purity in most cancers. Further, C16orf54 expression was positively correlated with immune cell infiltration and the expression of immune regulatory genes, including chemokines, receptors, major histocompatibility complexes, immune inhibitory, and immune stimulatory genes, in most cancers. Additionally, C16orf54 expression was significantly associated with tumour heterogeneity indicators, such as tumour mutation burden (TMB) and microsatellite instability (MSI), and was significantly correlated with DNAss and RNAss tumour stemness indicators. Moreover, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis, as well as Gene Set Enrichment analysis (GSEA), revealed that C16orf54 expression was closely linked to the signalling pathways of immune cells and factors. The integrated analysis of C16orf54 indicates it as a potential prognostic, diagnostic, and immune marker, which could be adopted as a novel target for adjuvant immunotherapy across pan-cancer.

PSMD12 promotes breast cancer growth via inhibiting the expression of pro-apoptotic genes.

Breast cancer (BC), the most frequent cancer in women worldwide, is extremely heterogeneous. For effective and precise treatment and to cope with drug resistance in BC, we need to find more therapeutic molecular targets. In this study, we found that the Proteasome 26S Subunit, Non-ATPase 12 (PSMD12) was upregulated in BC samples, its expression was heterogeneous among different cell lines, and high levels of PSMD12 were related to poor prognosis of BC patients. Notably, the expression of PSMD12 increased in the nucleus. Cytological experiments revealed that PSMD12 knockdown inhibited cell growth and migration, and a genome-wide CRISPR-Cas9 knockout (GeCKO) screen also confirmed that PSMD12 is a crucial gene for the growth of BC cells. Flow cytometry showed that cell apoptosis increased in the PSMD12 knockdown, and RNA-seq indicated that the apoptosis pathway was activated, and the TXNIP, GADD45A, GADD45B, RHOB, and CDKN1A pro-apoptotic genes were highly expressed, a result that was validated by RT-qPCR and Western blot. Furthermore, restoration of PSMD12 expression decreased the expression of pro-apoptotic genes. A tumor-bearing mice assay demonstrated that BC growth was arrested by reduced PSMD12 levels in vivo. Taken together, PSMD12, a subunit of 19S regulator of 26S proteasome, was identified as a potential prognostic and therapeutic molecular target for BC, which provides a new insight for developing anticancer drugs that promote apoptosis based on the targeting of the 26S proteasome complex.

Identification of Differently Expressed Genes Associated With Prognosis and Growth in Colon Adenocarcinoma Based on Integrated Bioinformatics Analysis.

Latest statistics showed that the morbidity and mortality of colon adenocarcinoma (COAD) ranked fourth and fifth, respectively, around the world. COAD was a heterogeneous disease, and the high rates of recurrence, metastasis, and drug resistance still posed great challenges for treatment, which needs to further develop therapeutic and prognostic targets. In this study, we got the top 3,075 differentially expressed genes (DEGs) and 1,613 potential prognostic genes by GEPIA 2 and identified 1,166 fitness genes in COAD based on genome-scale CRISPR-Cas9 knockout (GeCKO) screening data. Excluding the genes already reported in the literatures, a total of nine DEGs overlapping with prognostic and fitness genes were further analyzed. High expression of CCT6A, RHOQ, and RRP12 promoted COAD cell growth and were relative to lower survival rate of COAD patients, while high expression of UTP18, DDOST, YRDC, ACTG1, RFT1, and NLE1 also promoted COAD cell growth, but were relative to higher survival rate. In addition, CCT6A, UTP18, YRDC, RRP12, RFT1, NLE1, as well as DDOST were essential genes across pan-cancer including COAD cells, and ACTG1 and RHOQ were less essential genes in cancer cells. In a word, we discovered nine novel potential genes that could serve as anticancer targets and prognostic markers in COAD and its subtypes.