Invest Clin 64(2): 184 - 195, 2023 https://doi.org/10.54817/IC.v64n2a05
Corresponding author: Xingkui Tang, Department of General Surgery, Guangzhou Panyu Center Hospital,Guangzhou,
Guangdong Province, China; Tel: +86-020-87331714; Email: tangxingkui@126.com
Expression of MiR-20a-5p and its target
gene in colon cancer and its effect on the
proliferation and apoptosis of colon cancer
cells.
Xingkui Tang1,Yukun Lin2, Yaqiong Wang2, Jialin He1, Xijun Luo1, Jun Jie Liang1
and Xianjun Zhu1
1 Department of General Surgery, Guangzhou Panyu Center Hospital, Guangzhou,
Guangdong Province, China.
2 Department of Electron Microscopy, Zhongshan School of Medicine, Sun Yat-sen
University, Guangzhou, Guangdong Province, China.
Keywords: MicroRNA-20a-5p; similar gene of breast cancer metastasis suppressor
gene 1; colon cancer; proliferation; apoptosis.
Abstract. We investigated the expression of micro ribonucleic acid (miR)-
20a-5p and its target gene, breast cancer metastasis suppressor 1 like (BRMS1L),
in colon cancer tissues and their effects on the proliferation and apoptosis of colon
cancer cells. The dual luciferase assay was used to detect the targeted regulation
of miR-20a-5p on BRMS1L. The expression levels of miR-20a-5p and BRMS1L in
colon cancer tissues and cells were detected by quantitative real-time polymerase
chain reaction (qRT-PCR). MiR-20a-5p mimic and mimic negative control (NC)
were transfected into the colon cancer cell line SW480 by the liposome transient
transfection method. The MTT assay, monoclonal formation of cancer cells, and
flow cytometry were used to detect cell proliferation and apoptosis. The expres-
sion level of miR-20a-5p in colon cancer tissues was significantly higher than that
in adjacent tissues, and the expression level of BRMS1L was significantly lower
than that in adjacent tissues. The expression level of miR-20a-5p was significantly
correlated with tumor-node-metastasis (TNM) stage, lymph node metastasis, in-
vasion depth, and differentiation degree. The higher the expression level of miR-
20a-5p, the more advanced the TNM stage and invasion depth, and the easier
it is for lymph nodes to metastasize (p<0.05). Compared with the control and
the miR-NC groups, the miR-20a-5p group’s cell proliferation ability, expression
of CyclinD1 and B-cell lymphoma-2 (Bcl-2) were significantly increased, while
apoptosis ability and caspase-3 protein expression were significantly decreased
(p<0.05). The expression of miR-20a-5p in colon cancer tissues and cells in-
creased. Overexpression of miR-20a-5p could promote the proliferation of colon
cancer cells and inhibit their apoptosis.
MiR-20a-5p and colon cancer 185
Vol. 64(2): 184 - 195, 2023
Expresión del MiR-20a-5p y su gen objetivo en cáncer de colon
y sus efectos sobre la proliferación y apoptosis de células
cancerosas de colon
Invest Clin 2023; 64 (2): 184 – 195
Palabras clave: MicroARN-20a-5p; gen similar al gen supresor de metástasis del cáncer
de mama 1; cáncer de colon; proliferación; apoptosis.
Resumen. Investigamos la expresión del ácido microribonucleico (miR)-
20a-5p y su objetivo, el gen similar al supresor de metástasis del cáncer de mama
1 (BRMS1L), en tejidos de cáncer de colon y sus efectos sobre la proliferación y la
apoptosis de las células de cáncer de colon. Se utilizó el ensayo de luciferasa dual
para detectar la regulación específica del miR-20a-5p en BRMS1L. Los niveles
de expresión de miR-20a-5p y BRMS1L en tejidos y células de cáncer de colon
se detectaron mediante la reacción en cadena de la polimerasa cuantitativa en
tiempo real (qRT-PCR). El MiR-20a-5p mímico y el control mímico negativo
(NC) se transfectaron a la línea celular de cáncer de colon SW480 mediante el
método de transfección transitoria de liposomas. Se utilizaron el ensayo MTT,
la formación monoclonal de células cancerosas y la citometría de flujo para de-
tectar la proliferación celular y la apoptosis. El nivel de expresión de miR-20a-
5p en tejidos de cáncer de colon fue significativamente mayor que en los tejidos
adyacentes, y el nivel de expresión de BRMS1L fue significativamente menor
que en tejidos adyacentes. El nivel de expresión de miR-20a-5p se correlacionó
significativamente con el estadio tumor-ganglio-metástasis (TNM), la metásta-
sis al ganglio linfático, la profundidad de la invasión y el grado de diferencia-
ción. Cuanto mayor era el nivel de expresión de miR-20a-5p, más avanzada eran
la etapa TNM y la profundidad de la invasión, y más fácil era que los ganglios
linfáticos hicieran metástasis (p<0,05). En comparación con los grupos con-
trol y miR-NC, la capacidad de proliferación celular del grupo miR-20a-5p, la
expresión de CyclinD1 y el linfoma-2 de células B (Bcl-2) aumentaron signifi-
cativamente; mientras que la capacidad de apoptosis y la expresión de la pro-
teína caspasa-3 disminuyeron significativamente (p<0,05). La expresión del
miR-20a-5p en células y tejidos de cáncer de colon aumentó. La sobreexpresión
del miR-20a-5p podría promover la proliferación de células de cáncer de colon
e inhibir su apoptosis.
Received: 11-11-2022 Accepted: 04-12-2022
INTRODUCTION
Colon cancer (CC) is a common ma-
lignant tumor of the digestive tract, and
its morbidity and mortality are increasing
year by year. Every year, there are 1.2 mil-
lion new colon cancer patients in the world,
with 600,000 deaths 1. The pathogenesis of
colon cancer is complex, and the activation
of carcinogenic pathways and the inhibi-
tion of defense mechanisms are important
causes of the tumor, which not only affect
the function of the digestive system but also
involve the liver, lung, and other organs in
186 Tang et al.
Investigación Clínica 64(2): 2023
distant metastasis 2. When colorectal can-
cer is diagnosed at an early stage, it can be
cured by surgery, but when it is diagnosed
at an advanced stage, the curative effect of
chemotherapy drugs is very limited 3. There-
fore, finding new therapeutic methods and
targets is very important. Micro ribonucle-
ic acid (MiRNA) is an endogenous single-
stranded non-coding RNA with a length of
20-25 nt, which combines with the messen-
ger ribonucleic acid (mRNA) of the target
gene through base complementary pairing,
resulting in its degradation or translation
stop, thus down-regulating the expression of
the target gene 4. MiRNA plays the role of on-
cogene or tumor suppressor gene in tumors
and can regulate biological processes such
as proliferation, differentiation, invasion,
and apoptosis of tumor cells 5. It has been
reported that miR-20a-5p regulates tumor
growth, cell differentiation, and apoptosis
of many cancers. Luo Sheng et al. 6 pointed
out that up-regulation of the expression of
miR-20a-5p could promote the proliferation
of pancreatic cancer cells and inhibit their
apoptosis. Liu Xiao dong et al. 7 found that
HAGLROS inhibited the expression of miR-
20a-5p, thus inhibiting the proliferation of
cardiomyocytes induced by high glucose and
promoting their apoptosis. MiR-20a-5p was
sheared from miR-20a. At present, miR-20a
is widely studied in the serum of colon cancer
patients, but the expression of miR-20a-5p in
colon cancer tissues and its effect on colon
cancer cells are rarely reported. Therefore,
this study intended to explore the expres-
sion level of miR-20a-5p and its target gene
in colon cancer tissues and cells, as well as
its effects on proliferation and apoptosis of
colon cancer cells, so as to provide theoreti-
cal basis for early diagnosis and treatment of
colon cancer.
MATERIALS AND METHODS
Objectives
This study belongs to a single-center
study, and the research objects are collected
in a continuous way. Inclusive criteria: All
patients were diagnosed as colon cancer by
histopathology, and had not received radio-
therapy, chemotherapy or other anti-tumor
adjuvant therapy before operation. Exclu-
sion criteria: Other digestive diseases (colon
polyps, chronic enteritis, etc.) and other sys-
temic malignant tumors (lung cancer, breast
cancer, etc.). From January 2021 to October
2022, 87 cases of colon cancer patients in
our hospital were collected. The specimens
of colon cancer tissues and adjacent tissues
>5cm from the tumor edge were collected.
The collected specimens were kept in liquid
nitrogen. General clinical data include age,
sex, smoking history, drinking history, tumor
diameter, tumor location, tumor-node-me-
tastasis (TNM) stage, lymph node metasta-
sis, infiltration depth, differentiation degree
and other information were collected.
Cells
Human normal colon epithelial cell
lines FHC, colon cancer cell lines SW480,
HT-29 and HCT-116 (Beina Biotechnology
Co., Ltd.) (Wuhan, China).
Reagents and Equipment
MiR-20a-5p mimic (MiR-20a-5p mimic)
and mimic negative control (NC) (Shang-
hai Jima Pharmaceutical Technology Co.,
Ltd.); MiR-20a-5p and BRMS1L primers
(Shanghai Shenggong Bioengineering Co.,
Ltd.); Monoclonal antibodies against Cy-
clinD1, B-cell lymphoma-2(Bcl-2), cas-
pase-3 and glyceraldehyde-3-phosphate
dehydrogenase(GAPDH) (Cell Signal Tech-
nology Company, USA); Trizol ribonucleic
acid(RNA) extraction kit, Lipofectamine
TM3000 liposome and reverse transcrip-
tion kit (Dalian Bao Biotechnology Co.,
Ltd.); Bicinchoninic acid(BCA) protein as-
say kit (Shanghai Biyuntian Biotechnology
Co., Ltd.); Fetal serum, Dulbecco’s modifi-
cation of Eagle’s medium (DMEM) medium
and trypsin (GIBCO Company, USA); Net
Airtech clean bench (Thermo Fisher Com-
pany, USA); DYCZ 425D double vertical elec-
MiR-20a-5p and colon cancer 187
Vol. 64(2): 184 - 195, 2023
trophoresis apparatus (Beijing Liuyi Instru-
ment Factory); Carbon dioxide incubator
(Thermo Company, USA); SpectraMax i D5
microplate reader (Thermo, USA); IXplore
Standard inverted microscope (Olympus, Ja-
pan).
Cell culture
Colon cancer cell lines (SW480, HT-29,
HCT-116) and human normal colon epitheli-
al cell line FHC were resuscitated, then add-
ed into DMEM medium containing 10% fetal
bovine serum, and cultured in an incubator
at 37℃ and 5% CO2. When the cell fusion
rate reached 80%, the cells were digested
and passaged with trypsin.
Grouping processing
SW480 cells were divided into three
groups: control group: untransfected SW480
cells; MiR-NC group: SW480 cells transfected
with mimic NC; miR-20a-5p group: SW480
cells transfected with miR-20a-5p mimic.
10 mL SW480 cell fluid were inoculat-
ed into 6-well plate, 100 pmol of miR-20a-5p
mimic and mimic NC were added into DMEM
medium, and 4μL Lipofectamine 3000 were
added to incubate for 30 min. The cells were
washed with the mixture, cultured in DMEM
medium for 24 h, and the transfection effi-
ciency was detected by flow cytometry.
Bioinformatics prediction and double
luciferase experiment
Using bioinformatics database Tar-
getScanHuman (http://www.targetscan.org/
vert_72/) to predict the related target genes
of miR-20a-5p, it was found that BRMS1L
might be the target gene of miR-20a-5p.
The wild-type 3’UTR vector (pgl3-BRMS1L-
3’-UTR-wt) and the mutant 3’UTR lucifer-
ase reporter vector (pGL3-BRMS1L-3’UTR-
MUT) were constructed in turn. SW480 cells
in logarithmic growth phase were inoculat-
ed into 6-well plates, and the two plasmids
were mixed with mimic NC and miR-20a-5p
mimic respectively, and then co-transfected
into the cells by Lipofectamine TM3000 for
48 hours. Collect cells from each group to
prepare cell lysate, and detect luciferase ac-
tivity according to the requirements of the
kit, to determine whether miR-20a-5p binds
to 3’UTR of BRMS1L.Each group had three
parallel settings.
Detection of miR-20a-5p and BRMS1L in
colon cancer tissues and cells by qRT-PCR
Total RNA was extracted from tissues
and cells by Trizol reagent. RNA was reverse
transcribed into cDNA by reverse transcrip-
tion kit, and amplified by SYBR Green meth-
od. The U6 was used as internal reference.
The reaction conditions: pre-denaturation
at 95℃ for 5min, pre-denaturation at 95℃
for 30s and pre-denaturation at 60℃ for 30
s, a total of 40 cycles. The relative expres-
sion levels of miR-20a-5p and BRMS1L were
calculated by 2-ΔΔCT formula. PCR primers:
miR-20a-5p forward primer, 5’-AGTCTATA-
CAAGGGCAAGCTCTC-3’, reverse primer,
5’-CCCAATACGACCAAATCCGTT-3’. U6 for-
ward primer 5’-CTGCTTCGGCAGCACA-3’,
reverse primer 5’-AACGCTTCACGAATTT-
GCGT-3’. BRMS1L forward primer, 5’-GGCA-
CAGCATTGATATTACCTCA-3’, reverse prim-
er, 5’-TATGGACCTGAAACAACAACTGG-3’.
Each group had three parallel settings.
Detection of cell proliferation by MTT
assay and cell cloning
After 24 hours of transfection, the cells
were digested and inoculated in 96-well
plates at the density of 1×103/ well. Each
well was equipped with three duplicate wells,
which were cultured for 24, 48, 72 and 96
hours, respectively. MTT solution was added,
incubated at 37℃ for 4 hours, centrifuged to
remove supernatant, DMSO was added, and
the absorbance value was detected at 490
nm with enzyme-labeled instrument. Each
group had three parallel settings.
The cell density was adjusted to 1×103/
well, and the cells were inoculated in 6-well
culture plates, and each well was provided
with three duplicate wells. After culturing
for ten days, it was fixed with 4% paraformal-
188 Tang et al.
Investigación Clínica 64(2): 2023
dehyde solution, stained with crystal violet,
and the colony formation was observed un-
der microscope and counted. Each group
had three parallel settings.
Detection of the apoptosis of cell lines
by flow cytometry
The adjusted cell density was 1×105/
well, which was inoculated into 6-well plates,
cultured for 48 h, washed with PBS, and sus-
pended with binding buffer. AnnexinV FITC
and PI were added, incubated in the dark for
10 min, and cell apoptosis was detected by
flow cytometry. Each group had three paral-
lel settings.
Detection of CyclinD1, Bcl-2
and caspase-3 by Western blotting
Cell precipitation was harvested and
added with precooled protein lysate, fol-
lowed by incubation on ice for 30 min and
centrifugation at 10,000 rpm at 4°C. Sub-
sequently, the supernatant was taken for
protein quantification using the BCA Pro-
tein Assay Kit (Thermo Fisher), and the
total protein content was adjusted for sam-
ple preparation. After electrophoresis at
90 V, the proteins were transferred onto a
PVDF membrane. The target band was cut,
blocked with 5% skim milk, and incubated
on a shaker at room temperature for 1 h.
After antibody corresponding incubation
solution (CyclinD1, Bcl-2, caspase-3, GAP-
DH, 1: 2000) was added, the membrane was
incubated on a shaker at room temperature
for 30 min, and then 4°C overnight. On the
second day, after returning to room tem-
perature, the membrane was washed. Next,
the membrane was added with secondary
antibody corresponding incubation solu-
tion (1:5,000), and incubated on a shaker
at room temperature for 2 h. After wash-
ing, the membrane added with an appro-
priate amount of ECL solution, followed
by reaction for 5 min avoiding light. Then,
fluorescence results were gathered using a
quantitative imager. Each group had three
parallel settings.
Statistical analysis
The SPSS 26.0 software (International
Business Machines Corporation, New York,
USA) was utilized for statistical analysis, and
GraphPad Prism 5.0 software was employed
for plotting. The t-test was adopted for com-
parison between groups, and one-way ANO-
VA was used for comparison among groups.
A difference of p<0.05 was considered as
statistically significant.
RESULTS
Bioinformatics prediction and double
luciferase experiment
TargetScanHuman prediction showed
that miR-20a-5p and BRMS1L had targeted
binding sites. The double luciferase experi-
ment showed that the luciferase activity of
wild-type BRMS1L was significantly decreased
(p<0.05) after transfection of miR-20a-5p,
while that of mutant BRMS1L was no signifi-
cantly different (p>0.05) (Fig. 1), indicating
that there was a targeted regulation relation-
ship between miR-20a-5p and BRMS1L.
Fig. 1. Double luciferase verification experiment
(compared with miR-NC group, *: p<0.05).
MiR-20a-5p and colon cancer 189
Vol. 64(2): 184 - 195, 2023
Expression of miR-20a-5p and BRMS1L
mRNA in colon cancer tissues and colon
cancer cell lines
The results of qRT-PCR (Fig. 2) showed
that the expression of miR-20a-5p in colon
cancer tissue was significantly higher than
that in para-cancerous tissues (p<0.05), and
the expression of BRMS1L was significantly
lower (p<0.05). Compared with human nor-
mal colon epithelial cell line FHC, the ex-
pression of miR-20a-5p in colon cancer cell
lines SW480, HT-29 and HCT-116 increased
significantly, and the expression of BRMS1L
decreased significantly (p<0.05).
Relationship between the expression
of miR-20a-5p and clinicopathological
features of patients
To verify the role of miR-20a-5p in the
occurrence and development of colon can-
cer, 87 patients were divided into a high-ex-
pression group (n=43) and a low-expression
group (n=44) according to the p median
expression of miR-20a-5p. Results (Table 1)
showed that age, sex, smoking history, drink-
ing history, tumor size, and tumor location
had no significant differences between the
high-expression group and the low-expres-
sion group of miR-20a-5p (p>0.05), but had
Fig. 2. The expression of miR-20a-5p and BRMS1L in colon cancer tissues (A, B, Compared with normal tissues,
*:p<0.05, **: p<0.001) and colon cancer cells (C, D, Compared with FHC, *: p<0.05, **: p<0.001).
190 Tang et al.
Investigación Clínica 64(2): 2023
Table 1
Relationship between expression level of miR-20a-5p in colon cancer
and clinicopathological parameters of patients.
n
miR-20a-5p
χ2 p
Low
expression
n (%)
High
Expression
n (%)
Age 1.299 0.254
<65 22 13 (52.00) 9 (36.00)
≥65 28 12 (48.00) 16 (64.00)
Gender 0.725 0.395
Male 27 15 (60.00) 12 (48.00)
Female 23 10 (40.00) 13 (52.00)
History of smoking 0.368 0.544
Ye s 16 7 (28.00) 9 (36.00)
No 34 18 (72.00) 16 (64.00)
History of drinking 0.439 0.508
Ye s 12 5 (20.00) 7 (28.00)
No 38 20 (80.00) 18 (72.00)
Tumor diameter (cm) 1.389 0.239
≤518 11 (44.00) 7 (28.00)
>532 14 (56.00) 18 (72.00)
Tumor location 3.949 0.139
Gastric antrum 23 15 (60.00) 8 (32.00)
Gastric body 11 4 (16.00) 7 (28.00)
Gastric fundus and cardia 16 6 (24.00) 10 (40.00)
TNM 6.65 0.01
I～II 29 19 (76.00) 10 (40.00)
III～IV 21 6 (24.00) 15 (60.00)
Lymph node metastasis 5.195 0.023
Ye s 28 10 (40.00) 18 (72.00)
NO 22 15 (60.00) 7 (28.00)
Infiltration depth 6.876 0.009
Mucosa layer 31 11 (44.00) 20 (80.00)
Submucosal layer 19 14 (56.00) 5 (20.00)
Degree of differentiation 5.882 0.015
poorly 34 13 (52.00) 21 (84.00)
Medium/high 16 12 (48.00) 4 (16.00)
MiR-20a-5p and colon cancer 191
Vol. 64(2): 184 - 195, 2023
statistical differences with the TNM stage,
lymph node metastasis, infiltration depth,
and differentiation degree between the high-
expression group and the low-expression
group of miR-20a-5p.
Results of cell proliferation detected
by MTT assay and cell cloning
The results of the MTT assay (Fig. 3A)
showed that at 24h, 48h, 72h and 96h, the
OD value of the miR-20a-5p group was signifi-
cantly higher than that of the control and miR-
NC groups (p<0.05). However, there was no
significant difference in OD between the con-
trol group and the miR-NC group (p>0.05).
The monoclonal formation experiment (Fig.
3B) showed that at 48h, compared with the
control and miR-NC groups, the number of
cell monoclonal formation in the miR-20a-5p
group was significantly higher than that in
the control and miR-NC groups, and the dif-
ference was statistically significant (p<0.05).
However, there was no significant difference
in the number of cell clones between control
group and miR-NC group (p>0.05).
Results of cell apoptosis detected by flow
cytometry
The results of flow cytometry (Fig. 4)
showed that compared with control group
and miR-NC group, the apoptosis rate of
miR-20a-5p group decreased significantly
(p<0.05). However, there was no signifi-
cant difference in apoptosis between control
group and miR-NC group (p>0.05).
Results of CyclinD1, Bcl-2 and caspase-3
expressions detected by Western blotting.
The Western blot results (Fig. 5) showed
that compared with the control and miR-NC
groups, the expression levels of CyclinD1 and
Bcl-2 in the miR-20a-5p group were significant-
ly increased, and the expression levels of cas-
pase-3 were significantly decreased (p<0.05).
Fig. 3. MTT assay (A) and cell cloning (B) were used to detect the cell proliferation ability (compared with
miR-NC group, *: p<0.05).
Fig. 4. Fow cytometry was used to detect apoptosis
(compared with miR-NC group, *: p<0.05).
192 Tang et al.
Investigación Clínica 64(2): 2023
Mechanism of miR-20a-5p affecting
malignant phenotype of colon cancer cells
By detecting the levels of miR-20a-5p and
BRMS1L in colon cancer tissues and cells, we
found that the expression of miR-20a-5p in co-
lon cancer tissues and cells was up-regulated,
while that of BRMS1L was down-regulated,
and the level of miR-20a-5p was significantly
correlated with the TNM stage, lymph node
metastasis, invasion depth and differentiation
degree of patients. miR-20a-5 played the role
of an oncogene, and BRMS1L played the role
of tumor suppressor. It indicated that miR-
20a-5 and BRMS1L were related to the pro-
gression of colon cancer. To further observe
the effect of miR-20a-5p on the malignant
behavior of colon cancer cells, we tested the
proliferation and apoptosis of cancer cells,
and found that overexpression of miR-20a-
5p promoted cell proliferation and inhibited
cell apoptosis. At the same time, miR-20a-5p
could increase the protein levels of CyclinD1
and Bcl-2 and decrease the expression of cas-
pase-3. Therefore, we concluded that miR-
20a-5p could promote the proliferation of
colon cancer cells and inhibit the apoptosis
of colon cancer cells by targeted regulation of
BRMS1L (Fig. 6).
DISCUSSION
MiRNA can regulate the expression of
target genes, and participate in the prolif-
eration, migration, invasion and apoptosis of
tumor cells as oncogenes or tumor suppres-
sor genes 8,9.
Many studies have shown that miR-20a-
5p has the function of oncogene. The re-
search of Wang Xiaojing et al. 10 showed that
miR-20a-5p could alleviate the endothelial
cell injury induced by oxidized low density
lipoprotein (ox-LDL) by targeting and regu-
lating cardiac myosin-related transcription
factor A(MRTFA). Zheng Hui e et al. 11 found
that acupuncture can down-regulate the
expression of miR-20a-5p, thus promoting
Fig. 5. The expression levels of CyclinD1, Bcl-2 and caspase-3 were detected by protein blotting (compared
with miR-NC group, *: p<0.05).
Fig. 6. Mechanism of action of miR-20a-5p on ma-
lignant phenotype of colon cancer cells.
MiR-20a-5p and colon cancer 193
Vol. 64(2): 184 - 195, 2023
cell proliferation and inhibiting cell apop-
tosis in rats with cerebral ischemia-reperfu-
sion injury.
Bai et al. 12 found that miR-20a-5p was
highly expressed in triple-negative breast
cancer, and promoted the growth of tumor
cells by targeting human-related transcrip-
tion factor 3 (RUNX3).
However, Yu et al. 13 found that miR-
20a-5p showed low expression in neuroblas-
toma, which inhibited the proliferation of
tumor cells through targeted regulation
of autophagy related gene 7(ATG7). It was
suggested that miR-20a-5p had different
regulatory effects on the occurrence and
development of tumors. The results showed
that the expression of miR-20a-5p in colon
cancer was significantly higher than that
in adjacent tissues. MiR-20a-5p was sig-
nificantly correlated with the TNM stage,
lymph node metastasis, invasion depth and
differentiation degree, and the higher the
expression of miR-20a-5p, the more serious
the TNM stage and invasion depth, and the
easier it was for lymph nodes to metastasize
(p<0.05). The cell experiment showed that
after miR-20a-5p mimic was transfected,
the proliferation ability of SW480 cells was
obviously enhanced, but the apoptosis abil-
ity was obviously weakened, which indicated
that miR-20a-5p played a cancer-promoting
role in colon cancer.
BRMS1L is the homologous gene of
BRMS1, which can promote the deacety-
lation of histone deacetylase and combine
with specific transcription factors, so as to
regulate the expression of related genes
and inhibit the proliferation and metastasis
of various tumor cells. Cao et al. 14 found
that BRMS1L inhibited the invasion and
metastasis of ovarian cancer cells by inhib-
iting β-catenin-wnt signaling pathway. Lv et
al. 15 found that BRMS1L inhibited the inva-
sion of tumors of the nervous system and
played an anti-cancer gene role in these
tumors. Wang Jihong et al. 16 found that
miR-17-5p could promote the proliferation,
invasion, migration and apoptosis of CNE2
cells of nasopharyngeal carcinoma by down-
regulating the expression of BRMS1L. Chen
Jie 17 found that miR-20a-5p targeted and
regulated the expression of BRMS1L, inhib-
ited the proliferation of the human colorec-
tal cancer cell line SW480 and promoted its
apoptosis. In this study, the online database
of TargetScanHuman was used to predict
that miR-20a-5p has a binding site with
BRMS1L, and the double luciferase gene re-
porting experiment verified that miR-20a-
5p can effectively target the negative regu-
lation of BRMS1L.
The growth of a tumor is closely related
to cell proliferation and apoptosis. When the
expression of proliferation and apoptosis
genes that affect tumor growth changes, the
progress of cancer will also change accord-
ingly 18. CyclinD1 is a proliferation-promot-
ing gene, which is involved in the regulation
of cell cycle. Its coded products can bind to
the corresponding kinases, thus promoting
the development of cell cycle and enhancing
cell vitality 19. Bcl-2 is an apoptosis-inhibit-
ing gene, which plays a role by antagonizing
the apoptosis-promoting gene Bax 20. Cas-
pase-3 is a pro-apoptosis gene, which is acti-
vated by the mitochondrial and death recep-
tor pathways, and then causes cell apoptosis
21. In this study, the expression of CyclinD1,
Bcl-2 and caspase-3 in miR-20a-5p group
were significantly increased, which indicated
that miR-20a-5p induced the proliferation of
cancer cells and inhibited their apoptosis by
regulating the expression of CyclinD1, Bcl-2
and caspase-3.
To sum up, the expression of miR-
20a-5p in colon cancer tissues and cells
increased, and the expression of its target
gene BRMS1L in colon cancer tissues and
cells increased. Over-expression of miR-20a-
5p could enhance the proliferation of co-
lon cancer cells and inhibit their apoptosis,
which provided a theoretical and experimen-
tal basis for the treatment of colon cancer
by inhibiting the expression of miR-20a-5p.
194 Tang et al.
Investigación Clínica 64(2): 2023
Funding
None.
Conflicts of interest
The authors declare that they have no
conflicts of interest to report regarding the
present study.
Author’s ORCID numbers
• Xingkui Tang: 0009-0002-9418-3364
• Yukun Lin: 0009-0002-9261-3682
• Yaqiong Wang: 0009-0000-9568-1731
• Jialin He: 0009-0000-6708-0306
• Xijun Luo: 0009-0005-6134-4290
• Jun Jie Liang: 0009-0009-1589-3482
• Xianjun Zhu: 0009-0008-8618-4213
Author contributions
XT and YL performed the experiments
and wrote the article. YW, JH, XL, JL and XZ
performed the experiments.
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