Verified Rigor RecordRigor Reviewed

Rigor-Reviewed Manuscript

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: 71
Author: Αlpha¹

Paper source

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: 7.5.0Source: full text

Not verified in this report

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.

11 major claims checked against the paper's own evidence: 2 not fully backed by the presented evidence (unsupported or overstated).

Recomputed 1 test: 1 consistent, 0 inconsistent, 1 via agent-written checks.

3 integrity concerns flagged (0 high).

Rigor

Eight Dimensions2 pass · 5 warn · 1 fail

11 major claims2 overstated2 partial7 supported

Prior literature on LAYN, TLN1, and CREB1 is cited, including their roles in other cancers and relevant signaling pathways. The authors state that the role of LAYN in CHOL and the interaction between these molecules in CHOL metastasis have not been reported, establishing clear knowledge gaps. The hypothesis that CREB1 transcriptionally regulates LAYN to mediate the TLN1/ITGB1 axis in CHOL metastasis follows from the cited evidence. Limitations of prior work (e.g., no studies on LAYN in CHOL) are implicitly addressed by the study design but not explicitly discussed as limitations of the prior research.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedINADEQUATE

Per reviewer (2) — the votes behind the verdict

PASSReviewer 1· 90% conf

The manuscript adequately establishes the scientific premise by citing prior work on LAYN, TLN1, and CREB1 in other cancers, identifies knowledge gaps in cholangiocarcinoma, and provides a logical rationale for the study.

Evidence

paraphrase[Introduction, paragraphs 2-4]

“The introduction reviews known roles of LAYN in cell adhesion and motility, TLN1 in integrin activation and metastasis, and CREB1 as a proto-oncogene, while noting that their roles in CHOL have not been reported.”

direct quote[Introduction, final 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.”

paraphrase[Introduction]

“The paper states what has not been reported before (e.g., 'the role of LAYN has not been extensively studied in CHOL cells') but does not critically evaluate the methods or interpretations of the prior studies it cites.”

PASSReviewer 2· 90% conf

The paper sufficiently establishes its scientific foundation by citing prior work on LAYN, TLN1, and CREB1 in various cancers and identifying gaps in CHOL research.

Evidence

direct quote[Introduction, paragraphs 2-4]

“Notably, low levels of LAYN have been associated with reduced cell invasion and lymph node metastasis in lung cancer cells”

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.”

paraphrase[Introduction, paragraph 5]

“The study aims to investigate whether CREB1, LAYN, and TLN1 can serve as novel oncogenes in CHOL and explore their interactions.”

The in vivo model states 'The nude mice were randomly divided into two groups' but provides no randomization method. Blinding of investigators during outcome assessment is not mentioned. No a priori power/sample-size calculation is reported. Pre-specified inclusion/exclusion criteria for cell lines or animals are absent. Outlier handling is not described. The study uses positive controls (e.g., sh-NC, oe-NC) but no explicit negative control (e.g., a non-targeting scramble control) is described. Independent replication of key findings is not reported or planned. Biological vs. technical replicates are not distinguished; n=3 is stated for in vitro experiments, but it is unclear if these are independent biological replicates.

Sub-criteria

controlsINADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitINADEQUATE

inclusion exclusionNOT REPORTED

randomization methodNOT REPORTED

replicate distinctionNOT REPORTED

independent replicationNOT REPORTED

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 85% conf

The study design lacks explicit randomization and blinding details for the in vivo experiment, does not report a power analysis or pre-specified inclusion/exclusion criteria, and does not distinguish biological from technical replicates.

Evidence

direct quote[Section 2.7, Animal model]

“The nude mice were randomly divided into two groups of six mice each”

absence[Methods, Sections 2.4-2.11]

No mention of blinding for in vivo or in vitro assessments.

absence[Methods, Section 2.11 Statistical analysis]

Power analysis, inclusion/exclusion criteria, and outlier handling are absent.

WARNReviewer 2· 70% conf

The study uses cell-line and animal models with knockdown/overexpression, but lacks several key design elements such as randomization method, blinding, power analysis, and independent replication.

Evidence

direct quote[Methods, Section 2.7]

“The nude mice were randomly divided into two groups of six mice each”

absence[Methods, Sections 2.2-2.11]

No mention of blinding for animal experiments or cell-based assays.

absence[Methods, Section 2.7]

Sample size justification is not provided.

For the in vivo study, 'Four-week-old male BALB/c nude mice' are reported, meeting sex, age, and strain reporting. However, only males are used with no scientific justification (cost/convenience not stated, but no rationale is given for single-sex use). Housing conditions are implied only by 'after one week of acclimatization' but specific details (light cycle, temperature, enrichment, group housing) are absent. For human cell lines, cell type and catalog numbers are provided, satisfying species/strain/source. Demographics of the human cell line donors are not applicable (established cell lines).

Sub-criteria

demographicsN/A

sex reportedADEQUATE

sex justifiedNOT REPORTED

age weight healthADEQUATE

housing conditionsINADEQUATE

species strain sourceADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 80% conf

Sex, age, and health status are reported for the animal model, but the use of only male mice is not scientifically justified. Housing conditions are partially described. Human cell lines are identified but without detailed age/weight/health status of the human donors (not applicable for established cell lines).

Evidence

direct quote[Section 2.7, Animal model]

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

paraphrase[Section 2.7, Animal model]

“Mice underwent one week of acclimatization, but specific husbandry conditions (light cycle, temperature, enrichment) are not described.”

direct quote[Section 2.2, Cell culture and treatment]

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

PASSReviewer 2· 85% conf

The study adequately reports biological variables for both in vitro and in vivo components, including sex, age, weight, species/strain, and cell line sources. Housing conditions are briefly mentioned.

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)”

direct quote[Methods, Section 2.7]

“After one week of acclimatization”

The paper states 'This study was approved by Animal Ethics Committee of Xiangya Hospital, Central South University (No.202300195)', which is a named body with a protocol number, meeting the standard for IACUC-equivalent approval. Regulatory compliance (e.g., 'Guide for the Care and Use of Laboratory Animals') is not explicitly stated, but the approval by a named ethics committee implies adherence. For the human cell lines, they are commercially sourced (Pricella, BIOESN), so no IRB approval is needed. Informed consent is not applicable for commercially obtained cell lines.

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

Animal ethics approval from a named committee with a protocol number is provided. Regulatory compliance for animal care is implied but not explicitly named. Human cell lines are commercially obtained, so no human ethics is required.

Evidence

direct quote[Ethic approval section]

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

absence[Methods and Ethics sections]

Explicit regulatory compliance statement is absent.

PASSReviewer 2· 95% conf

Animal ethics approval is explicitly reported with a named committee and protocol number. The study involves no human subjects; informed consent is not applicable.

Evidence

direct quote[Ethic approval section (after Discussion)]

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

absence[Ethic approval section]

No mention of regulatory compliance frameworks (e.g., Guide for Care and Use of Lab Animals).

Antibodies are listed with vendor and catalog numbers, but RRIDs and validation data are not provided. Cell lines are identified with source and catalog numbers, but authentication (e.g., STR profiling) and mycoplasma testing are not reported. Organisms (mice) are identified by strain and source, but no RRID. Plasmids are identified with Honorgene catalog numbers, and primer sequences are given. Software (GraphPad Prism 8.0, GEPIA) is named; Prism version is provided, but GEPIA and STRING are not versioned. Microplate reader, microscope brands are named but version/software version not always given.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedADEQUATE

organisms identifiedINADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationNOT REPORTED

software tools identifiedINADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 85% conf

Antibodies, cell lines, and plasmids are identified with vendor/catalog numbers, but mycoplasma testing and RRIDs are not provided. Software is named without version numbers in the methods.

Evidence

direct quote[Section 2.3, Western blot]

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

absence[Section 2.2, Cell culture and treatment]

Cell line authentication and mycoplasma testing not reported.

direct quote[Section 2.11, Statistical analysis and Section 2.1]

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

PASSReviewer 2· 85% conf

Antibodies, cell lines, plasmids, and software are identified with vendors/catalog numbers. However, validation data for antibodies are not provided, and cell line authentication/mycoplasma testing are not stated.

Evidence

direct quote[Methods, 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[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)”

direct quote[Methods, Section 2.11]

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

The methods name Student's t-test, one-way and two-way ANOVA. However, normality and heteroscedasticity are not mentioned. Exact p-values are rarely given; the text uses symbolic cut-offs (e.g., *p < 0.05, **p < 0.01). Effect sizes (e.g., Cohen's d) and confidence intervals are absent. GraphPad Prism 8.0 is identified. Data are presented as bar graphs with SD error bars and per-group n stated, but individual data points are not shown. The GRIM test is not applicable here because data are continuous (absorbance readings) not integer. No mathematical implausibility detected.

Sub-criteria

tests namedADEQUATE

exact p valuesINADEQUATE

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedADEQUATE

assumptions verifiedNOT REPORTED

mathematical plausibilityPASS

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 80% conf

Statistical tests are named but assumptions (normality, equal variance) are not verified. Exact p-values are mostly reported as ranges (p < 0.05, p < 0.01) rather than exact values. Effect sizes and confidence intervals are not reported. Software is identified with version. Data presentation includes bar plots with error bars and n, but individual data points are not shown, and error bars are defined as SD but not justified for the test.

Evidence

direct quote[Section 2.11, Statistical analysis]

“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[Results, Section 3.1]

“we observed significantly elevated expression compared to that in the Normal group (A, p < 0.01).”

absence[Results, Sections 3.1-3.5]

Effect sizes and confidence intervals are not reported.

WARNReviewer 2· 75% conf

Statistical tests are named, and p-value thresholds are set, but effect sizes, confidence intervals, and exact p-values are largely missing. Assumptions are not verified; data presentation uses error bars but individual points are not shown.

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[Results, e.g., Section 3.1]

“p < 0.01”

paraphrase[Figures (described in Results)]

“Figures are described as showing bar charts; no scatter dot plots are mentioned.”

The 'Data available' section states 'All data can be obtained from the corresponding author.' This is a vague statement without a mechanism, platform, or conditions, which is rated inadequate. No datasets are deposited in a repository, no accession numbers are provided (except for databases used, which are public), and no custom code is shared (no mention of code availability). Even though public databases are cited, the experimental data generated (WB blots, cell assay data, animal data) are not deposited.

Sub-criteria

code sharingNOT REPORTED

accession numbersN/A

repository depositNOT REPORTED

data availability statementINADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 90% conf

A data availability statement is present but is vague ('All data can be obtained from the corresponding author' with no repository or mechanism). No code is shared. Bioinformatics databases are accessed but no accession numbers for the study-specific data are provided.

Evidence

direct quote[Data available section]

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

absence[Data available section and Methods]

No repository deposit or accession numbers.

absence[Methods sections]

No code sharing.

FAILReviewer 2· 90% conf

No data availability statement beyond a vague line; no repository deposit, accession numbers, or code sharing. The paper essentially offers no data access route.

Evidence

direct quote[Data available section (after Discussion)]

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

absence[Data available section, Methods]

No repository deposit for the experimental data.

absence[Data available section, Methods]

No code sharing statement or repository.

Methods describe cell culture, transfection, Western blot, CCK-8, Transwell, IF, animal model, H&E, Co-IP, ChIP, and statistical analysis in sufficient detail. No reporting guideline (e.g., ARRIVE for animal studies) is mentioned. The study does not transparently report negative outcomes; all reported outcomes are significant. Limitations are discussed (e.g., need for in vivo studies with more samples, differences from prior studies), but sources of bias, confounding, and the small sample size are not critically assessed. Conclusions are proportional to the correlational and in vitro/in vivo evidence; causation is not overclaimed. Funding source and conflict of interest statement are provided.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedINADEQUATE

limitations discussedINADEQUATE

conclusions proportionalADEQUATE

Per reviewer (2) — the votes behind the verdict

WARNReviewer 1· 85% conf

Methods are detailed enough for replication, but the study lacks trial registration (n/a for preclinical), does not reference a reporting guideline, does not report all outcomes transparently (some results are presented directionally without full data), limitations are discussed but incompletely, conclusions are generally proportional, and funding/COI statements are present.

Evidence

absence[Methods sections]

No reporting guideline referenced.

direct quote[Results, Sections 3.1-3.5]

“we observed significantly elevated expression... Knockdown of LAYN significantly inhibited proliferation, migration, invasion, and EMT”

paraphrase[Discussion, final paragraph]

“Authors discuss the need for further studies and acknowledge differences from prior work, but do not discuss potential biases, confounding factors, or the small sample size limitations.”

WARNReviewer 2· 80% conf

Methods are generally detailed enough for replication, limitations are discussed, conclusions are proportional, and funding/COI are stated. However, no reporting guideline (ARRIVE, CONSORT) is mentioned, and trial registration is not applicable for a preclinical animal study.

Evidence

paraphrase[Methods, Sections 2.2-2.11]

“All procedures for cell culture, transfections, assays, and animal experiments are described step-by-step.”

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[Funding and Declaration sections]

“This study was supported by...Natural Science Foundation of Hunan Province (No. 2023JJ30910).”

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

Checked 30 references: 29 verified — 1 unresolved.

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

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