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 línea celular AMC-HN-8 y la línea

celular epitelial laríngea 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 laríngeo. 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 laríngeas 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 características de regulación de

hsa-miR-548-3p en la glicoproteína 1 asociada a distrofina (DAG1). Se utilizó

inmunofluorescencia para analizar las características de expresión relativa de

la proteína DAG1. El experimento de clonación celular se utilizó para analizar

las características 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 proteína 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 proteína 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 Clínica 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 Clínica 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).

212 Chen et al.

Investigación Clínica 63(3): 2022

Plasmids had clear effects on the

expression of miR-548-3p

The results of RT-PCR analysis are

shown in Fig. 6. miR-548-3p is expressed

in normal cells, so there was a certain

amount of miR-548-3p in the NC group.

The expression of miR-548-3p in the Mod-

el group increased significantly, and the

expression of miR-548-3p increased and

decreased greatly through the influence of

overexpression plasmid and interference

plasmid. This was positively correlated

with DAG1 expression and the malignant

degree of laryngeal cancer cells, confirm-

ing the correlation of the three.

Expression of the protein to be analyzed

showed the same trend

The results of Western blot analysis are

shown in Fig. 7. The protein DAG1 is involved

in the assembly of laminin and basement

membrane, muscle membrane stability and

cell survival. Its overexpression will improve

the viability of cancer cells. The protein

LAMA2 is the main component of the base-

ment membrane and interacts closely with

other extracellular matrix components. Its

overexpression will increase the migration

and invasion ability of cancer cells. UTRN is

also a typical oncogene expressed protein.

Its overexpression will increase the activity

of cancer cells. Compared with model group,

the expression of DAG1, LAMA2 and UTRN

were significantly increased through over-

expression of miR-548-3p plasmid, thus the

expression of these proteins was obviously

decreased with the treatment of interfer-

ence plasmid. The expression of the above-

mentioned proteins is positively correlated

with malignant degree of laryngeal cancer

cells, and the results of this experiment also

show this.

Fig. 3. The results of cell migration experiment. There was no cloning phenomenon in the NC group. The

number of cell clones in the Ov-miR-548-3p group and Sh-miR-548-3p group was significantly different

from that in the Model group (p<0.05).

miR-548-3p/DAG1 axis promotes the development of laryngeal carcinoma 213

Vol. 63(3): 206 - 217, 2022

Fig. 4. The results of cell

scratch test. The data

of NC and Model group

are consistent with the

normal value.

214 Chen et al.

Investigación Clínica 63(3): 2022

Fig. 5. The results of cell migration test. The data of NC and Model group are consistent with the normal

value. The results of this experiment are consistent with the results of the cell scratch test.

Fig. 6. The results of RT-PCR analysis. The results of this experiment are consistent with the results of the

dual luciferase detection experiment and immunofluorescence analysis.

Fig. 7. The results of Western blot analysis. The data of NC and Model group are all consistent with the nor-

mal value. All the results of Ov-miR-548-3p group and Sh-miR-548-3p group were statistically diffe-

rent from the results of Model group (p<0.05). (A) Original gel electrophoresis image. (B) The ratio

of protein DAG1, LAMA2 and UTRN expression compared to NC group.

miR-548-3p/DAG1 axis promotes the development of laryngeal carcinoma 215

Vol. 63(3): 206 - 217, 2022

DISCUSSION

The essence of malignant tumors is

abnormal cell proliferation, which locally

invades surrounding normal tissues, and

even metastasizes to other parts of the body

through the circulatory system. From the

perspective of molecular pathology, the root

cause of malignant tumors is the accumu-

lation of DNA mutations. The accumulation

of mutations leads to the large expression

of proteins that promote cell growth, mak-

ing cell cycle control abnormal 12-14. Primary

laryngeal carcinoma is a kind of squamous

cell carcinoma, and its malignancy ranks in

the forefront of all cancers 15-16. This project

aims to promote the development of diag-

nosis and treatment of laryngeal cancer by

identifying the underlying molecular mecha-

nism of laryngeal cancer exacerbation.

miR-548-3p has been shown to be

widely involved in the regulation of cancer

genes. Luo et al pointed out that decrease

of miR-548c-3p could upregulate the expres-

sion of ITGAV and contribute to tumor pro-

gression 9. Observation of samples from oral

squamous cell carcinoma patients found

that miR-548-3p was increased in the OSCC

group compared with the control group 17.

This work has indicated that miR-548-3p in-

directly participates in the various processes

of malignant transformation of laryngeal

cancer by regulating the expression of DAG1.

In skeletal muscle, the DAG1 complex

works as a transmembrane linkage between

the extracellular matrix and the cytoskel-

eton. α-DAG1 is extracellular and binds to

merosin α-2 laminin in the basement mem-

brane, while β-DAG1 is a transmembrane

protein and binds to dystrophin, which is a

large rod-like cytoskeletal protein, absent in

Duchenne muscular dystrophy patients. Dys-

trophin binds to intracellular actin cables.

In this way, the DAG1 complex, which links

the extracellular matrix to the intracellular

actin cables, is thought to provide structural

integrity in muscle tissues 18,19. The DAG1

complex is also known to serve as an agrin

receptor in muscle, where it may regulate

agrin-induced acetylcholine receptor clus-

tering at the neuromuscular junction. There

is also evidence which suggests the function

of DAG1 as a part of the signal transduction

pathway because it is shown that Grb2, a

mediator of the Ras-related signal pathway,

can interact with the cytoplasmic domain of

DAG1 20. DAG1 also plays an important role

in the development and progression of can-

cer, and the regulation of DAG1 by miR-548-

3p is particularly important in the progres-

sion of laryngeal cancer.

α-DAG1 is extracellular peripheral gly-

coprotein that acts as a receptor for extracel-

lular matrix proteins containing laminin-G

domains and receptor for laminin-2 (LAMA2)

and agrin in peripheral nerve Schwann cells.

It also acts as a receptor for laminin LAMA5

21,22. β-DAG1 is transmembrane protein that

plays important roles in connecting the ex-

tracellular matrix to the cytoskeleton, which

acts as a cell adhesion receptor in both mus-

cle and non-muscle tissues. It is a receptor

for both DMD and UTRN and, through these

interactions, scaffolds axin to the cytoskel-

eton. β-DAG1 also functions in cell adhesion-

mediated signaling and implicated in cell

polarity 23,24. The direct action of DAG1 with

the proteins LAMA2 and UTRN enhances the

proliferation, migration and invasion ability

of laryngeal cancer cells, thereby increasing

the malignant degree of laryngeal cancer in

all directions.

In summary, this experiment con-

trolled the expression of miR-548-3p by add-

ing different plasmids, and then controlled

the expression of protein DAG1, and finally

achieved the effect of controlling the malig-

nant degree of laryngeal cancer. Cell cloning,

scratching and migration experiments were

used to determine the viability of laryngeal

cancer cells. At the same time, by measuring

the expression of cancer-related proteins,

the regulatory effect of protein DAG1 on la-

ryngeal cancer was further investigated. We

reported the novel function of miR-548-3p

in regulating laryngeal carcinoma cell mi-

216 Chen et al.

Investigación Clínica 63(3): 2022

gration and invasion though modulating the

expression of DAG1. This provides a poten-

tial strategy for the diagnosis and treatment

of laryngeal cancer. Nevertheless, more in-

depth research is still needed to sterenght

the conclusions of this experiment and la-

ryngeal cancer samples are also needed to

truly promote the conclusions of this work

for the future diagnosis and treatment of

lung cancer.

ACKNOWLEDGMENTS

We would like to acknowledge everyone

for their helpful contributions in this paper.

Funding

Key project of the First Affiliated Hos-

pital of Chengdu Medical College (No. CY-

FY2018YB05).

Ethics approval and consent to participate

The research protocol has been re-

viewed and approved by the Ethical Commit-

tee and Institutional Review Board of The

First Affiliated Hospital of Chengdu Medical

College and written informed consent was

obtained from all patients.

Competing interests

The authors declared that they have no

competing interests.

Author’s ORCID numbers

• Jia Chen: 0000-0002-9678-2523

• Yu Lin Zhou: 0000-0003-0034-093X

• Ke Wen: 0000-0002-0354-9985

• Shi Huang: 0000-0001-9811-4265

• Nan Hou: 0000-0001-9875-107X

• Ling Wang: 0000-0001-6367-2988

• Yi Wang: 0000-0001-5350-207X

Authors’ contributions

Each author has made an important

scientific contribution to the study and has

assisted with the drafting or revising of the

manuscript.

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