miR-19b-3p promotes colon cancer proliferation and oxaliplatin-based chemoresistance by targeting SMAD4: validation by bioinformatics and experimental analyses
Jiang et al. Journal of Experimental & Clinical Cancer Research
miR-19b-3p promotes colon cancer proliferation and oxaliplatin-based chemoresistance by targeting SMAD4: validation by bioinformatics and experimental analyses
Tao Jiang 0 2 5
Ling Ye 0 1 4
Zhongbo Han 0 3
Yuan Liu 1 4
Yinxue Yang 2 5
Zhihai Peng 1 4
Junwei Fan 1 4
0 Equal contributors
1 Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai 20080 , People's Republic of China
2 Department of Anal-Colorectal Surgery, General Hospital of Ningxia Medical University , Yinchuan 750004 , People's Republic of China
3 Department of General Surgery, Central Hospital of Zi Bo , Zi Bo 255000 , People's Republic of China
4 Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai 20080 , People's Republic of China
5 Department of Anal-Colorectal Surgery, General Hospital of Ningxia Medical University , Yinchuan 750004 , People's Republic of China
Background: As a disease with extremely complex molecular mechanisms, many deregulated miRNAs have been identified in colon cancer. Few studies have been performed by using Ingenuity Pathways Analysis (IPA) to predict miRNAs specifically expressed in colon cancer. Methods: A characteristic microRNA-target network of colon cancer was explored using IPA. Then the clinical significance of miR-19b-3p was evaluated in 211 colon cancer patients. The roles of miR-19b-3p and its candidate target gene, SMAD4, in colon cancer progression were examined both in vitro and in vivo. Results: Bioinformatics analysis showed that 15 microRNAs screened by IPA were significantly correlated with malignant biological behaviors of colon cancer. miR-19b-3p was the most significantly upregulated candidate based on the validation experiment using 211 colon cancer samples. High expression of miR-19b-3p was significantly associated with high N stage (P < 0.001), high AJCC stage (P < 0.001), poor histologic grade (P = 0.032), frequent venous and lymphatic invasion (P = 0.027), and liver metastasis (P < 0.001). Survival analysis revealed that miR-19b-3p was an independent prognostic factor associated with colon cancer patient's overall survival (OS) and disease-free survival (DFS). miR-19b-3p promoted proliferation and chemoresistance of colon cancer cells, but had no effect on invasion in vitro, along with tumorigenesis in vivo. In addition, we confirmed that miR-19b-3p mediates resistance to oxaliplatin-based chemotherapy via SMAD4. Conclusions: Our findings demonstrate the role of miR-19b-3p-SMAD4 axis in colon cancer progression, which may become a potential therapeutic target against chemotherapy resistance.
miR-19b-3p; SMAD4; Proliferation; Oxaliplatin; Chemoresistance
Colon cancer is the third most common cancer with
high cancer-related deaths worldwide [
recurrence and metastasis are the principal causes of
death, despite improvements in the multidisciplinary
and comprehensive treatment based on surgical operation
of colon cancer [
]. In spite of the in-depth studies of the
molecular mechanisms underlying colon cancer for the
last decades, chemoresistance remains a crucial challenge
for the treatment of colon cancer.
MicroRNAs (miRNAs) are a class of short, non-coding
]. miRNAs bind to the 3′-untranslated regions
(UTR) of partially complementary target messenger
RNAs (mRNAs) by base pairing mode, thereby suppressing
the expression of downstream target genes [
than 30% of mRNAs are regulated by miRNAs, which can
act as tumor suppressor genes or oncogenes, depending on
the microenvironment within the cell and the specific
downstream target genes they regulate . Therefore,
deregulation of miRNA expression influences many
biological functions of tumors such as angiogenesis,
differentiation, cell proliferation, and apoptosis [
]. As a disease with
extremely complex molecular mechanisms, many
deregulated miRNAs have been implicated in the pathogenesis of
colon cancer [
The powerful data analysis and search capabilities of
Ingenuity Pathways Analysis (IPA) contribute to the
understanding of existing data, target discovery, and
validation as well as analysis of biological networks
]. The application of IPA software has been widely
used in cancer research, and its powerful database makes it
easier to identify potential biomarkers for cancer diagnosis
and therapy [
]. Although many miRNAs have been
confirmed as biomarkers in colon cancer in a number of
studies, limited experimental research has been performed
by using IPA to better understand the miRNA/mRNA
network of colon cancer.
In this study, we found that miR-19b-3p was
significantly upregulated in colon cancer using IPA. To figure
out its role in colon cancer, we performed the current
study to evaluate the associations of miR-19b-3p
dysregulation with colon cancer progression in vitro and in vivo,
and further investigate its relationship with prognosis of
colon cancer patients.
IPA database is used for analyze and understand the
complex biological and chemical systems in life science
research. IPA provides exploratory investigation of genes,
proteins, and biological functions, creating customized
pathways or molecular interaction networks focused on
drug targets and identifying potential biomarkers. The
microRNA Target Filter associates microRNAs from a
dataset with experimentally observed mRNA targets which
is used to overlay microRNA data onto networks and
pathways, to add molecules to networks, and to
compare molecules from different experimental
observations. In this study, we used IPA 9.0 to screen miRNAs
specifically expressed in colon cancer to better understand
the biology around potential mRNA targets/diseases and
to identify the most biologically relevant targets.
The latest release of the miRTarBase database (http://
mirtarbase.mbc.nctu.edu.tw/) was used to collect
experimentally validated target genes of miRNAs, which were
screened by IPA. The miRNA-gene regulatory network
was based on the interactions of miRNAs and predicted
target genes. Functional annotations of the predicted target
genes of these miRNAs were obtained from R annotation
packages. Function enrichment analyses were performed by
Fisher’s exact test. P-values were adjusted for the multiple
testing corrections by R function “p.adjust”. We chose
functional terms whose adjusted P-value was smaller than 0.05
as significantly enriched functions.
Patients and tissue samples
A total of 211 tissue samples from colon cancer patients
with pathologically confirmed diagnosis were obtained
immediately after surgery and frozen at −80 °C in liquid
nitrogen before being deposited in the Anal-Colorectal
Surgery Department, General Hospital of Ningxia Medical
University. For each case, paired tumor and distal mucosae
were collected. All patients provided informed consent and
the study was approved by the institutional review boards
of General Hospital of Ningxia Medical University. The
pathologic verification of diagnosis and staging is
summarized according to the National Comprehensive Cancer
Network (NCCN) Practice guidelines. The follow-up of this
cohort ended on July 14, 2014 and the median duration of
follow-up was 59 (range, 12–83) months. Disease-free
survival (DFS) and overall survival (OS) rates were
defined as the interval from the initial surgery to
clinically or radiologically proven recurrence/metastasis and
Cell culture and transfection
The human colon cancer cell lines, SW620, LoVo, HCT
116, SW480, Caco-2, HT-29, RKO, DLD1, and human
normal colonic epithelial cells, NCM460, were obtained
from the Type Culture Collection of the Chinese Academy
of Sciences (Shanghai, China). Hsa-miR-193a-3p inhibitor
and negative control were purchased from Biomics Biotech
(Biomics Biotech, Nantong, China). Their sequences were
as follows: miR-19b-3p inhibitors, 5′ -UCAGUUUUGCAU
GGAUUUGCACA-3′; negative control, 5′ -CAGUACUU
UUGUGUAGUACAA-3′. The coding sequence of
miR19b-3p inhibitors or negative control was cloned into
pCDH-CMV lentivectors (SBI, Mountain View, CA, USA)
by BioLink Biotechnology (Shanghai, China). To package
the construct, 293 T cells were co-transfected with both
pPackH1 packaging plasmid mix (SBI, Mountain View, CA,
USA) and the lentivectors. Then the virus particles were
collected 48 h later. For transfection, cells were plated in
6well plates at a density of 2 × 105 cells/well and cultured
until they reached 80% confluence. Then RKO and SW480
cells were infected with viruses.
RNA extraction and quantitative real-time PCR
Total RNA, including miRNAs, was isolated from
clinical tissue specimens and cell lines using TRIzol reagent
(Invitrogen, Carlsbad, CA, USA) according to the
manufacturer’s protocol. The first strand cDNA was synthesized with
the RevertAid First Strand cDNA Synthesis Kit (MBI
Fermentas, Vilnius, Lithuania) using 1 μg of total RNA
as the template. miRNAs were prepared with the
HighSpecificity miRNA qRT-PCR Detection Kit (Stratagene,
Santa Clara, CA, USA) and U6 was used as an endogenous
control. The relative miR-19b-3p level was calculated from
this eq. 2-ΔCT[ΔCT = Ct (miR-19b-3p) - Ct (control)].
Realtime PCR of SMAD4 and miRNAs was carried out using
ViiA™ 7 system (Thermo Fisher Scientific, Waltham, MA,
USA) according to the manufacturer’s instructions. GAPDH
was used as endogenous control to normalize the
expression of SMAD4. The qRT-PCR primers are shown
in Additional file 1: Table S1.
Cell proliferation and invasion
Cell Counting Kit-8 (CCK8) assays (Dojindo, Kumamoto,
Japan) were used to evaluate the cell proliferation ability
according to the manufacturer’s protocol. The invasion
assays were implemented by using the transwell system.
The pore size of invasion chambers is 8.0 μm coated
with matrigel (Millipore, Billerica, MA, USA). The colon
cancer cells were suspended after being transfected for
48 h, and then seeded in the upper chambers of 24-well
transwell plates with serum and calcium-free solution
supplemented with 0.2% BSA. The lower chamber was filled
with 500 μL of medium containing 10% FBS. Cells were
incubated for 24 h at 37 °C with 5% CO2. Next, a cotton
swab was used to wipe off the matrigel and cells remaining
in the upper chambers. Cells that migrated to the lower
chambers were fixed with methanol for 20 min and then
stained with 1% crystal violet for 10 min. The number of
migrated cells was counted under a light microscope with
a 200× magnification. Five fields were randomly selected
on each membrane.
Cell viability and apoptosis
The Annexin V-PE and 7-AAD (BD Biosciences, San
Jose, CA, USA) double staining method was used to
examine cell viability. The frequency of apoptosis was
measured using the BD FACSCalibur™ Flow Cytometer
(BD Biosciences, San Jose, CA, USA) according to the
manufacturer’s instructions. Before assessing the
chemosensitivity of colon cancer cells to oxaliplatin, the optimal drug
concentration of oxaliplatin inducing cell death was
determined on untransfected cells. Cells were treated with
oxaliplatin (MedChem express, Monmouth Junction, NJ, USA)
at a final concentration of 50 μg/mL for 24 h. The
percentage of oxaliplatin-treated and untreated apoptotic cells was
calculated according to the number of cells positive or
negative for Annexin V-PE and 7-AAD. Results are presented as
the percentage of total cells that were living cells
(Ann−/7AAD-), early apoptotic cells (Ann+/7-AAD-), late apoptotic
cells, and dead cells (Ann+/7-AAD +).
Western blot was performed to assess SMAD4 expression
in miR-19b-3p inhibitor and negative control transfected
cells and tumors induced in SCID mice. SMAD4 was
detected with anti-SMAD4 (Abcam, Cambridge, UK) at a
1:5000 dilution. The level of SMAD4 was normalized to
the level of β-actin protein, which was detected by using
anti-β-actin (Abcam, Cambridge, UK) at a 1:2000 dilution.
Then horseradish peroxidase (HRP)-tagged anti-rabbit
or anti-mouse immunoglobulin (Abgent, San Diego,
CA, USA), at a dilution of 1:2000, was used to detect
the primary antibody. Enhanced chemiluminescence
reagent (Merck Millipore, Temecula, CA, USA) was applied
to reveal the protein bands. The Image J software (National
Institutes of Health, Bethesda, MD, USA) was used to
quantify the band intensity. To further explore the effects
of the interactions between miR-19b-3p and several mRNA
at the protein level, a series of western blot analyses was
performed following the aforementioned experimental
protocols. Primary antibodies included PRKACB, ATM,
CREB3L2, EGLN3, JUN, NR3C1, WEE1, RASSF1 and
TGFBR2 (Abcam, Cambridge, UK).
Twenty 6- to 8-week-old SCID mice were obtained from
the Shanghai Jiaotong University and maintained in specific
pathogen-free (SPF) conditions. Approximately 5 × 106
SW480 cells transfected with miR-19b-3p inhibitor or a
negative control were injected subcutaneously into the
opposite flanks of each mouse. Tumors grew in all animals
and the tumors size was measured at 4, 8, 12, 16, and
21 days. All nude mice were euthanized by cervical
dislocation at day 21 and their tumors were harvested, weighed,
and photographed. All procedures followed the Shanghai
Jiao tong University Affiliated Shanghai General Hospital
Animal Care guidelines.
Plasmid constructs and luciferase reporter assays
Dual-Luciferase reporter assays (Promega, Madison, WI,
USA) were used following the manufacturer’s instructions.
The putative miR-19b-3p complementary site in the
3′-UTR of SMAD4 mRNA (NM_005395; 3′-UTR:
1352–1358) or its mutant sequence were cloned into
the pmirGLO luciferase reporter vector. The novel
combined plasmid was named pmirGLO-SMAD4
3′’UTR-wt (wild type). A mutation of the 3′-UTR of SMAD4
was created by using a site-directed mutagenesis kit
(Thermo Fisher Scientific, Waltham, Massachusetts, USA)
and designated as pmirGLO-SMAD4 3′-UTR-mut
(mutant). SW480 cells were plated at 4000 cells per well in
100 μL DMEM in a 96-well microplate (BD Biosciences,
USA). Twenty-four hours after plating, cells were
cotransfected with 0.5 μg of pmirGLO-SMAD4
3′-UTRwt or pmirGLO-SMAD4 3′-UTR-mut, 0.01 μg of the
pMirGLO-Vector (Promega, Madison, WI, USA), 50 nM
miR-19b-3p mimics (Mimics), and miRNA mimics Negative
Control (NC) using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA, USA). The firefly and Renilla luciferase
activities were measured 24 h after transfection.
In situ hybridization (ISH) and immunohistochemistry on tissue microarray
The tissue microarray (TMA) including 120 pairs of colon
cancer and corresponding normal mucosa was obtained
from Outdo Biotech (Shanghai, China). The TMA slides
were dewaxed by xylene for 15 min twice. After being
dehydrated by immersion in 100% ethanol for 5 min, the
slides were air-dried and then incubated with pepsin at
37 °C for 15 min. Then the slides were fixed in 4%
paraformaldehyde, dehydrated in 90% ethanol, and
incubated with the digoxigenin-labeled probe (Exiqon,
Denmark) complementary to miR-19b-3p at 37 °C
overnight, according to the manufacturer’s instructions. The
slides were washed twice with 2× Saline-Sodium Citrate
Bufferat room temperature, and incubated with mouse
anti-digoxigenin monoclonal antibody according to the
manufacturer’s protocol. miR-19b-3p expression in the
TMA was assessed by 2 independent pathologists. The
proportion of positively stained tumor cells was graded
as follows: 0 (no positive cells), 1 (<10% positive cells),
2 (10–50% positive cells), 3 (>50% positive cells). The
intensity of the staining was recorded on a scale of 0
(no staining), 1 (weak staining), 2 (moderate staining), and
3 (strong staining). The staining index (SI) was defined as
the proportion of positively stained tumor cells × staining
To further confirm that SMAD4 is the target of
miR19b-3p, we detected SMAD4 protein expression levels by
immunohistochemistry on the same location of the TMA.
The scoring systems were similar to those used for ISH.
Statistical analyses were performed using the SPSS
statistical software program version 20 (SPSS Inc., Chicago,
IL, USA). Student’s t-test was applied to compare two
groups of quantitative data. The χ2 test or Fisher’s exact
test for enumeration data was used to analyze the
relationship between miR-19b-3p and clinicopathological
features. Kaplan-Meier method was used to analyze the
survival rates, and the differences between the survival
curves were examined by the log-rank test. Univariate
and multivariate survival analyses were performed using
Cox proportional hazards models. P < 0.05 was
considered statistically significant.
IPA and gene function analysis
We used IPA 9.0 to identify miRNAs specifically expressed
in colon cancer. A total of 15 miRNAs were identified in
the Ingenuity Knowledge Base by experimental evidence
(Additional file 2: Table S2). The 7 upregulated
miRNAs are: miR-19b-3p, miR-155-5p, miR-17-5p,
miR183-5p, miR-25-3p, miR-21-5p, and miR-196a-5p. The
8 downregulated miRNAs are: miR-29c-3p,
miR-34a5p, miR-542-5p, let-7a-5p, miR-126-3p, miR-143-3p,
miR-192-5p, and miR-194-5p. The experimentally
validated target genes of the 15 significant miRNAs were
searched with the miRTarBase database.
The predicted target genes in enriched functional
terms were used to construct a miRNA-target network.
The network was plotted by using R package “igraph”
(Fig. 1). The biological function analysis of the predicted
target genes based on IPA and GO annotation system
enabled us to comprehensively understand their functional
roles in colon cancer progression. The predicted target
genes were significantly involved in many biologic
processes relevant to cancer such as apoptosis, cell
proliferation, and cell cycle arrest (Fig. 2). Evidence indicated that
these malignant biological behaviors may lead to tumor
]. These bioinformatics analysis
suggested that the predicted target genes may control broad
biological functions associated with colon cancer.
miRNA expression profiling in colon cancer patients
Since gene therapy requires high expression levels of the
target gene, we selected the 7 upregulated miRNAs to
validate their expression patterns in the 211 colon cancer
samples using qRT-PCR. The expression profiles of the 7
miRNA are presented in Fig. 3. miR-19b-3p was the most
significantly upregulated candidate (P < 0.001).
miR-19b-3p expression is upregulated in colon cancer
and predicts poor prognosis of patients with colon cancer
Using qRT-PCR, we investigated miR-19b-3p expression
patterns in colon cancer specimens and paired normal
tissues. Results showed that miR-19b-3p expression levels
were associated with the clinicopathological data (Table 1),
which included high N stage (P < 0.001), high AJCC stage
(P < 0.001), poor histologic grade (P = 0.032), and liver
metastasis (P < 0.001).
Kaplan-Meier and Cox proportional hazard regression
analyses were performed and showed that miR-19b-3p
expression level was significantly associated with patient’s
survival. The results showed that high miR-19b-3p
expression correlated with lower OS (P < 0.001) and DFS
(P < 0.001) in 211 cases colon cancer patients (Fig. 4).
Similarly, univariate analysis showed that miR-19b-3p
expression was associated with OS (Table 2, P < 0.001)
and DFS (Table 3, P < 0.001). Using the parameters that
were significant in univariate analyses as covariates,
miR-19b-3p expression level was significantly
associated with OS (HR = 2.23, P = 0.008, Table 2) and DFS
(HR = 2.73, P = 0.016, Table 3). It demonstrates that
miR-19b-3p is an independent prognostic factor of
colon cancer patients.
miR-19b-3p is overexpressed in colon cancer cell lines
and promotes proliferation and chemoresistance in vitro
To further determine the potential function of miR-19b-3p
in promoting colon cancer progression, we first investigated
miR-19b-3p expression pattern in a panel of human colon
cancer cell lines and normal colonic epithelial cells. The
expression of miR-19b-3p was higher in the 8 colon cancer
cells than in normal colonic epithelial cells. Two out of
eight colon cancer cell lines, including SW480 and RKO,
reached the highest level of statistical significance versus
the control (Fig. 5a). We constructed a lentiviral vector
harboring a RNAi sequence targeting the human
miR-19b3p and investigated the effects of miR-19b-3p inhibitor on
a series of cancer-relevant in vitro assays, including
proliferation, invasion, apoptosis, and chemosensitivity in SW480
and RKO cells. miR-19b-3p downregulation suppressed the
proliferation of SW480 cells (Fig. 5b, left) and RKO cells
(Fig. 5b, right) but had no significant effect on invasion
using matrigel-coated transwell assays (Fig. 5c and d).
miR-19b-3p downregulation had little effects on cell
viability in the absence of oxaliplatin. However, the ability to
promote cell apoptosis was observed when cells were
treated with oxaliplatin (Fig. 5e and f ). These in vitro
findings showed that miR-19b-3p promotes proliferation and
chemoresistance in colon cancer cells and plays an
important role in colon cancer progression.
miR-19b-3p promotes proliferation of colon cancer in vivo
To evaluate the effect of miR-19b-3p on tumorigenesis
in vivo, SW480 cells were subcutaneously implanted in
SCID mice. Cells transfected with the miR-19b-3p inhibitor
or a negative control were injected into the opposite flanks
of each animal. miR-19b-3p inhibition resulted in less
spheroid tumors with clean edges, and the tumor growth
rate was decreased compared to negative control tumors
with more local proliferation phenotypes. The mice were
euthanized at day 21 and the tumors were immediately
harvested (Fig. 6a). The average tumor size of miR-19b-3p
inhibitor expressing tumors was significantly reduced
when compared to that of control tumors, especially at
day 21 (0.237 ± 0.091 cm3 versus 1.533 ± 0.231 cm3,
respectively, P < 0.05, Fig. 6b). Moreover, the average weight
of miR-19b-3p inhibitor expressing tumors was also
significantly reduced when compared to that of negative
controls (1.28 ± 0.23 g versus 2.74 ± 0.45 g, respectively,
P < 0.05, Fig. 6c). Furthermore, we performed western
blotting assays to investigate SMAD4 protein levels in
tumors induced in SCID mice. Results indicated that
miR19b-3p inhibitor group presented a significantly higher
SMAD4 expression pattern (Fig. 6d). These results
indicated that miR-19b-3p downregulation led to a significant
reduction in the proliferation ability of colon cancer cells
in vivo and miR-19b-3p was inversely correlated with
SMAD4 during tumorigenesis.
SMAD4 is targeted by miR-19b-3p in colon cancer
In order to investigate the direct target genes of
miR19b-3p that could explain the phenotype resulting from
miR-19b-3p upregulation in colon cancer, the miRTarBase
database (http://mirtarbase.mbc.nctu.edu.tw/), which provides
the most current and comprehensive information of
experimentally validated miRNA-target interactions was
utilized. SMAD4, PRKACB, ATM, CREB3L2, EGLN3,
JUN, NR3C1, WEE1, RASSF1, and TGFBR2 were
validated as candidate targets of miR-19b-3p in replicate
experiments in SW480 and RKO cells by using qRT-PCR.
SMAD4 was the top candidate gene, which was
significantly upregulated in SW480 cells transfected with the
miR-19b-3p inhibitor when compared with negative
control SW480 cells (Fig. 7a, top). The same pattern was
observed in RKO cells (Fig. 7a, bottom). Subsequent western
blotting analysis were performed and confirmed that
SMAD4 protein levels were significantly overexpressed in
transfected miR-19b-3p inhibitor colon cancer cells,
consistent with the results of qRT-PCR (Fig. 7b). In addition,
miRTarBase database was queried to search for binding
sites of miR-19b-3p in the 3′-UTR of SMAD4 (Fig. 7c).
Results indicated that SMAD4 possesses a putative
miR19b target sequence, which matched at 7mer-m8. To
verify that SMAD4 is directly targeted by miR-19b-3p, the
3′-UTR of SMAD4 containing a potential binding site for
miR-19b-3p was cloned into a luciferase reporter plasmid
and a dual luciferase reporter assay was performed. A
segment of pmirGLO-SMAD4 3′-UTR-wt (wild type),
pmirGLO-SMAD4 3′-UTR-mut (mutant), and
pMirGLOVector were transfected together with miR-19b-3p mimics
or miRNA mimics negative control into SW480 cells.
Results indicated that miR-19b-3p significantly inhibited
luciferase activity in the SMAD4 3′-UTR-wt transfected cells.
Meanwhile, no change in luciferase activity was observed
when the cells were transfected with the pMirGLO-Vector
lacking the miR-19b-3p binding sequence (Fig. 7d).
Furthermore, we carried out ISH and
immunohistochemistry using the TMA to investigate whether
miR19b-3p inversely correlated with SMAD4 expression.
Results indicated that patients whose localized colon
tumors were miR-19b-3p positive presented a
significantly lower SMAD4 expression pattern (Fig. 7e and
Additional file 3: Table S3).
miR-19b-3p mediates proliferation and resistance to
oxaliplatin-based chemotherapy via SMAD4
A significant increase of SMAD4 protein levels was
detected upon miR-19b-3p inhibition in SW480 and
RKO cell lines (Fig. 8a). In comparison with negative
control cells, miR-19b-3p inhibitor-transfected SW480
and RKO cells had an impaired proliferation ability.
Importantly, this phenotype could be reversed by transfection
of SMAD4 shRNA (P < 0.05, Fig. 8b and c). In addition,
miR-19b-3p downregulation promoted apoptosis when
cells were treated with oxaliplatin compared with
negative control cells. Cell apoptosis could also be reversed
by transfection of SMAD4 shRNA (P < 0.05, Fig. 8d–f ).
Together, these data suggest that miR-19b-3p-mediated
proliferation and resistance to oxaliplatin-based
chemotherapy are dependent on reduced SMAD4 expression levels.
Colon cancer is initiated by aberrant processing of genetic
information due to genetic changes involving tumor
suppressor genes and oncogenes, or altered epigenetic
mechanisms manifested in global or local changes of chromatin
]. Despite the continuous development of
novel tumor targeting therapeutics, chemoresistance
remains a challenge for the treatment of colon cancer [
At present, researchers have made great progress in the
area of chemoresistance-associated miRNAs in colon
cancer. However, the identification of novel miRNAs is
still pivotal in colon cancer therapy [
Our research used the IPA database to identify miRNAs
specifically expressed in colon cancer. The 7 upregulated
miRNAs were selected for further study, including
miR19b-3p, miR-155-5p, miR-17-5p, miR-183-5p, miR-25-3p,
miR-21-5p, and miR-196a-5p. Several of them are
associated with various human cancers. For example, miR-155-5p
is deregulated in colorectal cancer, osteosarcoma, and
triple-negative breast cancer [
annotations of the predicted target genes of these miRNAs were
obtained from R annotation packages, including GO
biological process. Genes in enriched functional terms were
used to construct a miRNA-target network. In this network
diagram, many genes were closely related with colon cancer
progression such as PTEN, STAT3, FOXO1, and SMAD4
]. The biological function analysis of the predicted
target genes based on IPA and GO annotation enabled us
to comprehensively understand their functional roles in
colon cancer progression. The predicted target genes were
significantly involved in many biologic processes relevant to
cancer such as apoptosis, cell proliferation, and cell cycle
arrest. These bioinformatics analyses suggested that the
predicted target genes may control broad biological
functions associated with colon cancer. miR-19b-3p was
identified as the pivotal oncogenic component of the miR-17-92
cluster of miRNAs because of its role in promoting
sustained cell survival and recognized as an onco-miR in some
types of cells and tissues [
]. Upregulated miR-19b-3p
plays a critical role in the tumorigenesis of Em-Myc-driven
B-cell lymphomas [
]. We found that high expression of
miR-19b-3p in colon cancer tissues was significantly
associated not only with tumorhistologic grade, but also with
AJCC stage. Furthermore, Kaplan-Meier and univariate
Cox proportional hazard regression analyses indicated that
patients with high miR-19b-3p expression displayed a lower
five-year OS and DFS. Multivariate analyses indicated that
miR-19b-3p expression in colon cancer was an independent
prognostic factor of survival. We also observed that
miR19b-3p downregulation had no effect on invasion, but
correlated with reduced cell proliferation and decreased
oxaliplatin-based chemoresistance. The proliferation ability
is associated with tumor size and the invasion ability is
correlated with distant metastasis. In other words,
miR19b-3p does not act as an enhancer of cancer metastasis,
but of tumorigenesis in vitro. We speculate that this
may be because of the complex tumor microenvironment
in vivo. Recent molecular and cellular biology studies
indicated that tumor growth and metastasis are not only
determined by cancer cells, but are also affected by various
stromal cells [
]. During metastasis, tumor cells need to
go through a series of complex steps, during which they
continually adapt to different microenvironments [
Thus, a stable in vivo model of colon cancer metastasis
will be necessary to further understand the underlying
mechanisms in our future study.
In this study, we demonstrated that SMAD4 is the direct
target of miR-19b-3p in colon cancer. As an important
mediator of the TGF-β signaling pathway, SMAD4 plays
an important role in suppressing tumor progression and
promoting apoptosis of tumor cells [
expression of SMAD4 is associated with attenuated sensitivity to
chemotherapy and poor prognosis of patients with colon
cancer in several clinical studies [
]. The inverse
relationship between miR-19b-3p and SMAD4 in colon cancer
cell lines and tumor samples was investigated in our study.
Importantly, since SMAD4 is targeted by this miRNA, we
showed that the proliferation ability and resistance to
oxaliplatin-based chemotherapy is reversed by the
transfection of SMAD4 shRNA.
In summary, our study shows that miR-19b-3p
overexpression plays an important role in promoting colon cancer
progression through enhanced proliferation and reduced
chemosensitivity. miR-19b-3p may be a clinical useful
prognostic molecular biomarker for prognosis and a target for
chemotherapy in colon cancer.
Additional file 1: Table S1. Oligonucleotide Sequence for the primers
used in the study. (DOCX 25 kb)
Additional file 2: Table S2. miRNAs specifically expressed in colon
cancer after being screened by IPA. (DOCX 26 kb)
Additional file 3: Table S3. The association between miR-19b-3p and
SMAD4 expression. (DOCX 19 kb)
AJCC: American Joint Committee on Cancer; CCK8: Cell counting kit-8;
DFS: Disease-free survival; GO: Gene Ontology; IPA: Ingenuity Pathways
Analysis; OS: overall survival; TMA: Tissue microarray
This project was supported by the National Natural Science Foundation of
China (No. 81272744, 81302111, 81560394 and 81460369) and the Scientific
Research Project grant funded by Ningxia high school (NGY2015096).
Conception and design: TJ, LY, ZH, JF. Development of methodology: TJ, LY,
ZH, YL. Acquisition of data: TJ, ZH, YL, YY. Analysis and interpretation of data:
TJ, ZH, YL, YY. Writing, review, and/or revision of the manuscript: TJ, LY, ZP,
JF. All authors read and approved the final manuscript.
The study was approved by the ethics committee of the General Hospital of
Ningxia Medical University. All animal procedures were performed according
to national guidelines and approved by the Institutional Committee of
Shanghai Jiao Tong University School of Medicine for Animal Research.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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