Sohn KM, et al. J Korean Med Sci. 2020 Aug;35(38):e343. https://doi.org/10.3346/jkms.2020.35.e343

The selection of reference genes is essential for quantifying gene expression. Theoretically they should be expressed stably and not regulated by experimental or pathological conditions. However, identification and validation of reference genes for human cancer research are still being regarded as a critical point, because cancerous tissues often represent genetic instability and heterogeneity. Recent pan-cancer studies have demonstrated the importance of the appropriate selection of reference genes for use as internal controls for the normalization of gene expression; however, no stably expressed, consensus reference genes valid for a range of different human cancers have yet been identified. In the present study, we used large-scale cancer gene expression datasets from The Cancer Genome Atlas (TCGA) database, which contains 10,028 (9,364 cancerous and 664 normal) samples from 32 different cancer types, to confirm that the expression of the most commonly used reference genes is not consistent across a range of cancer types. Furthermore, we identified 38 novel candidate reference genes for the normalization of gene expression, independent of cancer type. These genes were found to be highly expressed and highly connected to relevant gene networks, and to be enriched in transcription-translation regulation processes. The expression stability of the newly identified reference genes across 29 cancerous and matched normal tissues were validated via quantitative reverse transcription PCR (RT-qPCR). We reveal that most commonly used reference genes in current cancer studies cannot be appropriate to serve as representative control genes for quantifying cancer-related gene expression levels, and propose in this study three potential reference genes (HNRNPL, PCBP1, and RER1) to be the most stably expressed across various cancerous and normal human tissues.

Background: Lately, high-throughput RNA sequencing has been extensively used to elucidate the transcriptome landscape and dynamics of cell types of different species. In particular, for most non-model organisms lacking complete reference genomes with high-quality annotation of genetic information, reference-free (RF) de novo transcriptome analyses, rather than reference-based (RB) approaches, are widely used, and RF analyses have substantially contributed toward understanding the mechanisms regulating key biological processes and functions. To date, numerous bioinformatics studies have been conducted for assessing the workflow, production rate, and completeness of transcriptome assemblies within and between RF and RB datasets. However, the degree of consistency and variability of results obtained by analyzing gene expression levels through these two different approaches have not been adequately documented.Results: In the present study, we evaluated the differences in expression profiles obtained with RF and RB approaches and revealed that the former tends to be satisfactorily replaced by the latter with respect to transcriptome repertoires, as well as from a gene expression quantification perspective. In addition, we urge cautious interpretation of these findings. Several genes that are lowly expressed, have long coding sequences, or belong to large gene families must be validated carefully, whenever gene expression levels are calculated using the RF method.Conclusions: Our empirical results indicate important contributions toward addressing transcriptome-related biological questions in non-model organisms.

Neutropenic sepsis is a fatal consequence of chemotherapy, and septic complications are the principal cause of mortality. Chemotherapy-induced neutropenia leads to the formation of microscopic ulcers in the gastrointestinal epithelium that function as a portal of entry for intraluminal bacteria, which translocate across the intestinal mucosal barrier and gain access to systemic sites, causing septicemia. A cyclophosphamide-induced mouse model was developed to mimic the pathophysiologic sequence of events that occurs in patients with neutropenic sepsis. The TLR5 agonist bacterial flagellin derived from Vibrio vulnificus extended the survival of cyclophosphamide-treated mice by reducing the bacterial load in internal organs. The protective effect of flagellin was mediated by the antimicrobial protein lipocalin 2 (Lcn2), which is induced by TLR5-NF-κB activation in hepatocytes. Lcn2 sequestered iron from infecting bacteria, particularly siderophore enterobactin-dependent members of the Enterobacteriaceae family, thereby limiting their proliferation. Lcn2 should be considered for the treatment of neutropenic sepsis and gastrointestinal damage during chemotherapy to prevent or minimize the adverse effects of cancer chemotherapy.

The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3) is an epigenetic player that modulates chromatin accessibility to activate p53 during liver injury. Nonetheless, a precise tumor suppressive and epigenetic role of NR2E3 in hepatocellular carcinoma (HCC) development remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/β‐catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice consistently exhibits accelerated liver tumor formation accompanied by enhanced activation of Wnt/β‐catenin signaling pathway and inactivation of p53 signaling. At cellular level, the loss of NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates key genes in the WNT/β‐catenin pathway with increased chromatin accessibility. This event is mediated through increased formation of active transcription complex involving Sp1, β‐catenin, and p300, a histone acetyltransferase, on the promoters of target genes. These findings demonstrate that the loss of NR2E3 activates Wnt/β‐catenin signaling at cellular and organism levels and this dysregulation is associated with aggressive HCC development and poor clinical outcomes. In summary, NR2E3 is a novel tumor suppressor with a significant prognostic value, maintaining epigenetic homeostasis to suppress the Wnt/β‐catenin signaling pathway that promotes HCC development.

Cephalization refers to the evolutionary trend towards the concentration of neural tissues, sensory organs, mouth and associated structures at the front end of bilaterian animals. Comprehensive studies on gene expression related to the anterior formation in invertebrate models are currently lacking. In this study, we performed de novo transcriptional profiling on a proboscis-bearing leech ( Helobdella austinensis ) to identify differentially expressed genes (DEGs) in the anterior versus other parts of the body, in particular to find clues as to the development of the proboscis. Between the head and the body, 132 head-specific DEGs were identified, of which we chose 11 to investigate their developmental function during embryogenesis. Analysis of the spatial expression of these genes using in situ hybridization showed that they were characteristically expressed in the anterior region of the developing embryo, including the proboscis. Our results provide information on the genes related to head formation and insights into the function of proboscis-related genes during organogenesis with the potential roles of genes not yet characterized.