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Research Article

Clinical Significance, Immune Infiltration Landscape, and Functional Network Analysis of miR-192-5p in Colorectal Cancer

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DOI:

10.3791/71908

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September 1st, 2026

 ,  ,  ,  , 

* These authors contributed equally

In This Article

Summary

This study integrates public datasets, paired-tissue validation, cell migration assays, target network prediction, and immune signature analysis to evaluate miR-192-5p in colorectal cancer. The findings highlight discordance between TCGA/GDC expression data and local qRT-PCR results and caution against overinterpretation of its diagnostic, prognostic, functional, and immune-related significance.

Abstract

Colorectal cancer (CRC) is a heterogeneous malignancy in which microRNAs may contribute to tumor progression and biomarker discovery. This study evaluated the clinical significance, immune associations, and functional network of miR-192-5p in CRC by integrating TCGA/GDC and GEO bioinformatic analyses, paired tissue qRT-PCR validation, target prediction, immune signature analysis, and HT29 wound-healing experiments. Reanalysis of TCGA/GDC miRNA-seq data included 616 tumor and 11 normal COADREAD samples and showed higher database-level hsa-mir-192 signal in tumor samples than in normal samples (Mann-Whitney p = 9.96 x 10-8). The same direction was observed in 11 paired TCGA cases (median tumor-normal difference = 3.13 log2[RPM + 1], Wilcoxon p = 0.0029), whereas local qRT-PCR in paired clinical tissues supported lower mature miR-192-5p expression in CRC tissues. TCGA-based ROC analysis showed strong tumor-normal separation, but balanced resampling and leave-one-normal-out analyses confirmed that this result should be interpreted cautiously due to the limited number of normal samples. Clinicopathological analysis suggested associations with N stage and age in the TCGA cohort, whereas T stage and M stage were not significant in the summarized table, and overall survival analysis did not show a significant prognostic association. Candidate hub-gene analyses using TCGA RNA-seq provided exploratory expression, correlation, and survival evidence but did not establish direct miR-192-5p targeting. Immune signature analysis showed heterogeneous, mostly weak correlations. Functionally, miR-192-5p mimic transfection reduced HT29 wound closure. These findings indicate that miR-192-5p is biologically relevant to CRC, but its expression direction and clinical interpretation are platform-dependent and require further experimental validation.

Introduction

Colorectal cancer (CRC) is one of the most common malignancies worldwide and remains a leading cause of cancer-related mortality1,2. Although colonoscopy, fecal immunochemical testing, SEPT9 methylation testing, circulating tumor markers, and molecular profiling have improved CRC detection and clinical management3,4, substantial heterogeneity remains in tumor biology, treatment response, and patient outcome5,6,7. Biomarkers that help characterize CRC biology m....

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Protocol

This study was performed in accordance with the Declaration of Helsinki. Approval was granted by the Ethics Committee of Inner Mongolia Medical University on October 21, 2021. Written informed consent was obtained from all participants included in the study.

Clinical specimen collection

Paired CRC and adjacent noncancerous tissues were collected from 45 patients who underwent radical resection between December 2017 and February 2020. None of the patients received preoperative radiotherapy or chemotherapy, and patients with other malignancies were excluded. Adjacent noncancerous tissue was collect....

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Results

Expression analysis in public datasets and paired clinical tissues

The expression pattern of miR-192 was reassessed using GDC-derived TCGA-COAD and TCGA-READ miRNA-seq data (Figure 1). In these files, the database-level entry was annotated as hsa-mir-192 rather than directly as the mature hsa-miR-192-5p species; therefore, this public-database result was interpreted as a TCGA miR-192 signal. In the combined COADREAD cohort, 616 primary tumors and .......

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Discussion

This study evaluated the clinical and biological relevance of miR-192-5p in CRC by combining public dataset analysis, local tissue validation, target network prediction, immune signature analysis, and in vitro migration experiments. The analyses used verified TCGA sample definitions, separated COAD and READ where possible, used log2(RPM + 1) miRNA values, distinguished paired from unpaired comparisons, tested ROC robustness, and added hub-gene RNA-seq sensitivity analyses. The central finding is that the public TCGA/GDC .......

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Disclosures

The authors have no conflict of interest to declare.

Acknowledgements

The authors thank the Molecular Diagnostic Laboratory of Tumor of Inner Mongolia Medical University for technical support. This work was supported by the Scientific Research Project of the Yancheng Municipal Health Commission (Grant No. YK2023101), which also funded the article processing charge.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.25%+2:60 trypsin-EDTABiological Industries03-054-1Cell digestion/passaging
10% ammonium persulfateSolarbio Science & Technology Co., Ltd.A8090SDS-PAGE reagent
10% sodium dodecyl sulfateSolarbio Science & Technology Co., Ltd.SIS1070SDS-PAGE reagent
4x protein loading bufferSolarbio Science & Technology Co., Ltd.P1016Protein sample preparation
-80 C ultra-low temperature freezerQingdao Haier Biomedical Co., Ltd.https://www.haiermedical.com/Sample storage
Anti-Myc mycoplasma removal reagentWuhan Procell Life Science & Technology Co., Ltd.P-CMR-001Mycoplasma decontamination
ARL2, BATF2, MEN1, and beta-actin primer setsSangon Biotech Co., Ltd., Shanghai, ChinaCustom synthesizedmRNA qPCR primers
Automatic microplate readerPerkinElmer, USAVICTOR NivoAbsorbance measurement
BCA Protein Assay KitThermo Fisher Scientific23225Protein quantification
Biosafety cabinet / clean benchQingdao Haier Biomedical Co., Ltd.https://www.haiermedical.com/Aseptic cell culture
Cell counting chamber / hemocytometerShanghai Qiujing Biochemical Reagent Instrument Co., Ltd.http://www.shqiujing.net/Cell counting
Cell Counting Kit-8 (CCK-8)BiosharpBS350BCell proliferation/cytotoxicity assay
Cell culture flasks, culture plates, centrifuge tubes, and pipette tipsCorninghttps://www.corning.com/in/en.htmlGeneral cell culture consumables
Cell Cycle and Apoptosis Detection KitMeilun Biotechnology Co., Ltd., Dalian, Chinahttps://www.chemicalbook.com/ShowSupplierProductsList13366/0_EN.htmFlow-cytometric cell-cycle/apoptosis analysis
CO2 cell incubatorQingdao Haier Biomedical Co., Ltd.https://www.haiermedical.com/Cell culture
con238 negative-control oligonucleotideSangon Biotech Co., Ltd., Shanghai, ChinaCustom synthesizedTransfection control
Cytoscape softwareCytoscape Consortiumhttps://cytoscape.org/Network module and hub-gene visualization
Digital thermostatic magnetic stirrerChangzhou Saipu Experimental Instrument Factoryhttps://czxtyq.en.alibaba.com/Solution preparation
Electric thermostatic blast drying ovenNanjing Wohuan Technology Industry Co., Ltd.https://www.wtsensor.com/about-wt/aboutwt/Drying/sterilization support
Electrophoresis systemBio-Rad LaboratoriesMini-PROTEAN 3 CellGel electrophoresis
Electrotransfer systemHoeferTE77XPProtein transfer
Fetal bovine serumBiological Industries04-001-1ACSCell culture supplement
Flow cytometerACEA BiosciencesNovoCyteFlow cytometry
Fluorescence inverted microscopeNingbo Sunny Instruments Co., Ltd.https://www.sunny-instrument.com/enCell and wound imaging
GDC/TCGA portalNational Cancer InstituteOpen-access databasePublic miRNA-seq, RNA-seq, and clinical data
GEO databaseNational Center for Biotechnology InformationGSE89076 and GSE156355Public gene-expression datasets
GraphPad PrismGraphPad SoftwareVersion 9Statistical analysis and graphing
High-speed refrigerated centrifugeShanghai Huxiangyi Centrifuge Instrument Co., Ltd.https://www.bioridgecentrifuge.com/Sample preparation
HT29 cellsAuthors' laboratory cell stockNot applicableHuman colorectal cancer cell line
Image analysis softwareImageJNot applicableWound-width quantification
Imaging systemTanon Science & Technology Co., Ltd.Tanon-5200Gel/blot imaging
IncuCyte S3 dynamic cell observation and functional analysis systemBeijing Sensi Wantong Technology Co., Ltd.IncuCyte S3Live-cell imaging/function analysis
Intelligent thermostatic water bathLeicaHI1210Temperature-controlled incubation
Lentivirus and infection reagentShanghai GeneChem Co., Ltd.https://synapse.patsnap.com/organization/04ffbf33ff2a6311226a8fa05498cec4Stable cell transduction
Manual pipettesGilsonPIPETMAN PLiquid handling
McCoy's 5A basal mediumWuhan Procell Life Science & Technology Co., Ltd.PM150710HT29 cell culture medium
Medical centrifugeZhuhai manufacturer not specified3200Sample preparation
Medical refrigerator-freezerQingdao Haier Biomedical Co., Ltd.https://www.haiermedical.com/Reagent/sample storage
Meilunbio Fixer Supersensitive ECL Chemiluminescence SubstrateMeilun Biotechnology Co., Ltd., Dalian, ChinaMA0186Immunoblot detection
Mini centrifugeBeijing DLAB Scientific Co., Ltd.https://www.dlabsci.com/Sample preparation
miR-192-5p and U6 primer setsTIANGEN Biotech Co., Ltd., Beijing, ChinaCustom synthesizedmiRNA qPCR primers
miR-192-5p mimicSangon Biotech Co., Ltd., Shanghai, ChinaCustom synthesizedTransient transfection oligonucleotide
miRcute Enhanced miRNA First-Strand cDNA Synthesis KitTIANGEN Biotech Co., Ltd., Beijing, ChinaKR211miRNA reverse transcription
miRcute Enhanced miRNA Fluorescence Quantitation Kit (SYBR Green FP)TIANGEN Biotech Co., Ltd., Beijing, ChinaFP411miRNA qPCR
miRWalkPublic databaseWeb databasemiRNA-target prediction database
Nitrocellulose membraneMilliporeHATF00010Protein transfer membrane
Nonfat dry milkSolarbio Science & Technology Co., Ltd.D8340Immunoblot blocking reagent
Paired colorectal cancer and adjacent noncancerous tissuesAuthors' institutionNot applicableClinical specimens
PCR tubesCorningPCR-02-CPCR/qRT-PCR setup
Penicillin-streptomycin solutionBiological Industries03-031-1BCell culture antibiotic
PerfectStart Uni RT&qPCR KitTransGen Biotech Co., Ltd., Beijing, ChinaAUQ-01mRNA reverse transcription and qPCR
Phosphate-buffered saline (PBS)Biological Industries02-023-1ACell washing buffer
Prestained protein markerFermentas26619Protein molecular-weight marker
Pure water preparation systemChina MoerNot availablePure water preparation
PuromycinPhygenePH1143Selection of transduced cells
R softwareR Foundation for Statistical ComputingVersion 3.6.3Statistical analysis and bioinformatics
Rapid transfer bufferCrisbioNot applicableProtein electrotransfer
Real-time fluorescence quantitative PCR systemApplied Biosystems (ABI), USANot availableqRT-PCR detection
RIPA lysis bufferSolarbio Science & Technology Co., Ltd.R0020Protein extraction
RNase-free microcentrifuge tubesCorning3208RNA handling
Serum-free cell cryopreservation mediumNew Cell & Molecular Biotech Co., Ltd.C40100Cell cryopreservation
SPSS StatisticsIBMVersion 25Statistical analysis
Stabilized antibody diluentSolarbio Science & Technology Co., Ltd.Not applicablePrimary/secondary antibody dilution
Sterile pipette tipsWuhan Servicebio Technology Co., Ltd.TP-10-CWound-healing scratch tool
STRING databaseSTRING ConsortiumWeb databaseProtein-interaction network analysis
TEMEDSolarbio Science & Technology Co., Ltd.T8090SDS-PAGE reagent
TransZol UpTransGen Biotech Co., Ltd., Beijing, ChinaET111-01RNA extraction
Tris-HCl buffer, pH 6.8Solarbio Science & Technology Co., Ltd.T1020SDS-PAGE stacking-gel buffer
Tris-HCl buffer, pH 8.8Solarbio Science & Technology Co., Ltd.T1010SDS-PAGE resolving-gel buffer
Tween-20Solarbio Science & Technology Co., Ltd.T8220Immunoblot washing reagent
Vertical pressure steam sterilizerShanghai Boxun Medical Biological Instrument Corp.BXM-30RSterilization

References

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  4. Song, L., Jia, J., Peng, X., Xiao, W., Li, Y. The performance of the SEPT9 gene methylation assay and a comparison with other CRC screening tests: a meta-analysis. Sci Rep. 7 (1), 3032 (2017).
  5. Linnekamp, J. F., Wang, ....

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