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\begin{document}

\title{MiR-205-3p Modulates Hepatocellular Carcinoma-Derived Endothelial Cell
Progression through suppression of HINT1}



\author[1]{Mengnan Wang}%
\author[1]{qiqi mao}%
\author[1]{Jianqiang Xiang}%
\author[1]{Hong Li}%
\affil[1]{Ningbo Medical Centre Lihuili Hospital}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\selectlanguage{english}
\begin{abstract}
\textbf{Objectives:} We investigated the molecular underpinnings of
miR-205-3p in driving the proliferative behavior of endothelial cells
originating from hepatocellular carcinoma (HCC). \textbf{Methods:} In
our study, reverse transcription quantitative PCR analysis identified a
significant upregulation of miR-205-3p in endothelial cells from HCC.
This upregulation was consistently observed across various HCC cell
lines and tissue samples. Further investigation highlighted the
regulatory effect of miR-205-3p on the HINT1 gene. The interaction
between miR-205-3p and the 3' untranslated region of HINT1 was confirmed
using a dual-luciferase reporter assay. Additionally, experiments
involving MTT assays and flow cytometry demonstrated that
lentivirus-mediated suppression of HINT1 enhanced cellular proliferation
and reduced apoptotic activity in HCC cell models. \textbf{Results:}
This study reveals that miR-205-3p contributes to the progression of
endothelial cells from HCC by targeting and inhibiting HINT1, a tumor
suppressor. \textbf{Conclusions:} This study provides a theoretical
foundation suggesting that miR-205-3p may serve as a viable molecular
target for gene therapy interventions in clinical settings.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Annually, hepatocellular carcinoma (HCC) manifests globally with an
incidence exceeding 850,000 cases {[}1{]}. This malignancy ranks as the
second primary oncogenic cause of mortality, and its prevalence is
escalating {[}2{]}. HCC is clinically characterized by profound
cachexia, debilitating fatigue, icterus, extensive tissue infiltration,
and systemic metabolic dysregulation, all of which significantly impair
patient prognosis. The armamentarium for HCC management includes
surgical resection, hepatic arterial chemoembolization, vascular
ligation, cryoablation, thermal ablation via radiofrequency or
microwave, laser therapy, along with systemic chemotherapeutics and
ionizing radiation {[}3-5{]}. Despite these interventions, the
recurrence rate and resistance to therapy in HCC remain considerable
clinical impediments. Therefore, probing into the molecular etiologies
of hepatic oncogenesis and its progression is imperative for enhancing
diagnostic accuracy, therapeutic precision, and overall disease
outcomes.

MicroRNAs (miRNAs) are a subclass of endogenous non-coding RNAs that
orchestrate post-transcriptional gene regulation, either by repressing
translation or expediting the degradation of their target mRNAs {[}6,
7{]}. miR-205-3p, initially identified through comparative genomic
MiRscan analyses within conserved sequences between murine and the
red-finned oriental dolphin {[}8{]}; , has been corroborated in human
pathological investigations {[}9{]}. Its differential expression
profiles in various neoplasms suggest a pivotal role in tumorigenesis
and cancer progression. Diminished miR-205-3p expression characterizes
certain malignancies such as prostate {[}10{]} and breast cancer
{[}11{]} while an upregulation is documented in pathologies including
non-small cell lung carcinoma {[}12{]}, melanoma{[}13{]}, and head and
neck squamous carcinoma (HNSC) {[}14{]}. denoting a potential oncogenic
inducer in a tissue-specific context. Specifically, in NSCLC, miR-205-3p
has been demonstrated to instigate oncogenic pathways by modulating the
Akt signaling cascade through targeted inhibition of PHLPP2 {[}15{]}.
Moreover, the miR-205-mediated silencing of PTEN has been linked to
enhanced neoplastic cell proliferation in endometrial carcinomas
{[}16{]}. These cumulative research findings emphasize the oncogenic
facilitation by miR-205-3p. Nonetheless, its explicit mechanistic role
and the pathways through which it advances HCC pathophysiology require
further elucidation.

Endoglin, commonly designated as CD105, is a membrane glycoprotein
prominently expressed on the surface of vascular endothelial cells. This
protein critically engages in the binding of transforming growth
factor-beta (TGF-\selectlanguage{greek}β\selectlanguage{english}), playing a pivotal role in angiogenesis,
particularly within the context of tumor neovascularization. CD105 is
intricately associated with regulatory pathways that govern cell
proliferation.{[}17{]}. While endoglin exhibits minimal expression in
most normal tissues, its presence is pronounced in the vasculature of
the umbilical cord, underscoring its significance in angiogenesis. This
restricted expression profile renders CD105 a salient marker for the
identification and characterization of tumor-associated vascular
endothelial cells {[}18, 19{]}. Furthermore, a positive correlation
exists between CD105 expression and the proliferation markers Cyclin A
and Ki-67 within the endothelial cells of the tumor vasculature,
suggesting that CD105 expression is indicative of cellular proliferative
status {[}20{]}.

Existing in a dimeric form, the Histidine triad nucleotide-binding
protein 1 (HINT1) is a purine nucleotide-binding protein and is a part
of the ancient HIT superfamily. Through the mouse knockout technique,
Tao et al {[}21{]} first discovered that the HINT1 gene may exert tumor
suppressive effects. In addition, Li et al {[}22{]} discovered a
substantially higher probability of spontaneous tumorigenesis in
HINT1-deficient mice than wild-type mice by feeding HINT1 knockout mice
for 2 to 3 years. Existing studies indicate that HINT1 protein exerts
its tumor growth inhibitory effects by suppressing tumor cell growth,
promoting apoptosis, and affecting tumor cell signaling; nevertheless,
whether HINT1 can be regulated at the transcriptional level in liver
cancer remains unclear.

Prior investigations have suggested that the progression of
hepatocellular carcinoma (HCC) may be modulated by the interplay between
miR-205-3p and Histidine triad nucleotide-binding protein 1 (HINT1);
however, the intricacies of their interaction in the regulatory
landscape of hepatic oncogenesis remain ambiguous. In this study, we
employed flow cytometry, terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL), and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assays to delineate the expression dynamics of miR-205-3p in HCC and to
elucidate its biological implications. Concomitantly, luciferase
reporter assays were conducted to verify HINT1 as a putative target in
the miR-205-3p regulatory axis. We also examined the correlation between
HINT1 protein levels and tumor vascular endothelial cells, providing an
analysis of the influence of HINT1 on the proliferation of these cells.
Our findings present novel perspectives for the therapeutic targeting of
HCC.

\textbf{Materials and methods}

Cell culture

The normal human umbilical vein endothelial cells line (HUVEC)~and the
HCC cell lines HepG2 andHep3B were procured from the Cell Bank of the
Chinese Academy of Sciences in Shanghai, China. Dulbecco's modified
Eagle medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented
with 10\% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific,
Inc.), 100 mg/ml streptomycin and 100 IU/ml penicillin was employed to
culture these cells. The temperature of the culture was kept at 37 \selectlanguage{ngerman}° C,
and the CO2 level was kept at 5\%.

Clinical samples

Between January and October 2020, HCC tissue samples along with adjacent
normal tissues, situated about 1 cm from the margin of the tumor, were
gathered from 10 patients at the Ningbo Medical Center Li Huili
Hospital, Zhejiang, China. Before undergoing the surgical procedure,
every patient provided their signed informed consent, and the tissue
collection complied with the medical ethics standards of Ningbo Medical
Center Li Huili Hospital (approval no. KY2021PJ188). Importantly, before
the sample collection, none of the patients received any kind of
treatment with chemotherapy or radiation. Five of the samples were from
patients in more advanced stages of HCC (stages III and IV), while the
others were from patients in earlier stages (stages I and II). All
diagnoses were made following the Revised International System for
Staging Liver Cancer {[}23{]}. All patients have provided written
informed consent.

Cell transfection

HINT1-specific small interfering RNAs (50 nM; HINT1-siRNA1, 5' -ATU TUC
CAT CAC GCA TAT GTT-3'; HINT1-siRNA2, 5' -TTG CTT AAA TTA TTG TCA
GCT-3') and a nonspecific control (50 nM; si-NC, 5' -CAT CTG ATT TCA ATG
GTG CTT AT-3') were produced by Shanghai GenePharma Co., Ltd.
Additionally, a miR\selectlanguage{english}-205\selectlanguage{english}-3p inhibitor (50 nM; 5' -ATG TTC AGT GGG AGT GAG
TGT TC-3') and its negative controls (miR\selectlanguage{english}-NC; 50 nM; 5' -CTA CAA CCT CCT
AGA AAG AGT AGA-3') were procured from Changzhou Ruibo Bio\selectlanguage{english}-Technology
Co., Ltd. In a 6-well plate, 3x10\textsuperscript{5} cells were planted
per well and subjected to culture till~they reached \selectlanguage{english}[?]70\%~confluence.
Lipofectamine 3000 reagent was employed~to transfect the cells with the
miR205 inhibitor, HINT1 siRNA1, HINT1 siRNA2, and their matching NCs, as
per the package instructions. After 72 hours post-transfection, the
cells were harvested for additional examination. Both RT-qPCR and
Western blotting were conducted to verify the transfection efficiency.
The Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific,
Inc.) was applied in all transfection procedures.

RT\selectlanguage{english}-qPCR

Both tissues and cell lines were processed utilizing TRIzol reagent
(Invitrogen; Thermo Fisher Scientific, Inc.) to extract total RNA. The
PrimeScript RT reagent kit was employed for the~synthesis of~cDNA
following package guidelines (Takara Bio, Inc.). The SYBR Green
PrimeScript\selectlanguage{english} PLUS RT\selectlanguage{english}-PCR kit (Takara Bio, Inc.) was then utilized to
carry out qPCR. The procedure included an initial denaturation phase at
95\selectlanguage{english}@C for 30 seconds, followed by 40 cycles of 95@\selectlanguage{english}C denaturation for 15
seconds, 60@\selectlanguage{english}C annealing for 25 seconds, and 72@\selectlanguage{english}C extension for 1 minute,
ending with a final extension step at 72@\selectlanguage{english}C for 10 minutes. The internal
controls used were U6 for miRNA and \selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}actin for mRNA, with the relative
gene expression being determined via the 2-\selectlanguage{greek}ΔΔ\selectlanguage{english}Cq method{[}24{]}. The
primers employed were as follows: 5'-CTT GTC CTT CAT TCC ACC GGA-3' and
5'-TGC CGC CTG AAC TTC ACT CC-3' for miR-205-3p; 5'-CAC AGA GAA GA GTC
TGG CCC-3' and 5'-AGG TTG CTT GTG ACA GGT CC-3' for HINT1; 5'-GAT GGA
CTC TGG TGA TGG TGT GAC-3' and 5'-TTT CTC TTT CGG CTG GTG GTG TG-3' for
\selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}actin; and 5'-CTC GCT TCG GCA GCA CA-3' and 5'-AAC GCT TCA CGA ATT TGC
GT-3' for U6.

\emph{Luciferase assa}\textbf{y}

The 3'untranslated region (UTR) of wild type (WT) HINT1 and its mutant
(MUT) was amplified before being cloned into pGL3 luciferase vectors
(Promega Corporation) using the Q5 Site-Directed Mutagenesis kit (New
England BioLabs, Inc., cat. no. E0554S), as per the protocol provided by
the manufacturer. Cells were subjected to co-transfection with
miR-205-3p inhibitor (or miR-NC) at 10 nM concentration and 200 ng/well
of luciferase reporter vectors for the HINT1 gene with either the WT or
MUT type 3'UTR. This was carried out utilizing Lipofectamine 3000
(Invitrogen; Thermo Fisher Scientific, Inc.) as per the guidelines
stipulated by the manufacturer. Post-transfection, the relative
luciferase activities were measured for 48 hrs with the Dual-Luciferase
Reporter Assay kit (Promega Corporation), per package guidelines.
Renilla luciferase activity was utilized for normalization against
firefly luciferase activity.

MTT assay

The MTT test was employed~to ascertain cell viability.
Post-transfection, 96-well plates were used to culture the cells with
each well containing 1.0x10\textsuperscript{4} cells and subsequently
incubated at 37@\selectlanguage{english}C for time intervals of 24h, 48h, and 72h. Next, a 0.5
mg/ml MTT solution (Sigma; Merch KGaA) was introduced into the cells and
left to culture~for 4 hrs, immediately after which co-culturing with
100\selectlanguage{greek}µ\selectlanguage{english}l DMSO was performed at 37@\selectlanguage{english}C for 10 min. The optical density was
subsequently~calculated from the absorbance measurements taken at 490
nm.

Flow cytometric analysis of CD105 expression

For FACS analysis, cells were prepared at a density of
1x10\textsuperscript{6} cells mixed with 90?l of buffer and 10?l of
phycoerythrin-conjugated CD105 antibody, and then stained at 4[?]C for a
duration of 30 minutes. The acquired data were subsequently processed
and interpreted using EXPO32 Software.

Tube formation assay

The day before the experiment, Matrigel was left to thaw overnight in a
refrigerator set at 4 \selectlanguage{ngerman}°C. The frozen \selectlanguage{greek}μ-\selectlanguage{english}Slide plate and gun tip were also
removed from the refrigerator. Then, 10 \selectlanguage{greek}μ\selectlanguage{english}l/well of Matrigel gel was
transferred to the \selectlanguage{greek}μ-\selectlanguage{english}Slide plate, which was then placed into a Petri
dish of suitable size. Then, the entire Petri dish was allowed to
incubate at~37 \selectlanguage{ngerman}° C for 30 minutes. Following the digestion of T-ECs and
N-ECs from the culture flasks, the cell suspension was set to (1
\textasciitilde{} 2) × 10\textsuperscript{5} cells/ml. Prior to the
addition of cell suspension to the wells (50 \selectlanguage{greek}μ\selectlanguage{english}l/well), the \selectlanguage{greek}μ-\selectlanguage{english}Slide plate
was removed. Subsequently, 4 to 6 hours were spent incubating the plate
at 37 \selectlanguage{ngerman}° C. Thereafter, the medium was removed and the wells were washed
with buffer. Finally, the results of the experiment were examined under
a microscope.

DIl-ac-LDL assay

In 24-well plates, T-ECs and N-ECs were cultivated and then incubated
for 48 hours. Following this, a serum-free ECM medium was used to
replace the previous medium, which was allowed to incubate for an
additional 3 hours. Afterward, Dil-ac-LDL, which had been labeled with
rhodamine and prepared from serum-free ECM medium, was introduced into
the wells. Once a 4-hour incubation period had passed, the medium was
again aspirated. To remove any unbound Dil-ac-LDL, the wells were then
rinsed with 0.01 M PBS. Using a fluorescence microscope, the results of
the experiment were then examined.

Prediction of miR\selectlanguage{english}-205\selectlanguage{english}-3p target genes

Using two online tools, TargetScan version 7.0
(\emph{http://www.targetscan.org/vert\_72/}) and miRDB
(http://mirdb.org/index.html), the potential target genes downstream of
miR\selectlanguage{english}-205\selectlanguage{english}-3p were predicted.

In vivo tumor biology

Eight NOD/SCID mice, each four weeks old and weighing around 20+-2 grams,
were procured from Beijing Vital River Laboratory Animal Technology Co.,
Ltd. These animals were placed in a facility with a controlled
environment of 22+-1\selectlanguage{english}@C temperature, 55\selectlanguage{ngerman}±2\% humidity, and a 12/12\selectlanguage{english}-h
light/dark cycle, with unlimited water and food access. Each mouse
received a subcutaneous injection of 0.2 ml PBS containing 5x106 HepG2
cells on its right side. Post a fortnight, the mice were given an
intravenous injection of either miR\selectlanguage{english}-205\selectlanguage{english}-3p antagomir (100 nmol) or 200
\selectlanguage{greek}µ\selectlanguage{english}l antagomir NC in the tail. Every five days, a slide caliper was
utilized~to measure the size of the tumor. Following 42 days from the
initial injection, Intraperitoneal administration of a mix of 100 mg/kg
ketamine and 10 mg/kg xylazine anesthetized the mice. After euthanizing
the mice via cervical dislocation, the volume of their tumors was
measured. The presence of Ki67 was determined using immunofluorescence.

MiRNA targeting gene screening technique

Cells were processed for RNA extraction, which was then isolated using
Trizol (Sigma Corporation) and chloroform. The isolated RNA was
precipitated with isopropanol. Any DNA present was eliminated by
treating RNA with DNase I (Takara Corporation). The RNA was subsequently
converted to cDNA via RT-PCR. In parallel, DH5\selectlanguage{greek}α \selectlanguage{english}bacteria were cultured
in 2XYT media devoid of antibiotics, followed by their amplification and
harvesting. The bacterial cells were rendered competent using calcium
chloride, combined with glycerol, and preserved at -80\selectlanguage{ngerman}°C. Plasmid
presence was confirmed through colony PCR, which encompassed preparing a
PCR reaction system, programming, and initiating the PCR. Lastly, a
quantitative PCR (qPCR) was executed, which involved setting up the
reaction system for the first strand and primers, amalgamating and
centrifuging the components, and running the qPCR.

Statistical analysis

Data collection was conducted in triplicate, with the outcomes
represented as the mean ± SD. Statistical evaluation of RT-qPCR findings
from HCC tissues and their corresponding tissues utilized a paired
Student's t-test. To identify statistical variations in the remaining
data, we employed~either an unpaired Student's t-test or a one-way
analysis of variance (ANOVA) accompanied by Tukey's post-hoc test.
Kaplan-Meier analysis accompanied by a log-rank test was adopted to
ascertain differences in overall survival (OS) between the two groups of
patients. GraphPad Prism 6.0 (GraphPad Software, Inc.) facilitated the
statistical computations. A P value of less than 0.05 was seen as
statistically consequential.

\textbf{Results}

CD105\textsuperscript{+} cell purification and identification of
endothelial function

An adapted technique for the purification of vascular endothelial cells
was utilized to gather vascular endothelial cells
(CD105\textsuperscript{+} cells), independently from both liver cancer
tissue and normal liver tissue. The liver cancer vascular endothelial
cells (T-ECs, Tumor-Endothelial Cells) and normal hepatic vascular
endothelial cells (N-ECs, Normal-Endothelial Cells) microscopically
showed a goose-flint-like morphology (Fig. 1A). Based on flow cytometry,
CD105 positive rates of T-ECs and N-ECs were 95.6 ± 1.4 \% and 94.9 ±
2.3 \%, respectively (Fig. 1B), increasing the sorting efficiency of
positive cells to more than 95 \% to minimize the effect of negative
cells on subsequent experiments, using endothelial cell tube-forming
assay and endothelial cell. The endothelial cell function of T-ECs and
N-ECs was identified using endothelial cell tube formation and
endothelial cell phagocytosis assays. As a result, we discovered that
T-ECs and N-ECs could form tubular lumen-like structures on Matrigel gel
(Fig. 1C) and phagocytosed ac-LDL before emitting red fluorescence (Fig.
1D).

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
\selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}catenin, APC, Axin,
GSK-3\selectlanguage{greek}β, \selectlanguage{english}and CK1, is involved in facilitating the degradation of
\selectlanguage{greek}β-\selectlanguage{english}catenin at a reduced level. Upon the destruction of this complex, the
accumulation of \selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{greek}β-\selectlanguage{english}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, \selectlanguage{greek}β-\selectlanguage{english}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/\selectlanguage{greek}β-\selectlanguage{english}catenin pathway. Additionally, \selectlanguage{greek}β-\selectlanguage{english}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, \selectlanguage{greek}β-\selectlanguage{english}catenin
functions as a pivotal effector molecule in the Wnt/\selectlanguage{greek}β-\selectlanguage{english}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
\selectlanguage{greek}β-\selectlanguage{english}catenin {[}29{]}. Effectively suppressing tumorigenesis, progression,
invasion, and migration can be achieved by reducing intracellular levels
of \selectlanguage{greek}β-\selectlanguage{english}catenin and attenuating its capacity to translocate into~the
nucleus {[}30{]}.

\textbf{Discussion}

Previous research has examined the potential impact of abnormal miRNA
expression levels on the development of tumors. {[}31-33{]}. Given the
intimate connection between miRNAs and tumors, mounting data illustrate
that the management of miRNA expression could potentially introduce a
new and effective approach to tumor therapy. {[}34{]}. Our data show
that miR-205-3p downregulation remarkably suppresses vascular
endothelial cell proliferation and survival in liver cancer. It also
enhances apoptosis by focusing on the tumor suppressor HINT1.

miR-205-3p, which is encoded by the LOC642587 locus in the second intron
of chromosome 1, is associated with the onset and progression of
different types of cancer {[}35-37{]}. Past research suggests that both
tumor suppression and oncogenic activity may be attributed to
miR-205-3p. Overexpression of miR-205-3p inhibits carcinogenesis
substantially because of its role as a tumor suppressor {[}38{]}.
Furthermore, the expression levels of miR-205-3p are associated with the
existence of melanoma tissue. Downregulation of miR-205-3p leads to a
worse clinical prognosis {[}39, 40{]}. On the other hand, various types
of cancer may be contributed~by the miR-205-3p. For instance,~Niu et al
{[}41{]} observed an upregulation of miR-205-3p in ovarian cancer, its
upregulation directly correlating with advanced disease stages. Targets
in distinct tumor microenvironments may account for the various
functions of miR-205-3p. As such, this study set out to compare
miR-205-3p expression in patients with liver cancer and adjoining
non-cancerous~healthy tissues. We initially found markedly elevated
miR-205-3p levels in liver cancer vascular endothelial cells compared to
normal hepatic vascular endothelial cell tissue. Moreover, miR-205-3p
exhibits a high expression state in advanced (III and V) clinical
patients. In contrast, utilizing miR-205-3p inhibitors to inhibit
miR-205-3p resulted in enhanced apoptosis and reduced tumor size in
HepG2 and HepB3 cells in mice. In conclusion, the current findings
suggest that miR-205-3p modulates liver cancer tumorigenesis and hastens
its progression.

Investigating the potential mechanisms underpinning miR-205-3p's
promotion of tumor growth, this study focused on HINT1 as a possible
direct target in liver cancer vascular endothelial cells. We observed
HINT1 expression alterations by introducing a miR-205-3p inhibitor into
both liver cancer and healthy hepatic vascular endothelial cells. Our
findings highlighted a significant discrepancy in HINT1 expression
between these two types of cells. Furthermore, the miR-205-3p inhibitor
notably augmented HINT1 protein and mRNA expression across different
cellular and tissue models compared to their respective controls. This
consolidates the idea that miR-205-3p binds to the 3'-UTR region~of
HINT1, aligning with the outcomes from the luciferase reporter assay.
However, this study is not devoid of limitations. While studies suggest
that miR-205-3p can influence liver cancer progression by targeting
HINT1, further exploration is needed to understand how HINT1 modulates
cancer cell behavior. Additionally, the expression of HINT1 in vascular
endothelial cells in liver cancer and its link to clinical features and
the prognosis of liver cancer patients is still uncertain because of the
small number of clinical samples available. Future research should delve
into the potential roles of HINT1 in liver cancer. Despite these
constraints, our study contributes to the expanding knowledge of liver
cancer.

The prediction of our group through high-throughput sequencing results
shows that the target genes that may bind to miR-205-3p are PLAC8,
LRRQ4, and CLL15.Currently, it has been shown that PLAC8 can promote the
proliferation of hepatocellular carcinoma cells by down-regulating and
enhancing the Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling pathway {[}42{]}. And LRRIQ4 is a
gene in the human genome that belongs to the protein family of
Leucine-rich repeats (LRRs). Some studies have found that the Terc gene
cluster (LRRIQ4) has an effect on hepatic telomere length (aTL) using a
mouse model, which in turn may affect liver growth {[}43{]}. CCL15
belongs to the CC chemokine family, and it is moreover a serum biomarker
and an independent predictor of hepatocellular carcinoma survival {[}44,
45{]}, and it has been demonstrated that CCL15 can pass through the CCR1
axis for hepatocellular carcinoma metastasis {[}46{]}.

In summary, our investigation revealed a notable increase in miR-205-3p
levels within liver cancer vascular endothelial tissue, correlating with
a less favorable prognosis for patients suffering from liver cancer.
MiR-205-3p seems to have a function in driving liver cancer development
by regulating HINT1. These insights demonstrate that~miR-205-3p may be
involved in liver cancer pathogenesis. The biological value of
miR-205-3p/HINT1 interaction in liver cancer warrants
additional~investigation.

\textbf{Acknowledgments:} Not applicable.

\textbf{Informed consent:} All individuals in this study have given
their informed consent.

\textbf{Ethics approval and consent to participate:} The current study
received approval from the Medical Ethics Committee of Ningbo Medical
Center Li Huili Hospital (approval no. KY2021PJ188).

\textbf{Authors' contributions:} The experiments were conceptualized and
planned by LH and MQQ. The execution of the experiments was carried out
by MQQ, WMN, and XJQ. Data analysis and interpretation were performed by
MQQ and WMN. The manuscript was written by WMN and subsequently revised
by MQQ and LH. The authenticity of all raw data has been confirmed by
MQQ, LH, and WMN. The final manuscript was read and approved by all
authors.

\textbf{Competing interests:} The authors declare that they have no
competing interests.

\textbf{Funding:} The Project of Zhejiang Medical and Health Platform
Plan (No.2022KY1079) provided financial support for this research.

\textbf{Availability of data and materials:} The datasets utilized
and/or examined in the present study can be made accessible by the
corresponding author upon a reasonable request.

\textbf{Patient consent for publication:} Not applicable.

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2461-71.\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

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\end{figure}

\textbf{Figure 1. Purification of CD105\textsuperscript{+} cells and
identification of endothelial function.}

Morphology of CD105\textsuperscript{+} cells sorted by magnetic beads
under a microscope, Scale Bar=200\selectlanguage{greek}μ\selectlanguage{english}m. (B)The expression of CD105 in T-ECs
and N-ECs was detected by flow cytometry. (C) Light microscopy of T-ECs
and N-ECs in endothelial tube formation experiment, Scale Bar=200\selectlanguage{greek}μ\selectlanguage{english}m. (D)
Fluorescence microscopy of T-ECs and N-ECs under endothelial cell
phagocytosis experiment, Scale Bar=100\selectlanguage{greek}μ\selectlanguage{english}m.\selectlanguage{english}
\begin{figure}[H]
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\end{figure}

\textbf{Figure 2. Upregulation of miR-205-3p-3p in liver cancer vascular
endothelial cells.}

The statistical analysis showing the miR-205-3p expression in T-ECs and
N-ECs is depicted, *** P\textless{}0.001. (B) The representation of
miR-205-3p expression in HCC cell lines and human umbilical vein
endothelial cells (HUVEC) is provided. (C) A comparison illustrating the
miR-205-3p expressions between five patients with late-stage disease
(stages III and IV) and five patients with early-stage disease (stages I
and II) is given, *P\textless{}0.05.\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

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\textbf{Figure 3. Screening for miR-205-3p target genes.}

Displayed are the Colony PCR results, with marker bands at 100 bp, 250
bp, 500 bp, 750 bp, 1000 bp, 2000 bp, 3000 bp, 5000 bp, and 8000 bp. (B)
A segment of the HINT1 gene sequence findings is showcased. (C) The
anticipated miR-205-3p target location within the 3'UTR of HINT1 is
illustrated.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

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\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

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\end{figure}

\textbf{Figure 4. Identifying HINT1 as a miR-205-3p target gene.}

MiR-205 3p's putative target location in the 3'UTR of HINT1. (B) The
relative luciferase activity was measured 48 hours after co-transfection
of HepG2 and HepB3 cells with a miR NC or miR-205 3p inhibitor and a
luciferase reporter vector expressing either the WT or the MUT 3'UTR of
HINT1. *P\textless{}0.05 vs. miR NC. (C) The evaluation of mRNA
expression levels was conducted. The data represents the mean \selectlanguage{ngerman}± SD from
three separate tests. **P\textless{}0.01. Terminology: MUT, mutant; NC,
negative control; WT, wild type; miR, microRNA.\selectlanguage{english}
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\end{center}
\end{figure}

\textbf{Figure 5. The influence of miR-205-3p knockdown on the
proliferative capacity of ECs derived from HepG2 and HepB3 xenografts.}

Reverse transcription-quantitative PCR was conducted to analyze miR-205
3p expression in ECs from HepG2 and HepB3 xenografts that had been
transfected with miR-205-3p inhibitor or miR NC. (B) Using an MTT assay,
the impact of miR-205 3p suppression on cell survival was evaluated. (C)
To gauge the impact of miR-205 3p suppression on cell growth, a colony
formation test was conducted.\selectlanguage{english}
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\end{figure}

\textbf{Figure 6. Inhibition of miR-205-3p suppresses liver cancer
growth and enhances apoptotic capacity in mouse xenograft models.}

(A) Illustrative images of removed xenograft tumors are presented. (B)
Mice treated with either antagomir NC or miR-205 3p antagomir had~the
sizes of their~tumors measured. (C) Representative image showing Ki67
staining~with a scale bar of 100 m.

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