Stemness genes and miR-1247-3p expression associate with clinicopathological parameters and prognosis in lung adenocarcinoma.
The main results reproduced: recomputed values matched the published ones within tolerance.
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 were directly comparable
- ✓Reported values are derivable from the shared data
- ✓The central claim held under reproduction
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡The deviation was non-trivial in magnitude
- 🟡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.
▸Reproduction agent’s raw note
Described well enough to reproduce the PRIMARY result 1:1. The repo (Sashoss/LUAD_PLOS_data @ 8b6d7e1) ships data + derived outputs but NO authors' analysis code; per brief P16 we re-ran the paper-described pipeline (DESeq2 for TCGA, Limma for GEO; FC=tumor/normal) on the shipped inputs on «our HPC» («infra», conda DESeq2 1.42.0 / limma 3.58.1 / R 4.3). TCGA primary cohort (gene + miRNA DESeq2) reproduces EXACTLY: Pearson cor of log2FC = 1.000 over the whole transcriptome (34924 genes) and miRNA set (1359); all headline stemness genes (ORC1L 2.741, KIF20A 3.144, DLGAP5 3.671, RAB3B 4.242, CENPI 2.740) match Table 1 to 3 decimals, and the headline miR-1247-3p downregulation is exact (-2.325). The GEO validation cohorts (Limma) reproduce direction and ranking but NOT magnitude 1:1 (GSE31210 cor=0.9975 but 1.45x smaller; GSE40419 cor0.70-0.75, inconsistent) because the authors' microarray preprocessing/probe-collapsing is undocumented (no code shipped) - this is the hard ~20% and was not chased further. NOT attempted (out of scope): univariate/multivariate Cox survival (clinical survival not shipped as a runnable table), TIMER2 immune infiltration (external web tool output), miRWalk target prediction (external web DB output). One audit note: an early fabrication suspicion for ORC1L was OUR gene-ID error (ENSG00000113742=CPEB4 not ORC1; correct ORC1=ENSG00000085840) and was RESOLVED - ORC1L reproduces exactly. Net: the paper's core differential-expression claims are faithfully reproducible from its own shipped data via the stated standard pipeline.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessment Score 90assessed: 2026-06-15 ⛓ 093e294c34f8
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When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-15
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-15no human curator yet
- Last updated
- 2026-08-05
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 high mortality of lung adenocarcinoma (LUAD) is linked to acquisition of progenitor-like cells with stem-like characteristics that help the tumor regulate immune cell infiltration; the study tests whether stemness-related genes and miRNAs in LUAD associate with immune/accessory cell infiltration rates and patient survival.
- ★ Three stem cell-related genes (ORC1L, KIF20A, DLGAP5) are differentially expressed in LUAD and correlate with altered immune infiltration and reduced patient survival. finding
- ★ hsa-mir-1247-3p, via targeting of tumor suppressor SLC24A4 and oncogenes RAB3B and HJURP, together with MDSC infiltration modulation, primarily regulates LUAD patient survival. finding
- ★ Increased expression of stemness-related genes in LUAD correlates with reduced immune cell infiltration and reduced patient survivability. finding
- ★ Stemness genes ORC1L, KIF20A, DLGAP5 positively correlate with Th2 and MDSC infiltration, while ORC1L negatively correlates with HSC infiltration. finding
- hsa-mir-1247-3p and/or its gene targets are proposed as candidate biomarkers/therapeutic avenue to enhance LUAD patient survival. resource
- ★ A bioinformatics mining pipeline (differential expression, univariate/multivariate Cox survival, immune deconvolution, miRNA-3'UTR target prediction) was used to identify markers across TCGA and GEO cohorts. method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Bulk RNA-seq differential expression (DeSeq2) | LUAD tumor (537) vs normal (59) human samples | none (tumor vs normal observational) | differentially expressed genes (log2 fold change, BH adjusted p-value) | TCGA database |
| miRNA-seq differential expression (DeSeq2) | LUAD tumor vs normal human samples | none | differentially expressed miRNAs (LFC, BH adjusted p) | TCGA database |
| Microarray gene expression differential expression (Limma) validation | LUAD tumor vs normal (GSE40419: 87 tumor/77 normal; GSE31210: 226 tumor/20 normal) | none | differentially expressed genes (LFC, BH adjusted p) | GEO microarray datasets |
| Univariate Cox-proportional hazard survival analysis | LUAD patient clinical + gene expression data (high vs low expression groups) | none | hazard ratio, p-value for gene expression vs survival | Timer2.0 |
| Immune/accessory cell infiltration correlation analysis | LUAD TCGA tumor samples, 32 immune/accessory cell types | none | correlation between gene expression and cell infiltration rate; hazard ratio for infiltration vs survival | Timer2.0 (Timer, TIDE, MCP-counter, quanTIseq, xCell, CIBERSORT, Epic) |
| miRNA–3'UTR target binding prediction | differentially expressed stemness-related genes (3'UTR regions) | none | predicted/validated miRNA-gene binding (3p/5p variants) | mirWalk and miRTarBase |
| Multivariate Cox-proportional hazard survival analysis | LUAD patients; covariates = gene/miRNA expression, MDSC/Th2/HSC infiltration, age, cancer stage | none | hazard ratio, p-value for each covariate vs survival | R Survival Analysis package (MDSC from TIDE; HSC, Th2 from Timer2.0) |
- ▲ Four stemness genes (ORC1L, KIF20A, DLGAP5, RAB3B) differentially expressed in LUAD with hazard ratio >1 (DLGAP5 HR 1.314, KIF20A HR 1.314, ORC1L HR 1.263, RAB3B HR 1.181) DLGAP5 LFC 3.671 (TCGA DeSeq2); HR 1.314
- – ORC1L, KIF20A, DLGAP5 positively correlate (>0.6) with Th2 and MDSC infiltration; ORC1L negatively correlates (<-0.6) with HSC infiltration correlation >0.6 or <-0.6
- ▲ High MDSC infiltration strongly associated with lower patient survival HR=62.036 (p=0)
- ▲ High Th2 infiltration associated with lower patient survival HR=3.804 (p=0.006)
- ▼ High HSC infiltration associated with higher patient survival HR=0.167 (p=0.046)
- ▼ Five miRNAs (hsa-mir-197-3p, 1247-3p, 122-5p, 215-5p, 192-5p) differentially expressed targeting RAB3B 3'UTR; only hsa-mir-1247-3p met both DeSeq2 and Limma cutoffs hsa-mir-1247-3p DeSeq2 LFC -2.324, Limma LFC -3.151
- – Multivariate analysis: increased hsa-mir-1247-3p expression and increased MDSC infiltration significantly associated with loss of survival; no correlation between MDSC and miR-1247-3p miR-1247-3p HR 0.667653 p=0.02279; MDSC HR 0.655898 p=0.028825; Pearson r=-0.05
- – hsa-mir-1247-3p targets HJURP and RAB3B (LFC >2 across all datasets) and SLC24A4 (LFC <-2 across all datasets) LFC >2 or <-2
- fold_change hsa-mir-1247-3p DeSeq2 LFC -2.324 (BH p=3.51E-19), Limma LFC -3.151 (BH p=1.63E-11) (miRNA downregulation in LUAD)
- other Hazard ratio 62.036 (p=0) (MDSC high vs low infiltration univariate survival)
- other Hazard ratio 3.804 (p=0.006) (Th2 high vs low infiltration survival)
- other Hazard ratio 0.167 (p=0.046) (HSC high vs low infiltration survival)
- pvalue HR 0.667653, p=0.02279 (hsa-mir-1247-3p in multivariate Cox model)
- correlation Pearson = -0.05 (MDSC infiltration vs hsa-mir-1247-3p (no significant correlation))
- count 537 LUAD tumor and 59 normal samples (TCGA cohort)
- fold_change DLGAP5 LFC 3.671 (DeSeq2), 5.411/4.184/3.003 (Limma), HR 1.314 p=0.0 (top stemness gene differential expression and survival)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This retrospective bioinformatics study used TCGA RNA-seq data (537 tumor, 59 normal) and two GEO microarray validation datasets to identify stemness-related genes and miRNAs differentially expressed in lung adenocarcinoma. Differential expression was assessed with DESeq2 (RNA-seq) and limma (microarray), both with Benjamini-Hochberg FDR correction at adjusted p ≤ 0.05 and |log2FC| > 2. Univariate and multivariate Cox proportional hazard models were then used to evaluate associations between gene/miRNA expression, computationally predicted immune cell infiltration rates, and patient survival, with results reported as hazard ratios and exact BH-adjusted p-values.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| DESeq2 Wald test | Differential expression of genes and miRNAs in TCGA LUAD tumor vs. normal samples | 537 tumor + 59 normal (TCGA) | not stated |
| limma moderated t-test | Differential expression of genes in GEO microarray datasets GSE40419 and GSE31210 used as validation cohorts | 87 tumor + 77 normal (GSE40419); 226 tumor + 20 normal (GSE31210) | not stated |
| Univariate Cox proportional hazard model | Association between high vs. low gene expression or immune cell infiltration and LUAD patient survival, performed via Timer2.0 | — | not stated |
| Multivariate Cox proportional hazard model | Joint association of miRNA/gene expression, immune infiltration rates, patient age, and cancer stage with patient survival (R Survival package) | — | not stated |
| Pearson correlation | Association between stemness gene expression and computationally predicted infiltration rates of 32 immune/accessory cell types; also reported for MDSC vs. hsa-mir-1247-3p (r = -0.05) | — | not stated |
| Kaplan-Meier survival curve | Visual display of survival probability for high vs. low immune cell infiltration and gene expression groups, obtained from Timer2.0 | — | na |
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Patient samples were dichotomized into high and low expression (or infiltration) groups before Cox proportional hazard model analysis↳ Could also: Cox regression treating gene/miRNA expression as a continuous variable, or using a restricted cubic spline to model non-linear dose-response — Continuous Cox modeling preserves information across the full expression range, avoids sensitivity to the choice of cutpoint, and is generally more statistically efficient than a median-split dichotomization
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Pearson correlation was used to assess associations between gene expression and computationally predicted immune cell infiltration rates across 32 cell types↳ Could also: Spearman rank correlation — Spearman correlation does not assume linearity or bivariate normality, assumptions that may not hold for RNA-seq-derived values and computationally estimated infiltration scores
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Hazard ratios from Cox proportional hazard models were reported with p-values but without confidence intervals↳ Could also: Report 95% confidence intervals alongside each hazard ratio — Confidence intervals directly convey the precision and uncertainty of each hazard ratio estimate, complementing the p-value and making effect magnitude more interpretable
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Correlations between gene expression and 32 immune cell types were screened using a |r| > 0.6 threshold with nominal p ≤ 0.05, without an explicit multiplicity correction for the 32 cell-type tests↳ Could also: Apply BH-FDR correction across the family of 32 correlation tests per gene — Testing 32 cell types per gene increases the probability of finding at least one spurious correlation at a nominal threshold; a within-family correction would be consistent with the approach applied to the differential expression and survival analysis steps
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Univariate Cox models were used to pre-screen individual genes and immune cell types for survival associations before entry into the multivariate model↳ Could also: LASSO-penalized Cox regression for simultaneous covariate selection in a single multivariate model — LASSO-penalized Cox regression selects predictors jointly, reducing the potential for bias introduced by sequential univariate pre-screening and handling correlated predictors more explicitly
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DESeq2 was the sole method used for RNA-seq differential expression in the TCGA dataset↳ Could also: edgeR (quasi-likelihood F-test) or voom/limma applied to RNA-seq count data as a concordance check — DESeq2, edgeR, and voom/limma are all widely validated for RNA-seq differential expression; reporting genes that are significant across two methods is a common strategy for increasing confidence in findings, analogous to the cross-platform validation the paper applied to the microarray data
Citation network
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Data lineage
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What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-37948380
Paper: Limbu & McCloskey 2023, PLoS One. "Stemness genes and miR-1247-3p expression associate with clinicopathological parameters and prognosis in LUAD." DOI 10.1371/journal.pone.0294171 · PMCID PMC10637681.
Code/Data artifact: https://github.com/Sashoss/LUAD_PLOS_data @ commit
8b6d7e125fdbb20685a32a9246bc7f2f4846784f (2023-09-01). This is a data + derived-results
repository — it ships NO authors' analysis scripts (no .R/.py/.ipynb/README). Per
brief rule P16, reproduction = re-run the standard, paper-described pipeline on the
shipped inputs and compare to the shipped derived outputs + paper claims. This is
fully valid.
Pipeline (from paper Methods)
- Differential expression:
- TCGA gene & miRNA counts → DESeq2.
- GEO microarray/RNA-seq (GSE40419, GSE31210) → Limma.
- DEG cutoff: Benjamini-Hochberg adj p ≤ 0.05 AND |log2FC| > 2. FC = cancer/normal.
- Univariate & multivariate Cox proportional-hazard survival analysis (high vs low expr).
- TIMER2 immune-infiltration correlation; miRWalk target prediction.
Shipped inputs (on «infra», complete, sizes match GitHub)
data_gene.txt(99 MB): TCGA STAR raw gene counts, ENSG ids, 596 samples (537 Primary_Tumor + 59 Solid_Tissue_Normal; condition encoded in column-name prefix).data_mirna.txt(2.5 MB): TCGA miRNA raw counts, 565 samples (519 T + 46 N).GSE40419-GPL11154_withGeneName.csv(17 MB) +GSE40419_annotation.csv(87 T / 77 N).GSE31210-GPL57_withGeneName.csv(53 MB) +GSE31210_annotation.csv(226 T / 20 N).Metadata_LUAD_gene.tsv,Metadata_LUAD_mirna.tsv(clinical/sample metadata).
Shipped derived outputs (comparison ground-truth)
TCGA_diffExp_gene_DeSeq2.csv(full transcriptome),TCGA_diffExp_mirna_DeSeq2.csv.GSE40419_diffExp_Limma.csv,GSE31210_diffExp_Limma.csv,TCGA_diffExp_gene_Limma.csv(latter three restricted to a ~15-gene stemness panel).multivariate_survival_analysisData_Table1/2.csv,Timer2_TCGA_infiltration_data.csv.
IN SCOPE (attempted — clearly specified pipeline, inputs+outputs shipped)
- TCGA DESeq2 gene diff-exp (tumor vs normal) → compare to
TCGA_diffExp_gene_DeSeq2.csv. - TCGA DESeq2 miRNA diff-exp → compare to
TCGA_diffExp_mirna_DeSeq2.csv(key: hsa-mir-1247). - GSE40419 Limma diff-exp → compare to
GSE40419_diffExp_Limma.csv(stemness panel). - GSE31210 Limma diff-exp → compare to
GSE31210_diffExp_Limma.csv(stemness panel).
Key reported claims compared: 3 stemness genes ORC1L/KIF20A/DLGAP5 up in LUAD; hsa-mir-1247-3p down; targets SLC24A4 (down), RAB3B/HJURP (up).
OUT OF SCOPE (80/20 — not attempted, reasons)
- Univariate/multivariate Cox survival (Table 1/2): requires cBioPortal-sourced clinical survival + stage/age not fully shipped in a single runnable table; lower priority than diff-exp; skipped per 80/20.
- TIMER2 immune infiltration: TIMER2 is an external web tool; shipped CSV is its output, not reproducible without re-querying the web service. Out of scope.
- miRWalk target prediction: external web DB output (xlsx), not a runnable pipeline.
- Wet-lab / manual interpretation claims: out of scope by definition.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
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.
The paper's core differential-expression claims reproduce 1:1 from its own shipped TCGA data: whole-transcriptome and whole-miRNA log2FC correlate at 1.000 and every Table 1 headline stemness gene plus miR-1247-3p matches to 3 decimals, so q5/q7 are fully on the authors' side as confirmed. The only deviations sit in the GEO validation cohorts, where Limma magnitudes run ~1.45x low (GSE31210) or inconsistent (GSE40419 RAB3B 3.152→0.494) — direction and ranking still hold (GSE31210 cor=0.9975). This gap is on our-method/availability side, caused by the authors shipping no analysis code and not documenting their microarray preprocessing, not by fabrication (the early ORC1L suspicion was our own gene-ID error, resolved). Overall: a strong primary reproduction with moderate, fully explainable secondary-cohort magnitude deviations → yellow.
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