Received: 25 March 2019 | Accepted: 14 June 2019

DOI: 10.1002/jcp.29050

ORIGINAL RESEARCH ARTICLE

MicroRNA‐204‐3p represses colon cancer cells proliferation,

migration, and invasion by targeting HMGA2

Xiangpeng Xi1 | Mujian Teng1 | Liang Zhang2 | Lijian Xia3 | Jingbo Chen3 |
3
Zhonghui Cui

1
General Surgery Center, Shandong Provincial
Qianfoshan Hospital Affiliated to Shandong
University, Jinan, China
2
Department of Gastrointestinal Surgery,
Jinan Zhangqiu District Hospital of TCM,
Jinan, China
3
Department of Gastrointestinal Surgery,
Shandong Provincial Qianfoshan Hospital
Affiliated to Shandong University, Jinan, China
Correspondence
Zhonghui Cui, Department of Gastrointestinal
Surgery, Shandong Provincial Qianfoshan
Hospital Affiliated to Shandong University,
No. 16766 Jingshi Road, 250014 Jinan, China.
Email: cuizhonghui0048@sina.com
Funding information
Natural Science Foundation of Shandong
Province, Grant/Award Number:
ZR2015HL077
Abstract
Colon cancer is a detrimental neoplasm of the digestive tract. MicroRNAs (miRNAs)
as central regulators have been discovered in colon cancer. Nonetheless, the impact
of miR‐204‐3p on colon cancer remains indistinct. The research attempted to uncover
the impacts of miR‐204‐3p on colon cancer cells growth, migration, and invasion.
miR‐204‐3p expression level in colon cancer tissues and diverse colon cancer cell
lines were testified by the quantitative real‐time polymerase chain reaction.
Exploration of the impacts of miR‐204‐3p on cell growth, migration, invasion, and
their associated factors through assessment of CCK‐8, flow cytometry, Transwell, and
western blot, respectively. High mobility group AT‐hook 2 (HMGA2) expression was
then detected in Caco‐2 cells after miR‐204‐3p mimic and inhibitor transfection,
additionally dual‐luciferase activity was implemented to further uncover the
correlation between HMGA2 and miR‐204‐3p. The impact of HMGA2 on Caco‐2
cell growth, migration, and invasion was finally assessed. We found that repression of
miR‐204‐3p was discovered in colon cancer tissues and HCT116, SW480, Caco‐2,
HT29 and SW620 cell lines. MiR‐204‐3p overexpression mitigated Coca‐2 cell
viability, facilitated apoptosis, simultaneously adjusted CyclinD1 and cleaved caspase‐
3 expression. Cell migration, invasion, and the associated factors were all suppressed
by miR‐204‐3p overexpression. Reduction of HMGA2 was presented in Caco‐2 cells
with miR‐204‐3p mimic transfection, and HMGA2 was predicated to be a target gene
of miR‐204‐3p. Besides, HMGA2 silence showed the inhibitory effect on Caco‐2 cells
growth, migration, and invasion. In conclusion, miR‐204‐3p repressed colon cancer
cell growth, migration, and invasion through targeting HMGA2.

KEYWORDS
apoptosis, cell viability, HMGA2, invasion, microRNA‐204‐3p, migration

1 | INTRODUCTION cases of colon cancer is 71,402 in men, and 64,010 in women,

accounting for 8–9% of all cancer cases (Siegel et al., 2017). The signs
Colon cancer is a familiar detrimental tumor of the digestive tract, and symptoms of colon cancer include abnormality of defecation, blood
which accounts for second of the gastrointestinal tumors (Di et al., in the stool, weight loss, nausea and vomiting (Astin, Griffin, Neal, Rose,
2013). Colon cancer often occurs in adults older than 40 years, and the & Hamilton, 2011; Walter et al., 2016). Surgical resection is the first
incidence of this disease in men is slightly higher than in women (Farner choice for remedying colon cancer, supplemented by radiotherapy,
et al., 1998; Siegel, Miller, & Jemal, 2017). In 2017, the estimated new chemotherapy, and traditional Chinese medicine (Qureshi, Verma, Ross,
1330 | © 2019 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/jcp J Cell Physiol. 2020;235:1330–1338.
XI ET AL. | 1331

F I G U R E 1 miR‐204‐3p was repressed in colon cancer tissues and cell lines. (a) miR‐204‐3p expression in colon cancer tissues (n = 20) and in
their para‐carcinoma tissues (n = 20) were determined via RT‐qPCR; (b) miR‐204‐3p expression in the dissimilar cell lines of HCT116, SW480,
Caco‐2, HT29, and SW620 and in human intestinal epithelial cells line FHC was reassessed via RT‐qPCR. The outcomes were showcased as
mean ± SD with SPSS software statistical analysis. miR, microRNA; RT‐qPCR, quantitative real‐time polymerase chain reaction. *p < .05, **p < .01,
***p < .001

& Landau, 2010). Recently, many scholars have adopted endoscopic
resection for remedying early colon cancer (Kato et al., 2001; Veldkamp
et al., 2004). Nevertheless, the valid molecular biomarkers in colon
cancer are still absence.
MicroRNA (miRNA) is a short RNA molecule with a low number of
nucleotides (20–22), which can adjust gene expression (Low, Ho, Too,
Yap, & Ng, 2014). Recent study certifies that highly expressed miR‐21
and miR‐155 are connected with the metastasis and poor prognosis of
colon cancer (Shibuya, Iinuma, Shimada, Horiuchi, & Watanabe, 2010).
miR‐195 has been corroborated to function as a tumor suppressor in
colon cancer (Liu, Chen, Xu, Li, & Du, 2010). An momentous study
uncovered that miR‐378‐5p could repress colon cancer cell prolifera-
tion and accelerate apoptosis via targeting BRAF (Wang et al., 2015).
miR‐204‐3p and miR‐204‐5p are mature isoforms of miR‐204 gene.
Most research works related to miR‐204 are focused on probing the
functions of miR‐204‐5p (Zeng et al., 2016; Zhang et al., 2015). In
colon cancer, Yin et al. (2014) discovered the repressed effect of miR‐
204‐5p on colon cancer cell proliferation and invasion, meanwhile
demonstrated the improvement for chemotherapeutic sensitivity. But,
the influence of miR‐204‐3p in colon cancer is still vague.
High mobility group AT‐hook 2 (HMGA2) recognizes as a new
oncogene in the existing research (Lee & Dutta, 2007). Several striking
findings relate to the HMGA2, which has been verified to upregulate in
colon cancer, and to be closely linked to the occurrence, development,
and metastasis of colon cancer (Wang et al., 2011; Wisniewski et al.,
2015). Our study intended to probe the impact of miR‐204‐3p on colon
cancer cell growth, migration, and invasion. Whether HMGA2 is a latent
target gene of miR‐204‐3p, as well as whether HMGA2 participates in
regulating these biological processes are also explored.

2 | M A T E R I A L S AN D M E T H O D S

2.1 | Clinical specimens

F I G U R E 2 miR‐204‐3p expression was overexpressed and
silenced in Caco‐2 cells. Caco‐2 cells were one by one transfected
with miR‐204‐3p mimic, miR‐204‐3p inhibitor, and their severally
controls (NC mimic/inhibitor). miR‐204‐3p expression in Caco‐2 cells
with these plasmids transfection was detected through RT‐qPCR.
The outcomes were showcased as mean ± SD with SPSS software
statistical analysis. miR, microRNA; NC, negative control; RT‐qPCR,
quantitative real‐time polymerase chain reaction. ***p < .001
Colon cancer tissues and the para‐carcinoma tissues were attained from
20 patients with colon cancer aging from 37 to 64 years (10 females
and 10 males), which were obtained from Shandong Provincial
Qianfoshan Hospital Affiliated to Shandong University. These patients
were collected from October 2017 to June 2018. None of these
patients adopted any therapies before the operation. All participants
signed the informed consent. The research was supported by the
Medical Ethics Committee of the Shandong Provincial Qianfoshan
Hospital Affiliated to Shandong University.
1332 | XI ET AL.

F I G U R E 3 miR‐204‐3p repressed Caco‐2 cell proliferation and facilitated apoptosis. After miR‐204‐3p mimic/inhibitor and NC mimic/inhibitor
transfection for 12, 24, 48, and 96 hr, severally. (a) Cell growth curves were delineated according to cell viability determination through CCK‐8
trial. Then, (b) cell viability, (c) CyclinD1 expression in Caco‐2 cells with miR‐204‐3p mimic/inhibitor and NC mimic/inhibitor transfection for 48 hr
were tested through CCK‐8 and western blot; (d) cell apoptosis and (e and f) total or cleaved caspase‐3 in above‐transfected condition were
evaluated through flow cytometry and western blot. The outcomes were showcased as mean ± SD with SPSS software statistical analysis. CCK‐8,
Cell Counting Kit‐8; miR, microRNA; NC, negative control. *p < .05, **p < .01, ***p < .001

2.2 | Cell culture coincubated with 10 μl Cell Counting Kit‐8 (CCK‐8, Dojindo, Gaithers-
burg, MD) solution for extra 2 hr at 37°C in 5% CO2 incubator. The
Colon cancer cell lines, such as HCT116, SW480, Caco‐2, HT29, SW620,
absorbance at 450 nm was testified through utilizing a microplate
and human intestinal epithelial cells line FHC bought from American Type
Reader (Bio‐Rad, Hercules, CA). The cell growth curves were appraised
Culture Collection, cultured (ATCC, Rockville, MD) were cultivated in
after transfection for 12, 24, 48, and 96 hr in Caco‐2 cells.
Dulbecco’s modified Eagle medium (DMEM; Hyclone, Logan, UT) and
supplemented with 10% fetal bovine serum (FBS, Hyclone) in a 5% CO2
incubator under the conventional culture condition. 2.5 | Apoptosis assay
Cell apoptosis in transfected Caco‐2 cells was testified by Annexin

2.3 | Cell transfection
miR‐204‐3p mimic, inhibitor, and the severally controls (GenePharma
Co., Shanghai, China) were synthesized, as well as sh‐HMGA2 and
pEX‐HMGA2 were constructed through utilizing U6/GFP/Neo and
pEX‐2 vectors (GenePharma Co.). The sh‐NC and pEX‐2 were regarded
as control groups. Lipofectamine 3000 reagent (Life Technologies) was
mixed with these plasmids and transfected into Caco‐2 cells based on
the specification. After 48 hr posttransfection, Caco‐2 cells were
gathered and employed in the following experiments.
2.4 | Cell viability assay
Caco‐2 cells were cultivated in 96‐well plate with the initial density of
5 × 103 cells/well. After transfection, Caco‐2 cells were gathered and
V‐Phycoerythrin (PE) Kit (Beyotime Biotechnology, Shanghai, China).
The transfected Caco‐2 cells were collected and rinsed two times with
phosphate‐buffered saline (PBS; Beyotime). After centrifugation at
1,000 rpm for 5 min, the supernatant was removed, and 195 μl of
Annexin V‐PE was added to resuspend cells. Subsequently, 5 μl Annexin
V‐PE was mixed to the cell suspension and stained Caco‐2 cells for
15 min in the absence of light. Flow cytometry analysis was immediately
done by using a FACScan (Beckman Coulter, Fullerton, CA).
2.6 | Migration and invasion assays
The abilities of Caco‐2 cells migration and invasion were assessed by
using Transwell chamber (Corning, Cambridge, MA). The chamber was
coated with Matrigel (BD Biosciences, San Diego, CA) for cell invasion
assessment. After transfection with the plasmids of miR‐204‐3p mimic,
XI ET AL. | 1333

F I G U R E 4 miR‐204‐3p repressed Caco‐2 cells migration and invasion. After miR‐204‐3p mimic/inhibitor and NC mimic/inhibitor
transfection for 12 and 24 hr, severally. (a) The scratch width was appraised via executing wound scratch trial. (b) Cell migration and (c) MMP‐9
level, as well as (d) cell invasion and (e and f) Vimentin level in Caco‐2 cells with miR‐204‐3p mimic/inhibitor and NC mimic/inhibitor
transfection for 48 hr transfection were evaluated via Transwell and western blot. The outcomes were showcased as mean ± SD with SPSS
software statistical analysis. miR, microRNA; NC, negative control. *p < .05, **p < .01, ***p < .001

inhibitor, sh‐HMGA2, pEX‐HMGA2, and their severally controls, at 37°C. After cultivation, the wound scratch width was reckoned via
Caco‐2 cells were gathered and were adjusted the concentration to employing a microscope (Olympus).
2 × 105/ml. Afterward, 600 μl of complete medium containing 10% FBS
were supplemented into the lower chamber (the bottom of 24‐well
plate), meanwhile 200 μl cell suspension was added into the upper 2.8 | Dual‐luciferase activity assay
chamber. After 24 hr incubation, the chamber was carefully taken out
Caco‐2 cells were cultured into 24‐well plate, meanwhile cotrans-
by exploiting a tweezer, and drained the liquid of upper chamber, as
fected with the vector of miR‐204‐3p mimic or NC mimic together
well as transferred it to a hole prefilled with about 800 μl methanol.
with HMGA2 3′‐untranslated region (3′‐UTR) via exploiting Lipofec-
After fixation for 30 min, cells were stained with 0.5% crystal violet
tamine 3000 (Life Technologies). Afterward, the luciferase activity
(Sigma‐Aldrich, St. Louis, MO) for 20 min. Cells on the membrane
was appraised through utilizing the dual‐luciferase assay system
surface at the bottom of the upper chamber were removed by utilizing
(Promega Corporation, WI) on the basis of the manufacturer’s
a cotton swab. The migrating and invading cells were counted by
information.
adopting a microscope (Olympus, Tokyo, Japan).

2.9 | Real‐time quantitative PCR (RT‐qPCR)

2.7 | Wound scratch assay
After miR‐204‐3p mimic/inhibitor and NC mimic/inhibitor plasmids
transfection for 24 or 48 hr, Caco‐2 cells were trained in the routine
cultivated condition until 80–90% confluence, and then the wound
scratch trial was executed. The scratches were created via exploiting
20 μl pipette tips. After this, Caco‐2 cells were laundered with PBS
(Beyotime), and then fostered in an incubator encompassing 5% CO2
Total RNA isolated from colon cancer specimens or different cell cells
through exploiting the TRIzol reagent (Invitrogen), and 25 μg
templates were utilized to deploy reverse transcriptions system to
composite complementary DNA (cDNA) by based on the specifica-
tions of a miScript II RT Kit (Qiagen, Valencia, CA). miR‐204‐3p
expression was measured via utilizing a SYBR Green PCR Master Mix
(Applied Biosystems, Foster, CA). U6 served as endogenous control,
1334 | XI
F I G U R E 5 HMGA2 was a novel target gene of miR‐204‐3p.
(a) HMGA2 expression in Caco‐2 cells with miR‐204‐3p mimic/
inhibitor and NC mimic/inhibitor transfection for 48 hr were
appraised via western blot and RT‐qPCR; (b) relative luciferase
activity in Caco‐2 cells with HMGA2‐wt and HMGA2‐mut and miR‐
204‐3p mimic and NC mimic co‐transfection was assessed via dual‐
luciferase assay. The outcomes were showcased as mean ± SD with
SPSS software statistical analysis. miR, microRNA; NC, negative
control; RT‐qPCR, quantitative real‐time polymerase chain reaction.
*p < .05, **p < .01
and data were gauged through the classical 2−ΔΔCt method (Arocho,
Chen, Ladanyi, & Pan, 2006).
2.10 | Western blot assay
Total protein was collected in radioimmunoprecipitation assay lysis
buffer (Beyotime) containing a protease inhibitor cocktail (Calbio-
chem, Billerica, MA). After centrifugation, protein samples were
quantified by the bicinchoninic acid (BCA)™ Protein Assay Kit (Pierce,
Appleton, WI). Afterward, 20 μg protein lysates were electrophor-
esed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis,
and then transferred onto a polyvinylidene difluoride membrane.
After sealing with 5% bovine serum albumin, primary antibodies of
CyclinD1 (ab226977), total caspase‐3 (ab13847), cleaved caspase‐3
(ab49822), MMP‐9 (ab73734), Vimentin (ab137321), HMGA2
ET AL.
(ab109329), and β‐actin (ab8227, Abcam, Cambridge, MA) were
cocultivated with the membranes overnight at 4°C. Subsequently,
membranes were then rinsed in PBS, synchronously cocultivated
with a secondary antibody (ab205718, Abcam) for 1 hr at the
ambient temperature. The specific blots were visualized by exploiting
western blot analysis Luminol Reagent (Santa Cruz, CA). The gay
values of internal parameters were quantified via utilizing the Image
Lab™ Software (Bio‐Rad).
2.11 | Statistical analysis
All data from above‐mentioned experiments are expressed via the
mean ± standard deviation (SD) on the basis of three independent
experiments. SPSS® version 19.0 statistical software program (SPSS
Inc, Chicago, IL) was exploited to figure out the disparate group
results. p‐values were analyzed through exploiting an analysis of
variance (ANOVA) followed by Tukey post hoc test. A value of p < .05
was deemed to be a statistically significant result.
3 | RESULTS
3.1 | miR‐204‐3p was repressed in colon cancer
tissues and cell lines
The samples of 20 colon cancer tissues and 20 para‐carcinoma
tissues were collected, miR‐204‐3p expression in these procured
tissues were probed via quantitative real‐time polymerase chain
reaction (RT‐qPCR). The reduction of miR‐204‐3p in colon cancer
tissues was obviously discovered as relative to that in the para‐
carcinoma tissues (p < .05, Figure 1a). Then, we further studied miR‐
204‐3p expression in the disparate cell lines of HCT116, SW480,
Caco‐2, HT29, and SW620. Human intestinal epithelial cells line FHC
served as control group. In Figure 1b, we discovered that miR‐204‐3p
expression was memorably restrained in HCT116, SW480, Caco‐2,
HT29, and SW620 cells as compared with that in FHC cells (p < .001).
Data from above observations uncovered that miR‐204‐3p was
restrained in colon cancer tissues and cell lines, which might be a
biomarker of colon cancer. Further, the maximal inhibition of miR‐
204‐3p was explained in Caco‐2 cells, which was therefore adopted
for utilizing the follow‐up research.
3.2 | miR‐204‐3p repressed Caco‐2 cell
proliferation and triggered apoptosis
For further delving the influence of miR‐204‐3p in colon cancer cells
growth, miR‐204‐3p mimic and miR‐204‐3p inhibitor and the
severally controls were built and transfected into Caco‐2 cells. In
Figure 2, the enhancement of miR‐204‐3p was observed in miR‐204‐
3p mimic‐transfected cells, meanwhile, the repression of miR‐204‐3p
was discovered in miR‐204‐3p inhibitor‐transfected cells (p < .001).
Subsequent experiment results uncovered that cell viability was
decreased by miR‐204‐3p overexpression (p < .05, p < .01 or p < .001,
Figure 3a,b), meanwhile CyclinD1 level was also repressed by
XI ET AL. | 1335

F I G U R E 6 HMGA2 silence restrained Caco‐2 cells viability and facilitated apoptosis. Caco‐2 cells were respectively transfected with pEX‐
HMGA2, sh‐HMGA2, and their severally controls (pEX‐2 and sh‐NC). (a) HMGA2 expression in above‐involved transfected Caco‐2 cells was
detected via western blot and RT‐qPCR; (b) cell viability, (c) cell apoptosis, and (d) total caspase‐3 and cleaved caspase‐3 were evaluated via
CCK‐8, flow cytometry and western blot. The outcomes were showcased as mean ± SD with SPSS software statistical analysis. CCK‐8, Cell
Counting Kit‐8; NC, negative control; RT‐qPCR, quantitative real‐time polymerase chain reaction. **p < .01, ***p < .001

miR‐204‐3p overexpression (p < .05, Figure 3c). Further, the results in
Figure 3d–f displayed that cell apoptosis was hastened by
miR‐204‐3p overexpression (p < .001), and the improvement of
cleaved caspase‐3 level was also triggered by miR‐204‐3p over-
expression (p < .001). All above findings suggested that miR‐204‐3p
could obstruct cell growth in Caco‐2 cells.
3.3 | miR‐204‐3p reduced Caco‐2 cell migration
and invasion
The next experiments further delved the impacts of miR‐204‐3p on Caco‐
2 cell migration and invasion. The wound scratch experiment revealed
that the wound area was visibly expedited in miR‐204‐3p mimic‐
transfected cells (p < .05 or p < .001). Nonetheless, the adverse result was
presented in miR‐204‐3p inhibitor‐transfected cells (p < .05 or p < .001,
Figure 4a). Next, the prohibitive cell migration was explained in miR‐204‐
3p mimic‐transfected cells (p < .001), nevertheless the accelerative cell
migration was observed in miR‐204‐3p inhibitor‐transfected cells (p < .05,
Figure 4b). Concurrently, MMP‐9 expression was refrained by miR‐204‐
3p overexpression and boosted by miR‐204‐3p suppression (p < .05 or
p < .01, Figure 4c). Cell invasion assay results were similar with the
migration results. The results in Figure 4d–f revealed that cell invasion
and Vimentin expression were all repressed by miR‐204‐3p over-
expression as well as facilitated by miR‐204‐3p suppression (p < .05 or
p < .001). The observations corroborated that miR‐204‐3p could reduce
Caco‐2 cell migration and invasion.
3.4 | HMGA2 was a neoteric direct target of
miR‐204‐3p in Caco‐2 cells
The RT‐qPCR assay was executed to probe the dependency between
HMGA2 and miR‐204‐3p after Caco‐2 cells were transfected miR‐
204‐3p mimic/inhibitor and NC mimic/inhibitor. The interesting results
in Figure 5a emerged that HMGA2 was restrained by miR‐204‐3p
overexpression as relative to the NC mimic group (p < .01), concur-
rently accelerated by miR‐204‐3p suppression compared with NC
inhibitor group (p< .05). A negative correlation between HMGA2 and
miR‐204‐3p was ascertained according to the above observations.
Afterward, we executed dual‐luciferase activity experiment and
discovered that miR‐204‐3p overexpression reduced the luciferase
activity of HMGA2‐wt as relative to its corresponding control (p < .01,
Figure 5b). The luciferase activity of HMGA2‐wt had no obvious
change in Caco‐2 cells with miR‐204‐3p mimic or NC mimic
transfection. Data from above‐mentioned findings indicated that
HMGA2 might be a newfangled target of miR‐204‐3p in Caco‐2 cells.
3.5 | HMGA2 expedited Caco‐2 cell viability,
migration, and invasion
To further testify the influences of HMGA2 in colon cancer cells, pEX‐
HMGA2, sh‐HMGA2, and their correlative control (pEX‐2 and sh‐NC)
were transfected into Caco‐2 cells. Expression level of HMGA2 was
presented in Figure 6a, and indicated that the plasmids of
pEX‐HMGA2 and sh‐HMGA2 were both successfully transfected into
1336 | XI ET AL.

F I G U R E 7 HMGA2 silence repressed Caco‐2 cell migration and invasion. Caco‐2 cells were respectively transfected with pEX‐HMGA2, sh‐HMGA2,
and their severally controls (pEX‐2 and sh‐NC). (a) Cell migration and (b) MMP‐9 level in above‐mentioned transfected Caco‐2 cells were examined via
Transwell and western blot; meanwhile (c) cell invasion and (d) Vimentin level in above‐mentioned transfected Caco‐2 cells were appraised via Transwell
and western blot. The outcomes were showcased as mean ± SD with SPSS software statistical analysis. *p < .05, **p < .01, ***p < .001

Caco‐2 cells (p < .001). Results in Figure 6b revealed cell viability was
accelerated by HMGA2 overexpression, however retarded by HMGA2
silence (p < .01). On the side, HMGA2 silence evoked cell apoptosis
(p < .01, Figure 6c) and advanced cleaved caspase‐3 expression
(p < .001, Figure 6d) in Caco‐2 cells. Furthermore, Figure 7a,b disclosed
that HMGA2 overexpression facilitated cell migration (p < .001), as
well as increased MMP‐9 level (p < .05) in Caco‐2 cells. Likewise, cell
invasion and Vimentin level were also exalted by HMGA2 over-
expression (p < .001, Figure 7c,d). Nevertheless, these above‐men-
tioned results about cell migration and invasion were all inversed in sh‐
HMGA2 transfected Caco‐2 cells (p < .05, p < .01, or p < .001, Figure
7a–d). Data from above findings affirmed that HMGA2 could facilitate
Caco‐2 cell growth, migration, and invasion.
4 | D IS C U S S IO N
Colon cancer has become a familiar malignant tumor with a high
incidence in the world (Yang et al., 2014). Although the treatments of
colon cancer have improved, the prognosis is still not optimistic. In
clinic, many patients are diagnosed with advanced stage of colon
cancer, and the recurrence or metastasis may occur after surgery
(Xynos et al., 2016). Therefore, the identification of colon cancer
biomarkers has important clinical significance. Increasing evidence
has confirmed that miRNA is a new biomarker and can be used in
early diagnosis, treatment, and prognosis evaluation of colon cancer
(Luo, Burwinkel, Tao, & Brenner, 2011; Matsumura et al., 2015). We
intended to probe whether miR‐204‐3p is a novel biomarker for
colon cancer, and also explored the impacts of miR‐204‐3p on colon
cancer cell growth, migration, and invasion. Results corroborated the
reduction of miR‐204‐3p in colon cancer tissues and cell lines. In
addition, repression of cell proliferation, facilitation of apoptosis, and
inhibition of cell migration and invasion were discovered in Caco‐2
cells transfected with miR‐204‐3p mimic. Besides, HMGA2 was
considered to be a novel target gene of miR‐204‐3p, and HMGA2
silence repressed Caco‐2 cell growth, migration, and invasion.
Recently, abnormal expressed miRNAs has been found in colon
cancer in many research works (Li et al., 2014; Xiao, Deng, Zhang,
XI ET AL.
Zhang, & Huang, 2013). Study of Chiang et al. (2012) substantiated
that miR‐192, miR‐194 and miR‐215 were repressed in colon cancer
tissues as well as in HT‐29, HCT‐116, and SW‐620 cells. Nie et al.
(2012) demonstrated the reduction of miR‐365 in colon cancer
tissues, and restoration of miR‐365 could suppress colon cell cycle
progression, simultaneously induce cell apoptosis. We observed the
similar results to the previous research works, and the results
presented the downregulation of miR‐204‐3p in colon cancer tissues
and in HCT116, SW480, Caco‐2, HT29, and SW620 cell lines. The
findings certified that miR‐204‐3p was likely to be a novel biomarker
for colon cancer.
miR‐204‐3p has been discovered in various cancers, including
clear cell renal cell carcinoma (ccRCC; Han et al., 2018), hepatocel-
lular carcinoma (Cui, Shen, Chen, & Hu, 2014), and breast cancer (Li
et al., 2013). In addition, Chen et al. (2016) testified the prohibitive
impact of miR‐204‐3p on the development of glioma. Nevertheless,
the influence of miR‐204‐3p in colon cancer remains unaware. The
observations from the current study discovered that miR‐204‐3p
overexpression reduced Caco‐2 cell viability, boosted apoptosis,
synchronously regulated CyclinD1, and cleaved caspase‐3 expres-
sion. More interestingly, the restrained cell migration, invasion, as
well as MMP‐9 and Vimentin expression were also observed in Caco‐
2 cells with miR‐204‐3p mimic transfection. The observations
attested that miR‐204‐3p might be a tumor suppressor in the
carcinogenesis of colon cancer.
HMGA2 is a nonhistone and architectural transcription factor, which
exerts the oncogenic effect on multitudinous cancers. HMGA2 has also
been proved to be a direct target gene of different miRNAs. One study
corroborated that miR‐543 repressed colon cancer growth and
metastasis through targeting HMGA2 (Fan et al., 2016). Other study
revealed that miR‐204 regulated colon cancer cell sensitivity by targeting
HMGA2 (Wu, Liang, Shen, & Shen, 2016). On the basis of preceding
research works, we supposed that HMGA2 might be a direct of miR‐204‐
3p. In our study, the results testified that HMGA2 was restrained by miR‐
204‐3p in Caco‐2 cells. Dual‐luciferase activity assessment corroborated
that HMGA2 was a novel target gene of miR‐204‐3p. Further experiments
results revealed that cell viability, migration, and invasion were obviously
suppressed by HMGA2. This data indicated that HMGA2 might be a key
regulator in the development of colon cancer. More studies are merited
to affirm this hypothesis.
Taken together these results, this study discovered the repressive
effect of miR‐204‐3p on Caco‐2 cell proliferation, migration, and invasion
through targeting HMGA2. These observations attested that miR‐204‐3p
might be a novel tumor suppressor in the carcinogenesis of colon cancer.
A C K N O W L E D GE M E N T
This study was supported by the Natural Science Foundation of
Shandong Province (Grant no. ZR2015HL077).
CO NFLICT OF I NTERE STS
The authors declare no conflict of interests.
| 1337
AU TH OR CO NTRIB UTION S
Conceived and designed the experiments: X. X., M. T., and Z. C.;
performed the experiments and analyzed the data: X. X, M. T., L. Z.,
and L. X.; contributed reagents/materials/analysis tools: L. X. and
J. C.; wrote the manuscript: X. X. and Z. C.
D A TA AV A I L A B I L I TY
The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request.
ORCI D
Zhonghui Cui http://orcid.org/0000-0002-5791-0721
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How to cite this article: Xi X, Teng M, Zhang L, Xia L, Chen J,
Cui Z. MicroRNA‐204‐3p represses colon cancer cells
proliferation, migration and invasion by targeting HMGA2. J Cell
Physiol. 2020;235:1330–1338. https://doi.org/10.1002/jcp.29050