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Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis

  • Authors:
    • Jie Zhang
    • Kai Huang
    • Jianwu Zhou
    • Wenge Liao
    • Fei Li
    • Zhenzhen Zhao
    • Shan Wang
  • View Affiliations / Copyright

    Affiliations: Department of Pediatric Surgical Oncology, The Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatric Metabolism and Inflammatory Diseases, Chongqing 400014, P.R. China, Department of Pediatric Surgery, Guizhou Provincial People's Hospital, Guiyang, Guizhou 550002, P.R. China
    Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 23
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    Published online on: November 6, 2025
       https://doi.org/10.3892/ol.2025.15376
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Abstract

Long non‑coding RNAs (lncRNAs) are emerging as key regulators of neuroblastoma (NB) progression; however, their interplay with MYCN‑driven mechanisms remains to be elucidated. The present study aimed to characterize the expression profile of lncRNA RP11‑196G11.6 in NB, and to further explore its functional role and mechanism in the pathogenesis of this disease. Transcriptomics data from NB and disseminated tumor cell (DTC) samples (Gene Expression Omnibus; accession no. GSE94035) were analyzed via principal component analysis (PCA), differential expression analysis and CIBERSORT immune profiling. The biological function was assessed using gain‑ and loss‑of‑function experiments in IMR‑32 (MYCN+) and SH‑SY5Y (MYCN‑) cells. Dual‑luciferase reporter assays were performed to identify the interaction between RP11‑196G11.6, microRNA (miR)‑376a‑3p and RING1 and YY1‑binding protein (RYBP). Rescue experiments were performed by co‑transfecting RP11‑196G11.6‑overexpressing cells with a miR‑376a‑3p mimic to identify the hypothetical regulatory role of RP11‑196G11.6 in NB progression in vitro. The present analysis results demonstrated that the PCA could distinguish tumor and DTC samples, with MYCN amplification driving distinct clustering in tumors but not in DTCs. Differential expression analysis based on MYCN+/‑ in both tumor and DTC groups identified 161 differentially expressed lncRNAs (73 upregulated and 88 downregulated). Notably, RP11‑196G11.6 was highly expressed in MYCN+ tumors, and silencing RP11‑196G11.6 promoted the viability, migration, invasion and epithelial‑mesenchymal transition in MYCN+ cells, whereas RP11‑196G11.6 overexpression induced the opposite effects in MYCN‑ cells. Mechanistically, RP11‑196G11.6 directly inhibited miR‑376a‑3p, which targeted RYBP. Overexpression of miR‑376a‑3p reversed the tumor suppressor phenotype driven by RP11‑196G11.6. In summary, the present study demonstrated that RP11‑196G11.6 may inhibit NB progression by sponging miR‑376a‑3p, leading to the upregulation of RYBP expression and consequently inhibiting NB progression. These findings revealed a novel lncRNA‑miRNA axis involved in NB pathogenesis.
View Figures

Figure 1

Analysis of DElncRNAs in tumor and
bone marrow metastatic tissues. (A) Schematic diagram of the
bioinformatics analysis process. (B) PCA between tumor and DTC
samples, and between MYCN amplification statuses. (C) Analysis of
DEGs between MYCN+ and MYCN− samples in tumor
and DTC samples. (D) Venn diagrams depicting commonly upregulated
and downregulated DEGs in the four groups of samples. (E) GO
enrichment analysis showing the GO terms that were most
significantly enriched in 808 DEGs. The color scale represents the
enrichment intensity of gene sets across different groups, based on
the number of genes enriched in each pathway and normalized to a
range of −2 to 2. (F) PCA of the lncRNAs among the DEGs. (G)
DElncRNA analysis between MYCN+ and MYCN−
samples in tumor and DTC samples. (H) Venn diagrams indicating
commonly upregulated and downregulated DElncRNAs in the four groups
of samples. (I) Weighted gene co-expression network analysis and
co-expression analysis. Scatter plots of DElncRNAs and their
co-expressed DEmRNAs in MYCN+ vs. MYCN−
samples across tumor and DTC cohorts. Red points denote upregulated
lncRNAs involved in co-expression pairs and blue points denote
downregulated lncRNAs. The straight lines represent correlations
between the two variables. Cutoffs of P≤0.01 and Pearson
coefficient >0.8 were applied to identify the co-expression
pairs. (J) Top 10 most enriched GO terms (biological processes) in
DEmRNAs co-expressed with the DElncRNAs; the highlighted terms in
red are associated with immune and inflammatory responses. lncRNA,
long non-coding RNA; PCA, principal component analysis; DTC,
disseminated tumor cell; DEG, differentially expressed gene; GO,
Gene Ontology; DElncRNA, differentially expressed lncRNA; DEmRNA,
differentially expressed mRNA; FDR, false discovery rate.

Figure 2

Analysis of DElncRNAs associated with
Tregs and Tfhs. (A) CIBERSORT analysis of the proportion of each
immune cell type in MYCN+ and MYCN− samples
from tumors and DTCs. (B) PCA of the different types of cells. (C)
Specific proportions of Tregs in the four types of samples.
**P<0.01 vs. Tumor-MYCNnonamplified. (D) Specific
proportions of Tfhs in the four types of samples. ***P<0.001 vs.
Tumor-MYCNnonamplified. (E) lncRNA-immune cell
infiltration networks. (F) Expression levels of four DElncRNAs
associated with Tfh infiltration. lncRNA, long non-coding RNA;
DElncRNA, differentially expressed lncRNA; DTC, disseminated tumor
cell; Treg, regulatory T cell; Tfh, T follicular helper cell; PCA,
principal component analysis.

Figure 3

RP11is highly expressed in
MYCN+ NB tumor tissues and MYCN+ NB cells,
and RP11 upregulation inhibits NB cell viability. (A) FISH analysis
revealed the expression level of RP11 in MYCN+ and
MYCN− NB tumor tissue specimens (scale bar, 100 µm; U6,
control probe). *P<0.05 vs. MYCN+ group. (B)
Quantification of RP11 levels in SH-SY5Y and IMR-32 cells by
reverse transcription-quantitative PCR. *P<0.05 vs. SH-SY5Y. (C)
Quantification of RP11 levels in IMR-32 cells following
transfection with si-RP11 and RP11 levels in SH-SY5Y cells
following transfection with oe-RP11. *P<0.05 vs. si-NC,
@P<0.05 vs. oe-NC. (D) Viability of IMR-32 and
SH-SY5Y cells was assessed using a Cell Counting Kit-8 assay.
*P<0.05 vs. si-NC, @P<0.05 vs. oe-NC. (E) Levels
of apoptosis were assessed using flow cytometry in IMR-32 and
SH-SY5Y cells. (F) Apoptosis rate of IMR-32 and SH-SHY5Y cells
*P<0.05 vs. si-NC or @@P<0.01 vs. oe-NC. RP11,
RP11-196G11.6; si, small interfering; oe, overexpression; NB,
neuroblastoma; FISH, fluorescence in situ hybridization; NC,
negative control.

Figure 4

RP11 upregulation inhibits
neuroblastoma cell migration and invasion. (A) Cell scratch assay
results indicated that knockdown of RP11 expression promoted
migration in IMR-32 cells and oe-RP11 suppressed migration in
SH-SY5Y cells (scale bar, 200 µm). (B) Comparison of cell migration
rates in IMR-32 and SH-SY5Y cells in the cell scratch assay.
*P<0.05 vs. si-NC, @P<0.05 vs. oe-NC. (C)
Transwell assay results indicated that knockdown of RP11 expression
promoted invasion in IMR-32 cells and oe-RP11-196G11.6 suppressed
invasion in SH-SY5Y cells (scale bar, 200 µm). (D) Quantitative
analysis of cell invasion rate in the Transwell assay. *P<0.05
vs. si-NC, @P<0.05 vs. oe-NC. (E) Representative WB
images in IMR-32 cells. (F) Expression levels of vimentin,
E-cadherin and p-EGFR/EGFR in IMR-32 cells *P<0.05 vs. si-NC.
(G) Representative WB images in SH-SY5Y cells. (H) Expression
levels of vimentin, E-cadherin and p-EGFR/EGFR protein in SH-SY5Y
cells. @P<0.05 vs. oe-NC. RP11, RP11-196G11.6; si,
small interfering; oe, overexpression; WB, western blotting; p-,
phosphorylated; NC, negative control.

Figure 5

RP11 inhibits miR-376a-3p expression
in neuroblastoma cells. (A) Quantification of miR-376a-3p levels in
IMR-32 cells following transfection with si-RP11 and in SH-SY5Y
cells following transfection with oe-RP11. *P<0.05 vs. si-NC,
@P<0.05 vs. oe-NC. (B) Luciferase reporter assay was
performed to measure the activity of the WT or MUT RP11 3′UTR
vector in 293T cells that were co-transfected with either
miR-376a-3p or NC mimics. *P<0.05 vs. WT-RP11 + NC. (C) RYBP
protein levels detected by WB in IMR-32 and SH-SY5Y cells.
*P<0.05 vs. si-NC, @P<0.05 vs. oe-NC. (D)
Luciferase reporter assay was performed to measure the activity of
the WT or MUT RYBP 3′UTR vector in 293T cells that were
co-transfected with either miR-376a-3p or NC mimics. *P<0.05 vs.
WT-RYBP + NC. RP11, RP11-196G11.6; si, small interfering; oe,
overexpression; NC, negative control; WB, western blotting; WT,
wild-type; RYBP, RING1 and YY1-binding protein; MUT, mutant;
miR-376a-3p, microRNA-376a-3p.

Figure 6

RP11 inhibits SH-SY5Y cell viability,
migration and invasion by downregulating miR-376a-3p expression.
(A) SH-SY5Y cell viability was assessed using a Cell Counting Kit-8
assay. *P<0.05 vs. oe-NC + mimic NC; #P<0.05 vs.
oe-RP11 + mimic NC. (B) SH-SY5Y apoptosis was assessed using flow
cytometry to assess the levels of SH-SY5Y apoptosis. *P<0.05 vs.
oe-NC + mimic NC; #P<0.05 vs. oe-RP11 + mimic NC. (C)
SH-SY5Y cell migration ability was detected using a cell scratch
assay (scale bar, 200 µm). (D) Comparison of cell migration rates
between the four groups in the cell scratch assay. *P<0.05 vs.
oe-NC + mimic NC; #P<0.05 vs. oe-RP11 + mimic NC. (E)
SH-SY5Y cell invasive ability was detected using a Transwell assay
(scale bar, 200 µm). (F) Quantitative analysis of cell invasion
rate in the four groups. *P<0.05 vs. oe-NC + mimic NC;
#P<0.05 vs. oe-RP11 + mimic NC. (G) Representative
western blotting image. (H) Semi-quantification of RYBP, vimentin,
E-cadherin and p-EGFR/EGFR protein expression levels. *P<0.05
vs. oe-NC + mimic NC; #P<0.05 vs. oe-RP11 + mimic NC.
oe, overexpression; NC, negative control; RP11, RP11-196G11.6;
RYBP, RING1 and YY1-binding protein; miR-376a-3p, microRNA-376a-3p;
p-, phosphorylated.
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Copy and paste a formatted citation
Spandidos Publications style
Zhang J, Huang K, Zhou J, Liao W, Li F, Zhao Z and Wang S: Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis. Oncol Lett 31: 23, 2026.
APA
Zhang, J., Huang, K., Zhou, J., Liao, W., Li, F., Zhao, Z., & Wang, S. (2026). Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis. Oncology Letters, 31, 23. https://doi.org/10.3892/ol.2025.15376
MLA
Zhang, J., Huang, K., Zhou, J., Liao, W., Li, F., Zhao, Z., Wang, S."Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis". Oncology Letters 31.1 (2026): 23.
Chicago
Zhang, J., Huang, K., Zhou, J., Liao, W., Li, F., Zhao, Z., Wang, S."Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis". Oncology Letters 31, no. 1 (2026): 23. https://doi.org/10.3892/ol.2025.15376
Copy and paste a formatted citation
x
Spandidos Publications style
Zhang J, Huang K, Zhou J, Liao W, Li F, Zhao Z and Wang S: Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis. Oncol Lett 31: 23, 2026.
APA
Zhang, J., Huang, K., Zhou, J., Liao, W., Li, F., Zhao, Z., & Wang, S. (2026). Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis. Oncology Letters, 31, 23. https://doi.org/10.3892/ol.2025.15376
MLA
Zhang, J., Huang, K., Zhou, J., Liao, W., Li, F., Zhao, Z., Wang, S."Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis". Oncology Letters 31.1 (2026): 23.
Chicago
Zhang, J., Huang, K., Zhou, J., Liao, W., Li, F., Zhao, Z., Wang, S."Long non‑coding RNA RP11‑196G11.6 inhibits neuroblastoma progression by regulating the miR‑376a‑3p/RYBP signaling axis". Oncology Letters 31, no. 1 (2026): 23. https://doi.org/10.3892/ol.2025.15376
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