Pancreatic ductal adenocarcinoma (PDAC) remains a highly aggressive malignancy with limited effective therapies. Plant-derived exosome-like nanovesicles (ELNs) have emerged as natural nanocarriers able to cross kingdoms and deliver biologically active molecules. The functional cargos of plant ELNs and their mechanistic effects in PDAC, however, are not well defined. This study examined ELNs from Tetrastigma hemsleyanum to identify active small RNA cargos and characterize their effects on pancreatic cancer cell proliferation.
ELNs were isolated from T. hemsleyanum and characterized using nanoparticle tracking analysis and zeta potential measurements to assess particle size distribution and surface charge. Cellular uptake of the plant ELNs by pancreatic cancer cells was evaluated using PKH67 fluorescent labeling, demonstrating efficient internalization. The study used in vitro cell models to test biological activity after ELN uptake.
Small RNA sequencing of the isolated ELNs identified several small RNAs associated with the nanovesicles. Follow-up RT-qPCR validated that miR-396b was highly enriched among ELN-associated miRNAs, designating it as a candidate functional cargo mediating biological effects on recipient pancreatic cancer cells.
Biological effects were assessed using CCK-8 cell viability assays and colony formation assays. Treatment of pancreatic cancer cells with T. hemsleyanum ELNs inhibited cell proliferation in a dose-dependent manner. Similarly, experimental overexpression of miR-396b in pancreatic cancer cells produced a significant anti-proliferative effect comparable to that of ELN treatment. These findings indicate that ELN-delivered small RNAs, particularly miR-396b, contribute to the growth-suppressive activity observed in vitro.
To identify downstream effectors of miR-396b, the authors performed transcriptomic profiling and focused on interleukin 33 (IL33) as a candidate target. Both ELN treatment and miR-396b overexpression reduced IL33 expression at the mRNA and protein levels as measured by RT-qPCR and immunoblotting. Bioinformatic prediction identified a putative miR-396b binding site within the 3' untranslated region (3'UTR) of IL33. Dual-luciferase reporter assays showed that miR-396b suppressed reporter activity driven by the wild-type IL33 3'UTR, whereas mutation of the predicted binding site abolished the suppression. These results provide mechanistic evidence that miR-396b directly binds and represses IL33 expression.
The study performed functional rescue experiments to test the contribution of the miR-396b/IL33 interaction to the anti-proliferative phenotype. Inhibition of miR-396b lessened the growth suppression mediated by ELN treatment, indicating that miR-396b is a biologically relevant ELN cargo in this context. Conversely, re-expression of IL33 in cells overexpressing miR-396b partially reversed the anti-proliferative effect of miR-396b, supporting the idea that IL33 downregulation mediates a substantial portion of the growth-inhibitory activity. Together, these experiments substantiate a functional miR-396b/IL33 regulatory axis underlying ELN activity in pancreatic cancer cells.
This work identifies miR-396b as a major functional cargo of T. hemsleyanum-derived ELNs and demonstrates that ELN-delivered miR-396b suppresses pancreatic cancer cell proliferation, at least in part by direct repression of IL33. The findings characterize a cross-kingdom ELN–miRNA–IL33 regulatory pathway and support the potential of plant-derived ELNs as a bioactive nanoplatform for PDAC intervention.
Limitations reported by the authors include that additional ELN cargos were not fully explored, and the study did not include in vivo validation; both areas were noted as warranting further investigation. The experiments described are limited to in vitro cellular models and molecular assays (small RNA sequencing, RT-qPCR, immunoblotting, dual-luciferase reporter assays, and rescue experiments). No clinical or animal data were reported in the source, and details such as specific cell lines, exact ELN concentrations, sequencing read counts, or statistical values were not provided in the abstract.
Future work recommended by the authors includes broader profiling of ELN cargo beyond miR-396b and rigorous in vivo studies to assess therapeutic potential, biodistribution, safety, and efficacy of T. hemsleyanum ELNs and delivered miR-396b in PDAC models.
Overall, the study provides mechanistic in vitro evidence that plant-derived ELNs can deliver functional microRNA cargo to human cancer cells and that miR-396b-mediated suppression of IL33 contributes to reduced PDAC cell proliferation. These preclinical findings generate hypotheses for follow-up validation in animal models and for exploration of other ELN components that may have cooperative or independent bioactivity.