Cancer-cell-secreted miR-204-5p induces leptin signalling pathway in white adipose tissue to promote cancer-associated cachexia.
Part of the results reproduced; minor but material deviations remained.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values are derivable from the shared data
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
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- Reproduced
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no human curator yet
- Last updated
- 2026-07-29
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusThe study tests whether and how breast cancer-derived small extracellular vesicles (sEVs), specifically exosomal miR-204-5p, regulate white adipose tissue browning and fat loss to promote cancer-associated cachexia.
- ★ Breast cancer cell-secreted exosomal miR-204-5p induces HIF1A in white adipose tissue by targeting the VHL gene. mechanism
- ★ Elevated HIF1A protein induces the leptin signalling pathway and enhances lipolysis/browning in WAT. mechanism
- ★ Breast cancer-derived sEVs are taken up by WAT and directly drive fat loss and hypermetabolism during cancer-associated cachexia. finding
- ★ Exogenous VHL expression blocks the effect of exosomal miR-204-5p on WAT browning. mechanism
- ★ miR-204 directly binds the 3'UTR of human and mouse VHL/Vhl genes. finding
- Mice lacking cancer-derived miR-204-5p secretion show reduced plasma phosphatidyl ethanolamine levels. finding
- Rab27a knockout/knockdown impairs exosome secretion and attenuates cachexia phenotypes. method
- ★ Circulating miR-204 is closely related to hypermetabolism and energy consumption in vivo. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Orthotopic xenograft tumour model with Lck-GFP tracing | BALB/c mice (4T1, 4T1/Rab27a KO) and NOD/SCID/IL2Rγ-null (NSG) mice (MDA-MB-231, 231/Rab27a KD) | Rab27a KO/KD; tumour xenograft | GFP signal in iWAT/eWAT, tissue/body weight, fat morphology | — |
| Indirect calorimetry (metabolic cage) | BALB/c and NSG tumour-bearing mice; sEV-injected mice | tumour bearing / sEV intravenous injection | Oxygen consumption (VO2) and heat production over 48 h | — |
| sEV intravenous injection | NSG mice (MCF-10A/MDA-MB-231 sEVs) and BALB/c mice (4T1 sEVs) | ~10 μg sEVs twice weekly for 5 weeks | sEV uptake, body weight, fat deposition, food intake | IVIS Spectrum system; micro-CT |
| Bulk RNA-seq with GSEA | eWAT from MDA-MB-231 tumour-bearing/sEV-treated mice vs controls | tumour/sEV exposure | Hypoxia (HIF1) and leptin pathway enrichment (NES, FDR q) | — |
| qRT-PCR | iWAT and eWAT from tumour-bearing and sEV-injected mice | tumour/sEV/Rab27a KD | mRNA abundance of Vhl, Hif1α, leptin | — |
| Western blot / immunoblot | iWAT, eWAT, primary adipocytes, 3T3-L1, hypothalamus | sEVs, miR-204 mimic, Vhl cDNA overexpression | VHL, HIF1A, LEPTIN protein; STAT3 pTyr705 | — |
| Luciferase reporter assay | MCF-10A cells transfected with WT or mutated VHL 3'UTR constructs | miR-204 expression | Reporter responsiveness confirming direct 3'UTR targeting | — |
| ELISA | Serum, iWAT, and SVF cell culture medium | sEVs (MDA-MB-231/10A-miR-204), tumour bearing | Leptin concentration | ELISA kit |
- ▲ 4T1/Ctrl and 231/Ctrl mice showed elevated oxygen consumption and heat production vs tumour-free or Rab27a-impaired mice
- ▼ 4T1/Ctrl and 231/Ctrl mice lost weight after five weeks ~6.41% (4T1) and ~7.57% (231)
- ▼ Body weight change in NSG mice receiving MDA-MB-231 sEVs vs PBS/MCF-10A sEVs -7.48±6.95% (231 sEVs) vs 8.72±4.37% (PBS), 7.03±0.63% (MCF-10A)
- ▼ Body weight change in BALB/c mice receiving 4T1 sEVs vs PBS -2.35±1.43% (4T1 sEVs) vs 16.32±4.52% (PBS)
- – VHL protein suppressed and HIF1A induced in eWAT/iWAT of tumour-bearing and sEV-treated mice
- ▲ miR-204 overexpression yielded upregulated miR-204 in 10A/miR-204 sEVs vs MCF-10A sEVs 15-fold
- ▲ Plasma miR-204 enrichment in 10A/miR-204 sEV mice and tumour-bearing mice vs controls 10-fold (10A/miR-204) and 6-fold (tumour-bearing)
- ▲ Leptin (mRNA, protein, serum/tissue) elevated in WAT of miR-204 sEV and tumour-bearing mice
- fold_change 15-fold upregulation of miR-204 in 10A/miR-204 cells derived sEVs (10A/miR-204 sEVs vs MCF-10A sEVs)
- fold_change 10-fold plasma miR-204 enrichment in 10A/miR-204 sEVs mice; 6-fold in tumour-bearing mice (plasma vs control mice)
- fold_change 5-10 fold upregulation of miR-204 in iWAT/eWAT (high miR-204 sEVs or 4T1/Ctrl mice vs controls)
- mean -7.48±6.95% (MDA-MB-231 sEVs), 7.03±0.63% (MCF-10A sEVs), 8.72±4.37% (PBS) (body weight change in NSG mice)
- mean -2.35±1.43% (4T1 sEVs) vs 16.32±4.52% (PBS) (body weight change in BALB/c mice)
- other ~6.41% and ~7.57% weight loss after five weeks (4T1/Ctrl and 231/Ctrl mice)
- count n=7 BALB/c per group; n=5 NSG per group (metabolic measurements)
- count n=3 mice Control and n=3 mice MDA-MB-231 tumour (RNA-seq/GSEA groups)
Statistical methods review
Model: opusA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study is a series of controlled in vivo (mouse xenograft and sEV-injection) and in vitro experiments comparing tumour/sEV-treated groups against control groups, with most multi-group comparisons analysed by one-way ANOVA followed by Dunnett's multiple-comparison test against a reference control. Transcriptomic differences were assessed by RNA-seq with GSEA reporting normalized enrichment scores, nominal P-values and FDR q-values, while a luciferase reporter assay used an unpaired two-tailed t-test. Quantitative data are reported as mean ± s.e.m. with significance thresholds (*P<0.05 to ****P<0.0001), and exact P-values plus source data are provided in a Source data file.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| One-way ANOVA followed by Dunnett's multiple-comparison test | Oxygen consumption/heat production and body-weight/fat-distribution comparisons across groups (Fig. 1d,e,j,k,l) and qRT-PCR of Vhl/Hif1α/leptin (Fig. 2b,d) | n=7 (BALB/c) and n=5 (NSG) for metabolic cages; n=3 per group for micro-CT; n=5–6 mice per group for qRT-PCR | not stated |
| Unpaired two-tailed t-test | Luciferase reporter responsiveness of WT vs mutated VHL/Vhl 3'UTR to miR-204 (Fig. 2h) | n=3 biological replicates | not stated |
| GSEA (gene set enrichment) reporting NES, nominal P-value and FDR q-value | RNA-seq of eWAT for hypoxia/HIF1 and leptin signalling pathways (Fig. 2a, Fig. 3a) | n=3 mice per group | na |
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Multi-group comparisons used one-way ANOVA followed by Dunnett's test comparing each group to a single control.↳ Could also: Tukey's HSD (or Sidak) post-hoc could also be used when all pairwise comparisons among groups are of interest. — Tukey/Sidak would additionally provide every group-to-group contrast with family-wise error control, whereas Dunnett focuses power on comparisons against the reference control.
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Variability was summarized as mean ± s.e.m.↳ Could also: Standard deviation or a 95% confidence interval could also be reported, alongside plotting individual data points. — SD/CI convey the spread of the data and estimate precision directly, which is often favoured for the small per-group n used here.
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Several comparisons rely on small sample sizes (e.g., n=3) analysed with parametric tests.↳ Could also: Non-parametric tests (e.g., Mann-Whitney U or Kruskal-Wallis with Dunn's post-hoc) could also be applied. — Non-parametric approaches do not assume normality, which can be useful when group sizes are small and the distribution is hard to verify.
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The reporter assay (Fig. 2h) used an unpaired two-tailed t-test for WT vs mutant constructs across multiple binding sites.↳ Could also: A two-way ANOVA (construct × site) or a t-test with multiplicity adjustment across the sites could also be used. — A single model would jointly account for multiple constructs/sites and control the error rate across the related comparisons.
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Sample-size/power was described by stating per-group n without a power calculation, and randomization/blinding were not described.↳ Could also: An a priori power analysis and explicit reporting of randomization/blinding could also be included. — These additions would document how n was chosen and how allocation/assessment bias was managed, aiding reproducibility.
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Significance was emphasized with starred thresholds and exact P-values.↳ Could also: Reporting estimated effect sizes with confidence intervals could also accompany the P-values. — Effect sizes with CIs communicate the magnitude and precision of differences, complementing significance testing.
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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BALB/c mice injected with 4T1 sEVs lose ~2.4% body weight versus ~16% weight gain in PBS controlsother balb/c-mouse down 2023×1papers★ This paper is the founder (earliest)
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Tumor-bearing BALB/c (4T1) and NSG (MDA-MB-231) mice lose ~6–8% body weight over five weeks compared to tumor-free controlsother mouse down 2023×1papers★ This paper is the founder (earliest)
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Oxygen consumption and heat production are elevated in tumor-bearing mice relative to tumor-free or Rab27a-impaired controlsother mouse up 2023×1papers★ This paper is the founder (earliest)
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NSG mice injected with MDA-MB-231 sEVs lose ~7.5% body weight versus ~8.7% weight gain in PBS controlsother nsg-mouse down 2023×1papers★ This paper is the founder (earliest)
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miR-204 is enriched ~15-fold in sEVs secreted by MCF-10A cells overexpressing miR-204qPCR mcf-10a up 2023×1papers★ This paper is the founder (earliest)
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Circulating miR-204 is elevated 6–10-fold in plasma of tumor-bearing mice and miR-204 sEV-injected mice versus controlsqPCR mouse plasma up 2023×1papers★ This paper is the founder (earliest)
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LEP mRNA, protein, and secreted leptin are elevated in white adipose tissue of tumor-bearing and miR-204 sEV-treated miceqPCR mouse white-adipose-tissue up 2023×1papers★ This paper is the founder (earliest)
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VHL protein is suppressed and HIF1A protein is induced in white adipose tissue of tumor-bearing and sEV-treated micewestern-blot mouse white-adipose-tissue down 2023×1papers★ This paper is the founder (earliest)
Citation network
Where this publication sits in the reproducibility-weighted citation graph — what it is built on, and what is built on it. Citation data from OpenAlex.
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Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-37620316
Paper: Hu et al. 2023, Nat Commun — "Cancer-cell-secreted miR-204-5p induces leptin signalling pathway in white adipose tissue to promote cancer-associated cachexia." DOI 10.1038/s41467-023-40571-9 · PMCID PMC10449837.
Data / code artifacts
- GEO GSE222380 — bulk RNA-seq of mouse epididymal white adipose tissue (eWAT),
Illumina NovaSeq 6000, Mus musculus, 9 samples in 3 groups (n=3 each):
- Control / untreated eWAT (GSM6921889–891)
- MDA-MB-231 sEV (EV)-treated eWAT (GSM6921892–894)
- Tumor-bearing ("231/Ctrl") eWAT (GSM6921895–897)
- Raw reads: SRA PRJNA921843. Processed:
GSE222380_Raw_gene_counts_TPM.xlsx(5.4 MB).
- Code link (per registry): https://github.com/jstjohn/SeqPrep — this is the generic FASTQ adapter-trim/merge tool used as the first pipeline step, not an authors' analysis repo. Per brief rule 2 (P16) applying a third-party tool to the paper's own data is a valid reproduction. The full analysis pipeline is described in the Methods (Majorbio-style boilerplate).
Pipeline (from Methods)
raw PE reads → SeqPrep + Sickle (adapter+quality trim, default params) → HISAT2 (align to reference, orientation mode) → RSEM (gene abundance, TPM) → DESeq2 (differential expression) → GSEA (pathway enrichment).
In scope (pipeline-derived → attempt to reproduce)
| # | Reported result | Location | Pipeline step |
|---|---|---|---|
| C1 | eWAT gene-level quantification (raw gene counts / TPM per sample) | GSE222380 processed file | SeqPrep+Sickle→HISAT2→RSEM |
| C2 | GSEA: "signalling by leptin" upregulated in 231/sEV and 231/Ctrl eWAT vs control | Fig 3a | DESeq2 + GSEA |
| C3 | GSEA: HIF1 & hypoxia metagene pathway upregulated in 231/Ctrl eWAT vs control | Fig 2a | DESeq2 + GSEA |
Out of scope (wet-lab / not pipeline-derived → NOT attempted)
- qRT-PCR / Western / IHC / luciferase reporter / ELISA leptin measurements.
- miR-204-5p sEV loading, in-vivo cachexia phenotyping (body/muscle weight), cell assays.
- Any result with no shipped data + pipeline to regenerate it.
Reproduction strategy (two jobs)
- Downstream (quick): use authors' published count matrix → DESeq2 + GSEA → reproduce C2/C3 pathway directions (reproduces DE+GSEA steps).
- Full pipeline (heavy): download the 9 SRR, run SeqPrep+Sickle→HISAT2→ featureCounts/RSEM → reconstruct a gene-count matrix → correlate vs authors' published counts (C1), then DESeq2+GSEA on reconstructed counts (C2/C3).
Note: the paper reports the GSEA results qualitatively (direction of enrichment in figure panels); it does not print discrete DEG counts, per-gene fold-changes, or exact thresholds. Grading of C2/C3 is therefore direction/sign agreement, not a numeric tolerance.
Assessments & scoring basis
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An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Reproduction ran on the authors' own deposited count matrix (GSE222380), so input data is identical and the reported qualitative directions are fully derivable — no fabrication concern. Both figure claims reproduce by sign of GSEA enrichment: hypoxia/HIF1 up (NES=+1.80, padj=2.9e-4) and leptin up in the tumor-bearing arm (NES=+1.91, padj=0.048). The one explainable shortfall is the sEV-vs-control leptin enrichment, which holds in direction but not significance (NES=+0.995, padj=0.67), attributable to our side — a proxy hypoxia set, an unspecified ranking, and a low-power 10-gene leptin set — rather than an authors' defect. Overall a solid, sign-agreement reproduction with method-dependent caveats; C1 full-pipeline quantification is still pending.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.