Pathway analysis revealed that TNRC6A predominantly regulates RNA processing and miRNA-mediated silencing, whereas TNRC6B modulates the mitogen-activated protein kinase and inflammatory responses, indicating that TNRC6A and TNRC6B function cooperatively and distinctly in miRNA-mediated gene regulation.
Abstract
Trinucleotide repeat-containing 6A (TNRC6A) and TNRC6B—members of the GW182 protein family—are essential components of the microRNA (miRNA)-induced silencing complex that post-transcriptionally regulate gene expression. Although both proteins interact with argonaute proteins to mediate miRNA-directed gene silencing, their distinct subcellular localizations and tissue-specific expression patterns indicate functional divergence. We compared their expression profiles across 19,616 samples from 54 tissues and demonstrated that TNRC6A exhibited a 4.1-fold higher mean transcript expression level than that of TNRC6B, with a moderate correlation (r = 0.686). Additionally, TNRC6A was predominantly expressed in the brain, reproductive tissues, and endocrine organs, whereas TNRC6B is expressed uniformly across most tissues. Knockout (KO) and rescue analyses revealed distinct regulatory targets—412 and 309 genes were upregulated in TNRC6A- and TNRC6B-KO cells, respectively, and 164 and 136 genes were downregulated in cells overexpressing TNRC6A and TNRC6B, respectively. Pathway analysis revealed that TNRC6A predominantly regulates RNA processing and miRNA-mediated silencing, whereas TNRC6B modulates the mitogen-activated protein kinase and inflammatory responses. Shared targets were associated with apoptosis and proteostasis, indicating that TNRC6A and TNRC6B function cooperatively and distinctly in miRNA-mediated gene regulation.
Background/Objectives: Long non-coding RNAs (lncRNAs) are important regulators of tumor biology through their interactions with DNA, proteins, and non-coding RNAs. Although ENST00000615487.1 (also known as CTD-2396E7.11/AC010503.4) has been associated with multiple malignancies, its biological role in colorectal cancer (CRC) remains poorly characterized. This study aimed to investigate the expression pattern, cellular and subcellular localization, and potential functional role of ENST00000615487.1 in CRC using integrated in vitro and in silico approaches. Methods: Molecular characteristics of the transcript were obtained with the CPC2 and RNA Analyzer 3 tools. Differential expression of ENST00000615487.1 across 10 tumor types was analyzed using the UCSC Xena Browser. Transcript expression was experimentally evaluated in normal, tumor, and fibroblastic colon cell lines by PCR, while subcellular localization was assessed through the lncATLAS, lncLocator, and iLoc-LncRNA tools, and experimentally confirmed by qRT-PCR. Single-cell RNA sequencing data from the GSE161277 dataset were analyzed to determine cell type-specific expression patterns. Potential interactions with DNA, miRNAs, and proteins were investigated using Fasim-LongTarget, miRDB, and AnnoLnc2, followed by functional enrichment analyses using STRING and Enrichr. Results: ENST00000615487.1 was identified as a structurally stable non-coding transcript with a highly organized secondary structure. Differential expression analysis demonstrated significant downregulation in CRC compared with that in normal colon tissue. Single-cell transcriptomic analysis revealed predominantly epithelial-specific expression. In silico and experimental analyses demonstrated predominant nuclear localization in normal colon cells, whereas cytoplasmic enrichment was observed in CRC cells. Functional analyses identified potential interactions with HIP1R, RPH3AL, specific miRNAs, and proteins involved in transcriptional regulation and RNA processing pathways, as well as functional connections with proteins involved in vesicular transport. Conclusions: ENST00000615487.1 is a structurally stable lncRNA exhibiting context-dependent expression and localization patterns in CRC, suggesting a potential shift from nuclear transcriptional regulation toward cytoplasmic post-transcriptional functions during colorectal carcinogenesis.
Nataša Đokić, Anastasija Bubanja, Jelena Karanović et al.· Non-Coding RNA· 0 citations
Ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2), an enzyme involved in extracellular nucleotide metabolism and purinergic signaling, has been linked to tumor–immune interactions, although its role in colorectal cancer (CRC) remains unclear. This study examined the expression pattern and regulatory context of ENTPD2 through integrative analysis of transcriptomic, proteomic, microRNA (miRNA), and single-cell transcriptomic datasets. Transcriptomic analyses showed that ENTPD2 mRNA levels are elevated in colorectal tumors compared with normal tissues and that higher expression is associated with shorter relapse-free survival. In contrast, proteomic analyses indicated reduced ENTPD2 protein abundance in tumor samples, suggesting a divergence between transcript and protein expression. Analysis of candidate miRNAs identified miR-708-5p as a potential post-transcriptional regulator, supported by its increased expression in CRC and a predicted binding site within the ENTPD2 3′-untranslated region (UTR). Single-cell transcriptomic datasets further indicated that ENTPD2 transcripts are mainly detected in malignant epithelial cells. We performed a functional validation using dual-luciferase reporter assays, qRT-PCR, and Western blot analysis in CRC cell lines. Experimental analyses demonstrated that miR-708-5p directly targets the ENTPD2 3′UTR in HCT116 cells and suppresses ENTPD2 expression in both HCT116 and HT-29 cells. These findings support a potential contribution of miR-708-5p to ENTPD2 regulation in CRC.
Leishmaniasis is a systemic zoonotic disease caused by Leishmania and can be fatal if left untreated. Recent evidence has shown that long non-coding RNAs (lncRNAs) play a crucial role in regulating the immune response to resist microbial infections. In this study, RNA-seq was performed to analyze the transcriptomic profiles of raw264.7 cells infected with Leishmania donovani ( L. donovani ) for 7.5 hours. A total of 50 differentially expressed (DE) lncRNAs and 287 DE mRNAs were identified, revealing the expression characteristics of lncRNAs in raw264.7 cells following L. donovani infection. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the predicted potentially associated genes of these lncRNAs were primarily enriched in pathways associated with host immune responses. Notably, NEAT1 and SNHG17, which have been reported to regulate inflammatory responses in multiple pathological conditions, were among the identified DE lncRNAs. These findings suggest that they may play important roles in the host response to L. donovani infection and could serve as potential targets for the prevention and treatment of leishmaniasis.
Grass carp (Ctenopharyngodon idella) reovirus (GCRV), the causative agent of hemorrhagic disease, causes substantial economic losses in the grass carp industry every year. However, the biological regulatory roles of long non-coding RNAs (lncRNAs) during GCRV infection remain largely unclear. In this study, high-throughput sequencing technology was used to analyze the expression profiles of lncRNAs in GCRV-infected and mock-infected liver and intestinal tissues of grass carp. In this study, compared with those in the control group, the transcriptomic profiles of grass carp after GCRV genotype II (HZ08 strain) infection revealed 742 upregulated and 1,259 downregulated lncRNA transcripts in the intestines, whereas 709 upregulated and 1,513 downregulated lncRNA transcripts were identified in the liver. GO and KEGG pathway analyses indicated that these lncRNAs were enriched primarily in pathways associated with biological process regulation, including "Pyruvate metabolism" and the "AMPK signaling pathway". Notably, we revealed that lncRNA50877 (PX600394) was upregulated in grass carp tissues and CIK cells following GCRV infection. CIK cells infected with equal titers of GCRV in vitro for 24 h exhibited aggravated cytopathic effect (CPE) upon lncRNA50877 overexpression. Furthermore, ChIRP-MS and RNA pull-down assays confirmed its direct interaction with KRT8 (keratin 8, PX531115). Our results demonstrated that lncRNA50877 disrupts mitochondrial homeostasis by negatively regulating KRT8 expression, thereby modulating ROS levels, apoptosis, and autophagy; promoting oxidative stress-induced cell injury; and providing favorable conditions for GCRV replication. In summary, lncRNA50877 may act as an autophagy-related lncRNA that regulates mitochondrial function and contributes to innate immune responses, offering new insights into the crosstalk between autophagy and innate immunity.
Yexuan Zhang, Shuai Liu, Zhiwei Sun et al.· Developmental and Comparativ...· 0 citations
ObjectiveTriple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer. HSPA8 is a chaperone protein involved in the proper folding or degradation of many proteins that are implicated in various types of cancers, while its role in TNBC remains unclear.MethodsIn this study, we extensively explored the cellular function and molecular targets of HSPA8 in TNBC cells by performing small interfering RNA (siRNA)-mediated knockdown, followed by cytological assays and transcriptome sequencing (RNA-seq).ResultsThe results of HSPA8 expression levels across distinct breast cancer subtypes indicated that TNBC exhibits higher HSPA8 expression. Then, cytological experiments demonstrated that knockdown of HSPA8 significantly inhibited TNBC cell proliferation, invasion and migration, and induced apoptosis. Mechanistically, RNA-seq showed that HSPA8 globally regulates gene expression and alternative splicing (AS), particularly modulating expression of genes in PI3K/AKT signaling pathway, and AS of genes in protein phosphorylation. Finally, we experimentally confirmed that knockdown of HSPA8 significantly suppressed the activation of the PI3K/AKT axis and AKT phosphorylation in TNBC cells.ConclusionsOur findings highlight an important pro-oncogenic role of HSPA8 in TNBC progression, mediated in part through transcriptomic remodeling and activation of the PI3K/AKT signaling pathway. These results may inform the development of novel targets and strategies for future targeted therapy in TNBC.
Wenting Xu, Yongtao Li, Hu Wang et al.· Science in progress· 0 citations
Timely and efficient degradation of maternal mRNA is essential for early embryonic development, which occurs from fertilization through the initiation of zygotic genome activation (ZGA). Yet, the regulatory mechanisms governing this process remain poorly characterized. In the present study, we investigated the function of CCR4-NOT transcription complex subunit 1 (CNOT1) during goat embryogenesis. We found that CNOT1 was upregulated during mammalian ZGA, and that its knockdown led to developmental arrest and a marked reduction in blastocyst formation. Moreover, CNOT1 knockdown impaired nascent RNA activity, resulting in 814 upregulated and 1014 downregulated genes, which were enriched for RNA splicing, regulation of chromosome organization, and RNA localization. RNA splicing analysis revealed differential splicing events in 2959 genes, of which 259 were downregulated following CNOT1 knockdown. Notably, CNOT1 was predicted to crosstalk with the m6A reader YTHDF2. Knockdown of YTHDF2 resulted in CNOT1 downregulation at the 8-cell stage in goats and increased transcription levels around polyadenylation sites during ZGA in mice. Together, these findings indicate that CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and ZGA during goat embryogenesis. Our work provides new insight into the complex regulatory landscape underlying ZGA and may inform strategies to improve the efficiency of goat embryogenesis.