A long noncoding RNA called KCNMB2-AS1 acts as a powerful oncogenic driver in clear cell renal cell carcinoma, the most common form of kidney cancer, according to a new study published in the journal Cell Division. The research, led by Geng Huang, Dingwen Gui, Yankuang Guo, Shuai Luo, and Zuwei Xu, reveals that the RNA is activated by a transcription factor known as FOXP3 and promotes tumor progression through a molecular pathway involving microRNA-744-3p and the protein CD1D. The findings offer a previously unrecognized regulatory axis that may contribute to the development of new biomarkers and therapeutic strategies for a disease that accounts for roughly 400,000 new cases and nearly 175,000 deaths worldwide each year.
Clear cell renal cell carcinoma, or ccRCC, represents about 70 percent of all renal cell carcinoma cases. Although radical or partial nephrectomy remains the primary treatment for patients with localized or early-stage disease, up to 30 percent of individuals eventually develop distant metastases or local recurrence following surgery, even with the integration of multimodal therapies such as chemotherapy, radiotherapy, and targeted treatments. Major risk factors contributing to RCC development include chronic kidney disease, hypertension, obesity, and smoking. Because of this persistent clinical challenge, researchers have been searching for molecular mechanisms that could be exploited to improve patient survival and clinical outcomes.
Long noncoding RNAs are transcripts longer than 200 nucleotides that lack protein-coding potential. Increasing evidence demonstrates that they exert diverse roles across various cancers, functioning as oncogenes or tumor suppressors through mechanisms such as RNA–RNA interactions, chromatin remodeling, and post-transcriptional regulation. KCNMB2-AS1 has recently gained attention for its context-dependent oncogenic activities in different malignancies. In bladder cancer, its elevated expression is linked to advanced tumor stages and enhanced malignancy. In cervical cancer, it acts as a competing endogenous RNA, promoting tumorigenesis by sequestering tumor-suppressive microRNAs. However, its role in ccRCC had remained largely unexplored until now.
The research team began by analyzing publicly available datasets from The Cancer Genome Atlas and starBase databases. They found that KCNMB2-AS1 expression was significantly increased in ccRCC tumor tissues compared to normal renal tissues, with 538 tumor samples showing markedly higher levels than 72 normal samples. Kaplan–Meier survival analysis based on TCGA data demonstrated that patients with high KCNMB2-AS1 expression exhibited significantly poorer overall survival. The finding was further validated by quantitative real-time PCR in an independent cohort of 60 ccRCC clinical specimens, where KCNMB2-AS1 levels were markedly higher in tumor tissues compared with adjacent normal tissues. Similar upregulation was also observed in ccRCC cell lines, including 786-O, A498, and 769-P, relative to normal renal epithelial cells.
Importantly, correlation analysis of KCNMB2-AS1 expression with clinicopathological parameters in these patients revealed that elevated levels were significantly associated with advanced tumor stage, presence of distant metastasis, higher AJCC stage, and elevated Fuhrman grade. In contrast, no significant correlations were identified with patient age, sex, tumor size, or lymph node metastasis. These clinical associations suggest that KCNMB2-AS1 may serve as a potential prognostic biomarker for disease progression and patient outcomes, although the authors caution that the clinical validation cohort was derived from a single-center study with a relatively modest sample size, which may limit the broader applicability of the findings.
To investigate the functional role of KCNMB2-AS1 in ccRCC, the researchers transfected both A498 and 769-P cells with short hairpin RNAs targeting the transcript. Functional assays demonstrated that KCNMB2-AS1 knockdown markedly reduced cell viability and proliferation. Similarly, silencing KCNMB2-AS1 significantly impeded cell migration and invasion. At the molecular level, depletion of KCNMB2-AS1 reversed epithelial-mesenchymal transition, as evidenced by downregulation of N-cadherin and Vimentin, coupled with upregulation of E-cadherin. In vivo xenograft experiments using A498 cells corroborated these findings, as KCNMB2-AS1 knockdown markedly inhibited tumor growth, reflected by reduced tumor volume and weight. Conversely, enforced overexpression of KCNMB2-AS1 enhanced proliferation, migration, invasion, and EMT of ccRCC cells, and xenograft experiments further validated the tumor-promoting effects in vivo.
Mechanistically, the team sought to identify the upstream regulators responsible for KCNMB2-AS1 overexpression. Using the PROMO database, they identified 26 potential transcription factors predicted to interact with the KCNMB2-AS1 promoter. Among these, FOXP3 emerged as a compelling candidate owing to its well-documented role in modulating lncRNA expression across various malignancies. TCGA data analysis revealed that FOXP3 was significantly upregulated in ccRCC tissues compared with normal kidney tissues. Moreover, correlation analysis using the GEPIA platform demonstrated a strong positive relationship between FOXP3 and KCNMB2-AS1 expression in ccRCC specimens. Functional assays confirmed this regulatory link: enforced expression of FOXP3 led to a marked increase in KCNMB2-AS1 transcript levels in both A498 and 769-P cells.
Bioinformatic analysis using the JASPAR database identified three potential FOXP3-binding motifs within the KCNMB2-AS1 promoter region. Subsequent chromatin immunoprecipitation assays confirmed that FOXP3 specifically bound to the P2 promoter site, with a 13.42-fold enrichment compared to the IgG control in A498 cells. Consistent with these findings, dual-luciferase reporter assays revealed that FOXP3 overexpression markedly enhanced the transcriptional activity of the wild-type KCNMB2-AS1 promoter, whereas mutation of the P2 binding site completely abolished this activation. Collectively, these results demonstrate that FOXP3 directly binds to the KCNMB2-AS1 promoter to transcriptionally activate its expression in ccRCC, establishing a previously unrecognized FOXP3–KCNMB2-AS1 regulatory axis that contributes to ccRCC oncogenesis.
The researchers then investigated how KCNMB2-AS1 exerts its oncogenic effects downstream. Subcellular fractionation experiments confirmed that KCNMB2-AS1 is predominantly localized in the cytoplasm of ccRCC cells, suggesting a post-transcriptional regulatory role. RNA immunoprecipitation using anti-Ago2 antibodies revealed a strong enrichment of KCNMB2-AS1 within RNA-induced silencing complexes compared with IgG controls, further supporting its interaction with microRNAs. Bioinformatic prediction via the LncRNASNP2 database identified five potential miRNA response elements within the KCNMB2-AS1 sequence. Of these, miR-744-3p emerged as the most relevant candidate, displaying significantly reduced expression in ccRCC cell lines relative to normal renal epithelial cells. TCGA data analysis confirmed marked downregulation of miR-744-3p in ccRCC tissues compared with normal samples. Correlation analysis using the StarBase database revealed a significant negative association between KCNMB2-AS1 and miR-744-3p expression in clinical samples.
Functional validation using dual-luciferase reporter assays demonstrated that co-transfection with miR-744-3p mimics markedly suppressed the luciferase activity of the wild-type KCNMB2-AS1 3′UTR construct, whereas miR-744-3p inhibitors exerted the opposite effect. In contrast, mutation of the predicted binding site abolished these regulatory effects. Together, these findings indicate that KCNMB2-AS1 directly binds and sponges miR-744-3p, functioning as a competing endogenous RNA in ccRCC. This ceRNA mechanism means that KCNMB2-AS1 acts as a molecular sponge, sequestering miR-744-3p and thereby relieving its inhibitory effect on downstream target genes.
To delineate the molecular mechanism linking the KCNMB2-AS1/miR-744-3p axis, the team combined bioinformatic predictions with experimental validation to identify downstream targets of miR-744-3p. Venn diagram analysis revealed 11 overlapping genes among the predicted miR-744-3p targets and the transcripts upregulated in ccRCC tissues. Among these, CD1D was prioritized as a key candidate due to its pronounced upregulation following miR-744-3p inhibition in ccRCC cells. Clinical data from the GEPIA database corroborated this finding, showing significantly elevated CD1D expression in ccRCC tumors compared to normal kidney tissues. Consistent results were observed in ccRCC cell lines, where RT-qPCR analysis confirmed increased CD1D mRNA expression. TargetScan analysis identified a conserved miR-744-3p binding site within the CD1D 3′UTR.
Functional validation through dual-luciferase reporter assays demonstrated that miR-744-3p mimics markedly suppressed the luciferase activity of the wild-type CD1D 3′UTR construct, while miR-744-3p inhibitors significantly enhanced reporter activity. Mutation of the predicted binding site abolished these effects. Furthermore, the reduction in CD1D protein levels caused by KCNMB2-AS1 silencing was rescued upon co-transfection with a miR-744-3p inhibitor. RIP assays confirmed that KCNMB2-AS1, miR-744-3p, and CD1D were co-enriched in Ago2-containing RISC complexes, supporting a direct ceRNA interaction. Correlation analyses further revealed an inverse relationship between miR-744-3p and CD1D expression, alongside a strong positive correlation between KCNMB2-AS1 and CD1D levels in ccRCC samples. Together, these findings establish CD1D as a direct and functional target of miR-744-3p in the KCNMB2-AS1-driven ceRNA network.
To determine whether CD1D mediates the oncogenic function of KCNMB2-AS1, rescue experiments were conducted in ccRCC cells. Successful ectopic expression of CD1D was confirmed following transfection with a CD1D construct. Functional assays revealed that CD1D overexpression effectively reversed the proliferative suppression induced by KCNMB2-AS1 knockdown, as shown by restored colony-forming ability. Furthermore, CD1D overexpression abrogated the suppressive effects of KCNMB2-AS1 depletion on cell migration and invasion. At the molecular level, CD1D overexpression restored the EMT marker alterations induced by KCNMB2-AS1 knockdown, restoring the mesenchymal phenotype. Collectively, these findings demonstrate that CD1D acts as a downstream effector through which KCNMB2-AS1 promotes ccRCC progression.
CD1D is a classical MHC class I–like glycoprotein that has been reported to exhibit elevated expression in anaplastic thyroid carcinoma compared with normal thyroid tissue. Reduced CD1D expression has been confirmed in specific cancer types, including cervical carcinoma and breast cancer. Interestingly, in RCC, CD1D expression is upregulated and correlates with poorer overall and relapse-free survival. This context-dependent expression pattern suggests that tumor-specific epigenetic alterations and microenvironmental factors may differentially regulate CD1D transcription across cancer types. In the present study, the researchers observed that CD1D was markedly overexpressed in ccRCC cells and identified it as a direct downstream target of miR-744-3p, highlighting its potential as a therapeutic target in renal cancer.
Despite providing strong mechanistic and functional evidence, the study is limited by its single-center clinical cohort and reliance on in vitro and immunodeficient in vivo models, which may not fully capture the complexity of human disease. The in vivo assays were conducted in immunodeficient mice, which may not fully reflect the complexity of the human ccRCC tumor microenvironment, particularly in the context of immune-mediated interactions. The investigation also primarily focused on transcriptional regulation, such as FOXP3-mediated activation of KCNMB2-AS1, without examining potential post-transcriptional mechanisms, including mRNA stability or degradation dynamics. Future research should include multi-center validation studies and the development of physiologically relevant models to confirm these findings. Moreover, exploring post-transcriptional regulation of KCNMB2-AS1 and CD1D-directed therapeutic strategies will be essential for translating this mechanistic insight into clinical benefit.
Subject of Research: The role of the FOXP3-activated long noncoding RNA KCNMB2-AS1 and the miR-744-3p/CD1D axis in clear cell renal cell carcinoma progression
Article Title: FOXP3-activated KCNMB2-AS1 promotes clear cell renal cell carcinoma through the miR-744-3p/CD1D axis
Article References: Huang, G., Gui, D., Guo, Y., Luo, S., & Xu, Z. (2025). FOXP3-activated KCNMB2-AS1 promotes clear cell renal cell carcinoma through the miR-744-3p/CD1D axis. Cell Division, 21(1), Article 4. https://doi.org/10.1186/s13008-025-00177-7
Image Credits: AI Generated
DOI: 10.1186/s13008-025-00177-7
Keywords: KCNMB2-AS1, clear cell renal cell carcinoma, FOXP3, miR-744-3p, CD1D, long noncoding RNA, competing endogenous RNA, kidney cancer, oncogenic lncRNA, epithelial-mesenchymal transition, xenograft model, TCGA
News Source: Nathaniel Bowman. (November 4, 2026). Long Noncoding RNA KCNMB2-AS1 Drives Clear Cell Renal Cell Carcinoma Growth. Scienmag.
