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

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 mortality^1,2. Although colonoscopy, fecal immunochemical testing, SEPT9 methylation testing, circulating tumor markers, and molecular profiling have improved CRC detection and clinical management^3,4, substantial heterogeneity remains in tumor biology, treatment response, and patient outcome^5,6,7. Biomarkers that help characterize CRC biology may therefore contribute to risk stratification and to the development of more individualized therapeutic strategies⁸.

MicroRNAs (miRNAs) are small non-coding RNAs, approximately 19–24 nucleotides in length, that regulate gene expression mainly through post-transcriptional mechanisms⁹. Dysregulated miRNA expression can influence cancer cell proliferation, migration, invasion, apoptosis, immune interactions, and treatment response¹⁰. miR-192-5p has been reported to act in a context-dependent manner across different diseases and cancers, with either oncogenic or tumor-suppressive associations depending on tissue type, molecular background, and experimental system^{11,12,13,14,15,16}. In CRC, previous studies have reported altered miRNA profiles in clinical specimens and functional regulation of malignant phenotypes in colon cancer cells^12,14,15. However, public database analyses may not always show the same expression direction, and the clinical significance, target network, and immune associations of miR-192-5p in CRC require cautious evaluation.

The present study evaluated miR-192-5p in CRC by integrating TCGA/GDC and GEO bioinformatic analyses, qRT-PCR validation in paired clinical tissues, in vitro migration assays, candidate target-gene prediction, pathway enrichment, and immune signature correlation analysis. The study was based on the hypothesis that mature miR-192-5p may be downregulated in CRC tissues and may exert a tumor-suppressive effect. Because the reanalyzed TCGA/GDC miRNA-seq signal showed the opposite expression direction, the results are interpreted as exploratory and platform-dependent rather than as a single definitive expression pattern.

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 collected at least 5 cm from the tumor margin. Samples were frozen promptly after surgical removal and stored in liquid nitrogen until RNA extraction. The cohort included 27 male and 18 female patients; 13 patients were younger than 60 years, and 32 were 60 years or older. Clinicopathological variables included tumor diameter, TNM stage, differentiation, vascular tumor thrombus, lymph node metastasis, and preoperative CEA level.

Public data acquisition and preprocessing

Open-access miRNA-seq, RNA-seq, and clinical data for TCGA-COAD and TCGA-READ were obtained from the Genomic Data Commons portal. Primary tumors and normal solid tissue samples were identified according to GDC sample type. Metastatic and recurrent tumor samples were excluded from tumor-normal differential expression analysis. For TCGA miRNA-seq files, the database-level entry detected for this analysis was hsa-mir-192; expression values were analyzed as log2(RPM + 1), where RPM indicates reads per million mapped miRNA reads. Both unpaired and paired tumor-normal analyses were performed when matched samples were available. GEO datasets GSE89076 and GSE156355 were used for candidate target-gene screening. Differentially expressed genes from GEO were screened using adjusted p < 0.05 and absolute log2 fold-change ≥ 2. Dataset names were checked for consistency throughout the manuscript.

Diagnostic, clinicopathological, and survival analyses

Receiver operating characteristic (ROC) analysis was used to assess exploratory tumor-normal separation in TCGA-COAD, TCGA-READ, and the combined COADREAD cohort. Because the normal-sample number was small, additional sensitivity analyses were performed, including 5,000-iteration balanced tumor-normal resampling and leave-one-normal-out analysis. For clinicopathological association analyses, primary tumor samples were divided into high- and low-expression groups by the median TCGA miR-192 expression level. Analyses were repeated for the combined COADREAD cohort and for COAD and READ separately when sample size permitted. Overall survival was evaluated using Kaplan-Meier analysis with the log-rank test and univariable Cox regression. Diagnostic, clinicopathological, and survival analyses were reported separately to avoid overstating prognostic conclusions.

Candidate target-gene and hub-gene analyses

Candidate target genes were identified by integrating miRWalk prediction with differentially expressed genes from GSE89076 and GSE156355. The overlap among these three sources was retained for downstream exploratory analysis. A protein-protein interaction network was constructed using STRING, and hub genes were selected according to node degree and module connectivity using Cytoscape-based cytoHubba and MCODE analyses. The retained hub genes (KIF20A, TPX2, CDCA5, CCNB1, CDK1, PLP1, NRXN1, GRIK3, and KIF5C) were further evaluated using TCGA RNA-seq data. Tumor-normal expression differences, Spearman correlations with TCGA miR-192 expression, and overall-survival associations were summarized as supplementary exploratory analyses. These genes were considered candidate targets only; no direct binding, rescue effect, or downstream protein-level regulation was inferred from bioinformatic data.

GO and KEGG enrichment analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed for the candidate gene set and significant network modules. GO terms were grouped into biological process, cellular component, and molecular function categories. Enrichment results with p < 0.05 were considered statistically notable and were interpreted as hypothesis-generating pathway signals rather than experimentally validated mechanisms.

RNA extraction and qRT-PCR analysis

Total RNA was extracted from 45 paired CRC and adjacent noncancerous tissue samples using TRIzol reagent according to the manufacturer's protocol. RNA purity and concentration were assessed spectrophotometrically, and samples with A₂₆₀/A₂₈₀ values between 1.8 and 2.1 were used for reverse transcription. qRT-PCR was performed using a miRNA reverse transcription system and SYBR Green-based miRNA qPCR reagents from TransGen Biotech. Reverse transcription and qPCR amplification were performed according to the reagent manufacturer's instructions. Each qRT-PCR reaction was run in technical triplicate. RNU6B was used as the internal reference. The miR-192-5p primers were 5'-GGGGCTGACCTATGAATTGA-3' (forward) and 5'-CAGTGCAGGGTCCGAGGT-3' (reverse). The RNU6B primers were 5'-ATTGGAACGATACAGAGAAGATT-3' (forward) and 5'-GGAACGCTTCACGAATTTG-3' (reverse). Melt-curve analysis was used to assess amplification specificity when supported by the qPCR platform. Relative expression was calculated using the 2^-ΔΔCt method. Paired tumor and adjacent tissue results were analyzed as paired clinical measurements.

Cell culture and transfection

The human CRC cell line HT29 was obtained from the authors' laboratory cell stock and had been authenticated and confirmed to be free of mycoplasma contamination. HT29 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 ^oC in a 5% CO₂ atmosphere. Cells were passaged when they reached approximately 80% confluence. miR-192-5p mimics and the negative-control oligonucleotide con238 were purchased from Sangon Biotech (Shanghai, China) and used for transient transfection. HT29 cells were seeded in 6-well plates and transfected when the cell density reached approximately 20–30%, using a final oligonucleotide concentration of 50 nM and a lipid-based transfection protocol performed according to the reagent manufacturer's instructions. The exact reagent-to-oligonucleotide ratio and reagent amount followed the manufacturer's recommended protocol used in the authors' laboratory records. Cells were incubated with transfection complexes under standard culture conditions, and downstream transfection imaging and wound-healing assays were performed after transfection according to the experimental schedule. Only HT29 cell data were included in the present study.

Wound-healing assay

Transfected HT29 cells were seeded in 6-well plates and cultured until a confluent monolayer formed. A linear scratch was generated using a sterile 200 µL pipette tip, and detached cells were removed by washing with PBS. The medium was then replaced with serum-free medium to reduce proliferation-related effects during migration assessment. Wound images were captured at 0 h, 24 h, and 48 h. The wound-healing assay was performed in three independent biological replicates, and multiple microscopic fields were measured for each group and time point. Wound width was quantified from microscopy images using image-analysis software; the exact software version used for the original measurements was not available in the source records. Wound-healing rate was calculated as [(wound width at 0 h - wound width at the indicated time point) / wound width at 0 h] x 100%. Because the available manuscript data do not include a separate proliferation-control experiment, the wound-healing results were interpreted as migration-related but not as a complete exclusion of proliferation effects.

Immune signature correlation analysis

The association between TCGA miR-192 expression and immune-related signatures was reassessed using primary tumor RNA-seq data. A total of 425 tumor samples with matched miRNA and RNA-seq data were included in the signature-score correlation analysis. miR-192 expression showed statistically significant but weak negative correlations with several immune signatures after false-discovery rate correction, including Tfh cells (rho = -0.205, FDR = 0.00049), activated dendritic cells (rho = -0.186, FDR = 0.00098), Treg cells (rho = -0.185, FDR = 0.00098), plasmacytoid dendritic cells (rho = -0.169, FDR = 0.0027), and macrophages (rho = -0.149, FDR = 0.0092). Positive correlations were observed mainly for eosinophil (rho = 0.139, FDR = 0.0155) and Th17-cell signatures (rho = 0.121, FDR = 0.0296). These findings indicate heterogeneous and generally weak immune associations rather than a uniform positive immune-infiltration pattern.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.0, SPSS 25.0, and R software 3.6.3. Continuous variables were summarized as mean ± standard deviation or median with interquartile range, as appropriate. Paired tissue comparisons were analyzed using paired tests when tumor and adjacent tissue samples were matched. Nonparametric tests were used when normality assumptions were not met. ROC curves, balanced resampling, and leave-one-normal-out analysis were used for exploratory diagnostic discrimination; Chi-square tests were used for categorical clinicopathological associations; log-rank and Cox models were used for survival analyses; and Spearman correlation was used for immune and hub-gene analyses. Two-sided p < 0.05 was considered statistically significant, with false-discovery rate correction reported for immune signature correlations.

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 ...

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25%+2:60 trypsin-EDTA	Biological Industries	03-054-1	Cell digestion/passaging
10% ammonium persulfate	Solarbio Science & Technology Co., Ltd.	A8090	SDS-PAGE reagent
10% sodium dodecyl sulfate	Solarbio Science & Technology Co., Ltd.	SIS1070	SDS-PAGE reagent
4x protein loading buffer	Solarbio Science & Technology Co., Ltd.	P1016	Protein sample preparation
-80 C ultra-low temperature freezer	Qingdao Haier Biomedical Co., Ltd.	https://www.haiermedical.com/	Sample storage
Anti-Myc mycoplasma removal reagent	Wuhan Procell Life Science & Technology Co., Ltd.	P-CMR-001	Mycoplasma decontamination
ARL2, BATF2, MEN1, and beta-actin primer sets	Sangon Biotech Co., Ltd., Shanghai, China	Custom synthesized	mRNA qPCR primers
Automatic microplate reader	PerkinElmer, USA	VICTOR Nivo	Absorbance measurement
BCA Protein Assay Kit	Thermo Fisher Scientific	23225	Protein quantification
Biosafety cabinet / clean bench	Qingdao Haier Biomedical Co., Ltd.	https://www.haiermedical.com/	Aseptic cell culture
Cell counting chamber / hemocytometer	Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.	http://www.shqiujing.net/	Cell counting
Cell Counting Kit-8 (CCK-8)	Biosharp	BS350B	Cell proliferation/cytotoxicity assay
Cell culture flasks, culture plates, centrifuge tubes, and pipette tips	Corning	https://www.corning.com/in/en.html	General cell culture consumables
Cell Cycle and Apoptosis Detection Kit	Meilun Biotechnology Co., Ltd., Dalian, China	https://www.chemicalbook.com/ShowSupplierProductsList13366/0_EN.htm	Flow-cytometric cell-cycle/apoptosis analysis
CO2 cell incubator	Qingdao Haier Biomedical Co., Ltd.	https://www.haiermedical.com/	Cell culture
con238 negative-control oligonucleotide	Sangon Biotech Co., Ltd., Shanghai, China	Custom synthesized	Transfection control
Cytoscape software	Cytoscape Consortium	https://cytoscape.org/	Network module and hub-gene visualization
Digital thermostatic magnetic stirrer	Changzhou Saipu Experimental Instrument Factory	https://czxtyq.en.alibaba.com/	Solution preparation
Electric thermostatic blast drying oven	Nanjing Wohuan Technology Industry Co., Ltd.	https://www.wtsensor.com/about-wt/aboutwt/	Drying/sterilization support
Electrophoresis system	Bio-Rad Laboratories	Mini-PROTEAN 3 Cell	Gel electrophoresis
Electrotransfer system	Hoefer	TE77XP	Protein transfer
Fetal bovine serum	Biological Industries	04-001-1ACS	Cell culture supplement
Flow cytometer	ACEA Biosciences	NovoCyte	Flow cytometry
Fluorescence inverted microscope	Ningbo Sunny Instruments Co., Ltd.	https://www.sunny-instrument.com/en	Cell and wound imaging
GDC/TCGA portal	National Cancer Institute	Open-access database	Public miRNA-seq, RNA-seq, and clinical data
GEO database	National Center for Biotechnology Information	GSE89076 and GSE156355	Public gene-expression datasets
GraphPad Prism	GraphPad Software	Version 9	Statistical analysis and graphing
High-speed refrigerated centrifuge	Shanghai Huxiangyi Centrifuge Instrument Co., Ltd.	https://www.bioridgecentrifuge.com/	Sample preparation
HT29 cells	Authors' laboratory cell stock	Not applicable	Human colorectal cancer cell line
Image analysis software	ImageJ	Not applicable	Wound-width quantification
Imaging system	Tanon Science & Technology Co., Ltd.	Tanon-5200	Gel/blot imaging
IncuCyte S3 dynamic cell observation and functional analysis system	Beijing Sensi Wantong Technology Co., Ltd.	IncuCyte S3	Live-cell imaging/function analysis
Intelligent thermostatic water bath	Leica	HI1210	Temperature-controlled incubation
Lentivirus and infection reagent	Shanghai GeneChem Co., Ltd.	https://synapse.patsnap.com/organization/04ffbf33ff2a6311226a8fa05498cec4	Stable cell transduction
Manual pipettes	Gilson	PIPETMAN P	Liquid handling
McCoy's 5A basal medium	Wuhan Procell Life Science & Technology Co., Ltd.	PM150710	HT29 cell culture medium
Medical centrifuge	Zhuhai manufacturer not specified	3200	Sample preparation
Medical refrigerator-freezer	Qingdao Haier Biomedical Co., Ltd.	https://www.haiermedical.com/	Reagent/sample storage
Meilunbio Fixer Supersensitive ECL Chemiluminescence Substrate	Meilun Biotechnology Co., Ltd., Dalian, China	MA0186	Immunoblot detection
Mini centrifuge	Beijing DLAB Scientific Co., Ltd.	https://www.dlabsci.com/	Sample preparation
miR-192-5p and U6 primer sets	TIANGEN Biotech Co., Ltd., Beijing, China	Custom synthesized	miRNA qPCR primers
miR-192-5p mimic	Sangon Biotech Co., Ltd., Shanghai, China	Custom synthesized	Transient transfection oligonucleotide
miRcute Enhanced miRNA First-Strand cDNA Synthesis Kit	TIANGEN Biotech Co., Ltd., Beijing, China	KR211	miRNA reverse transcription
miRcute Enhanced miRNA Fluorescence Quantitation Kit (SYBR Green FP)	TIANGEN Biotech Co., Ltd., Beijing, China	FP411	miRNA qPCR
miRWalk	Public database	Web database	miRNA-target prediction database
Nitrocellulose membrane	Millipore	HATF00010	Protein transfer membrane
Nonfat dry milk	Solarbio Science & Technology Co., Ltd.	D8340	Immunoblot blocking reagent
Paired colorectal cancer and adjacent noncancerous tissues	Authors' institution	Not applicable	Clinical specimens
PCR tubes	Corning	PCR-02-C	PCR/qRT-PCR setup
Penicillin-streptomycin solution	Biological Industries	03-031-1B	Cell culture antibiotic
PerfectStart Uni RT&qPCR Kit	TransGen Biotech Co., Ltd., Beijing, China	AUQ-01	mRNA reverse transcription and qPCR
Phosphate-buffered saline (PBS)	Biological Industries	02-023-1A	Cell washing buffer
Prestained protein marker	Fermentas	26619	Protein molecular-weight marker
Pure water preparation system	China Moer	Not available	Pure water preparation
Puromycin	Phygene	PH1143	Selection of transduced cells
R software	R Foundation for Statistical Computing	Version 3.6.3	Statistical analysis and bioinformatics
Rapid transfer buffer	Crisbio	Not applicable	Protein electrotransfer
Real-time fluorescence quantitative PCR system	Applied Biosystems (ABI), USA	Not available	qRT-PCR detection
RIPA lysis buffer	Solarbio Science & Technology Co., Ltd.	R0020	Protein extraction
RNase-free microcentrifuge tubes	Corning	3208	RNA handling
Serum-free cell cryopreservation medium	New Cell & Molecular Biotech Co., Ltd.	C40100	Cell cryopreservation
SPSS Statistics	IBM	Version 25	Statistical analysis
Stabilized antibody diluent	Solarbio Science & Technology Co., Ltd.	Not applicable	Primary/secondary antibody dilution
Sterile pipette tips	Wuhan Servicebio Technology Co., Ltd.	TP-10-C	Wound-healing scratch tool
STRING database	STRING Consortium	Web database	Protein-interaction network analysis
TEMED	Solarbio Science & Technology Co., Ltd.	T8090	SDS-PAGE reagent
TransZol Up	TransGen Biotech Co., Ltd., Beijing, China	ET111-01	RNA extraction
Tris-HCl buffer, pH 6.8	Solarbio Science & Technology Co., Ltd.	T1020	SDS-PAGE stacking-gel buffer
Tris-HCl buffer, pH 8.8	Solarbio Science & Technology Co., Ltd.	T1010	SDS-PAGE resolving-gel buffer
Tween-20	Solarbio Science & Technology Co., Ltd.	T8220	Immunoblot washing reagent
Vertical pressure steam sterilizer	Shanghai Boxun Medical Biological Instrument Corp.	BXM-30R	Sterilization