circ_0029343 promotes malignant phenotypes in clear cell renal cell carcinoma and is associated with miR-490-5p/NAT10 signaling and pyroptosis-related pathways.

Background

Clear cell renal cell carcinoma (ccRCC) is a highly metastatic and aggressive form of kidney cancer with limited treatment options and poor patient outcomes. CircRNAs are key regulators in tumor development, but the specific function of circ_0029343 in ccRCC has not been fully elucidated. The objective of this study was to determine the functional significance and regulatory pathways of circ_0029343 in ccRCC.

Methods

The expression of Circ_0029343 was examined in ccRCC tissues and cells using qRT-PCR assay. To validate circ_0029343 as a circular RNA, we confirmed the back-splice junction, assessed RNase R resistance and transcript stability, and determined subcellular localization. Clinical associations were evaluated using chi-square tests, univariate and multivariable logistic regression for postoperative recurrence/metastasis, and a nomogram model with bootstrap calibration and ROC/AUC assessment. Functional assays, including Cell Counting Kit-8 (CCK-8), Transwell invasion, and immunofluorescence staining, were performed to assess the effects of Circ_0029343 knockdown on proliferation, invasion, and epithelial-mesenchymal transition (EMT). Dual-luciferase and RNA pull-down assays were conducted to confirm the interaction between Circ_0029343 and miR-490-5p. The downstream target of miR-490-5p, NAT10, was validated through bioinformatics and functional analyses. The impact of circ_0029343 on pyroptosis-associated signaling was assessed by measuring NLRP3, cleaved caspase-1, IL-1β, and gasdermin D (GSDMD-N) expression levels, LDH release, and phase-contrast microscopy to evaluate pyroptosis-like lytic cell death characteristics. Additional evaluation of inflammasome/caspase-1-dependent activation of the caspase-1/GSDMD axis involved caspase-1 inhibition rescue (VX-765) and time-course analyses of cleaved caspase-1 and GSDMD-N. A xenograft model was utilized to assess tumor growth in vivo.

Results

High circ_0029343 expression was also associated with postoperative recurrence/metastasis risk, and a multivariable logistic regression-based nomogram demonstrated good discrimination (AUC = 0.835) with satisfactory calibration. Circ_0029343 exhibited a back-splice junction, RNase R resistance, greater stability than linear SCARB1 transcripts, and cytoplasmic localization. Knockdown of circ_0029343 suppressed proliferation, invasion, and EMT, while promoting pyroptosis-associated signaling and lytic cell death characteristics indicative of pyroptosis, demonstrated by heightened LDH release, activation of the NLRP3/caspase-1/GSDMD pathway, and pyroptosis-like morphology. In vitro, circ_0029343 functioned as a ceRNA for miR-490-5p, correlating with elevated NAT10 levels, thus implicating the miR-490-5p/NAT10 axis in malignant characteristics. In a Caki-1 subcutaneous xenograft model, circ_0029343 knockdown decreased tumor growth and was accompanied by changes in miR-490-5p/NAT10 and pyroptosis-related markers.

Conclusions

Circ_0029343 promotes ccRCC malignant phenotypes and is associated with pyroptosis-related signaling; in vitro data support that circ_0029343 may exert these effects, at least in part, through miR-490-5p/NAT10 signaling.