In recent years, non-coding RNAs—especially microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)—have emerged as powerful regulators of cellular behavior. These molecules modulate gene expression, often by targeting mRNAs for translational suppression or degradation. Two recent studies—one focused on osteoarthritis and the other on 5-Fu-resistant colorectal cancer—illustrate how these non-coding, regulatory RNAs operate within disease-relevant signaling networks, providing new points for therapeutic intervention.

Both studies use the pmirGLO Dual-Luciferase miRNA Target Expression Vector to evaluation predicted miRNA activity.  This dual-luciferase system offers a clean and quantifiable way to validate miRNA–mRNA interactions using a simple bioluminescent readout. By cloning the 3´ untranslated regions (UTRs) of suspected targets downstream of a firefly luciferase reporter and normalizing against Renilla luciferase, researchers can rapidly confirm whether a miRNA directly regulates its target.

Study One: Rewiring Autophagy in Osteoarthritis

In a model of inflammatory osteoarthritis, Li et al. (2025) found that miR-103-3p was upregulated in chondrocytes exposed to interleukin-1β, promoting apoptosis and extracellular matrix degradation—key features of cartilage destruction. The authors suspected that miR-103-3p might act by repressing CPEB3, a translational regulator. They cloned the wild-type and mutant 3′UTRs of CPEB3 into the pmirGLO vector and co-transfected them with miR-103-3p mimics into chondrocytes.  Li et al. observed that firefly luciferase activity dropped when the wild-type UTR was present, but not when mutations disrupted the binding site, providing strong evidence that miR-103-3p directly targets CPEB3. Mechanistically, this interaction activated the PI3K/Akt/mTOR pathway, suppressed autophagy, and exacerbated cartilage damage. Importantly, blocking miR-103-3p reversed these effects, suggesting a novel therapeutic angle.

Study Two: Cracking 5-Fu Resistance in Colorectal Cancer

In a separate but conceptually related study, Lu (2025) explored how colorectal cancer cells evade the cytotoxic effects of 5-fluorouracil (5-Fu). NOP14-AS1, a long non-coding RNA, was observed to be overexpressed in 5-Fu-resistant cells. Bioinformatics analyses predicted that NOP14-AS1 contained a binding site for the miR-30a-5p, a miRNA predicted to bind to LDHA gene 3´UTR; LDHA is key enzyme in glycolysis that catalyzes the conversion of pyruvate to lactate. LDHA expression is observed to be upregulated in several cancer types including the 5-Fu resistant colorectal cancer cell model, and glucose uptake and lactate production were observed to be remarkedly increased these cells. Confirmation of the predicted miRNA binding sites were obtained by luciferase assays and RNA pull-down assays. For the luciferase assays, either the 3′UTR of LDHA or the lncRNA sequence of NOP14-AS1 was inserted downstream of the luciferase gene in the pmirGLO vector These results suggest a model where NOP14-AS1 acts as a molecular sponge formiR-30a-5p, decreasing its ability to regulate LDHA expression in 5-Fu-resistant cells. Overexpression of miR-30a-5p suppressed LDHA levels, dampened glycolysis, and restored drug sensitivity. This lncRNA-miRNA-mRNA axis (NOP14-AS1 → miR-30a-5p → LDHA) revealed a metabolic checkpoint exploitable for therapy.

Read more about the connection between cellular energy and metabolism in cancer cells.

Summary

Both studies followed a workflow that began with an observed functional phenotype (apoptosis, drug resistance). Candidate non-coding RNAs that might be involved were predicted using bioinformatics tools. 3´ UTRs of potential target genes were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector to validate the interactions, and overexpression and inhibition experiments were used to confirm the downstream effects of the disruption of non-coding RNA regulation of gene expression in disease models, providing insight into potential therapeutic targets.

Learn more about solutions for RNA targeting therapeutics at Promega.com

Literature Cited

1. Li J. et al. (2025) MiR-103-3p regulates chondrocyte autophagy, apoptosis, and ECM degradation through the PI3K/Akt/mTOR pathway by targeting CPEB3. J Orthop Surg Res. 20:324. DOI
2. Lu YN. (2025) Blocking lncRNA NOP14-AS1 overcomes 5-Fu resistance of colon cancer cells by modulating miR-30a-5p-LDHA-glucose metabolism pathway. Discov Oncol. 16:458. DOI

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Michele Arduengo

Supervisor, Digital Marketing Program Group at Promega Corporation

Michele earned her B.A. in biology at Wesleyan College in Macon, GA, and her PhD through the BCDB Program at Emory University in Atlanta, GA where she studied cell differentiation in the model system C. elegans. She taught on the faculty of Morningside University in Sioux City, IA, and continues to mentor science writers and teachers through volunteer activities. Michele manages the digital marketing program team at Promega.

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