xploration of the Expression and Preliminary Mechanisms of Estrogen and Progesterone Responsive Molecules in Progesterone Resistance

Description

Data Description: Molecular Profiling of Progesterone Resistance in Atypical Endometrial Hyperplasia Research Objective & Hypothesis This study investigates the molecular basis of progesterone resistance in atypical endometrial hyperplasia (AEH). We hypothesized that resistance arises from synergistic dysregulation across three pathways: estrogen signaling overactivation, progesterone signaling impairment, and sustained cellular proliferation. Data Collection Methods · Source: Endometrial tissues from 20 AEH patients after 6-month progestin therapy, stratified into progesterone-resistant (n=10) and sensitive (n=10) groups. · Immunohistochemistry (IHC): Quantified protein expression and localization via specific antibodies and ImageJ analysis (Mean Optical Density). · Western Blot (WB): Validated protein levels through SDS-PAGE and densitometric quantification. · Statistical Analysis: Unpaired t-tests (GraphPad Prism 9); significance at p < 0.05. Key Findings & Data Interpretation 1. IHC Data · What it shows: Visual and quantitative protein expression in tissue context. · Estrogen Axis: Significant upregulation of ERα, pS2, and MUC1 in resistant tissues, indicating sustained estrogenic activity. · Progesterone Axis: Coordinated downregulation of PR, its co-chaperones (FKBP4, FKBP5), and downstream effector FOSL2, demonstrating comprehensive progesterone signaling failure. · Proliferation Markers : Elevated SOX7 and Ki-67 in resistant group, confirming persistent proliferation despite treatment. 2. Western Blot Data · What it shows: Biochemical validation of protein expression trends. · Figure 3A-C: Confirmed significant downregulation of progesterone pathway components (FKBP4, FKBP5, FOSL2). · Figure 3C-D: Validated upregulation of SOX7 and estrogen-responsive effectors (pS2, MUC1). Notable Discoveries · Multi-pathway Dysregulation: Progesterone resistance involves synergistic failure across estrogen, progesterone, and proliferative pathways. · Novel SOX7 Involvement: First identification of SOX7 upregulation in AEH progesterone resistance, suggesting its role in sustaining proliferation. · Methodological Consistency: Concordant results from IHC (tissue context) and WB (biochemical validation) strengthen findings. Data Usage Guidance This dataset provides: · A molecular signature for identifying progesterone-resistant AEH · Validated targets for combination therapies (e.g., ERα or SOX7 inhibitors) · Foundation for functional studies to establish causality

Steps to reproduce

Data Acquisition and Methodology 1. Study Cohort and Tissue Collection · Source: Endometrial tissue samples were obtained from 20 pathologically confirmed AEH patients at Liuzhou Maternal and Child Health Hospital (2019-2022). · Treatment Protocol: All patients received oral medroxyprogesterone acetate (MPA) at 160 mg/day for 6 months. · Group Classification: Patients were stratified into progesterone-resistant (n=10) and progesterone-sensitive (n=10) groups based on post-treatment pathological evaluation. 2. Immunohistochemistry (IHC) Workflow · Tissue Processing: Tissues were fixed in 4% paraformaldehyde for 24 hours, paraffin-embedded, and sectioned at 4 μm thickness. · Antigen Retrieval: Performed using citrate buffer (pH 6.0) with heat-mediated epitope retrieval. · Staining Protocol: · Blocking: 3% H₂O₂ for endogenous peroxidase, 5% goat serum for nonspecific binding · Primary Antibodies: Incubated overnight at 4°C with optimized dilutions: · ERα (1:200), PR (1:300), MUC1 (1:500), SOX7 (1:400) · Detection: HRP-conjugated secondary antibody (1 hour, room temperature) · Visualization: DAB chromogen with hematoxylin counterstaining · Quantification: Three representative 200× fields per section were imaged. Mean Optical Density (MOD) was measured using ImageJ software (National Institutes of Health). 3. Western Blot Analysis · Protein Extraction: Total protein extracted using RIPA lysis buffer supplemented with protease inhibitors · Quantification: Protein concentration determined by BCA assay · Electrophoresis: 20 μg protein/lane separated by 10% SDS-PAGE · Transfer: Proteins transferred to PVDF membranes · Blocking and Incubation: · Blocking: 5% non-fat milk in TBST (2 hours, room temperature) · Primary Antibodies: Overnight incubation at 4°C · Secondary Antibodies: HRP-conjugated (2 hours, room temperature) · Detection: ECL substrate (Millipore) with imaging via ChemiDoc MP System (Bio-Rad) · Quantification: Band intensities analyzed using ImageJ software 4. Instrumentation and Reagents · Key Instruments: · Microtome for sectioning · ChemiDoc MP Imaging System (Bio-Rad) · Standard electrophoresis and transfer apparatus · Critical Reagents: · Primary antibodies against all target proteins · HRP-conjugated secondary antibodies · DAB substrate kit · ECL detection reagent (Millipore) · RIPA lysis buffer with protease inhibitors 5. Statistical Analysis · Software: GraphPad Prism 9 · Methods: Normality testing (Shapiro-Wilk), between-group comparisons (unpaired t-test) · Significance Threshold: p < 0.05 (two-tailed) · Data Presentation: Mean ± standard deviation 6. Quality Control Measures · Experimental Controls: Included positive and negative controls for IHC · Loading Controls: Used GAPDH/β-actin for Western blot normalization · Blinded Analysis: IHC quantification performed without group identity · Technical Replicates: Multiple fields per sample and repeated Western blot experiments

Institutions

• Guilin Medical University

  Guangxi, Guilin

Categories

Funders

• Pinxiu Huang

  Grant ID: ORCID 0000-0002-9011-4970

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