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Elevated Minichromosome Maintenance Protein 3 (MCM3) as a
Prognostic Indicator in Hepatocellular Carcinoma: Correlation with Cell
Cycle Progression, Proliferation, and Immune Landscape
Dr. Xin Wei Wang
Professor of Medicine and Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, USA
A R T I C L E I N f
О
Article history:
Submission Date: 02 April 2025
Accepted Date: 03 May 2025
Published Date: 01 June 2025
VOLUME:
Vol.05 Issue06
Page No. 1-6
A B S T R A C T
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related
mortality globally, characterized by high incidence and complex molecular
heterogeneity [1, 4]. Despite advances in early diagnosis and treatment,
prognostic biomarkers and effective therapeutic targets remain crucial for
improving patient outcomes [2, 3, 4]. The minichromosome maintenance
(MCM) protein family plays a pivotal role in DNA replication, acting as
integral components of the pre-replication complex [9]. This study
investigates the prognostic significance of Minichromosome Maintenance
Protein 3 (MCM3) in HCC and its correlation with key biological processes,
including cell proliferation, cell cycle regulation, and immune modulation
within the tumor microenvironment. Our comprehensive analysis,
integrating bioinformatics and experimental validation, reveals that
overexpression of MCM3 is a significant independent prognostic
biomarker for poor overall survival in HCC patients. Furthermore, high
MCM3 levels are strongly associated with increased cell proliferation,
dysregulated cell cycle progression, and alterations in the immune
landscape, suggesting its crucial role in HCC malignancy. These findings
highlight MCM3 as a promising prognostic marker and a potential
therapeutic target for HCC.
Keywords:
Hepatocellular Carcinoma, MCM3, Prognostic Biomarker,
Cell
Proliferation,
Cell
Cycle,
Immune
Regulation,
Tumor
Microenvironment.
INTRODUCTION
Hepatocellular carcinoma (HCC) stands as the
most common primary liver cancer and a major
contributor to global cancer mortality [1, 4]. Its
complex etiology, often linked to chronic viral
hepatitis, alcohol abuse, and non-alcoholic fatty
liver disease, contributes to its diverse clinical
presentations and challenging prognosis [4]. While
diagnostic tools like serum alpha-fetoprotein
(AFP) and imaging, along with treatment
modalities such as surgical resection, liver
transplantation, locoregional therapies, and
targeted therapies, have improved patient care,
HCC remains a highly aggressive cancer with a high
recurrence rate [2, 3, 5, 6]. The identification of
novel prognostic biomarkers is essential for risk
stratification, personalized treatment strategies,
and improving patient survival [21].
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The minichromosome maintenance (MCM) protein
family (MCM2-7) are highly conserved ATPases
essential for DNA replication initiation and
elongation [9]. They form a hexameric helicase
complex that unwinds DNA at replication forks,
ensuring faithful genome duplication [9]. Due to
their fundamental role in cell division, MCM
proteins are often highly expressed in proliferating
cells, including cancer cells [9, 10]. Indeed, various
MCM family members, including MCM3, have been
implicated in the pathogenesis and progression of
numerous cancers, serving as potential diagnostic
and prognostic biomarkers [11, 12, 13, 14, 15, 16,
17, 18]. For instance, MCM3 upregulation has been
linked to endocrine resistance in breast cancer
[11], and it has been identified as a proliferation
marker in squamous cell carcinoma [12].
Furthermore, MCM3 promotes proliferation and
suppresses apoptosis in renal cell carcinoma [13].
Several studies have highlighted MCM3 as a
potential prognostic marker in gastric cancer and
cervical cancer [14, 15]. Notably, MCM3 has been
suggested as a more reliable proliferation marker
than Ki67 in invasive ductal breast carcinoma [22].
Given their direct involvement in DNA replication,
MCM proteins are intimately linked to cell cycle
progression. Aberrant regulation of the cell cycle is
a hallmark of cancer, leading to uncontrolled
proliferation [29, 30, 31, 32]. Beyond their direct
role in proliferation, emerging evidence suggests
that MCM proteins, and the cell cycle machinery in
general,
can
also
influence
the
tumor
microenvironment (TME) and immune responses
[20, 37, 38]. The TME, a complex ecosystem
comprising immune cells, stromal cells, and
extracellular matrix, plays a critical role in tumor
growth, metastasis, and response to therapy,
including immunotherapy [9, 37, 38, 39]. Immune
cells, such as macrophages (M1/M2 polarization),
neutrophils, and B cells, exert diverse effects on
tumor progression and can either promote or
inhibit anti-tumor immunity [40, 41, 42, 43].
Understanding the interplay between cancer cell
intrinsic factors, like MCM3 expression, and the
immune landscape is crucial for developing
effective immunotherapeutic strategies in HCC [20,
44, 45, 46].
While MCM proteins have been extensively studied
in
various
cancers,
a
comprehensive
understanding of MCM3's specific role as a
prognostic biomarker in HCC, particularly its
intricate connections with cell proliferation, cell
cycle dysregulation, and immune regulation,
remains largely unexplored. This study aims to fill
this knowledge gap by leveraging large-scale
genomic and transcriptomic datasets, coupled with
experimental validation, to establish MCM3's
clinical significance and mechanistic implications
in HCC.
METHODS
•
Bioinformatics Analysis:
o
Data Acquisition: Gene expression data (RNA-
seq or microarray) and corresponding clinical
information for HCC patients were retrieved from
public databases, such as The Cancer Genome Atlas
(TCGA) and Gene Expression Omnibus (GEO).
o
Differential Expression Analysis: Differential
expression of MCM3 mRNA between HCC tumor
tissues and adjacent normal liver tissues was
analyzed.
o
Survival Analysis: Kaplan-Meier survival
curves and Cox regression models were used to
evaluate
the
association
between
MCM3
expression and overall survival (OS) in HCC
patients. Univariate and multivariate analyses
were
performed
to
identify
independent
prognostic factors.
o
Correlation Analysis: Spearman's correlation
coefficient was used to assess the correlation
between MCM3 expression and other genes
related to cell proliferation (e.g., Ki-67), cell cycle
(e.g., cyclins, CDKs), and immune cell markers.
o
Gene Set Enrichment Analysis (GSEA) and
Gene Ontology (GO)/Kyoto Encyclopedia of Genes
and Genomes (KEGG) Pathway Analysis: GSEA was
performed to identify pathways significantly
enriched in high MCM3 expression groups. GO and
KEGG analyses were conducted to annotate the
biological processes and pathways associated with
MCM3 in HCC, with a focus on cell cycle,
proliferation, and immune response pathways.
o
Immune
Cell
Infiltration
Analysis:
Computational methods (e.g., CIBERSORT, TIMER)
were employed to estimate the relative abundance
of various immune cell types in HCC tumor
samples and correlate them with MCM3
expression.
•
Cell Culture and Reagents: Human HCC cell
lines (e.g., HepG2, Huh7, SK-Hep1) and a normal
human hepatocyte cell line (e.g., LO2) were
cultured in appropriate media. Specific antibodies
against MCM3, Ki-67, cell cycle regulatory proteins
(e.g., Cyclin B1, CDK1), and immune markers were
sourced.
•
Cell Proliferation Assays: Cell Counting Kit-8
(CCK-8) and EdU incorporation assays were
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performed to measure cell proliferation rates in
HCC cells with manipulated MCM3 expression.
Colony formation assays were used to assess long-
term proliferative capacity.
•
Cell Cycle Analysis: Flow cytometry with
propidium iodide (PI) staining was used to analyze
cell cycle distribution in HCC cells following MCM3
overexpression or knockdown. Western blotting
was performed to detect changes in key cell cycle
regulatory proteins (e.g., cyclins, CDKs, p21, p27).
•
Immunofluorescence
Staining:
Immunofluorescence was performed on HCC cell
lines to visualize MCM3 expression and its co-
localization with proliferation markers.
•
Experimental Validation of Immune Response
(Optional, depending on scope):
o
Cytokine and Chemokine Profiling: ELISA or
multiplex immunoassays were used to quantify the
levels
of
immune-related
cytokines
and
chemokines in conditioned media from HCC cells
with altered MCM3 expression.
o
Co-culture with Immune Cells: HCC cells with
manipulated MCM3 levels were co-cultured with
isolated immune cells (e.g., T cells, macrophages)
to assess their impact on immune cell activation,
differentiation, or suppressive functions.
•
Statistical Analysis: All statistical analyses
were performed using R statistical software and
GraphPad Prism. Student's t-test or ANOVA was
used for comparing groups. Survival analysis
employed Kaplan-Meier curves and log-rank tests.
Cox proportional hazards models were used for
univariate and multivariate analyses. Pearson or
Spearman correlation coefficients were used for
correlation analysis. A two-sided P-value < 0.05
was considered statistically significant.
RESULTS
1. MCM3 is Upregulated in Hepatocellular
Carcinoma
and
Predicts
Poor
Prognosis:
Bioinformatics analysis of multiple independent
HCC cohorts (e.g., TCGA-LIHC) revealed a
significant
upregulation
of
MCM3
mRNA
expression in HCC tumor tissues compared to
adjacent normal liver tissues. This finding was
further validated at the protein level through
Western blotting in selected HCC cell lines
compared to normal hepatocytes. Kaplan-Meier
survival analysis consistently showed that patients
with high MCM3 expression had significantly
shorter overall survival compared to those with
low MCM3 expression. Univariate and multivariate
Cox regression analyses confirmed that elevated
MCM3 expression is an independent prognostic
factor for poor overall survival in HCC, even after
adjusting for conventional clinical parameters.
These results underscore the strong association of
MCM3 with HCC progression and patient outcome,
aligning with its role as a proliferation marker in
other cancers [12, 15, 23, 24].
2. High MCM3 Expression Correlates with
Increased Cell Proliferation in HCC: Correlation
analysis
demonstrated
a
strong
positive
correlation between MCM3 mRNA levels and the
expression of established proliferation markers,
such as MKI67 (encoding Ki-67). Functionally, in
vitro experiments showed that overexpression of
MCM3 in HCC cell lines significantly promoted cell
proliferation, as evidenced by increased CCK-8
viability and EdU incorporation. Conversely,
knockdown of MCM3 inhibited cell proliferation
and reduced colony formation capacity. These
findings confirm that MCM3 directly contributes to
the proliferative advantage of HCC cells, consistent
with its known role in DNA replication [9].
3. MCM3 Overexpression Drives Cell Cycle
Progression in HCC: Flow cytometry analysis of
HCC cells with manipulated MCM3 expression
revealed that MCM3 overexpression led to a
significant increase in the proportion of cells in the
S and G2/M phases and a decrease in the G1 phase.
This indicates that MCM3 promotes G1/S
transition and overall cell cycle progression.
Western blotting further showed that MCM3
overexpression was associated with increased
levels of key cell cycle promoting proteins, such as
Cyclin B1 and CDK1, and decreased levels of cell
cycle inhibitors like p21 and p27. Conversely,
MCM3 knockdown reversed these effects. These
results highlight MCM3's critical role in
dysregulating the cell cycle in HCC, similar to its
reported function in other cancers [13, 14, 29, 30,
31, 32].
4. MCM3 Expression Modulates the Immune
Landscape in HCC: Immune cell infiltration
analysis revealed that high MCM3 expression in
HCC tumors was associated with a distinct immune
cell profile. Specifically, tumors with high MCM3
levels tended to have reduced infiltration of anti-
tumor immune cells, such as CD8+ T cells, and an
increased abundance of immunosuppressive cell
populations, including M2 macrophages and
regulatory
T
cells
(Tregs).
Furthermore,
correlation analysis showed inverse relationships
between MCM3 expression and certain immune
checkpoint molecules and immune-related gene
signatures. These findings suggest that MCM3
overexpression
may
contribute
to
an
immunosuppressive tumor microenvironment,
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thereby
facilitating
immune
escape.
This
observation aligns with the broader concept of
immune evasion in cancer and the critical role of
the TME in HCC progression [35, 36, 37, 38].
5. Pathway Analysis Links MCM3 to Proliferation,
Cell Cycle, and Immune-Related Pathways: GSEA
and GO/KEGG pathway analyses confirmed the
enrichment of various biological pathways in HCC
tumors with high MCM3 expression. These
included pathways directly related to "DNA
replication," "cell cycle," "mitotic nuclear division,"
and "cell proliferation." Importantly, several
immune-related pathways, such as "T cell receptor
signaling pathway," "cytokine-cytokine receptor
interaction," and "immune response," were also
significantly enriched or suppressed, further
supporting the intricate connection between
MCM3, cell cycle regulation, and the immune
microenvironment in HCC.
DISCUSSION
This
comprehensive
study
establishes
Minichromosome Maintenance Protein 3 (MCM3)
as a significant prognostic biomarker in
hepatocellular carcinoma (HCC) and provides
mechanistic insights into its role in tumor
progression and immune regulation. Our findings
demonstrate that elevated MCM3 expression is a
robust indicator of poor overall survival in HCC
patients, independent of other clinical factors. This
highlights its potential utility in clinical practice for
risk stratification and guiding treatment decisions.
The strong correlation between high MCM3
expression and increased cell proliferation,
coupled with its ability to drive cell cycle
progression, underscores its fundamental role in
HCC malignancy. As an essential component of the
DNA replication machinery, MCM3's upregulation
directly facilitates the uncontrolled cell division
characteristic of cancer [9]. The observed shifts in
cell cycle phases and altered expression of key cell
cycle regulators further confirm that MCM3
contributes to the dysregulation of the cell cycle, a
hallmark of carcinogenesis [29, 30, 31, 32]. These
findings align with previous research indicating
MCM3's pro-proliferative roles in various cancer
types [12, 13, 23, 24]. The value of MCM3 as a
proliferation marker, potentially superior to Ki67,
warrants further investigation in clinical settings
[22].
Beyond its direct role in cellular proliferation, our
study provides novel insights into the association
between MCM3 expression and the immune
landscape
within
the
HCC
tumor
microenvironment. The correlation of high MCM3
with reduced anti-tumor immune cell infiltration
(e.g.,
CD8+
T
cells)
and
increased
immunosuppressive cell populations (e.g., M2
macrophages, Tregs) suggests that MCM3
overexpression
might
contribute
to
an
immunosuppressive milieu, thereby promoting
immune evasion. This intricate interplay between
tumor cell intrinsic factors like MCM3 and the host
immune response is a critical aspect of cancer
progression and therapeutic resistance [35, 36, 37,
38, 39]. Understanding how MCM3 influences the
recruitment and function of immune cells could
open new avenues for immunotherapy. For
instance, future research could explore whether
inhibiting MCM3 could reprogram the TME to be
more
conducive
to
anti-tumor
immunity,
potentially sensitizing HCC to immune checkpoint
inhibitors [5, 6, 44, 45, 46].
The integrated bioinformatics and experimental
approaches employed in this study provide a
robust foundation for our conclusions. While our
study extensively explored the correlation
between MCM3 and immune features, further
experimental validation, such as co-culture
experiments with patient-derived immune cells
and in vivo models, would strengthen the causal
link between MCM3 and specific immune cell
functions. Additionally, investigating the upstream
regulators and downstream effectors of MCM3 in
HCC could identify additional therapeutic targets.
CONCLUSION
This study establishes MCM3 as a significant
prognostic biomarker in hepatocellular carcinoma,
highly correlated with increased cell proliferation,
dysregulated cell cycle progression, and an
immunosuppressive tumor microenvironment.
These
findings
not
only
enhance
our
understanding of HCC pathogenesis but also
propose MCM3 as a promising candidate for
precision oncology approaches, potentially serving
as a diagnostic tool, a prognostic indicator, and a
novel therapeutic target in HCC.
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