Introduction
Colorectal cancer (CRC) is one of the most common
types of malignant tumors of the digestive tract and ranks third
among new estimated cases and deaths among men and women globally.
At first, most patients with CRC in stages I and II undergo only
partial or total colectomy, and then approximately two-thirds with
stage III undergo chemotherapy to reduce the risk of recurrence.
Ultimately, chemotherapy is the main treatment of patients with CRC
in stage IV. With ~2 million new cases and 1 million deaths
worldwide in 2020, CRC ranks as the third and second most frequent
cause of cancer incidence and mortality, respectively (1). Based on research, targeted drugs can
prevent the growth of cancer cells through the action of specific
molecular targets necessary for cancer development and tumor growth
and can also be used to treat metastatic CRC. Activation or
inactivation of oncogenes and tumor suppressor genes are classic
markers of these tumors, and the relevant mechanisms have been the
core target of cancer research (2). Studying key molecular mechanisms
required for growth and metastasis will greatly assist in the
advancement of targeted therapies to improve the condition and
prognosis of patients with CRC (3).
MicroRNAs (miRNAs or miRs) are a class of
evolutionarily conserved small non-coding RNAs, which are 17-25
nucleotides in length. They bind to the 3'-untranslated regions
(UTRs) of their target mRNAs, affecting mRNA stability or
inhibiting its translation. miRNAs regulate gene expression at the
post-transcriptional level (4).
Abnormalities in miRNA expression are linked to diverse biological
changes, including cell proliferation, apoptosis, differentiation,
and tumorigenesis (5). Numerous
studies have shown that miRNAs are closely related to the
occurrence, development, and progression of tumors (6,7).
Therefore, they may serve as new and promising therapeutic
targets.
Research has found that miR-539 inhibits human CRC
progression by targeting RUNX2(8).
miR-205 can be upregulated or downregulated in human CRC, and p53
has been shown to regulate CRC metastasis by inhibiting SQLE
expression by inducing miR-205(9).
Cisplatin was demonstrated to inhibit the proliferation of Saos-2
cells by upregulating miR-376c and downregulating TGFA expression
(10). miR-539 is located on human
chromosome 4q32.31 and it is reportedly downregulated in a variety
of human cancers, including nasopharyngeal, prostate, and thyroid
cancers. Furthermore, miR-539 has been shown to be a tumor
suppressor in human malignancies (11). Combining the present research and
reviewing relevant literature, miR-205, miR-376c, and miR-539
(which are related to tumor necrosis factor-α induced protein 8)
were selected as research targets, and their roles in the
development and progression of CRC were explored. The purpose of
the presen study was to analyze the expression patterns of these
three miRNAs in CRC, determine their clinical significance, and
combine their expression characteristics in CRC to delineate their
roles in the development of CRC and provide a novel therapeutic
target for prognosis. The research protocol is shown in a schematic
flowchart (Fig. 1).
 | Figure 1Overview of the study design and
analytical workflow. The study employed a research framework of
‘computer simulation analysis-experimental validation-integrated
interpretation’ to systematically investigate the expression
patterns, clinical significance, and potential mechanisms of
miR-205, miR-376c, and miR-539 in CRC. The flowchart clearly
illustrates the entire process including: Obtaining data from
public databases (GEO, UALCAN, miRBase, miRDB) for bioinformatics
mining, collecting 25 clinical sample pairs for qPCR validation,
and finally integrating analyses using statistical and
bioinformatics tools. Key findings include: All three miRNAs were
significantly downregulated in cancer tissues; miR-376c was
associated with liver metastasis (particularly synchronous
metastasis); miR-205 and miR-539 were associated with tumor
differentiation, staging, and lymph node metastasis, respectively;
target gene prediction revealed 53 common potential targets shared
among the three miRNAs, suggesting their potential involvement in
CRC progression through a synergistic regulatory network. This
integrated flowchart visually represents the overall study approach
from hypothesis validation to mechanistic exploration and
highlights its main conclusions. CRC, colorectal cancer; miRNA or
miR, microRNA. |
Materials and methods
Bioinformatics analyses
MiRNA microarray data of solitary CRC were obtained
from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) and the
University of Alabama at Birmingham CANcer data analysis Portal
(UALCAN) (https://ualcan.path.uab.edu/analysis-mir.html). The
GSE41655 and GSE81581 datasets were used to investigate the
expression of miR-205, miR-376c, and miR-539 in CRC (12,13).
miRNA sequences and predicted target gene data were primarily
sourced from miRDB (https://mirdb.org/) and miRBase (https://www.mirbase.org/).
Relevant miRNA expression data were downloaded from
the GEO database based on the GSEE41655 and GSE81581 datasets
(13). The downloaded raw data
were preliminarily processed and standardized in RStudio
2025.5.1.513 using the Bioconductor 3.18 package (14). Statistical analysis was performed
using GraphPad Prism 10.6.1 software (Dotmatics). After performing
a normality test on the expressed data, the Kruskal-Wallis test was
selected for nonparametric analysis. Subsequently, Dunn's post hoc
pairwise multiple comparisons were conducted for groups showing
significant differences to obtain the results. The significance
level was then set and Bonferroni correction was applied for
multiple comparison adjustments. Concurrently, Spearman's ρ
correlation analysis was employed to perform pairwise correlation
analyses on the acquired data, generating correlation heatmaps.
Bonferroni correction was applied to each correlation test.
The Cancer Genome Atlas (TCGA) analysis portal in
UALCAN was selected, the colorectal adenocarcinoma (COAD) dataset
was chosen, and the differences in the expression between cancerous
and normal tissues were compared (15,16).
The database automatically generated box-and-whisker plots and
provided P-values for significance based on Kruskal-Wallis test.
The differences in the expression of target miRNAs in subgroups
were further analyzed based on clinical stages and lymph node
metastasis status (N0/N1/N2). When generating expression maps for
multi-group comparisons, the statistical engine of the UALCAN
platform automatically performs one-way analysis of variance
(ANOVA) to assess overall differences in gene expression levels
between groups. When ANOVA results indicate significant differences
(P<0.05), the platform further employs Tukey's Honestly
Significant Difference (HSD) post hoc test for pairwise comparisons
to control for error rates arising from multiple comparisons.
UALCAN was used to evaluate the relationship between target miRNA
expression levels and overall survival in patients with COAD based
on the Kaplan-Meier method (15).
Patients were divided into high-expression and low-expression
groups based on median expression values, and the log-rank test was
used to compare survival curves between the two groups. All
analytical results were exported directly from the platform and
underwent partial formatting integration and annotation in GraphPad
Prism.
Using the miRBase database via NCBI, the mature
sequences of three miRNAs were retrieved and their corresponding ID
numbers and standard nomenclature were collected (17,18).
The miRNA IDs or standard nomenclature obtained was used,
performing a search in the miRDB database and compiling the
predicted target genes into an XLSX worksheet (17) for convenient further analysis of
the associations among the three microRNAs. Target gene data were
read and organized using the readxl package 1.4.5 (https://cran.r-project.org/package=readxl). Missing
values were filtered for each column of miRNA target genes using
the na.omit() function to construct the target gene set (19). The Reduce() and intersect()
functions were used to calculate the common target points of the
target gene sets, and output the number of genes in the
intersection along with their specific list. Finally, the plotrix
package 3.8.4 (https://cran.r-project.org/package=plotrix) was used
to generate the Venn diagram. The target gene data was imported
using the readxl package and the acquired data was processed and
aggregated using the dplyr package 1.1.4 (https://cran.r-project.org/package=dplyr). The
na.omit() function was applied to clean missing values in each
target gene column (18). The
cleaned data was created into a list object as the foundational
data structure for subsequent UpSet plot analysis. The list was
organized with corresponding miRNA names as keys and their target
gene vectors as values to facilitate set operations and
visualization mapping (20). The
upset() function from the UpSetR package 1.4.0 (https://cran.r-project.org/package=UpSetR) was used to
create an UpSet plot, converting data into the appropriate format
via fromList(). Sets were sorted in descending order by
intersection frequency (order.by=‘freq’), preserving the original
set order (keep.order=TRUE), with set sizes indicated in steel blue
(sets.bar.color=‘steelblue’). Finally, the axis labels to
‘Intersection Size’ (Y-axis of the main bar plot) and ‘Set Size’
(X-axis of the set size bar plot) were explicitly set to generate
the UpSet plot.
Patients and tissue samples
Surgically resected CRC tissues were obtained from
25 patients at the Department of Gastrointestinal Surgery,
Zhongshan Hospital, Xiamen University (Xiamen, China). Patient
recruitment took place from January 2022 to October 2022, with all
tissue samples collected in October 2022. The sample age ranged
from 53 to 87 years old, with a median age of 66 years. The sample
included 18 males and 7 females. All patient samples were obtained
with written informed consent and approved [approval no. xmzsyyky
(2024-703)] by the Ethics Committee of Zhongshan Hospital, Xiamen
University (Xiamen, China). A total of 25 pairs of tissue specimens
were collected, all from the initial surgical operations, without
preoperative chemotherapy or radiotherapy. Each specimen was
collected from CRC tissue and distal colorectal mucosa. Tissue
samples were immediately frozen at -196˚C and subsequently stored
in a refrigerator at -80˚C. The reverse transcription reaction
mixture, consisting of total RNA templates extracted from the
aforementioned colorectal cancer tissue and distal colorectal
mucosa, reverse transcriptase (RT) enzyme, deoxyribonucleotide
triphosphates (dNTPs), and RT reaction buffer, all of which were
individually procured components included in the PrimeScript™ RT
Reagent Kit (cat. no. RR037A, Takara Bio, Inc.), was gently mixed
and incubated at 37˚C for 1 h to complete the reverse transcription
reaction.
Reverse transcription-quantitative PCR
(RT-qPCR)
Total RNA was extracted from fresh frozen tissue
samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific,
Inc.) according to the manufacturer's instructions. RNA
concentration and purity were assessed using a NanoDrop 2000
spectrophotometer (Thermo Fisher Scientific, Inc.), with the
A260/A280 ratio maintained between 1.8 and 2.0. RNA integrity was
verified by 1.5% agarose gel electrophoresis. A total of 1 µg of
total RNA was used as a template and reverse transcription was
performed using the TransScript® Green miRNA Two-Step
qRT-PCR Super Mix kit (TransGen Biotech Co., Ltd.) to synthesize
cDNA, according to the manufacturer's instructions.
qPCR reactions were performed using the Applied
Biosystems QuantStudio 5 Real-Time Fluorescent Quantitative PCR
System (Thermo Fisher Scientific, Inc.). The reaction system
consisted of 20 µl in total, including 10 µl TransStart®
Tip Green qPCR SuperMix (TransGen Biotech Co., Ltd.), 0.4 µl
forward primer, 0.4 µl universal reverse primer, 2 µl cDNA
template, and 7.2 µl nuclease-free water. The thermocycling
conditions were as follows: Pre-denaturation at 95˚C for 30 sec,
followed by 40 cycles of denaturation at 95˚C for 5 sec and
annealing/extension at 60˚C for 30 sec. Each sample was processed
in triplicate, with a no-template control (NTC) included to rule
out contamination. U6 snRNA was used as the reference gene, and the
relative expression levels of miRNAs were calculated using the
2-ΔΔCq method as previously described (21).
Data summary and statistical
analysis
All statistical analyses were performed using
Graphpad Prism 10.6.1 statistical software (Dotmatics). In the
GSE41655 dataset, a normality test on the data was first performed.
Subsequently, nonparametric Kruskal-Wallis test was used to analyze
the results. If the test results were significant, Dunn's post hoc
test was further performed for pairwise multiple comparisons. Based
on the GSE81581 dataset and qPCR data results, paired t-test
analysis was performed on the mean values of each group. Moreover,
the results were combined with clinical data for comprehensive
analysis according to sex, age, location, tumor stage, depth of
invasion, and lymph node metastasis in patients with CRC and
analyzed using Fisher's exact test. Univariate logistic regression
was used to analyze miR-205 and miR-539 as potential risk factors
associated with tumor progression. Two-tailed P<0.05 was
considered to indicate a statistically significant difference.
Spearman's ρ and Pearson's r correlation analyses were performed
separately on the GSE41655 dataset and clinical sample qPCR data,
with Bonferroni correction applied to the results.
Results
Differential expression and
correlation of miR-205, miR-376c, and miR-539 between cancer and
adjacent tissues in the GSE41655 dataset
To investigate the roles of the three miRNAs,
miR-205, miR376c and miR-539, in CRC and normal colorectal tissues,
their involvement in CRC pathogenesis was first analyzed using
publicly available data from the GEO database. Based on the
GSE41655 dataset, the Kruskal-Wallis test was employed to
demonstrate significant differences among the three miRNA groups.
Subsequently, using 15 normal colon tissue samples as controls,
pairwise multiple comparisons were performed among the three miRNA
groups via Dunn's test. It was found that miR-205 (P<0.05;
Fig. 2A) and miR-376c (P<0.05;
Fig. 2B) were downregulated in CRC
tumor tissues compared with normal colon tissue. Furthermore,
compared with normal colonic tissue, miR-539 expression was reduced
in CRC tumor tissue (P<0.05; Fig.
2C), while no significant changes were observed in
adenocarcinoma tissue (P<0.05; Fig.
2C). To further investigate the expression correlations among
these three miRNAs in CRC, correlation heatmap analysis was
performed using Pearson's r correlation analysis (Fig. 2D). The results indicated that there
was no significant correlation in the expression of these three
miRNAs in CRC (Fig. 2D).
 | Figure 2Expression profiles and correlation
analysis of miR-205, miR-376c, and miR-539 in colorectal cancer and
non-cancerous tissues from the GSE41655 dataset. The expression
levels and correlation of miR-205, miR-376c and miR-539 in normal
tissues and colorectal cancer tissues were analyzed in the GSE41655
dataset, which contained 35 colorectal tumor tissues, 15 matched
adjacent tissues, and 57 colorectal adenoma tissues. (A) miR-205
expression. (B) miR-376c expression. (C) miR-539 expression. (D)
Correlation analysis of the expression of miR-205, miR-376 and
miR-539 in cancer tissues. N, normal tissues; A, adenoma tissues;
C, colorectal cancer tumor tissues. *P<0.05,
***P<0.001 and ****P<0.0001. miR,
microRNA; ns, not statistically significant. |
miR-205, miR-376c, and miR-539
expression in tumor and normal tissues from clinical CRC
specimens
To increase the credibility of the results, the
expression levels of miR-205-5p, miR-376c-3p, and miR-539-5p were
further detected among 25 patients with CRC by RT-qPCR. The
expression levels of the miRNAs in cancer tissues were
significantly decreased compared with corresponding adjacent
healthy tissues: miR-205 (P<0.05; Fig. 3A), miR-376c (P<0.01; Fig. 3B) and miR-539 (P<0.05; Fig. 3C). To further investigate the
correlation between the expression levels of the three miRNAs and
their expression in CRC tumor tissues and adjacent normal tissues,
correlation analysis was also performed using Pearson's r
correlation analysis. The results revealed that miR-205 and miR-539
exhibited a strong correlation in their expression levels within
cancerous tissues (Fig. 3D).
miR-376c is closely related to liver
metastases, especially in the case of synchronous liver
metastasis
In order to further explore the role of the
aforementioned three microRNAs in the development of CRC, the
GSE81581 dataset was used. Compared with primary CRC tissues,
miRNA-376c expression was increased in CRC liver metastases
(P<0.05; Fig. 4B), especially
in the case of synchronous liver metastasis (Fig. 4E), while the expression levels of
miR-205 (P>0.05; Fig. 4A) and
miR-539 (P>0.05; Fig. 4C) were
not significantly altered in CRC liver metastases. In addition,
miR-376c expression exhibited no significant changes in CRC
metachronous liver metastasis (P>0.05; Fig. 4C).
miR-205, miR-376c, and miR-539 are
associated with CRC progression and prognosis
CRC is characterized by frequent metastasis, high
invasiveness, and rapid development and high mortality. The
expression of miR-205, miR-376c, and miR-539 was analyzed in the
TCGA database via UALCAN. As revealed Fig. 5A-C, miR-376 expression was
upregulated in primary cancer, while the expression levels of
miR-205 and miR-539 were relatively high in normal tissues. The
expression of miR-205, miR-376c, and miR-539 was closely associated
with individual cancer stage (Fig.
5D-F) and lymph node metastasis (Fig. 4G-I), but showed no direct
association with late survival (P>0.05; Fig. 5J-L).
Correlation analysis of miR-205,
miR-376c, and miR-539 target genes
To further investigate the relationships among
miR-205, miR-376c, and miR-539, the 5' and 3' mature sequences of
these three miRNAs were obtained from the miRBase database. No
significant common sequences identified among the mature forms of
these three miRNAs (Fig. 6A).
Subsequently, the predicted target genes of miR-205, miR-376c, and
miR-539 were retrieved from the miRDB database and the
relationships among these target genes were analyzed. Venn diagram
analysis indicated that there were no common target genes among the
six distinct sequences, miR-205-3p, miR-205-5p, miR-376c-3p,
miR-376c-5p, miR-539-3p, and miR-539-5p (Fig. 6B). However, the UpSet plot revealed
that the mature sequences of miR-205, miR-376c, and miR-539 (5p or
3p end sequences) share a total of 53 predicted target genes
(Fig. 6C).
 | Figure 6Sequences of miR-205, miR-376, and
miR-539 collected from the miRBase database, and predicted target
genes for miR-205, miR-376, and miR-539 mined from the miRDB
database for correlation analysis. (A) 5p and 3p mature sequences
of miR-205, miR-376c, and miR-539. (B) UpSet plot of the target
gene intersection analysis for the 5p and 3p mature sequences of
miR-205, miR-376, and miR-539. (C) Venn diagram of the target gene
intersection analysis for the 5p and 3p mature sequences of
miR-205, miR-376, and miR-539. miR, microRNA. |
Distribution of miR-205, miR-376c, and
miR-539 in patients with CRC and clinicopathological
characteristics
To investigate the association of the miRNAs with
clinicopathological characteristics, non-parametric tests were
performed to analyze the relationships between miRNA expression and
clinicopathological features in CRC samples. Based on the
experimental data obtained, the median expression levels of
miR-205, miR-376c, and miR-539 in cancer tissues were calculated,
and grouped by age (<60 years old, ≥60 years old), sex, tumor
stage (stages I and II vs. III and IV), presence or absence of
lymph node metastasis, tumor location (left or right colon), and
degree of differentiation (low, moderate, or high). As shown in
Tables I and III, miR-205 and miR-539 significantly
differed according to tumor stage (P=0.0154) and lymph node
metastasis (P=0.0154) in cancer tissues respectively, while no
statistically significant differences were observed for the other
clinicopathological factors. No significant associations were
observed between the expression of miR-376c and any of the six
clinicopathological characteristics (Table II). In addition, logistic
regression analysis indicated that miR-205 may be a potential risk
factor associated with tumor progression and differentiation, while
miR-539 may be a potential risk factor associated with tumor
progression (Table IV).
 | Table IDistribution of miR-205 expression
relative to normal adjacent tissue and clinicopathological
characteristics in patients with colorectal cancer. |
Table I
Distribution of miR-205 expression
relative to normal adjacent tissue and clinicopathological
characteristics in patients with colorectal cancer.
|
Characteristics | No. of
patients | Downregulated
(%) | Upregulated
(%) | P-value |
|---|
| Age (years) | | | | >0.9999 |
|
<60 | 6 | 3(12) | 3(12) | |
|
≥60 | 19 | 11(44) | 8(32) | |
| Sex | | | | 0.6564 |
|
Female | 7 | 3(12) | 4(16) | |
|
Male | 18 | 11(44) | 7(28) | |
| TNM stage | | | | 0.0154a |
|
Ⅰ, Ⅱ | 12 | 10(40) | 2(8) | |
|
Ⅲ, Ⅳ | 13 | 4(16) | 9(36) | |
| Lymphatic
metastasis | | | | 0.0154a |
|
N=0 | 12 | 10(40) | 2(8) | |
|
N≠0 | 13 | 4(16) | 9(36) | |
| Location | | | | >0.9999 |
|
Left | 17 | 9(36) | 8(32) | |
|
Right | 8 | 5(20) | 3(12) | |
|
Differentiation | | | | 0.3500 |
|
Low | 6 | 2(8) | 4(16) | |
|
Middle | 19 | 12(48) | 7(28) | |
| Table IIIDistribution of miR-539 expression
relative to normal adjacent tissue and clinicopathological
characteristics in patients with colorectal cancer. |
Table III
Distribution of miR-539 expression
relative to normal adjacent tissue and clinicopathological
characteristics in patients with colorectal cancer.
|
Characteristics | No of patients | Downregulated
(%) | Upregulated
(%) | P-value |
|---|
| Age (years) | | | | >0.9999 |
|
<60 | 6 | 3(12) | 3(12) | |
|
≥60 | 19 | 11(44) | 8(32) | |
| Sex | | | | 0.6564 |
|
Female | 7 | 3(12) | 4(16) | |
|
Male | 18 | 11(44) | 7(28) | |
| TNM stage | | | | 0.0154a |
|
Ⅰ, Ⅱ | 12 | 10(40) | 2(8) | |
|
Ⅲ, Ⅳ | 13 | 4(16) | 9(36) | |
| Lymphatic
metastasis | | | | 0.0154a |
|
N=0 | 12 | 10 (40%) | 2 (8%) | |
|
N≠0 | 13 | 4 (16%) | 9 (36%) | |
| Location | | | | >0.9999 |
|
Left | 17 | 10(40) | 7(28) | |
|
Right | 8 | 4(16) | 4(16) | |
|
Differentiation | | | | 0.3500 |
|
Low | 6 | 2(8) | 4(16) | |
|
Middle | 19 | 12(48) | 7(28) | |
| Table IIDistribution of miR-376c expression
relative to normal adjacent tissue and clinicopathological
characteristics in patients with colorectal cancer. |
Table II
Distribution of miR-376c expression
relative to normal adjacent tissue and clinicopathological
characteristics in patients with colorectal cancer.
|
Characteristics | No. of
patients | Downregulated
(%) | Upregulated
(%) | P-value |
|---|
| Age (years) | | | | 0.5623 |
|
<60 | 6 | 4(16) | 2(8) | |
|
≥60 | 19 | 16(64) | 3(12) | |
| Sex | | | | 0.1130 |
|
Female | 7 | 4(16) | 3(12) | |
|
Male | 18 | 16(64) | 2(8) | |
| TNM stage | | | | 0.1602 |
|
Ⅰ, Ⅱ | 12 | 8(32) | 4(16) | |
|
Ⅲ, Ⅳ | 13 | 12(48) | 1(4) | |
| Lymphatic
metastasis | | | | 0.1602 |
|
N=0 | 12 | 8(32) | 4(16) | |
|
N≠0 | 13 | 12(48) | 1(4) | |
| Location | | | | >0.9999 |
|
Left | 17 | 13(52) | 4(16) | |
|
Right | 8 | 7(28) | 1(4) | |
|
Differentiation | | | | >0.9999 |
|
Low | 6 | 5(20) | 1(4) | |
|
Middle | 19 | 15(60) | 4(16) | |
| Table IVLogistic regression analysis between
miR-205, miR-539 and pathological characteristics. |
Table IV
Logistic regression analysis between
miR-205, miR-539 and pathological characteristics.
| | miR-205
(upregulated) | miR-539
(upregulated) |
|---|
| Pathological
characteristics | OR | 95% CI | P-value | OR | 95% CI | P-value |
|---|
| Advanced TNM stage
(III, IV) | 11.25 | 1.905-100.1 | 0.0136 | 11.25 | 1.905-100.1 | 0.0136a |
| N≥1 | 11.25 | 1.905-100.1 | 0.0136 | 11.25 | 1.905-100.1 | 0.0136a |
| Differentiation
(poor) | 10.83 | 1.362-233.3 | 0.0474 | 3.429 | 0.5273-29.75 | 0.2124 |
Discussion
The current prognosis for CRC is still not
optimistic, and its tumor progression is a multi-step process
involving changes at the genetic and epigenetic levels (1,2).
miRNAs are widely distributed in numerous eukaryotes, and the
transcription of miRNA genes is synchronized with other genes
(22). Notably, >60% of human
protein-coding genes are predicted to contain conserved targets of
miRNA, mainly in the 3' UTR. Therefore, miRNAs play important
regulatory roles in cell differentiation, proliferation, apoptosis,
and other pathological processes (23). miRNAs can function as oncogenes or
tumor suppressor genes based on their corresponding regulated mRNA
sequences. miRNAs exert regulatory functions by binding to mRNA
targets, disrupting mRNA stability or inhibiting translation
(2-4).
There are numerous studies on miRNAs in various cancers (3,4).
However, it is unclear whether various miRNAs play different roles
in CRC.
miRNAs also play important roles in tumor biology,
including tumor evolution, invasion, metastasis, and angiogenesis
(24). In the present study, the
differences in the expression levels of miR-205, miR-376c, and
miR-539 between CRC and adjacent tissues in 25 CRC patients were
analyzed and found significant differences in the expression levels
of the three miRNAs in cancer and matched normal tissues.
According to previous studies, miR-205 expression is
significantly reduced in glioma (25), head and neck squamous cell
carcinoma (26), thyroid cancer
(27), prostate cancer (28), renal cell carcinoma (29), pancreatic cancer (30) and hepatocellular carcinoma
(31). miR-205 has also been
reported to contribute to distinguishing between high-grade and
low-grade endometrioid tumors (32). High expression of mature miR-205
was revealed to be associated with lymph node-positive status in
patients with esophageal squamous cell carcinoma (33), and experiments have shown that
upregulation of miR-205 in vitro reduces cell viability,
migration, and invasion in gliomas. In the clinical tissues of the
present study, RT-qPCR showed that the expression levels of miR-205
in CRC tissues were significantly downregulated compared with
adjacent tissues (P<0.05). These results are consistent with
previous studies and demonstrate that miR-205 could act as a
potential therapeutic target for CRC (25-33).
As a large miRNA cluster, members of the miR-376
family have been found to be susceptible to changes in various
human cancers. For instance, POU2F2-mediated upregulation of lncRNA
PTPRG-AS1 inhibited ferroptosis in breast cancer via the
miR-376c-3p/SLC7A11 axis (34),
and MEG3 suppressed angiogenesis through the miR-376a/RASA1 axis in
ovarian carcinoma-derived microvascular endothelial cells (35). miR-376a expression was lower in
glioma cells than in normal astrocytes. miR-376a mimic inhibited
SIRT1, YAP1, and VEGF expression and suppressed the proliferation,
migration and angiogenesis abilities of the glioma cell lines LN229
and A172, whereas miR-376a inhibitor exerted the opposite effects
(36). Since substantial progress
has been recorded in research on miR-376a and miR-376b, miR-376c
was selected as a research target (35-37).
The present study showed that miR-376c was downregulated in CRC
tissues, with significant differences in expression between tumor
and adjacent tissues, indicating that miR-376c may be involved in
the development of CRC. Further bioinformatics analyses suggested
that it is potentially associated with CRC liver metastasis.
Furthermore, the expression of miR-539 in CRC was
evaluated. It is well-known that aberrant expression of miR-539
plays an important role in the initiation and development of
several cancers. For instance, miR-539 was revealed to act as a
tumor suppressor in hepatocellular carcinoma (HCC) (38), thyroid cancer (39), breast cancer (40), and osteosarcoma (41). Furthermore, miR-539 was shown to be
upregulated in hepatocellular carcinoma and may be involved in
regulating autophagy gene expression (42). Consistent with the aforementioned
previous research, the present study showed that miR-539 is
downregulated in CRC tissues, with significant differences in its
expression according to tumor stage and lymph node metastasis. In a
previous study, miR-539 was demonstrated to promote ferroptosis in
CRC by directly targeting TIPE to activate the SAPK/JNK signaling
pathway (43). Zhao et al
(44) reported that miR-539 was
downregulated in CRC tissues and cell lines, but was negatively
associated with advanced clinical stage and lymph node metastasis.
Therefore, further research is needed to clarify its role.
In the present study, significant differences were
found in the expression of the three miRNAs between tumor tissues
and corresponding normal tissues in the GEO database. In the
GSE41655 dataset, the overall expression trends of miR-376c,
miR-539, and miR-205 between tumor and normal tissues were
consistent with the results from our 25 clinical samples. In
addition, in the GSE81581 dataset, only the expression trend of
miR-376c was correlated with CRC liver metastasis. To investigate
whether the three miRNAs interact with one another during the
development of CRC, the expression levels of miR-205, miR-376c, and
miR-539 were analyzed and varying degrees of positive correlation
was found. Moreover, the ratios of expression levels in cancer
tissues to adjacent tissues also showed a certain degree of
positive correlation. These findings revealed the simultaneous
expression pattern of the three miRNAs in CRC, and suggest that
miR-205, miR-376c, and miR-539 may play different roles in the
occurrence and development of CRC. While the relevant molecular
mechanisms remain unclear, their target genes should be validated
in future studies.
Additionally, the mature sequences of the three
miRNAs were collected from the miRBase database and found no
significant common sequences among them. To further investigate the
relationships among their mechanisms of action, bioinformatics
analysis was employed to reveal potential associations between
miR-205, miR-376c, and miR-539 at the target gene level. The Venn
diagram analysis indicated that no common target genes were shared
among the six distinct sequences, miR-205-3p, miR-205-5p,
miR-376c-3p, miR-376c-5p, miR-539-3p, and miR-539-5p. However, the
UpSet plot revealed that the mature sequences of miR-205, miR-376c,
and miR-539 (5p or 3p end sequences) share a total of 53 predicted
target genes. This finding suggests that despite differences in
their expression patterns and functions, these three factors may
exert synergistic effects in the development of CRC by regulating
partially overlapping downstream gene networks. These common target
genes may be involved in classical tumor-related pathways such as
Wnt/β-catenin and PI3K/AKT (45,46),
but their specific functions and regulatory mechanisms require
further experimental investigation and validation.
miR-205 and miR-539 upregulation predicted advanced
TNM stage and poor tumor differentiation according logistic
regression analysis. However, they were downregulated in tumor
tissues compared with normal tissues. It is speculated that they
may be downregulated in early tumor development, and become
upregulated during tumor progression, thus exhibiting different
roles at different stages of CRC. Given that numerous miRNAs are
altered in CRC, these three miRNAs were randomly selected based on
previous studies (43-46),
as they exhibited significant correlations with clinicopathological
characteristics. Future studies should included larger sample sizes
and additional miRNAs.
The primary strength of this study lies in its
cross-referencing of multiple databases, integrating bioinformatics
with experimental validation methods to systematically investigate
the expression patterns, clinical significance, and target gene
networks of miR-205, miR-376c, and miR-539 in CRC. Additionally,
advanced visualization tools such as UpSet plots were employed to
identify 53 predicted target genes common these three miRNAs,
suggesting potential synergistic regulatory interactions among
them. However, the present study has several limitations, including
a small sample size that may affect statistical power, lack of
experimental validation on the relevant functions and pathways of
the predicted target genes, and insufficient functional mechanism
studies. Future studies should expand the sample size, and further
validate and explore the target genes and functional mechanisms of
these miRNAs through in vitro and in vivo
experiments. Additionally, future studies may explore the specific
mechanisms underlying the role of miRNA methylation patterns in CRC
survival and staging through investigation of miR-205, miR-376c,
and miR-539 miRNA methylation levels.
In conclusion, growing evidence indicates that miRNA
signatures are associated with diagnosis, prognosis, progression,
metastasis, and drug resistance of CRC. The present study revealed
the expression patterns of miR-205, miR-376c, and miR-539 in CRC
and their relationships with clinical features, such as sex and
age, and provided evidence of the role of these three miRNAs, and
that co-expression of these miRNAs exerts a synergistic regulatory
role in the pathogenesis of CRC. miR-205, miR-376c, and miR-539 may
participate in the development and progression of CRC. miR-205 and
miR-539 may act as potential risk factors predicting advanced
pathological stage and tumor differentiation. Overall, the findings
confirmed that miR-205, miR-376c, and miR-539 are aberrantly
expressed in CRC and closely associated with clinical and
pathological characteristics. Target gene intersection analysis
further suggests that these three miRNAs may synergistically
regulate CRC progression by sharing common downstream targets.
Therefore, targeting these miRNAs or their co-regulated pathways
may provide novel therapeutic strategies for CRC. Future research
should focus on validating the functions of these shared target
genes and their specific mechanisms of action in CRC.
Acknowledgements
The authors would like to thank the Zhongshan
Hospital of Xiamen University for providing clinical samples and
the contributions and support of the entire research team.
Funding
Funding: The present study was supported by the Natural Science
Foundation of Fujian Province (grant no. 2021J01013), Natural
Science Foundation of Xiamen (grant no. 3502Z20227104) and the the
Natural Science Foundation of Jiangxi Province, China (grant no.
20242BAB25511).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
JZ, GZ, ZL and CY contributed to the conception of
this study and performed the preliminary documentation. All authors
participated in the design of the study and implemented the
research. JZ, CY, ZL, HR, KQ, ZC, and AB examined the archives,
identified the cases included in the study, and collected the
pathological information. ZL, XC, and CW enrolled patients in the
study, performed clinical diagnosis, and collected clinical data.
LS is responsible for bioinformatics database analysis, manuscript
revisions, and responses to the comments of the reviewers. All
authors participated in the statistical analysis and contributed to
the interpretation of the results as well as the writing of the
study. JZ and ZL confirm the authenticity of all the raw data. All
authors reviewed all data, as well as read and approved the final
manuscript.
Ethics approval and consent to
participate
The present study abides by international and
national regulations in accordance with the Declaration of
Helsinki, and was approved [approval no. xmzsyyky (2024-703)] by
the Ethics Committee of Zhongshan Hospital, Xiamen University
(Xiamen, China). All patients provided written informed consent
before being included in the study.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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