Upregulation of miR-205-3p expression in liver cancer
vascular endothelial cells
Liver cancer vascular endothelial cells were discovered to have
upregulated miR-205-3p expression levels. Additionally, the miR-205-3p
levels expressed in liver cancer vascular endothelial cells and adjacent
normal cells from patient specimens were evaluated using Real-Time
Fluorescence Quantitative PCR (RT-qPCR). In ten samples of liver cancer
vascular endothelial cells, miR-205-3p was observed to be significantly
augmented. (Fig. 2A). Given these outcomes, RT-qPCR was conducted to
further probe the potential alterations in miR-205 expression in HUVECs
and among vascular endothelial cells (HepG2, HepG3B, and huh7) in
varying liver cancer transplantation tumors. Consequently, the
miR-205-3p levels were considerably elevated in vascular endothelial
cells of liver cancer compared to those in HUVECs (Fig. 2B).
Five patients with advanced stages (stages III and IV) and five patients
with early stages (stages I and II) were examined to understand the
changes in miR-205-3p expression. Our findings illustrated a significant
upregulation of miR-205-3p expression in the advanced stages relative to
the early stages (Fig. 2C).
Screening for
target genes of miR-205-3p.
The candidate miR-205-3p target genes were screened utilizing the miRNA
targeting gene screening technique, and the colony PCR result map (Fig.
3A) was obtained using Total RNA extraction, and RT-PCR. Each band
represented a potential miR-205-3p gene target. Luciferase reporter and
TargetScan were applied to validate the candidate miR-205-3p target
genes. Subsequent to this, specific sequences from the candidate
miR-205-3p target genes were extracted, leading to the selection of
HINT1, the target gene correlated with liver cancer. (Fig. 3B). By
analyzing the miR-205-3p sequence and the 3’UTR region of HINT1, the
complementary binding site (Fig. 3C) was determined. Hence, miR-205-3p
has the potential to suppress HINT1 expression by interacting with the
3’UTR.
Identifying HINT1
as a miR-205-3p target gene.
It should be noted that miR-205-3p specifically targets HINT1 as its
gene of interest. MiRNAs suppress the biological functioning of their
gene targets by regulating their expression in cells [25].
Furthermore, a single miRNA has the capability to target numerous genes
simultaneously, potentially reaching thousands of gene targets. In
addition, based on our findings, there is a possibility that HINT1 and
miR-205-3p interact with one another at the 3’UTR region, as depicted in
Figure 4A. To confirm our hypothesis, we employed a dual luciferase
assay by inserting the wild-type (WT) or mutant (MUT) 3’UTR of HINT1
downstream of the firefly luciferase gene (Figure 4B). After 48 hrs,
luciferase expression data were extracted from HepG2 and HepB3 cells
post-transfection with MiR-205-3p. These cells were then co-transfected
with either the WT or MUT 3’UTR of the HINT1 plasmid. Remarkably, when
contrasted to the miR-NC group, the luciferase activity of the WT 3’UTR
was considerably elevated by the miR-205-3p inhibitor. However, there
was no significant variation between the miR-NC and MUT 3’UTR groups
(Figure 4B). Moreover, as illustrated in Figure 4C, after transfecting
HepG2 and HepB3 cells with an inhibitor of miR-205-3p, the levels of
mRNA and protein expression of HINT1 were observed to be elevated. These
data strongly imply that HINT1 potentially represents a direct
miR-205-3p target in vascular endothelial cells derived from liver
cancer tissues.
The impact of
miR-205-3p knockdown on the proliferative capacity of ECs derived from
HepG2 and HepB3 xenografts.
We sought to investigate the impact of miR-205-3p knockdown on the
proliferative capacity of vascular endothelial cells (ECs) derived from
liver cancer. To accomplish this, to probe the impact that miR-205-3p
has on the proliferative capacity of HepG2 and Hep3B cells, we used an
miR-205-3p inhibitor. These cell lines were specifically chosen for
their upregulated miR-205-3p expression in liver cancer xenograft tumor
vessels.
When contrasted to the group that was treated with miR-NC, the level of
miR-205-3p was remarkably reduced in the group that had been transfected
with an inhibitor of miR-205-3p (Fig. 5A). We applied MTT tests to
determine whether or not miR-205 had any effect on the proliferative
capacity of HepG2 and Hep3B cells. As depicted in Fig. 5B, at 72 hours
post-transfection, cell viability was remarkably reduced due to the
miR-205-3p inhibitor-induced downregulation of miR-205-3p expression.
Furthermore, we conducted colony formation experiments to determine
whether knocking down miR-205-3p affected cellular proliferation. The
data confirmed a reduction in the total count of clones in the
miR-205-3p inhibitor group as opposed to the control group (Fig. 5C).
Inhibition of
miR-205-3p inhibits the proliferative capacity of liver cancer cells and
enhances apoptosis in mouse xenograft models.
The tumor growth inhibition and apoptosis promotion effects of
miR-205-3p antagomir were investigated in mouse xenograft models of
liver cancer. To assess the mid-term tumor suppression activity of
miR-205-3p, HepG2 cells were subcutaneously injected into mice before
treatment using miR-205-3p antagomir (Fig. 6A and Fig. 6B). Notably, a
reduction in tumor weight and size was observed in the miR-205-3p
antagomir treatment group as opposed to the antagomir NC treatment
group. Moreover, miR-205-3p antagomir treatment significantly suppressed
tumor cell proliferation, as evidenced by a decrease in Ki67 expression
(Fig. 6C).
Preceding research revealed that in liver cancer, HINT1 suppresses the
β-catenin protein’s functionality[26], a crucial protein entity
within the Wnt signaling pathway. The translocation of this protein from
the cytoplasm to the nucleus signifies the activation of the Wnt
signaling pathway and its corresponding function. In the cell, the
cytoplasmic destruction complex, which includes β-catenin, APC, Axin,
GSK-3β, and CK1, is involved in facilitating the degradation of
β-catenin at a reduced level. Upon the destruction of this complex, the
accumulation of β-catenin is accompanied by its movement into the
nucleus, where it activates its target genes. Previous research suggests
that around 70% of liver cancer cases exhibit cytoplasmic accumulation
of β-catenin.[27]. Precise regulation of cell invasion and migration
is critical to prevent cancer cells from acquiring high invasiveness and
metastatic potential. In this process, β-catenin serves dual roles.
Firstly, it acts as a crucial linker in adherens junctions, facilitating
cell-cell adhesion. Moreover, the AJ or tight junction complex, mediated
by E-cadherin, maintains the adhesive function of E-cadherin as a key
component in the Wnt/β-catenin pathway. Additionally, β-catenin is
involved in the adherens junction, AJ, or tight junction complex, where
it acts as a bridge between E-cadherin and the cytoskeleton, thereby
preserving the adherence function of E-cadherin. Furthermore, β-catenin
functions as a pivotal effector molecule in the Wnt/β-catenin pathway,
translocating into the nucleus and promoting the expression of tumor
cell metastasis-related genes [28]. Hence, the invasion and
migration processes are regulated by the intracellular levels of
β-catenin [29]. Effectively suppressing tumorigenesis, progression,
invasion, and migration can be achieved by reducing intracellular levels
of β-catenin and attenuating its capacity to translocate into the
nucleus [30].