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CREB1 promotes cholangiocarcinoma metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis · 10.1016/j.heliyon.2024.e36595

Name: Verified Rigor Record — CREB1 promotes cholangiocarcinoma metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis · 10.1016/j.heliyon.2024.e36595
Item: CREB1 promotes cholangiocarcinoma metastasis through transcriptional regulation of the LAYN-mediated TLN1/β1 integrin axis · 10.1016/j.heliyon.2024.e36595
Rating: 75
Author: Αlpha¹

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Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Interventional Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: moderateReviewer agreement: moderateEngine: 7.6.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.
Declared data/code links were not checked for liveness or content.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

This preclinical study on the CREB1/LAYN/TLN1 axis in cholangiocarcinoma has a clear scientific premise and fairly detailed methods, but is weakened by several standard rigor omissions: no blinding, no power analysis, vague data availability, and incomplete statistical reporting. The animal ethics approval is adequate but lacks a compliance statement. Copyedit errors (e.g., FADS2 and Meningioma remnants) suggest a hasty final editing pass. The paper also cites a reference that could not be found in any registry, raising a possible fabrication signal.

Two independent reviewers scored all eight dimensions; full-text of the manuscript was available. The statistics verification component checked zero tests (only threshold p-values reported, no test statistics or CIs provided), so mathematical validity is not confirmed; the citation component found 1 not-found reference (possible fabrication). The study was classified as interventional based on its primary experimental design.

10 major claims checked against the paper's own evidence: all adequately supported.

Rigor

Eight Dimensions3 pass · 4 warn · 1 fail

10 major claims10 supported

The introduction cites relevant prior research on LAYN in cell adhesion and metastasis, TLN1 in integrin activation and bone metastasis, and CREB1 as a transcription factor in various cancers, while explicitly stating that the roles of LAYN, TLN1, and CREB1 in CHOL have not been reported. The study aims to investigate whether these genes can serve as novel oncogenes or prognostic factors and explores potential interactions, which follows logically from the cited gaps.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedINADEQUATE

Per reviewer (2) — the votes behind the verdict

PASSReviewer 1· 95% conf

The study clearly establishes its scientific foundation by citing prior work on LAYN, TLN1, and CREB1 in other cancers, noting the knowledge gap in cholangiocarcinoma, and providing a logical rationale for the hypothesis.

Evidence

direct quote[Introduction, paragraphs 2-4]

“However, the role of LAYN has not been extensively studied in CHOL cells, and reported studies on LAYN in CHOL are limited.”

direct quote[Introduction, paragraph 5]

“This study aimed to investigate whether CREB1, LAYN, and TLN1 can serve as novel oncogenes or prognostic factors for CHOL and to explore the potential interactions between them.”

paraphrase[Introduction, paragraphs 2-4]

“The paper notes gaps (e.g., 'not been reported', 'limited') but does not describe how its design addresses specific methodological weaknesses of prior studies.”

PASSReviewer 2· 90% conf

The scientific premise is well-established, with adequate prior work cited and a logical rationale for the study, but limitations of prior research are not specifically addressed.

Evidence

direct quote[Introduction, paragraphs 2-4]

“Research has linked LAYN to tumor necrosis factor-α (TNF-α)-induced epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells []. Notably, low levels of LAYN have been associated with reduced cell invasion and lymph node metastasis in lung cancer cells []. However, the role of LAYN has not been extensively studied in CHOL cells, and reported studies on LAYN in CHOL are limited.”

direct quote[Introduction, last paragraph]

“This study aimed to investigate whether CREB1, LAYN, and TLN1 can serve as novel oncogenes or prognostic factors for CHOL and to explore the potential interactions between them.”

absence[Discussion]

Limitations of prior research are not specifically addressed beyond stating gaps.

Only 1 of 9 applicable sub-criteria (controls) is adequate. The animal experiment lacks blinding and randomization method details. No a priori sample size calculation is provided. Replicates (n=3 for in vitro, n=6 for in vivo) are stated but not explicitly distinguished as biological or technical. No outlier handling or independent replication is reported.

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitINADEQUATE

inclusion exclusionNOT REPORTED

randomization methodINADEQUATE

replicate distinctionINADEQUATE

independent replicationNOT REPORTED

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 90% conf

The study design has several strengths (use of controls, multiple cell lines, in vivo model) but lacks a power/sample-size calculation, explicit randomization method for in vitro assays, and independent replication of key findings.

Evidence

direct quote[Methods, section 2.7]

“The nude mice were randomly divided into two groups of six mice each: sh-NC ... and sh-LAYN ...”

absence[Entire methods]

Blinding is not mentioned anywhere in the manuscript.

absence[Methods, section 2.11 (statistical analysis)]

No power or sample-size calculation is reported.

FAILReviewer 2· 90% conf

The study design lacks critical rigor elements: no blinding, no power analysis, no pre-specified inclusion/exclusion criteria, and no distinction between biological and technical replicates.

Evidence

direct quote[Section 2.7]

“The nude mice were randomly divided into two groups of six mice each: sh-NC (RBE cells transfected with sh-NC) and sh-LAYN (RBE cells transfected with sh-LAYN).”

absence[Section 2.7, 2.8]

Blinding of investigators or assessors is not mentioned.

absence[Section 2.11]

No power analysis or sample size justification.

Mice are reported as male BALB/c nude, 4-week-old, but weight and health status are not provided. Housing conditions (light cycle, temperature, enrichment) are not described. Cell lines are adequately identified with source and catalog numbers.

Sub-criteria

demographicsN/A

sex reportedADEQUATE

sex justifiedINADEQUATE

age weight healthINADEQUATE

housing conditionsNOT REPORTED

species strain sourceADEQUATE

Per reviewer (2) — the votes behind the verdict

PASSReviewer 1· 95% conf

Sex, age, weight, species/strain, health status, and housing conditions are adequately reported for the animal model; cell lines are identified by source and catalog number.

Evidence

direct quote[Methods, section 2.7]

“Four-week-old male BALB/c nude mice (Hunan SJA Laboratory Animal Co. Ltd.) were used.”

direct quote[Methods, section 2.2]

“Human normal bile duct cells HIBEpiC (CP–H175, Pricella), human CHOL cells QBC939 (BES-2522HC, BIOESN), and RBE (CL-0191, Pricella)”

absence[Methods, section 2.7]

Housing conditions (e.g., light cycle, temperature) are not described.

WARNReviewer 2· 80% conf

Sex, species, and strain are reported, but age/weight/health status and housing conditions for mice are incomplete, and no justification is given for using only male mice.

Evidence

direct quote[Section 2.7]

“Four-week-old male BALB/c nude mice (Hunan SJA Laboratory Animal Co. Ltd.) were used.”

absence[Section 2.7]

Mouse weight not reported at baseline; health status not described.

absence[Section 2.7]

Housing conditions not reported.

The paper includes an animal ethics committee approval (Xiangya Hospital, No.202300195), which is adequate for the animal study. However, no explicit statement of compliance with specific regulations (e.g., NIH Guide, EU Directive) is given, making the regulatory_compliance sub-criterion inadequate.

Sub-criteria

iacuc statementADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceINADEQUATE

Per reviewer (2) — the votes behind the verdict

PASSReviewer 1· 95% conf

Ethical approval for the animal experiment is provided by a named body with a protocol number. No human subjects were involved, so human ethics are not applicable.

Evidence

direct quote[Back matter, 'Ethic approval']

“This study was approved by Animal Ethics Committee of Xiangya Hospital, Central South University (No.202300195).”

absence[Back matter, 'Ethic approval']

Regulatory compliance (e.g., Guide for Care and Use) is not explicitly stated.

WARNReviewer 2· 90% conf

Animal ethics approval is provided with a protocol number, but regulatory compliance (e.g., Guide for the Care and Use of Laboratory Animals) is not stated.

Evidence

direct quote[Ethic approval section]

“This study was approved by Animal Ethics Committee of Xiangya Hospital, Central South University (No.202300195).”

absence[Ethic approval section]

No statement of compliance with specific animal care regulations.

Antibodies are listed with vendor and catalog number (e.g., 'LAYN (1:1000, ab192610, Abcam)'), but validation data or specificity references are not included. Cell lines are identified with source and catalog number; authentication (e.g., STR) is not reported. Mycoplasma testing is not mentioned. Plasmids are identified by source and catalog number. Software (GraphPad Prism 8.0) is named with version.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedADEQUATE

organisms identifiedADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationNOT REPORTED

software tools identifiedADEQUATE

Per reviewer (2) — the votes behind the verdict

PASSReviewer 1· 90% conf

All key resources (antibodies, cell lines, plasmids, software) are identified with vendors and catalog numbers. Validation data for antibodies are not provided, but the paper is adequate for an exploratory study.

Evidence

direct quote[Methods, section 2.3]

“Primary antibodies including LAYN (1:1000, ab192610, Abcam) ... TLN1 (1:1000, ab71333, Abcam) ... CREB1 (1:1000, ab32515, Abcam)”

direct quote[Methods, section 2.2]

“Human CHOL cells QBC939 (BES-2522HC, BIOESN), and RBE (CL-0191, Pricella)”

direct quote[Methods, section 2.11]

“Statistical analyses were conducted using GraphPad Prism 8.0 software.”

PASSReviewer 2· 80% conf

Most key resources are adequately identified, with vendor and catalog numbers for antibodies, cell lines, plasmids, and software. However, cell line authentication and mycoplasma testing are not reported.

Evidence

direct quote[Section 2.3]

“Primary antibodies including LAYN (1:1000, ab192610, Abcam), N-cadherin (1:1000, ab76011, Abcam), Snai1 (1:1000, 13099-1-AP, Proteintech), ...”

direct quote[Section 2.2]

“Human normal bile duct cells HIBEpiC (CP–H175, Pricella), human CHOL cells QBC939 (BES-2522HC, BIOESN), and RBE (CL-0191, Pricella) were cultured...”

absence[Section 2.2]

Mycoplasma testing not reported.

Tests named: Student's t-test, one-way ANOVA, two-way ANOVA. Software: GraphPad Prism 8.0. Data presentation: bar charts with error bars, but the definition of error bars (SD) is given in the methods. Per-group N is stated (n=3 for in vitro, n=6 for in vivo). However, assumptions (normality, equal variance) are not verified. P-values are mostly reported as ranges (e.g., p < 0.01, p < 0.001), not exact values. Effect sizes and confidence intervals are not provided. Mathematical plausibility: some percentages (e.g., 96.8%, 193.6%) are reported from protein expression changes, but the raw data to verify the arithmetic (e.g., GRIM test) are not available in the text. No arithmetic errors are detected.

Sub-criteria

tests namedADEQUATE

exact p valuesINADEQUATE

effect sizes ciNOT REPORTED

data presentationADEQUATE

software identifiedADEQUATE

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 90% conf

Statistical tests are named, software is identified, and data are presented with error bars and p-values. However, test assumptions are not verified, exact p-values are often given as ranges (p < 0.05, etc.), and effect sizes/confidence intervals are not reported.

Evidence

direct quote[Methods, section 2.11]

“Student's t-test was used to evaluate the differences between two groups, while one-way ANOVA and two-way ANOVA were used to analyze multiple groups.”

direct quote[Figure legends (throughout Results)]

“*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.”

absence[Results and figure legends]

Effect sizes and confidence intervals are not reported.

WARNReviewer 2· 70% conf

Statistical tests are named and software is identified, but assumptions are not verified, exact p-values are not reported (only thresholds), effect sizes and confidence intervals are absent, and individual data points are not shown.

Evidence

direct quote[Section 2.11]

“Student's t-test was used to evaluate the differences between two groups, while one-way ANOVA and two-way ANOVA were used to analyze multiple groups.”

direct quote[Figures and Section 2.11]

“Statistical significance was set at p < 0.05.”

absence[Figures (e.g., Fig 1D-F)]

Individual data points not shown; bar graphs only.

The data availability statement says 'All data can be obtained from the corresponding author.' This is a vague statement without a platform, conditions, or timeframe, and thus is 'reported_but_inadequate.' No raw data are deposited in a repository. No code is shared. No accession numbers are provided.

Sub-criteria

code sharingNOT REPORTED

accession numbersNOT REPORTED

repository depositNOT REPORTED

data availability statementINADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 95% conf

A data availability statement is present ('All data can be obtained from the corresponding author') but is vague and does not provide a concrete access route, such as a repository or managed-access platform.

Evidence

direct quote[Back matter, 'Data available']

“All data can be obtained from the corresponding author.”

absence[All sections]

No repository deposit or accession numbers.

absence[All sections]

No code sharing.

WARNReviewer 2· 90% conf

A data availability statement is present but vague ('available from the corresponding author'), lacking a concrete repository or access mechanism. No code sharing or repository deposits are mentioned.

Evidence

direct quote[Data available section]

“All data can be obtained from the corresponding author.”

absence[Data available section]

No repository deposit mentioned.

Methods include detailed protocols for cell culture, transfection, WB, CCK-8, Transwell, IF, animal model, H&E, Co-IP, and ChIP. The Discussion acknowledges limitations (e.g., 'may be due to the differences in the downstream targets of LAYN action in CHOL cells compared to other tumor cells'). Conclusions are framed appropriately ('suggests', 'may serve as'). Funding (Natural Science Foundation of Hunan Province) and COI (none declared) are reported.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineN/A

methods completenessADEQUATE

all outcomes reportedINADEQUATE

limitations discussedADEQUATE

conclusions proportionalADEQUATE

Per reviewer (2) — the votes behind the verdict

PASSReviewer 1· 90% conf

Methods are largely detailed enough for replication. Limitations are discussed in the Discussion, and conclusions are proportional to the evidence. Funding and COI are reported. Trial registration and reporting guidelines are not applicable.

Evidence

paraphrase[Methods, sections 2.1–2.10]

“Detailed protocols for cell culture, transfection, WB, CCK-8, Transwell, IF, animal model, H&E, Co-IP, and ChIP are provided.”

direct quote[Discussion, paragraph 2]

“This may be due to the differences in the downstream targets of LAYN action in CHOL cells compared to other tumor cells.”

direct quote[Back matter, 'Funding' and 'Declaration of competing interest']

“This study was supported by ... Natural Science Foundation of Hunan Province (No. 2023JJ30910). The authors declare that they have no known competing financial interests or personal relationships...”

PASSReviewer 2· 80% conf

Methods are sufficiently detailed, all outcomes are reported, and conclusions are proportional. However, no reporting guideline (e.g., ARRIVE) is referenced, and limitations are only briefly discussed.

Evidence

direct quote[Section 2]

“We utilized the GEPIA, STRING, and hTFtarget databases for bioinformatics analysis. Overexpression or knockdown cell lines were constructed by transfecting the cells with different plasmids. Western blot (WB) was performed to detect LAYN, TLN1, and CREB1 expression.”

absence[Section 2]

No reporting guideline mentioned.

direct quote[Discussion, paragraph 3 and 5]

“This may be due to the differences in the downstream targets of LAYN action in CHOL cells compared to other tumor cells.”

8 copyedit issues flagged: mostly typo, consistency, clarity.

Checked 30 references: 29 verified — 1 unresolved.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

In this post-publication audit, the paper's reproducibility is substantially limited by missing design details (blinding, randomization method, power analysis) and a vague data availability statement. An informed reader should weigh these gaps heavily; the missing reference and copyedit errors (FADS2/Meningioma remnants) further erode confidence. A correction or independent re-analysis would be warranted to validate the key findings.

1.
HIGHreporting
State the exact randomization method (e.g., random number table) for the in vivo experiment and for any randomized in vitro experiments (Methods, section 2.7 and all in vitro methods).
Randomization is claimed but unverifiable, undermining internal validity of group comparisons.
2.
HIGHreporting
State explicitly whether blinding was used during outcome assessment (e.g., counting metastatic nodules) and, if not, justify its infeasibility (Methods, section 2.7, 2.8).
Lack of blinding is the most common cause of inflated effect sizes in preclinical studies.
3.
HIGHdata code
Replace the vague data availability statement with a repository link (e.g., Figshare, Zenodo) containing raw Western blot images, cell-count data, and any other underlying data, or specify a managed-access mechanism with conditions (Data available section).
The current statement (available from the author) is not verifiable and violates journal data policies.
4.
HIGHstatistics
Report exact p-values (e.g., p = 0.023) instead of threshold ranges in all figure legends (Results, all figures).
Threshold-only p-values (p < 0.05, p < 0.01, etc.) do not permit exact interpretation or meta-analysis.
5.
HIGHreporting
Investigate and resolve the 1 reference ('A novel screening test for colon cancer: talin-1') that could not be found in any registry — it may be fabricated or incorrectly cited (References section).
A reference that cannot be located in any registry is a fabrication signal that undermines the integrity of the paper.
6.
HIGHrigor
Provide a power analysis or sample-size justification for the animal experiment and, if available, for the in vitro experiments (Methods, section 2.7, 2.11).
Without power analysis, it is unclear whether the study had sufficient sensitivity to detect meaningful effects and whether the sample size was pre-specified.
7.
HIGHrigor
State whether test assumptions (normality, homogeneity of variance) were verified for the t-tests and ANOVAs, and if violated, describe the alternative tests used (Methods, section 2.11).
False-positive rates inflate when assumptions are violated and not accounted for.
8.
HIGHrigor
Distinguish biological vs. technical replicates in all experiments (e.g., 'n=3 independent biological replicates') in Methods sections 2.3-2.10.
Confusing biological and technical replicates invalidates statistical inference and is a common reproducibility barrier.
9.
HIGHrigor
Pre-specify inclusion/exclusion criteria for the animal model (e.g., health status at baseline, any exclusions during the study) (Methods, section 2.7).
Without pre-specified criteria, selective exclusion can bias results.
10.
HIGHrigor
Report mycoplasma testing results for all cell lines, or state whether testing was done; if not, explain its absence (Methods, section 2.2).
Mycoplasma contamination can alter cell behavior and invalidate experimental results.
11.
HIGHrigor
Provide STR authentication for the cell lines (QBC939, RBE, HIBEpiC) or cite the most recent authentication (Methods, section 2.2).
Cell line misidentification is a major source of irreproducibility in cancer research.
12.
HIGHreporting
Clarify animal housing conditions: light/dark cycle, temperature, cage enrichment, and welfare assessments (Methods, section 2.7).
Housing conditions affect stress, behavior, and experimental outcomes; reproducibility requires their specification.
13.
HIGHethics
Add a statement of compliance with the Guide for the Care and Use of Laboratory Animals or equivalent national standards in the Ethics section.
Many journals require an explicit compliance statement beyond the local ethics committee name and number.
14.
HIGHcopyedit
Fix 'FADS2' to 'LAYN' and 'Meningioma cells' to 'CHOL cells' in Methods sections 2.1 and 2.5; fix 'oe-LAYN + si-TLN1' to 'oe-LAYN + si-CREB1' in section 2.2.
These are copy-paste errors from unrelated manuscripts/analyses that indicate a hasty final edit and undermine credibility.
15.
HIGHstatistics
Include individual data points (e.g., dot plots) alongside bar charts in all figures, or replace bar charts with box plots or dot plots showing all points (Figures).
Bar charts obscure the distribution and sample size, making it impossible to assess variability or identify outliers.
16.
MEDIUMcopyedit
Fix the prime symbol in the ChIP primer sequence: '5′-TAACAGCGGTTGTGGCAGGA3.′' → '5'-TAACAGCGGTTGTGGCAGGA-3'' (Methods, section 2.10, last line).
Incorrect notation may cause confusion during primer synthesis or for readers replicating the experiment.
17.
MEDIUMreporting
Justify the use of only male mice (e.g., to avoid hormonal confounds, or note that sex difference was not the focus) (Methods, section 2.7).
NIH and many journals require consideration of sex as a biological variable; unexplained single-sex studies may be questioned.
18.
MEDIUMstatistics
Include effect sizes (e.g., Cohen's d) and 95% confidence intervals alongside p-values for all primary comparisons (Results, figures).
Effect sizes and CIs convey the magnitude and precision of effects, which p-values alone do not.

Full text (40,953 chars)Analyzed: Jul 6, 2026

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