Invest Clin 63(3): 206 - 217, 2022 https://doi.org/10.54817/IC.v63n3a01
Corresponding author. Yi Wang, Otorhinolaryngology Head and Neck Surgery, The First Affiliated Hospital of
Chengdu Medical College, No. 278, Middle Section of Baoguang Avenue, Xindu District, Chengdu, 610500, China.
Tel: +86-8301-6131. Fax: +86-8301-6131. E-mail: y_wang5566@126.com
Role and mechanism of miR-548-3p/
DAG1 in the occurrence and malignant
transformation of laryngeal carcinoma.
Jia Chen, Yu Lin Zhou, Ke Wen, Shi Huang, Nan Hou, Ling Wang and Yi Wang
Otorhinolaryngology Head and Neck Surgery, The First Affiliated Hospital of Chengdu
Medical College, Chengdu, 610500, China.
Key words: laryngeal cancer; hsa-miR-548-3p; DAG1.
Abstract. The AMC-HN-8 cell line and the primary human laryngeal epi-
thelial cell lines were utilized in this work to explore the molecular mecha-
nism of miR-548-3p regulating the gene DAG1 to induce the occurrence and
malignant transformation of laryngeal carcinoma. Non-coding RNA miR-548-
3p overexpression plasmid, interference plasmid and blank plasmid were con-
structed, and the plasmids were transfected into AMC-HN-8 cells, respectively.
Meanwhile, a non-transfected plasmid group and a human laryngeal epithelial
primary cell group were set up. Five groups of cells were named as NC (Nor-
mal control), Model, Ov-miR-548-3p, Sh-miR-548-3p and Blank-plasmid group.
The luciferase reporter experiment was used to analyze the regulation charac-
teristics of hsa-miR-548-3p on dystrophin-associated glycoprotein 1 (DAG1).
Immunofluorescence was used to analyze the relative expression characteris-
tics of the protein DAG1. The cell cloning experiment was used to analyze the
proliferation characteristics of AMC-HN-8. The scratch healing test was used
to analyze the migration ability of AMC-HN-8. The transwell test was used to
analyze the invasion ability of AMC-HN-8. The RT-PCR was used to analyze the
expression level of miR-548-3p. Western blot experiments were used to analyze
the expression of protein DAG1, laminin α2 (LAMA2) and utrophin (UTRN).
The luciferase report experiment and immunofluorescence test found that the
expression of DAG1 and miR-548-3p are positively correlated. Cell cloning,
scratching and migration experiments identified that the activity of laryngeal
cancer cells was positively correlated with the expression of DAG1. The results
of Western blot analysis further strengthened the above conclusions. Through
carrying out research on the cellular levels, our work has demonstrated that
miR-548-3p regulated the content of protein DAG1, and then further induced
malignant transformation of laryngeal carcinoma.
miR-548-3p/DAG1 axis promotes the development of laryngeal carcinoma 207
Vol. 63(3): 206 - 217, 2022
Papel y mecanismo de miR-548-3p/DAG1 en la aparición
y transformación maligna del carcinoma de laringe.
Invest Clin 2022; 63 (3): 206 – 217
Palabras clave: cáncer de laringe; hsa-miR-548-3p; DAG1.
Resumen. En este trabajo se utilizaron la lÃnea celular AMC-HN-8 y la lÃnea
celular epitelial larÃngea humana primaria, para explorar el mecanismo mole-
cular regulador del miR-548-3p sobre el gen DAG1 para inducir la aparición y
la transformación maligna del carcinoma larÃngeo. Se construyeron el plásmido
de sobreexpresión de miR-548-3p de ARN no codificante, el plásmido de interfe-
rencia y el plásmido en blanco, y los plásmidos se transfectaron en células AMC-
HN-8 respectivamente. Mientras tanto, se establecieron un grupo de plásmidos
no transfectados y un grupo de células primarias epiteliales larÃngeas humanas.
Se nombraron cinco grupos de células como NC (control normal), modelo, Ov-
miR-548-3p, Sh-miR-548-3p y grupo de plásmido en blanco. El experimento in-
dicador de luciferasa se utilizó para analizar las caracterÃsticas de regulación de
hsa-miR-548-3p en la glicoproteÃna 1 asociada a distrofina (DAG1). Se utilizó
inmunofluorescencia para analizar las caracterÃsticas de expresión relativa de
la proteÃna DAG1. El experimento de clonación celular se utilizó para analizar
las caracterÃsticas de proliferación de AMC-HN-8. Se utilizó la prueba de cica-
trización por rascado para analizar la capacidad de migración de AMC-HN-8. La
prueba de transwell se utilizó para analizar la capacidad de invasión de AMC-
HN-8. Se utilizó RT-PCR para analizar el nivel de expresión de miR-548-3p. Se
usó un experimento de transferencia Western (Western blot) para analizar las
expresiones de la proteÃna DAG1, laminina α2 (LAMA2) y utrofina (UTRN). El
experimento de reporte de luciferasa y la prueba de inmunofluorescencia en-
contraron que la expresión de DAG1 y miR-548-3p están positivamente corre-
lacionadas. Los experimentos de clonación celular, rascado y migración, iden-
tificaron que la actividad de las células cancerosas de laringe se correlacionó
positivamente con la expresión de DAG1. Los resultados del análisis de transfe-
rencia Western fortalecieron aún más las conclusiones anteriores. A través de la
investigación a nivel celular, nuestro proyecto ha demostrado que miR-548-3p
regula el contenido de la proteÃna DAG1 y luego induce la transformación ma-
ligna del carcinoma de laringe.
Received: 23-11-2021 Accepted: 27-03-2022
INTRODUCTION
Laryngeal cancer is a common malig-
nant tumor among head and neck tumors.
According to statistics from various places in
China, it accounts for 7.9% to 35% of malig-
nant tumors in the ear, nose and throat, and
ranks third in head and neck malignancies.
The treatment of laryngeal cancer in China
is still based on surgery 1. Surgical resection
combined with radiotherapy and chemother-
apy is mainly used. Laryngeal cancer is di-
208 Chen et al.
Investigación ClÃnica 63(3): 2022
vided into primary and secondary 2. Primary
laryngeal carcinoma refers to tumors whose
primary site is in the larynx, and squamous
cell carcinoma is the most common. Second-
ary laryngeal cancer refers to the metastasis
of malignant tumors from other parts to the
larynx, which is relatively rare 3. Symptoms
of laryngeal cancer are mainly hoarseness,
dyspnea, cough, dysphagia, neck lymph node
metastasis, etc.4.
The function of DAG1 is to participate
in the assembly of laminin and basement
membrane, muscle membrane stability,
cell survival, peripheral nerve myelination,
lymph node structure, cell migration and
epithelial polarization 5,6.
miRNA is a type of endogenous small
RNA with a length of about 20-24 nucleo-
tides, which has a variety of important regu-
latory effects in cells. Each miRNA can have
multiple target genes, and several miRNAs
can also regulate the same gene. The high
degree of conservation of miRNA is closely
related to the importance of its function.
miRNA is closely related to the evolution of
its target genes, and studying its evolution-
ary history helps to further understand its
mechanism and function 7. miR-548 is a larg-
er, poorly conserved primate-specific miRNA
gene family, consists of 69 human miR-548
genes located in almost all human chromo-
somes, and the miR-548 gene family enrich-
ment pathway analysis showed they played
important roles in various human diseases 8.
miR-548c is a member of miR-548 and the
mature miR-548c-3p is obtained from it.
miR-548c-3p is low expressed in hypo-
pharyngeal carcinoma tissues and cell lines,
inhibits the proliferation, cloning, migration
and invasion of FaDu cells, and promotes cell
apoptosis 9. Its expression pattern is consis-
tent with the expression pattern of tumor
suppressor genes 10. miR-548c-3p targets
TP53BP2, and the molecular axis of miR-548-
3p/TP53BP2 affects the biological functions
of hypopharyngeal carcinoma cells such as
proliferation, colonization, migration, inva-
sion, cycle and apoptosis 11.
To explore the role and mechanism of
miR-548-3p/DAG1 in the occurrence and
malignant transformation of laryngeal carci-
noma, the human laryngeal carcinoma cell
line AMC-HN-8 and the primary human la-
ryngeal epithelial cell line were utilized here.
We found that the non-coding RNA miR-548-
3p can target and regulate the gene DAG1,
and then further induce malignant transfor-
mation of laryngeal carcinoma.
MATERIALS AND METHODS
Experiment design
The culture the human laryngeal car-
cinoma cell line AMC-HN-8 and the primary
human laryngeal epithelial cell line were
strictly in accordance with the requirements
of aseptic cultures. The non-coding RNA miR-
548-3p overexpression plasmid, interference
plasmid and blank control plasmid were con-
structed, and the plasmids were transfected
into AMC-HN-8 cells respectively. At the
same time, a group of non-transfected plas-
mid group and a human laryngeal epithelial
primary cell group were set up. Five groups of
cells were named as NC group, Model group,
Ov-miR-548-3p group, Sh-miR-548-3p group
and Blank-plasmid group. The luciferase re-
porter experiment was used to analyze the
regulation characteristics of miR-548-3p on
gene DAG1. Immunofluorescence was used
to analyze the relative expression character-
istics of the protein DAG1. The cell cloning
experiment was used to analyze the prolifer-
ation characteristics of laryngeal carcinoma
cell lines. The scratch healing test was used
to analyze the migration ability of laryngeal
cancer cell lines. The transwell test was used
to analyze the invasion ability of laryngeal
cancer cell lines. RT-PCR was used to analyze
the expression level of miR-548-3p. A West-
ern blot was used to analyze the expression
of protein DAG1, LAMA2 and UTRN.
Luciferase reporter experiment
Recombinant plasmid preparation: a re-
combinant plasmid containing the gene to be
miR-548-3p/DAG1 axis promotes the development of laryngeal carcinoma 209
Vol. 63(3): 206 - 217, 2022
tested DAG1/miR-548-3p was prepared. The re-
porter gene with DAG1/ miR-548-3p label and
the target gene were co-transfected for 48h.
Cell processing: the dual luciferase detection
kit was operated according to the protocol.
Fluorescence detection: a microplate reader
was used for fluorescence intensity detection,
and finally data analysis was performed.
Immunofluorescence analysis
In each group of cells, 1% BSA was ap-
plied for blocking at room temperature for
30 min to block non-specific epitopes. The
specific primary antibody was incubated ac-
cording to the recommended instructions
for the antibody and let it stand overnight in
a humidified box at 4°C. The slices were tak-
en out the next day and rewarmed at room
temperature for 30 min. The corresponding
immunofluorescence secondary antibody
was selected and then incubated at 37°C for
30 min in the dark. The nucleus was stained
with DAPI under dark conditions. Anti-fluo-
rescence quencher was added for mounting.
Finally, a fluorescence microscope was used
to observe and take pictures.
Cell cloning experiment
Each group of cells were taken in the
logarithmic growth phase, digested with
0.25% trypsin and pipetted into single cells,
and suspended in a culture medium of 10%
fetal bovine serum for later use. The cell sus-
pension was diluted in gradient multiples,
and each group of cells was inoculated into a
dish containing 10 mL of pre-warmed culture
medium at 37°C at a gradient density of 50,
100, and 200 cells per dish, and gently ro-
tated to make the cells uniformly dispersed.
Placed it in a cell incubator at 37°C, 5% CO2
and saturated humidity for 2-3 weeks. It was
frequently observed that when there were
visible clones in the petri dish, the culture
were stopped. The supernatant was discarded
and washed carefully with PBS twice. To fix
the cells 5 mL of 4% paraformaldehyde was
added for 15 minutes. The fixative solution
was then removed, an appropriate amount of
GIMSA was added and the dye solution was
applied to dye for 10-30 minutes, then slowly
washed off with running water.
Cell scratch test
After the cells of each group were di-
gested and counted, 8×105 cells were divided
into 35mm2 culture dishes. A marker was
used to draw a line on the bottom of the dish
as a mark, the culture medium was aspirat-
ed, and a 10 μL pipette tip was used to mark
the cells in the dish perpendicularly to the
marker. A rinse with PBS was used to remove
the marked cells, and serum-free culture me-
dium was added to continue culturing. Pic-
tures were taken at 24h, and the intersection
of the line drawn by the marker was selected
and the cell scratch as the observation point,
and then observed at a fixed point.
Cell migration test
Twenty-four hours before the experi-
ment, the cells of different groups were re-
placed with serum-free medium, and the cul-
ture was continued. Before inoculation, the
24-well plate and transwell chamber were
soaked with 1×PBS for 5 min to moisten the
chamber. The cells were digested, washed
with serum-free medium, resuspended in se-
rum-free medium, counted and diluted to ad-
just the cell density to 5×105/ mL. Then 0.2
mL cell suspension (5×104 cells) were inocu-
lated into the transwell chamber, and 0.7 mL
of RPMI-1640 medium containing 10% PBS
was added to the lower 24-well plate, 3 rep-
licate holes per group, and placed in a 37°C
incubator for 24 hours to finish the culture.
To each well of the above cells 1 mL of 4%
formaldehyde solution was added, and fixed
at room temperature for 10 min. The fixative
solution aspirated, washed once with 1x PBS.
To each well, 1mL 0.5% crystal violet solution
was added, washed with 1×PBS three times
after dyeing for 30 min. A cotton swab was
used to carefully wipe off the cells that have
not migrated in the transwell, and placed un-
der a 200× microscope for observation.
210 Chen et al.
Investigación ClÃnica 63(3): 2022
RT-PCR analysis
The AMC-HN-8 cell and primary human
laryngeal epithelial cell were treated with
TRIzol reagent to extract the total RNA in the
cells. Revert Aid TW first Strand cDNA Synthe-
sis Kit was used to synthesize the first chain
of DNA. QuantiNova SyBr Green PCR Kit was
used to perform PCR analysis. Reaction condi-
tions: pre-denaturation at 95°C for 1 minute,
denaturation at 95°C for 30 seconds, annealing
at 60°C for 30 seconds, and elongation at 72°C
for 30 seconds, running 40 cycles.
Western blot analysis
Cells were collected from each group,
and 200 μL of cell lysate was added to each
six-well plate. After sonication, the cells
were lysed on ice for 1 hour. The lysed cell
sample was centrifuged at 12,500 rpm for 15
minutes at 4°C. Then, the supernatant was
transfered in the centrifuge tube to a clean
centrifuge tube. β-actin protein quantifica-
tion kit was used to quantify protein concen-
tration. The measured protein samples were
stored at -80°C. In Western blot electropho-
resis, the protein loading concentration was
50 μg per well. After SDS-PAGE electropho-
resis, the membrane was transferred and
blocked. Proteins DAG1, LAMA2 and UTRN
primary antibody (1: 500, anti-human, Ther-
mo-Fisher, USA) were diluted to use concen-
tration. The samples were incubated over-
night on a shaker at 4°C. After washing with
PBS, the samples were incubated with the
secondary antibody (1: 1000, anti-human,
Thermo-Fisher, USA) for 30 minutes at room
temperature in the dark. Finally, a developer
was used for development and photography.
Statistical analysis
The experimental results are expressed
as means ± standard deviations. Quanti-
tative variables between two groups were
compared by the Student’s t test (normal
distribution) or Mann-Whitney U test (non-
normal distribution), and one-way or two-
way ANOVA was used for comparing multiple
groups. Pearson χ2 test or Fisher’s exact test
were used to compare qualitative variables.
Pearman’s analysis was conducted for corre-
lation analysis. Statistical analysis was per-
formed using the SPSS 22.0 software. The
figures were produced with Origin 2021 and
Adobe Illustrator 2020 software.
RESULTS
miR-548-3p had a significant regulatory
effect on gene DAG1
The results of this experiment are
shown in Fig. 1. It can be seen from the
figure that miR-548-3p was significantly in-
creased with the expression of DAG1-wild
type (WT) (p=0.0003) and was remarkably
decreased with the expression of DAG-mu-
tant type (MUT) (p=0.008), indicating that
miR-548-3p has a significant regulatory ef-
fect on the gene DAG1.
The results of immunofluorescence
analysis are shown in Fig. 2. The green flu-
orescence in the figure was emitted by the
DAG1 protein A. This experiment visually
indicated the difference in DAG1 protein ex-
pression between the groups, and the experi-
mental results were consistent with the re-
sults of the luciferase report experiment B.
Protein DAG1 induced malignant
transformation of laryngeal carcinoma
The cell clone formation experiment
is an important technical method used to
detect cell proliferation, invasiveness, and
sensitivity to killing factors. The clone for-
mation rate reflects the two important traits
of cell population dependence and prolifera-
tion ability. The results of the cell cloning ex-
periment are shown in Fig. 3. It can be seen
from the figure that when miR-548-3p was
overexpressed, the number of cell clones in-
creased significantly. When miR-548-3p was
interfered, cell cloning was also inhibited.
This indicated that the expression of miR-
548-3p was positively correlated with the
malignant degree of laryngeal cancer cells.
The results of the cell scratch test are
shown in Fig. 4. Cell scratch is a typical meth-
miR-548-3p/DAG1 axis promotes the development of laryngeal carcinoma 211
Vol. 63(3): 206 - 217, 2022
od used to detect the invasion ability of tumor
cells. After 24 hours, the narrower the scratch,
the stronger the cell invasion ability. From the
results, we can know that overexpression of
miR-548-3p significantly enhanced the migra-
tion ability of laryngeal cancer cells, while in-
terference with the expression of miR-548-3p
could inhibit the migration of laryngeal cancer
cells. The blank plasmid had no effect on the
expression of miR-548-3p, so the cell scratch
of Model group and Blank-plasmid group was
very serious.
The cell migration test is similar to the
cell scratch test, which detects the strength
of cell invasion. The more cells, the stronger
the tumor cell’s malignant metastasis and
invasion ability. The results of the cell migra-
tion test are shown in Fig. 5. Comparing the
results of Fig. 4 and Fig. 5, we can find that
the results of the cell migration test are con-
sistent with the results of the cell scratch
test. The three experiments of cell clon-
ing, scratching and migration all reached a
common conclusion: the overexpression of
miR-548-3p enhanced the malignant trans-
formation of laryngeal carcinoma, whereas
miR-548-3p knockdown lead to inhibition of
malignant transformation.
Fig. 1. The results of the dual luciferase detection experiment. miR-548-3p has a significant regulatory effect
on gene DAG1 (p<0.05).
Fig. 2. The results of immunofluorescence analysis of the expression characteristics of DAG1. The expression of
DAG1 protein in the Ov-miR-548-3p group and Sh-miR-548-3p group changed significantly (p< 0.05).