In the vast world of molecular biology, there is a class of genetic players known as long non-coding RNAs (lncRNAs). These molecules, typically spanning over 200 nucleotides in length, make up a huge part of our genome and differ from our traditional genes—they don’t make proteins. Despite this, they have emerged as pivotal regulators in various biological processes, influencing gene expression through diverse mechanisms (i.e., chromatin remodeling, transcriptional and translational regulation, RNA and protein stability modulation, RNA alternative splicing, and protein localization). Recently, scientists have been intrigued by how these lncRNAs may be involved in the progression of diseases like cancer in response to environmental stressors like heavy metals.
The Symposium Session “Long Non-coding RNA Dysregulations in Metal Toxicity and Carcinogenesis” aimed to highlight cutting-edge science looking at this intricate interplay between lncRNAs, metal toxicity, and carcinogenesis. With a focus on arsenic, cadmium, and chromium, the speakers delved into the exciting new frontier of how certain non-coding RNAs might go haywire when our bodies encounter these metals, giving us a peek into the tiny but mighty molecules that may govern our wellbeing.
Xenobiotic Responsive Hepatic lncRNAs
Dr. David J. Waxman, a Professor of biology, medicine, and biomedical engineering at Boston University, kicked off the session by highlighting hepatic lncRNAs responsive to xenobiotics—chemical compounds not typically found in the body—underscoring their role in the liver’s response to environmental pollutants. In Dr. Waxman’s talk, titled “Xenobiotic-Responsive Hepatic Long Non-coding RNAs: Dysregulated Expression, Liver Zonation, and Intercellular Communication within the Liver Lobule at the Single Cell Level,” he unveiled several intriguing findings
Firstly, Dr. Waxman discussed the discovery of a comprehensive set of murine liver lncRNAs (about 48,000!), which served as a blueprint for scrutinizing single cell transcriptomes of mouse livers, sex-specific expression patterns, and responses to xenobiotics. Moreover, he was able to characterize 30,000 of them which can serve to identify tissue and cell specificity, which is crucial for clarifying their functional roles and potential implications in disease progression.
Dr. Waxman elaborated on how 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dysregulated the expression and zonation of hundreds of lncRNAs across the liver lobule, notably linked to liver fibrosis and nonalcoholic steatohepatitis (NASH) progression. He also found that TCDD perturbed numerous nuclear receptors (NR) and transcription factors and single cell analysis also unveiled alterations in cell-to-cell communication following TCDD exposure.
Dr. Waxman also identified 60 master regulatory lncRNAs governing both healthy and fibrotic livers through gene regulatory network analysis. Many of these lncRNA gene targets showed significant enrichment for specific biological pathways, confirming the regulatory networks established from intricate single-cell transcriptomic datasets.
Although not covered during the session due to time constraints, Dr. Waxman introduced the notion of RNA:DNA triplex binding. This refers to a molecular interaction where a single-stranded RNA molecule binds to the double-stranded DNA in a specific region, forming a three-stranded structure. This interaction occurs when the RNA molecule pairs with the complementary DNA sequence, usually in the major groove of the DNA helix. Triplex formation can influence gene expression by affecting the accessibility of the DNA to other molecules involved in transcription and regulation. In the context of lncRNAs, RNA:DNA triplex binding can serve as a mechanism for regulating gene expression and controlling cellular processes, and can serve in predicting regulatory interactions between lncRNAs and their target genes, providing a roadmap for future experimental validation in the lab.
Notably, his research not only shed light on the intricate workings of hepatic lncRNAs in response to xenobiotics but also set the stage for future studies in this burgeoning field of lncRNA research. His discoveries pave the way for deeper exploration into how lncRNAs orchestrate the liver’s response to foreign substances.
Role of lncRNAs in Chromium-Induced Lung Carcinogenesis
The next speaker, Dr. Zhishan Wang is a Professor of research at the Renaissance School of Medicine at Stony Brook University and presented her research on the mechanisms of lncRNA involvement in hexavalent chromium (Cr (VI))–induced lung cancer. In Dr. Wang’s talk, titled “Long Non-coding RNA ABHD11-AS1 Interacts with Spliceosome-Associated Factor 3 to Promote Hexavalent Chromium Carcinogenesis,” she revealed several findings pertaining to the important role of this lncRNA in Cr (VI) carcinogenesis.
Dr. Wang opened her talk by highlighting the environmental ubiquity of Cr (VI)—a group one carcinogen and the most toxic form of chromium (Cr). She emphasized the risk of Cr exposure in occupational workers and certain communities living near hazard sites and discussed additional sources of exposure, which include cigarette smoke and contaminated water.
The lncRNA ABHD11-AS1 was the focus of her study as it has been found to be upregulated in 10 different types of cancer; however, the mechanism of this lncRNA in lung carcinogenesis is largely unknown. In her research, she has found that ABHD11-AS1 expression levels are upregulated in Cr (VI)-transformed cells and Cr (VI)-exposed mouse cells. Additionally, in knock-out (KO) experiments of ABHD11-AS1 in Cr (VI)-transformed cells, Dr. Wang found the absence of this lncRNA reduces the cell’s tumor formation activity. More excitingly, a ABHD11-ASI KO in non-transformed cells can prevent the effects of Cr (VI) exposure. Moreover, KO of ABHD11-ASI in lung cancer cells reduces their cancer stem cell–like property and tumor growth.
Mechanistically, Dr. Wang discovered that lncRNA ABHD11-AS1 directly binds to SART3 (spliceosome associated factor 3, U4/U6 recycling protein) and affects CD44 alternative splicing. To explain, this process is like editing a movie where unnecessary scenes are removed and the remaining ones are pieced together to create the final film. In gene expression, RNA splicing works similarly: it removes non-coding regions (called introns) from a molecule called pre-mRNA and stitches together the coding regions (called exons) to form mRNA, which contains the instructions for making proteins in our cells.
What’s fascinating is that RNA splicing isn’t just a simple cut-and-paste job. It’s more like mixing and matching different scenes to create alternate versions of the movie. This is called alternative splicing. By including or excluding different exons, cells can make different protein “versions” from the same gene, akin to obtaining multiple movies from the same footage. This diversity increases the range of proteins our cells can produce.
In Dr. Wang’s research, she observed that the binding of ABHD11-AS1 and SART3 prompts the nuclear localization (meaning to move into the cell’s nucleus) of USP15 (ubiquitin specific peptidase 15). Once in the nucleus, USP15 interacts with pre-mRNA processing factor 19 (PRPF19), thereby promoting CD44 RNA alternative splicing, thus affecting how the CD44 gene is processed. Consequently, this splicing leads to activation of β-catenin and enhances cancer stemness. In conclusion, these findings suggest that lncRNA ABHD11-AS1's interaction with SART3 regulates CD44 RNA alternative splicing, promoting cell malignant transformation and lung carcinogenesis.
circRNAs in Arsenic-Mediated Carcinogenesis:
Dr. Yvonne Fondufe-Mittendorf, an epigenetics expert at the Van Andel Institute in the Department of Epigenetics, concluded the Symposium with a fascinating talk about circular RNAs (circRNAs). circRNAs are a distinct class of non-coding RNAs (ncRNAs), just like lncRNAs. While circRNAs and lncRNAs share similarities in their roles and functions within cells, they are fundamentally different in their structure and formation. circRNAs are characterized by their circular structure, formed through a backsplicing process where a downstream splice donor site joins with an upstream splice acceptor site, forming a closed loop. Unlike circRNAs, lncRNAs are linear RNA molecules that do not form a closed loop structure.
In Dr. Fondufe-Mittendorf’s talk, titled “Unraveling the Role of the circSATB2 Gene Regulatory Nexus in the iAs-Mediated Carcinogenesis,” she introduced the audience to widespread contamination of arsenic (As) in water, food, and coal mining industrial areas. Inorganic arsenic (iAs) in particular is linked to higher lung cancer rates, and while iAs does not directly alter our DNA, it can influence gene expression to promote carcinogenesis. In Dr. Fondufe-Mittendorf’s work, she studies ncRNAs linked to SATB proteins, crucial for proper gene regulation during cellular differentiation and susceptible to disruption by iAs exposure.
SATB2 is a gene that can regulate transcription by reorganizing chromatin and influencing gene expression. Depending on the cell type and gene environment, SATB2 can act either as an oncogene or tumor suppressor. Recently, Dr. Fondufe-Mittendorf discovered that during iAs-induced carcinogenesis, SATB2 is upregulated and generates a novel circRNA called circSATB2. This circRNA may interact with a specific microRNA, providing protection to linear SATB2 transcripts.
In KO cell experiments, Dr. Fondufe-Mittendorf observed that SATB2 expression recapitulates oncogenic KRAS signatures induced by iAs exposure. However, KO of both SATB2 and circSATB2 reversed these oncogenic signatures. Additionally, the overexpression of SATB2 is associated with a poor prognosis in lung adenocarcinoma cancers. Finally, it was discussed that the circSATB2 RNA is translated into a peptide that may lead to irregular SATB2 function. These findings have significant implications for understanding gene expression dysregulation and circRNA roles in iAs-induced lung carcinogenesis.
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