Silenced microRNA-135b-5p Inhibits Tongue Squamous Cell Carcinoma Proliferation and Apoptosis by Regulating ARID1A and the PI3K/AKT Axis

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0884-6812/21/4304-0151/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
A
RTICLES
An Official Periodical of The International Academy of Cytology and the Italian Group of Uropathology
AQCH
ANALYTICAL and
QUANTITATIVE
CYTOPATHOLOGY and
HISTOPATHOLOGY®
OBJECTIVE: Tongue squamous cell carcinoma (TSCC)
is a prominent type of oral cancer. Despite the numer-
ous research studies on SCC and microRNAs (miRs),
the relation between TSCC and miR-135b-5p is poorly
discussed. This experiment aims to find out the possible
effect of miR-135b-5p on TSCC with the network of its
downstream genes.
STUDY DESIGN: TSCC tissues and adjacent normal
tissues were harvested. Then, expression of miR-135b-5p
and AT-rich interactive domain‑containing protein 1A
gene (ARID1A) and the phosphatidyl inositol 3-kinase/
protein kinase B (PI3K/AKT) pathway was analyzed.
After the transfection of miR-135b-5p inhibitor and its
negative control into TSCC cells, functional assays were
employed to measure cell proliferation, apoptosis, and
cycle. Next, the target relation between miR-135b-5p
and ARID1A was confirmed. In addition, the fact that
miR-135b-5p promoted TSCC development via medi-
Analytical and Quantitative Cytopathology and Histopathology®
Silenced microRNA-135b-5p Inhibits Tongue
Squamous Cell Carcinoma Proliferation and
Apoptosis by Regulating ARID1A and the
PI3K/AKT Axis
Chao Guo, M.M., Wenjing Yi, M.M., Bin Sun, M.M., Xiaoyu Zha, M.M.,
Xiaoxue Chen, M.M., Zheng Zhou, M.M., and Changxue Li, M.M.
From the Stomatology Department, The First Affiliated Hospital of Shihezi University School of Medicine, Shihezi City, China; and
Shenzhen Stomatological Hospital of Southern Medical University (Pingshan), Shenzhen, China.
Chao Guo is Attending Physician, Stomatology Department, The First Affiliated Hospital of Shihezi University School of Medicine.
Wenjing Yi is Attending Physician, Shenzhen Stomatological Hospital of Southern Medical University (Pingshan).
Bin Sun is Professor, Stomatology Department, The First Affiliated Hospital of Shihezi University School of Medicine.
Xiaoyu Zha is Professor, Stomatology Department, The First Affiliated Hospital of Shihezi University School of Medicine.
Xiaoxue Chen is Attending Physician, Stomatology Department, The First Affiliated Hospital of Shihezi University School of Medicine.
Zheng Zhou is Professor, Stomatology Department, The First Affiliated Hospital of Shihezi University School of Medicine.
Changxue Li is Professor, Stomatology Department, The First Affiliated Hospital of Shihezi University School of Medicine.
Chao Guo and Wenjing Yi are co–first authors.
This study was supported by Scientific Research Project of Shihezi University (ZZZC201962A).
Address correspondence to: Zheng Zhou, M.M., Stomatology Department, The First Affiliated Hospital of Shihezi University School
of Medicine, No. 107 Beier Road, Shihezi City, 832000, China (15909935188@163.com), or to Changxue Li, M.M., Stomatology Depart-
ment, The First Affiliated Hospital of Shihezi University School of Medicine, No. 107 Beier Road, Shihezi City, 832000, China (lichang
xue100@163.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

ating ARID1A was demonstrated by functional rescue
experiment.
RESULTS: miR-135b-5p was upregulated in TSCC
tissues and cells, while ARID1A was suppressed (p<
0.05). Silenced miR-135b-5p discouraged TSCC cell
proliferation, improved apoptosis, induced cell cycle
arrest, and increased ARID1A expression while inac-
tivating the PI3K/AKT axis (p<0.05). Furthermore,
knockdown of ARID1A reversed the impacts on TSCC
cell proliferation and apoptosis exerted by silencing
miR-135b-5p.
CONCLUSION: This research supported that silenced
miR-135b-5p impeded TSCC proliferation and apoptosis
by promoting ARID1A and inactivating the PI3K/AKT
axis, which may provide some indications for TSCC
alleviation. (Anal Quant Cytopathol Histpathol 2021;
43:151–160)
Keywords: apoptosis; ARID1A; ARID1A protein,
human; carcinoma, squamous cell; cell line, tu-
mor; cell proliferation; drug resistance, neoplasm;
microRNA-135b-5p; microRNAs; PI3K/AKT path-
way; neoplasm metastasis; neoplastic stem cells;
proliferation; protein binding; tongue; tongue squa-
mous cell carcinoma.
Tongue squamous cell carcinoma (TSCC) refers to
a prevailing type of oral SCC which is character-
ized by frequent metastasis of lymph node.1 Alter-
ations of tumor microenvironment, immune cells,
and checkpoints destruct immune order, making
tumors more evasive to medical observation.2 As
an oral cancer with high aggressiveness, TSCC
takes responsibility for about 41% of oral cancers.3
A mounting range of population at or under the
age of 40, young adults and women are especial-
ly at risk of being susceptible to TSCC globally.4
The pathogenesis of TSCC is tightly associated
with tobacco abuse, excessive alcohol, and infec-
tion with human papillomavirus.5 Immune thera-
pies such as durvalumab, pembrolizumab, atezo
lizumab, and nivolumab currently appear to be
promising and efficient treatments for TSCC.2
Although there are plenty of technologies, TSCC
remains a fatal malignancy in humans.3 In this
circumstance, we undertook a microRNA (miR)-
based approach to understand the underlying
mechanism in TSCC development in order to de-
velop novel intervention strategies.
To learn more about the biomarkers during
TSCC progression would be conducive to early di-
agnosis, proper treatment, and constructive prog-
nosis.6 miRs are suggested to be reliable biomark-
ers in cancer mitigation with their ability of varying
from oncogenes to tumor suppressors in various
tumor progression.7 As major modulators in piv-
otal biological activities, miRs also significantly
participate in cutaneous SCC (CSCC), making them
indispensable biomarkers in other SCC malignan-
cies.1,8 Although there are multiple discussions
about the protective or detrimental impacts of
different miRs on TSCC, the exact role of miR-
135b-5p in this type of cancer remains to be elu
cidated. In fact, in esophageal SCC (ESCC), miR-
135b-5p is already ranked as a classifier whose col-
laboration with clinicopathological prognostic tar-
gets could motivate the accuracy to prognosis pre-
diction.9 miR-135b is highly expressed in several
CSCC cell lines.10 Furthermore, evidence has sug-
gested that miR-135b-5p overexpression predicts
a poor survival time in ESCC patients.11 As an
oncogene in a large number of other severe can-
cers, including pancreatic cancer, gastric cancer,
and breast cancer, miR-135b-5p gives a push to
tumor metastasis and growth by suppressing its
target genes.12-14 It is also worth mentioning that
miR-135b upregulates a downstream factor to en-
courage colony formation, angiogenesis, and sur-
vival of head and neck SCC (HNSCC).15 Accord-
ing to the evidence mentioned above, we make a
hypothesis that miR-135b-5p may play an import-
ant role in TSCC development through interacting
with other genes.
Materials and Methods
Ethics Statement
This study was approved and supervised by
the ethics committee of First Affiliated Hospital,
Shihezi University School of Medicine. All of the
subjects signed the informed consent. The proto-
col was also approved by the Institutional Animal
Care and Use Committee of First Affiliated Hospi-
tal, Shihezi University School of Medicine.
Clinical Samples
A total of 52 subjects with TSCC who were di-
agnosed and received tumor resection at First
Affili
ated Hospital, Shihezi University School of
Medicine, from February 2016 to February 2019
were recruited in this study, with their TSCC
tumor tissues and adjacent normal tissues har-
vested. Inclusion criteria were that no patient
should have received radiotherapy, chemotherapy,
or any other targeted therapies before surgery,
152 Analytical and Quantitative Cytopathology and Histopathology®
Guo et al

and all the tissues should have been removed into
−80°C liquid nitrogen as soon as the surgery was
performed.
Cell Cultivation and Transient Transfection
Following a previous research, normal human
oral keratinocytes and TSCC cell lines, Cal27, SCC4,
and UM1 (American Type Culture Collection
[ATCC], Rockville, Maryland) were all cultured in
Dulbecco’s Modified Eagle medium (Gibco, Grand
Island, New York), consisting of fetal bovine se-
rum (HyClone, Logan, Utah), penicillin, and strep-
tomycin.16
Inhibitor negative control (NC), miR-135b-5p
inhibitor, small interfere (si)-NC or si-AT-rich
interactive domain‑containing protein 1A gene
(ARID1A) (GenePharma Co., Ltd. Shanghai, China)
were respectively transfected into Cal27 cells by
a Lipofectamine 2000 (Invitrogen, Thermo Fisher
Scientific Inc., Waltham, Massachusetts, USA) at
50 nM at 5×106 cells. After 48 hours of transfec-
tion, the remaining operations were conducted.17
Cell Counting Kit-8 (CCK-8) Method
Cells (2.5×104 cells/mL) were seeded into 96-well
plates and cultivated in a 37°C incubator with 5%
CO2 at 0 hour, 24 hours, 48 hours, and 72 hours,
respectively. Cells were incubated with 10 μL
CCK-8 (Dojindo Laboratories, Kumamoto, Japan)
in each well for 2 hours. After that, the optical den-
sity at 450 nm was observed.
Flow Cytometry
Cells were harvested and then washed twice with
phosphate-buffered saline (PBS). The cell cycle
assessment went as follows: cells were treated in
4°C 70% ethyl alcohol for 2 hours, washed by PBS,
cultured with dye buffer, interacted with propidi-
um iodide, and supplemented with RNase A with
out exposure to the light for 30 minutes. Then the
cells were evaluated via fluorescence-activated cell
sorting (FACS) by flow cytometry (AceaBio, San
Diego, California, USA), and cell apoptosis was
analyzed in accordance with the instructions of
Annexin V-FITC apoptosis detection kit (Beyotime
Biotechnology Co. Ltd., Shanghai, China). FACS
was also applied in cell apoptosis analysis.
Reverse Transcription–Quantitative Polymerase
Chain Reaction (RT-qPCR)
Trizol (Takara Biotechnology Ltd., Dalian, China)
was utilized to extract the total RNA in tissues
and cells to determine the concentration and purity
of RNA. RNA was reversely transcribed to cDNA
using PrimeScript RT kits (Takara Biotechnolo-
gy). Fluorescence qPCR was conducted with the
ABI PRISM 7300 system (Applied Biosystems, Inc.,
Carlsbad, California, USA) and the SYBR Premix
Ex Taq kits (Takara Biotechnology). PCR condi-
tions were as follows: pre-denaturating at 94°C
for 4 minutes (30 cycles of denaturating at 94°C
for 30 seconds, annealing at 59°C, and extension
at 72°C for 1 minute), and then extension at 72°C
for 5 minutes. And 2-ΔΔCt method was applied for
expression calculation of genes with glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH) or U6
as the internal reference.18 The primers (Table I)
used were designed at Sangon Biotech Co., Ltd.
(Shanghai, China).
Western Blot Analysis
Cells were collected, and their proteins were
extracted using radio-immunoprecipitation assay.
Then, protein concentration was detected by bi-
cinchoninic acid protein assay kits (Beyotime).
Then, the total proteins were transferred onto the
polyvinylidene fluoride membranes (Amersham
Pharmacia Biotech, Piscataway, New Jersey, USA)
after being separated by 8% sodium dodecyl sul-
fate polyacrylamide gel electrophoresis. Subse-
quently, the membranes were sealed by 5% skim
milk powder for 2 hours and then incubated with
the primary antibodies: ARID1A (1:2000, ab264171),
GAPDH (1:1000, ab8245) (both from Abcam Inc.,
Cambridge, Massachusetts, USA), protein kinase
B (AKT) (1:1000, AF6261), p-AKT (1:1000, AF016)
(both from Affinity Biosciences, Cincinnati, Ohio,
USA), phosphatidyl inositol 3-kinase (PI3K) (1:1000,
CY5355), and p-PI3K (1:1000, CY6427) (both from
Volume 43, Number 4/August 2021 153
miR-135b-5p in TSCC Progression
Table I Primers Sequence of RT-qPCR
Gene Sequence (5’-3’)
miR-135b-5p F: 5’-GGTATGGCTTTTCATTCCT-3’
R: 5’-GCGAGCACAGAATTAATACGAC-3’
U6 F: 5’-GCTTCGGCAGCACATATACTAAAAT-3’
R: 5’-CGCTTCACGAATTTGCGTGTCAT-3’
ARID1A F: 5’-ACTCCATGGGGAGCTAGGT-3’
R: 5’-CACCCATGGGGTTTATGCCT-3’
GAPDH F: 5’-ACCCAGAAGACTGTGGATGG-3’
R: 5’-TCTAGACGGCAGGTCAGGTC-3’
ARID1A = AT-rich interactive domain‑containing protein 1A gene,
F = forward, GAPDH = glyceraldehyde 3-phosphate dehydrogenase,
miR = microRNA, R = reverse, RT-qPCR = reverse transcription–
quantitative polymerase chain reaction.

Abways, Shanghai, China) at 4°C overnight. Next,
the membranes were washed with tris-buffered
saline–tween buffer 3 times and cultured with
horse-radish peroxidase labeled goat anti-rabbit
immunoglobulin G antibody (1:2000; ab6721) (Ab
cam) for 1 hour. In addition, proteins were detect-
ed by strengthened electrochemiluminescence.
Dual Luciferase Reporter Gene Assay
Bioinformatics software was performed to deter-
mine the binding site between miR-135b-5p and
ARID1A 3’ untranslated region (3’UTR), on which
ARID1A wild-type (WT) and ARID1A mutant-type
(MUT) were constructed. 293T cells (ATCC) at
logarithmic growth phase were seeded into 96-
well plates. When the cell density approached 70%,
ARID1A-WT or ARID1A-MUT was mixed with
mimic NC and miR-135b-5p mimic (GenePharma)
and then co-transfected into Cal27 cells. After 48
hours transfection, cells were collected and lysed.
Luciferase activity was observed using luciferase
assay kits (BioVision, San Francisco, California,
USA) and a GloMax 20/20 Luminometer fluo-
rescence detector (Promega, Madison, Wisconsin,
USA). The experiment was repeated 3 times.
Statistical Analysis
SPSS 21.0 (IBM Corp., Released 2012, IBM SPSS
Statistics for Windows, Version 21.0. Armonk, New
York, USA) was employed to analyze data. The
data were shown in mean±standard deviation.
The t test was used for analyzing comparisons be-
tween two groups, one-way analysis of variance
(ANOVA) for comparing different groups, and
Tukey’s multiple comparisons test for pairwise
comparisons after ANOVA. The p value was at-
tained using a two-tailed test and p<0.05 referred
to significant difference.
Results
miR-135b-5p Is Highly Expressed in TSCC Tumor
Tissues and Cells
As the results of RT-qPCR represented, miR-135b-
5p expression was overtly higher in tissues of the
TSCC patients than in those of the adjacent nor-
mal tissues (p<0.05) (Figure 1A). Besides, com-
pared to that of the normal human oral keratino-
cytes, miR-135b-5p was strongly expressed in the
TSCC cell lines, Cal27, SCC4, and UM1, among
which the Cal27 cells showed the mostly differen-
tial expression (all p<0.05) (Figure 1B). Thus, Cal27
was selected in the following steps.
Silenced miR-135b-5p Sabotages TSCC Cell
Proliferation and Improves Apoptosis
miR-135b-5p was silenced in Cal27 cells, with the
results of RT-qPCR suggesting a successful trans-
fection (p<0.05) (Figure 2A). As it was observed
Guo et al
Figure 1 miR-135b-5p is highly expressed in TSCC tumor tissues and cells. (A–B) miR-135b-5p expression in TSCC tissues (A) and
TSCC cell lines (B) detected by RT-qPCR. Data are expressed as mean±standard deviation. The t test was used for pairwise comparison,
and one-way ANOVA was used to determine statistical significance. Tukey’s multiple comparisons test was applied for post hoc test.
**p<0.01. Repetition=3.

by CCK-8 method (Figure 2B) and flow cytometry
(Figure 2C–D), silenced miR-135b-5p could sup-
press TSCC cell proliferation, promote apoptosis,
and induce cell cycle arrest (all p<0.05).
miR-135b-5p Targets ARID1A
To further explore the mechanism of miR-135b-5p
in TSCC, a bioinformatics site (https:/
/cm.jefferson.
edu/rna22/Precomputed/) was employed to re-
veal that there were many gene-shared binding
sites with miR-135b-5p, such as ARID1A, PROX1,
FAM76A, SCMH1, and RUNX3. According to a
literature, ARID1A degradation led to a rapid de-
velopment of TSCC.19 Moreover, mice with con-
Figure 2 Silenced miR-135b-5p sabotages TSCC cell proliferation and improves apoptosis. (A) miR-135b-5p expression in Cal27 cells
assessed using RT-qPCR. (B) Cal27 cell proliferation measured by CCK-8 method. (C–D) Cal27 apoptosis (C) and cell cycle (D) evaluated
through flow cytometry. Data are expressed as mean±standard deviation. The t test was used for pairwise comparison. **p<0.01.
Repetition=3.

ditional ARID1A knockout were susceptible to
TSCC or esophageal SCC.20 Therefore, ARID1A
was applied in dual luciferase reporter gene as-
say, from which we learned that as compared with
that transfected with mimic NC luciferase activi-
ty of ARID1A 3’UTR WT vector transfected with
miR-135b-5p mimic was significantly decreased,
while that of ARID1A 3’UTR MUT vector exhib-
ited no difference (p<0.05) (Figure 3A). On the
other hand, miR-135b-5p underexpression en-
hanced ARID1A expression in TSCC cells (p<0.05)
(Figure 3B). Then, as it was unveiled by RT-qPCR
and western blot analysis, ARID1A expression
was greatly quenched in TSCC tumor tissues
Guo et al
Figure 3 miR-135b-5p targets ARID1A in TSCC. (A) Binding site between miR-135b-5p and ARID1A 3’UTR as well as the luciferase
activity of ARID1A-WT and ARID1A-MUT co-transfected with mimic NC or miR-135b-5p mimic into 293T cells. (B) miR-135b-5p
expression in Cal27 cells detected by RT-qPCR and western blot analysis after silencing miR-135b-5p. (C–D) ARID1A expression in TSCC
tissues (C) and TSCC cell lines (D) evaluated by RT-qPCR and western blot analysis. Data are expressed as mean±standard deviation.
The t test was used for pairwise comparison, and one-way ANOVA was used to determine statistical significance. Tukey’s multiple
comparisons test was applied for post hoc test. *p<0.05, **p<0.01. Repetition=3.

and cells (all p<0.05) (Figure 3C–D). Simply put,
miR-135b-5p targeted ARID1A in TSCC.
ARID1A Knockdown Reverses the Inhibited TSCC
Cell Proliferation and Promoted Apoptosis Induced
by Silencing miR-135b-5p
miR-135b-5p and ARID1A were degraded in Cal27
cells, and RT-qPCR and western blot analysis
found that, compared with the miR-135b-5p in-
hibitor+si-NC group, the miR-135b-5p inhibitor+
si-ARID1A had declined ARID1A expression (p<
0.05) (Figure 4A).
According to CCK-8 method (Figure 4B) and
flow cytometry (Figure 4C), the miR-135b-5p in-
hibitor+si-ARID1A had elevated TSCC cell prolif-
eration and decreased apoptosis as compared with
the miR-135b-5p inhibitor+si-NC group (p<0.05).
Silenced miR-135b-5p Inactivates the PI3K/AKT Axis
by Regulating ARID1A
A previous research supported that the PI3K/AKT
axis activation encouraged gastric carcinoma cell
proliferation and invasion.21 It was also discov-
ered that the PI3K/AKT axis would by upregu-
lated by ARID1A knockout in pancreatic cancer.22
Western blot analysis was performed to assess the
PI3K/AKT axis activation of TSCC cells in each
group, and it found that silencing miR-135b-5p
contributed to the downregulation of the PI3K/
AKT axis; while ARID1A underexpression result
ed in activation of the PI3K/AKT axis (all p<0.05)
(Figure 5).
Discussion
TSCC represented one of the indomitable oral
cancers despite the clinical progress made during
the last few decades.23 It was discovered that miRs
with aberrant expression worked as tumor pro
motors or suppressors in oral tumorigenesis by
actively participating in cell activities such as dif
ferentiation, proliferation, survival, and death, in-
dicating their valuable role in oral carcinoma di-
agnosis and prognosis.24 miR-135b-5p experienced
an evident activation in individuals with ESCC.11
In the current study we discussed the mechanism
of miR-135b-5p and ARID1A in biological pro-
cesses of TSCC with the involvement of the PI3K/
AKT pathway. Consequently, we found silenced
miR-135b-5p blocked TSCC cell growth by nega-
tively modulating ARID1A and inactivating the
PI3K/AKT axis.
The first and foremost finding in this experi-
Figure 4 ARID1A knockdown reverses the inhibited TSCC cell proliferation and promoted apoptosis induced by silencing miR-135b-5p.
(A) ARID1A expression in Cal27 cells measured using RT-qPCR and western blot analysis. (B) Cal27 cell proliferation determined by
CCK-8 method. (C) Cal27 cell apoptosis evaluated by flow cytometry. Data are expressed as mean±standard deviation. The t test was
used for pairwise comparison. **p<0.01. Repetition=3.

ment was that miR-135b-5p expression was ele-
vated in TSCC. Similarly, miR-135b-5p expressed
higher in tissues from patients with oral cancer
than those from the healthy volunteers.25 Highly
expressed miR-135b-5p in HNSCC motivated cell
colony formation and biological behaviors.15 On
the other hand, silenced miR-135b-5p quenched
TSCC proliferation, improved apoptosis, and in-
duced cell cycle arrest. In ESCC, miR-135b-5p ap-
peared to be related to the patients’ survival rate,
lymph node metastasis, and tumor differentia-
tion.11 miR-135b knockdown resulted in the slow-
down in cellular invasion and activity in CSCC.10
A previous literature documented that in SCCs,
the fact that apoptosis was strengthened was
beneficial to the repression of potential cancer de-
velopment and enhancement of tumor sensitiza-
tion to radiotherapy and chemotherapy.26 Besides,
downregulation of miR-135b encouraged the
growth of apoptosis factors,27 which indicated the
negative relation between miR-135b and apoptosis.
Importantly, we noticed that miR-135b-5p target-
ed ARID1A. ARID1A referred to a subunit gene
which was likely to mutate in different kinds of
cancers whose pathogenesis could be easily trig-
gered by the absence of ARID1A as this gene was
potent in maintaining enhancer activity and nor-
mal gene expression.28 ARID1A was reversely mod-
ulated by another oncogene in HNSCC so as to
amplify the stemness and carcinogenicity and con-
tribute to a poor survival rate of HNSCC.29 Like-
wise, as a target gene of a tumor promoter gene
in gastric cancer, ARID1A knockout could lead
to cancer gene invasion and growth.30 Besides,
ARID1A was able to enhance apoptosis to ensure
pancreatic cancer would be more sensitive to the
radiotherapy.22 That is to say, miR-135b-5p inhibi
tion was largely associated with different oral
malignancies with the involvement with ARID1A.
ARID1A knockdown reversed the sabotaged
TSCC cell proliferation and promoted apoptosis
caused by silencing miR-135b-5p, as revealed by
functional assays. As a famous and popular tumor
suppressor, ARID1A sabotaged cancer stem cells,
which was identified as the major cause of tumor
progression, and reduced the chemoresistance in
SCCs, thus generating a favorable prognosis.20 Put
in another way, stabilized expression of ARID1A
curbed SCC development and chemoresistance.19
It was reported that ARID1A loss brought about
growth of colorectal cancer via stimulating apop
tosis.31 In addition, silenced miR-135b-5p inacti-
vated the PI3K/AKT axis by negatively regulating
ARID1A. Data have supported that a lot of can-
cers, ranging from ovarian cancer to lung cancer,
witnessed AKT upregulation, which boosted tu-
Guo et al
Figure 5 Silenced miR-135b-5p inactivates the PI3K/AKT axis by regulating ARID1A. Protein levels of AKT, p-AKT, PI3K, and p-PI3K
of the PI3K/AKT axis in Cal27 cells were measured by western blot analysis. Data are expressed as mean±standard deviation. One-way
ANOVA was used to determine statistical significance. Tukey’s multiple comparisons test was applied for post hoc test. **p<0.01.
Repetition=3.

mor cell activity and mobility.32 According to a
recent study, the PI3K/AKT axis inhibitor con-
tributed to a better therapy for multiple cancers,
hinting at the detrimental role that the PI3K/
AKT axis played in human tumors.33 Importantly,
the PI3K/AKT axis and dysregulation of cell cycle
were both important players in ESCC progres-
sion.34 According to Jia et al, miR-135b directly
mediated the PI3K/AKT axis, an axis related to
tumor, to enhance colorectal cancer adhesion,
migration, and angiogenesis.35 Moreover, in pan-
creatic cancer with ARID1A knockout, the PI3K/
AKT axis was activated to reduce the efficiency of
regular radiotherapy.22
The main limitation of this study was that me
tastases and the prognosis of the 52 TSCC patients
were not analyzed. All in all, our study implied
that suppression of miR-135b-5p could suppress
TSCC cell growth by targeting ARID1A and inac-
tivating the PI3K/AKT pathway. These results are
promising in the prospect of promoting future al-
leviation of TSCC, thereby providing references for
optimizing individual treatment for patients with
TSCC. Though our findings offer therapeutic impli-
cation to TSCC therapy, the experimental results
and effective application into clinical practice still
need in-depth validation and exploration.
Ethical Approval
This study is approved by Ethics Committee of the
First Affiliated Hospital, Shihezi University School
of Medicine (2019-11-15,2019-089-01).
Consent for Publication
Informed consent was obtained from all individual
participants included in the study.
Availability of Data and Material
The datasets used or analyzed during the current
study are available from the corresponding author
on reasonable request.
Author Contributions
Guarantor of integrity of the entire study: Chao
Guo
Study concepts: Changxue Li
Study design: Chao Guo, Wenjing Yi
Definition of intellectual content: Chao Guo, Wen-
jing Yi, Changxue Li
Literature research: Wenjing Yi, Bin Sun, Xiaoyu
Zha
Clinical studies: Wenjing Yi, Xiaoxue Chen
Experimental studies: Chao Guo, Wenjing Yi, Bin
Sun, Xiaoyu Zha
Data acquisition: Xiaoxue Chen, Wenjing Yi
Data analysis: Xiaoyu Zha, Xiaoxue Chen
Statistical analysis: Bin Sun
Manuscript preparation: Chao Guo
Manuscript editing: Chao Guo, Zheng Zhou
Manuscript review: Zheng Zhou
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Silenced microRNA-135b-5p Inhibits Tongue Squamous Cell Carcinoma Proliferation and Apoptosis by Regulating ARID1A and the PI3K/AKT Axis

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Silenced microRNA-135b-5p Inhibits Tongue Squamous Cell Carcinoma Proliferation and Apoptosis by Regulating ARID1A and the PI3K/AKT Axis