Circular RNA_LARP4 inhibits cell proliferation and invasion of gastric cancer by sponging miR-424-5p and regulating LATS1 expression
Zhang et al. Molecular Cancer
Circular RNA_LARP4 inhibits cell proliferation and invasion of gastric cancer by sponging miR-424-5p and regulating LATS1 expression
Jing Zhang 0 1 3
Hui Liu 0 1 3
Lidan Hou 2
Ge Wang 1 3
Rui Zhang 1 3
Yanxia Huang 1 3
Xiaoyu Chen 1 3
Jinshui Zhu 1 3
0 Equal contributors
1 Department of Gastroenterology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital , No. 600 Yishan Road, Shanghai 200233 , China
2 Department of Gastroenterology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai , China
3 Department of Gastroenterology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital , No. 600 Yishan Road, Shanghai 200233 , China
Background: Non-coding RNAs (ncRNAs) have been shown to regulate gene expression involved in tumor progression of multiple malignancies. Our previous studies indicated that large tumor suppressor kinase 1 (LATS1), a core part of Hippo signaling pathway, functions as a tumor suppressor in gastric cancer (GC). But, the underlying molecular mechanisms by which ncRNAs modulate LATS1 expression in GC remain undetermined. Methods: The correlation of LATS1 and has-miR-424-5p (miR-424) expression with clinicopathological characteristics and prognosis of GC patients was analyzed by TCGA RNA-sequencing data. A novel circular RNA_LARP4 (circLARP4) was identified to sponge miR-424 by circRNA expression profile and bioinformatic analysis. The binding site between miR-424 and LATS1 or circLARP4 was verified using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The expression and localization of circLARP4 in GC tissues were investigated by fluorescence in situ hybridization (FISH). MTT, colony formation, Transwell and EdU assays were performed to assess the effects of miR-424 or circLARP4 on cell proliferation and invasion. Results: Increased miR-424 expression or decreased LATS1 expression was associated with pathological stage and unfavorable prognosis of GC patients. Ectopic expression of miR-424 promoted proliferation and invasion of GC cells by targeting LATS1 gene. Furthermore, circLARP4 was mainly localized in the cytoplasm and inhibited biological behaviors of GC cells by sponging miR-424. The expression of circLARP4 was downregulated in GC tissues and represented an independent prognostic factor for overall survival of GC patients. Conclusion: circLARP4 may act as a novel tumor suppressive factor and a potential biomarker in GC.
Circular RNA_LARP4; miR-424-5p; LATS1; Invasion; Gastric cancer
Gastric cancer (GC) is the fourth common
gastrointestinal malignancy and the third leading cause of
cancerrelated deaths worldwide [
]. Despite a steady decline in
GC incidence and mortality rates in recent years due to
improved nutritional compositions and H. pylori
], this disease still yields a great threat to human
health, leading to a poor prognosis for GC patients, with
a 5-year overall survival (OS) rate of less than 30% duo
to tumor metastasis and recurrence [
]. Therefore, to
discover novel molecular mechanisms and critical
signaling pathways, activated or inactivated in GC, is required
for developing effective therapeutic strategies for
anticancer therapy in GC.
Hippo signaling pathway was previously known to
control organ size and growth, and accumulating
evidence shows that this pathway acts a pivotal role in the
regulation of cell proliferation, metastasis and
]. Large tumor suppressor kinase 1 (LATS1) as
a core member of this pathway dominates breast cell fate
 and modulates liver progenitor cell proliferation and
]. Decreased LATS1 expression is
associated with unfavorable prognosis and contributes to
glioma progression . Our previous study showed that
loss of LATS1 is correlated with poor survival and
recurrence and promotes growth and metastasis of GC cells
]. But, LATS1/2 is proved to inhibit tumor immunity
and provides a concept for targeting LATS1/2 in cancer
Considerable studies highlight the regulatory
mechanisms by which non-coding RNAs (ncRNAs) participate
in the development of diseases including cancer [
microRNAs (miRNAs), an evolutionarily conserved group
of small regulatory ncRNAs, negatively modulate the
expression of protein-coding genes [
]. Moreover, some
miRNAs are implicated in carcinogenesis by regulating
Hippo signaling. For example, miR-130a-YAP positive
feedback loop facilitates organ size and tumorigenesis
], while miR-129 suppresses ovarian cancer survival via
repression of Hippo signaling effectors YAP and TAZ [
miR-135b, miR-31 and miR-181c function as oncogenes
boosting tumor metastasis and chemo-resistance by
targeting Hippo signaling members MST1, LATS2, MOB1
and SAV1 [
], thereby providing a novel mechanism
for Hippo signaling inactivation in cancer.
Circular RNAs (circRNAs) as a novel type of ncRNAs
derived from exons, introns or intergenic regions have a
covalently closed continuous loop, display cell or
tissuespecific expression and are conserved across species due
to resistance to RNase R [
], The expression of
circRNAs is highly stable in comparison with their linear
counterparts, and is predominantly localized in the
cytoplasm, indicating important functions for circRNAs in
human diseases [
]. Emerging evidence shows that
some circRNAs as miRNA sponges modulate gene
transcription and interact with RNA binding proteins (RBPs)
involved in tumorigenesis [
]. ciRS-7 serves as
miR7 sponge regulating the expression of several oncogenes
, and circHIPK3 as miR-124 sponge suppresses cell
proliferation in multiple caners [
]. circRNA expression
profiles reveal a tumor-promoting role of
TCF25-miR103a-3p/miR-107 axis in bladder cancer [
circRNA_001569/miR-145 axis in colorectal cancer [
providing novel promising markers for cancer diagnosis
In the present study, we identified an oncogenic
miR424, which was upregulated in GC tissues and was
negatively correlated with LATS1 expression. High expression
of miR-424 or low expression of LATS1 was closely
associated with pathological staging, poor survival and
recurrence of GC patients, and miR-424 overexpression
promoted cell growth and invasion by targeting LATS1
gene. Furthermore, we characterized a circRNA derived
from LARP4 gene locus, termed as circLARP4, which
was downregulated in GC tissues, and suppressed cell
proliferation and invasion by sponging miR-424 and
upregulating LATS1 gene. Therefore, circLARP4 might
act as a tumor suppressive factor and an independent
prognostic factor for survival of GC patients.
The clinical and pathological data of 387 cases of GC
patients and 41 adjacent normal tissues as well as the
relative expression levels of LATS1 and miRNAs
(hasmiR-16-5p, has-miR-15a-5p, has-miR-15b-5p,
has-miR590-3p and has-miR-424-5p) were downloaded from
The Cancer Genome Atlas 2015 RNA sequencing
database (http://xena.ucsc.edu/getting-started/). The
human tissue microarray of 80 paired GC patients (Cat No.
STC1602) was purchased from the shanghai Superbiotek
Pharmaceutical Technology Co., Ltd. (Shanghai, PR,
China). The protocols used in our study were approved
by the Ethics Committee of Shanghai Jiao Tong
University Affiliated Sixth People’s Hospital. The GC patients’
specimens were classified according to the 2004 WHO
criteria and TNM staging system, and
clinicopathological characteristics of GC patients from TCGA and tissue
microarray were shown in Additional file 1: Table S1–2.
Identification of miRNAs targeting LATS1 gene in cancer tissues
We identified the miRNAs that target LATS1 gene in
cancer by using the StarBase v2.0
(http://starbase.sysu.edu.cn) and the strict screening conditions including two
prediction algorithms (Pctar and miRanda), very high
stringency (>5) and being expressed in at least three
cancer types were limited to predict the miRNAs targeting
Normal human gastric epithelial cell line GES-1 and GC
cell lines (SGC-7901, MKN-45, MKN-28, HGC-27,
MGC803, AGS, BGC-823) were from Digestive Disease
Laboratory of Shanghai Sixth People’s Hospital. Cells were
cultured in Dulbecco’s Modified Eagle medium (DMEM)
medium supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 100 U/ml of penicillin, and 100 μg/ml
of streptomycin (HyClone). Cells in this medium were
placed in a humidified atmosphere containing 5% CO2 at
37 °C. All cells were used for study within 6 months.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol and reverse
transcription was performed using M-MLV and cDNA
amplification using the SYBR Green Master Mix kit (Takara,
Otsu, Japan). In addition, total RNA was isolated using a
High Pure miRNA isolation kit (Roche) and RT-PCR
using a TaqMan MicroRNA Reverse Transcription kit
(Life Technologies). The nuclear and cytoplasmic
fractions were isolated using NE-PER Nuclear and
Cytoplasmic Extraction Reagents (Thermo Scientific). The
primers were listed in Additional file 1: Table S3.
Western blotting analysis
HGC-27 and MKN-28 cells were harvested and extracted
using lysis buffer (100 mM Tris-HCl, 2% SDS, 1 mM
Mercaptoethanol, 25% Glycerol). Cell extracts were boiled in
loading buffer and equal amount of cell extracts were
separated on 15% SDS-PAGE gels. Separated protein bands
were transferred into polyvinylidene fluoride (PVDF)
membranes. The primary antibodies-anti-LATS1 (ab70561,
Rabbit polyclonal antibody, Abcam, Cambridge, MA, USA),
anti-YAP (ab52771, Rabbit monoclonal antibody, Abcam,
Cambridge, MA, USA), anti-p-YAP (S127) (ab76252, Rabbit
monoclonal antibody, Abcam, Cambridge, MA, USA) and
anti-GAPDH (ab153802, Rabbit polyclonal antibody, Abcam,
Cambridge, MA, USA) were diluted at a ratio of 1:1000
according to the instructions and incubated overnight at 4 °C.
Horseradish peroxidase-linked secondary antibodies were
added at a dilution ratio of 1:10,000, and incubated at room
temperature for 1 h. The membranes were washed with PBS
for three times and the immunoreactive bands were
visualized using ECL-PLUS/Kit (GE Healthcare, Piscataway, NJ,
USA) according to the kit’s instruction.
Luciferase reporter assay
HGC-27 and MKN-28 cells were seeded into 96-well
plates and were co-transfected with a mixture of 60 ng
of firefly luciferase reporter, 6 ng of pRL-CMV Renilla
luciferase reporter, and miR-424 mimic or inhibitor.
After 48 h of incubation, the firefly and Renilla luciferase
activities were measured with a dual-luciferase reporter
assay (Promega, Madison, WI, USA).
Plasmid, siRNAs and miRNA mimic and inhibitor
Plasmid mediated LATS1 or circLARP4 overexpression
vector, siRNA targeting LATS1 or circLARP4 vector,
miR-424 mimic and inhibitor were purchased from
Genechem (Shanghai, PR, China) and an empty vector
used as a control. The siRNA sequences were shown as
below, si-LATS1: ATCCTCGACGAGAGCAGA and
sicircLARP4: GGGCAGGCTCCCTTTCCCAAT. HGC-27
and MKN-28 cells were planted in 6-well plates 24 h
prior to si-LATS1, si-circLARP4, miR-424 mimic or
inhibitor transfection with 50–60% confluence, and then
were transfected with Lipofectamine 2000 (Invitrogen,
Carlsbad, CA, USA) according to the manufacture
Colony formation assay
HGC-27 and MKN-28 cells were trypsinized, and 1 × 103
cells were plated in 6-well plates and incubated at 37 °C
for 7 days. Colonies were dyed with dyeing solution
containing 0.1% crystal violet and 20% methanol. Cell colonies
were then counted and analyzed.
circRNA microarray analysis
Total RNA from three GC and adjacent normal tissues
was quantified using the NanoDrop ND-1000. The
sample preparation and microarray hybridization were
performed based on the Arraystar’s standard protocols.
Briefly, total RNAs were digested with RNase R to
eliminate linear RNAs and enrich circular RNAs. Then, the
enriched circular RNAs were amplified and transcribed
into fluorescent cRNA utilizing a random priming
method (Arraystar Super RNA Labeling Kit; Arraystar).
The labeled cRNAs were hybridized onto the Arraystar
Human circRNA Array (8x15K, Arraystar). After having
washed the slides, the arrays were scanned by the
Agilent Scanner G2505C.
Actinomycin D and RNase R treatment
Transcription was prevented by the addition of 2 mg/ml
Actinomycin D or DMSO (Sigma-Aldrich, St. Louis,
MO, USA) as the negative control. Total RNA (2 μg)
was incubated for 30 min at 37 °Cwith 3 U/μg of RNase
R (Epicentre Technologies, Madison, WI, USA). After
treatment with Actinomycin D and RNase R, the RNA
expression levels of LARP4 and circLARP4 were
detected by qRT-PCR.
5-Ethynyl-20-deoxyuridine (EdU) incorporation assay
The EdU assay was carried out with a Cell-Light EdU
DNA Cell Proliferation Kit (RiboBio, Shanghai, PR,
China). 1 × 104 cells were seeded in 96-well plate. After
incubation with 50 mM EdU for 2 h, the cells were fixed
in 4% paraformaldehyde and stained with Apollo Dye
Solution. Hoechst-33,342 was used to stain the nucleic
acid within the cells. Images were acquired with an
Olympus FSX100 microscope (Olympus, Tokyo, Japan),
and the percentage of EdU-positive cells was calculated.
RNA immunoprecipitation (RIP)
RIP assay was carried out by using a Magna RIP
RNABinding Protein Immunoprecipitation Kit (Millipore)
according to the manufacturer’s instructions. Antibodies
for RIP assays against AGO2 and IgG were purchased
from Abcam (ab5072, Rabbit polyclonal antibody,
Cambridge, MA, USA).
Cell viability and transwell invasion assays
Cell viability and Transwell assays were performed as
previously described [
RNA fluorescence in situ hybridization (FISH)
Oligonucleotide modified probe sequence for human
TAGTCC) was applied for FISH. First, the probe of
circLARP4 was marked with DIG-UTP (Roche,
11,209,256,910) for RNA labeling. The cell suspension
was pipetted onto autoclaved glass slides, which were
washed with PBS and fixed in 4% paraformaldehyde. After
dehydration with 70, 95 and 100% ethanol, hybridization
was carried out at 37 °C overnight in a dark moist
chamber. After hybridization, slides were washed three times in
50% 60 ml formamide/2X SSC for 5 min, and was
incubated with anti-DIG-HRP(PerkinElmer, NEF832001EA)at
4 °C overnight, After being washed for 3 times for 10 min
at room temperature, the slides were incubated with TSA
fluorescent signal reaction solution(PerkinElmer, NEL701
001KT, TSA Fluorescein system)for 30 min and was sealed
with tablets containing DAPI. The images were acquired
using a fluorescence microscopy (Leica, SP8 laser confocal
microscopy). The analysis software Image-pro plus 6.0
(Media Cybernetics, Inc., Rockville, MD, USA) was
applied to acquire the Immunofluorescence Accumulation
Optical Density (IOD) for evaluating the expression level
of circLARP4 in GC tissues.
Statistical analyses were carried out by using SPSS 20.0
(IBM, SPSS, Chicago, IL, USA) and GraphPad Prism.
Student’s t-test or Chi-square test was used to assess the
statistical significance for comparisons of two groups.
The Pearson’s correlation coefficient analysis was used
to analyze the correlations. Overall survival (OS) was
defined as the interval between the dates of surgery and
death and OS and disease-free survival (DFS or
recurrence) curves were analyzed with the Kaplane-Meier
method and log-rank test. Univariate analysis and
multivariate models were performed by using a Cox
proportional hazards regression model. Receiver operating
characteristic (ROC) curves were obtained using cutoff
finder online software (http://molpath.charite. de/cutoff/
load.jsp). P < 0.05 was considered statistically significant.
MiR-424-5p was negatively correlated with LATS1 expression in GC
Our previous studies showed that LATS1 is
downregulated in GC compared with the pair-matched normal
]. We further validated a decreased expression
of LAST1 in human gastric adenocarcinoma (GAC)
tissues (n = 387) and adjacent normal tissues (n = 41) as
well as in paired GAC tissues (n = 41) by using The
Cancer Genome Atlas (TCGA) sequencing data (Fig. 1a).
To dissect the molecular mechanism of LATS1
downregulation in GC, we first assessed the genetic or
epigenetic dysregulation of LATS1 in GC. But, we discovered
little evidence regarding the dysregulation of LATS1 at
the genetic (Additional file 2: Figure S1a and b) and
methylation levels (Additional file 2: Figure S1c),
suggesting that genetic alterations (amplification, deletion,
mutation and copy number deletion) and methylation
modification were not the main cause of the
downregulation of LATS1 in GC. Numerous studies show that
miRNAs exhibit a pivotal role in GC development by
repressing the expression of their target genes [
indicating that LATS1 might be regulated by miRNAs in
GC. Then, we utilized the StarBase v2.0, Pictar and
miRanda software to forecast the potential miRNAs that
target LATS1 gene 3′ UTR, and identified 5 miRNAs
that could bind to LATS1 gene 3′ UTR with very high
stringency (Fig. 1b). Furthermore, we investigated the
expression levels of these miRNAs in GAC, which were
highly expressed in GAC (n = 387) compared with
adjacent normal tissues (n = 41) as well as in paired GAC
(n = 41) (Fig. 1c1–5). The correlation analysis showed
that, except for the has-miR-15b-5p (Additional file 2:
Figure S1d), the other miRNAs displayed the negative
correlation with LATS1 expression, of which
miR-4245p (miR-424) had the strongest correlation with LATS1
expression (r = −0.2598, P < 0.0001) and was selected
for further observation.
MiR-424 and LATS1 expression were associated with clinicopathological characteristics and prognosis of GC patients
To evaluate whether the expression levels of LATS1 and
miR-424 were associated with clinical and pathological
characteristics and prognosis of GC patients, as shown
in Fig. 2a, based on the cutoff values of LATS1 and
miR424, which were obtained according to their expression
levels, OS time and OS status in GAC tissues, the 315
GAC patients were divided into two groups: LATS1 high
expression and LATS1 low expression or miR-424 high
expression and miR-424 low expression. Receiver
operating characteristic (ROC) curve and area under curve
(AUC) were determined to assess the sensitivity and
specificity of the survival prediction. The sensitivity,
specificity and AUC of LATS1 were 37.1%, 75.7% and 0.54
and those of miR-424 were 25.7%, 90.2% and 0.52,
indicating that LATS1 and miR-424 might be the potential
biomarkers for OS of GAC patients.
As indicated in Additional file 1: Table S4, LATS1 low
expression or miR-424 high expression was positively
correlated with pathological stage (P = 0.004; P = 0.036),
but harbored no association with other clinical
parameters (P > 0.05). Kaplan Meier analysis illustrated that
GAC patients with LATS1 low expression or miR-424
high expression developed more frequent recurrence
(Fig. 2b and c), but had no significant difference in OS
compared with the patients with LATS1 high expression
or miR-424 low expression (Additional file 3: Figure S2a
and b). According to the pathological stage, the patients
of early stage (stage I + II) or late stage (stage III + IV)
with miR-424 high expression possessed the higher
recurrence rate compared with those with miR-424 low
expression (Fig. 2d), but the patients of early stage (stage
I + II) or late stage (stage III + IV) with LATS1 low
expression had no obvious difference in tumor recurrence
compared with those with LATS1 high expression
(Additional file 3: Figure S2c). To define whether LATS1
or miR-424 expression was independent of other risk
factors related to the recurrence of GAC, multivariate
analyses were performed by using a Cox proportional
hazard model. As shown in Additional file 1: Table S5, 6,
in the univariate analysis, gender, LATS1 low expression
and miR-424 expression were associated with recurrence
of GC patients, but in the final multivariate Cox
regression model, it was miR-424 not LATS1 expression that
represented an independent prognostic factor for
recurrence of GAC.
Furthermore, by using the online bioinformatics tool
Kaplan-Meier plotter [
], we found that GC patients
with LATS1 low expression developed poorer survival
(Fig. 2e) and more frequent recurrence (Fig. 2g). In
addition, the patients of stage I or stage III with LATS1
low expression had poorer survival (Fig. 2f ) and those of
stage I or stage IV harbored higher recurrence (Fig. 2h)
compared with those with LATS1 high expression, but
the patients with LATS1 low expression had no
significant difference in OS for stage II or stage IV (Additional
file 3: Figure S2d) and in recurrence for stage II or stage
III (Additional file 3: Figure S2e) compared with those
with LATS1 high expression.
LATS1 Was validated as a target gene of miR-424
Having confirmed the negative correlation of miR-424
with LATS1 expression in GC tissues, we were curious
about whether LATS1 was a target gene of miR-424 in
GC cells. We first utilized the mirPath v.3 software to
verify whether miR-424 was related with LATS1
signaling pathway. Consequently, KEGG enrichment analysis
demonstrated that miR-424 was principally enriched by
Hippo signaling pathway in which LATS1 was a nuclear
member (Fig. 3a). Then, we examined the expression
levels of miR-424 and LATS1 in GC cell lines by
qRTPCR, which demonstrated that LATS1 had lower
expression, while miR-424 exhibited higher expression and
negative correlation with LATS1 expression in all GC
cell lines compared with the GES-1, of which MKN-28
cell line had the relatively higher miR-424 expression
level but HGC-27 cell line had the relatively lower
miR424 expression level (Fig. 3b). Thus, miR-424 mimic
(60 μM) and miR-424 inhibitor (80 μM) were
respectively transfected into HGC-27 cells with miR-424 low
expression and MKN-28 cells with miR-424 high
expression. After transfection for 48 h, qRT-PCR and western
blot analysis displayed substantially increased expression
of miR-424 and decreased expression of LATS1 in
miR424 mimic group in HGC-27 cells (Fig. 3c), and
indicated noticeably decreased expression of miR-424 and
increased expression of LATS1 in miR-424 inhibitor
group in MKN-28 cells (Fig. 3d). Luciferase reporter
vectors containing the wild type or mutant LATS1
3’UTR (Fig. 3e) and miR-424 mimic or inhibitor were
co-transfected into HGC-27 or MKN-28 cells.
Intriguingly, the luciferase activity of wild type LATS1
3’UTR evidently decreased in miR-424 mimic group
in HGC-27 cells (Fig. 3f ), but increased in miR-424
inhibitor group in MKN-28 cells compared with the
NC group (Fig. 3g). However, the luciferase activity of
mutant LATS1 3’UTR had no difference between
miR-424 mimic or inhibitor group and NC group.
Ectopic expression of miR-424 promotes GC cell growth and invasion via targeting LATS1 gene
To further observe the effects of miR-424 on LATS1
expression in GC cells, we performed the functional
experiments such as MTT, cell colony formation and
Transwell assays. First, the expression level of LATS1
was examined after transfection with miR-424 mimic
and (or) LATS1 in HGC-27 cells, and miR-424
inhibitor and (or) sh-LATS1 in MKN-28 cells indicated by
qRT-PCR (Additional file 4: Figure S3a). Then, the
promoting effects of miR-424 mimic on cell
proliferation (Fig. 4a), colony formation (Fig. 4c) and invasive
potential (Fig. 4e) were reversed by co-transfection of
LATS1 overexpression vector in HGC-27 cells, but
the inhibitory effects of miR-424 inhibitor on cell
proliferation (Fig. 4b), colony formation (Fig. 4d) and
invasive potential (Fig. 4f ) were rescued by
cotransfection of LATS1 shRNA vector in MKN-28
cells. These results suggested that miR-424 might
promote GC growth and invasion by targeting LATS1
Identification and characteristics of circLARP4 in GC cells
Mounting evidence shows that circRNAs as a novel type of
ncRNAs sponge miRNAs, regulate gene transcription and
interact with RBPs involved in tumorigenesis [
identify the circRNAs that sponge miR-424 in GC, we
applied the circRNA expression profile and circBase and
microRNA.org software to screen out 30 circRNAs that
could sponge and bind to the miR-424 (Fig. 5a, Additional
file 1: Table S7). Given the upregulation of miR-424 in GC,
15 circRNAs that were downregulated in GC were selected
for further analysis (Fig. 5a, Additional file 1: Table S7).
According to the restrictive conditions (P < 0.001, Fold
change > 2), among 15 circRNAs, only has_circ_101057
conformed to this requirement (Additional file 1: Table S7).
We noted that circ_101057 (chr12:50,848,096–50,855,130,
Fig. 5b) is derived from exon 9, 10 regions within La
ribonucleoprotein domain family member 4 (LARP4) locus,
which is located on chromosome 12q13.12. We termed
circ_101057 as circLARP4, whose genomic sequence is
7034 nt and spliced mature sequence length is 317 nt. The
genomic position reveals that the ninth and tenth exons
from the LARP4 gene are intermediated by long introns
(Fig. 5b). According to the qRT-PCR analysis, compared
with the linear LARP4, circLARP4 gave rise to resistance to
digestion induced by RNase R exonuclease, indicating that
circLARP4 harbors a loop structure (Fig. 5c). We next
observed the stability and localization of circLARP4. After
treatment with Actinomycin D, an inhibitor of transcription
at the indicated time points, total RNA was separated from
HGC-27 and MKN-28 cells. As a result, qRT-PCR analysis
showed that the transcript half-life of circLRP4 exceeded
24 h, while that of linear LARP4 displayed about 6 h in
HGC-27 and MKN-28 cells (Fig. 5d), indicating that
circLARP4 is highly stable in GC cells.
Cytoplasmic and nuclear RNA analysis by qRT-PCR
showed that circLARP4 was preferentially localized in the
cytoplasm in HGC-27 and MKN-28 cells (Fig. 5e).
Furthermore, we utilized FISH to assess circLARP4
expression level and localization in GC tissue cells, and found
that circLARP4 had low expression in GC tissues
compared with the adjacent normal (Fig. 5f ) and the green
fluorescent distribution position indicated that circLARP4
was mainly localized in the cytoplasm of both GC and
normal tissue cells (Fig. 5g). Collectively, these results
suggest that circLARP4 is a highly stable and cytoplasmic
circRNA derived from the LARP4 gene locus.
The effects of circLARP4 on GC cell proliferation and invasion in vitro
The special characteristics of circLARP4 spurred our
interest in assessing the biological functions of
circLARP4 in GC cells. We first detected the expression
level of circLARP4 in GC cell lines by qRT-PCR and
found circLARP4 was downregulated in GC cell lines
compared with GES-1 cells but had no correlation with
the expression of miR-424 and LATS1 in GC cells
(Additional file 4: Figure S3b). Then, we designed the
circLARP4 overexpression and interference sequences
against the back-splicing sequence of circLARP4 (Fig. 6a).
After circLARP4 overexpression or siRNA vector was
respectively transfected into HGC-27 and MKN-28 cells
for 48 h, their transfection efficiency was determined as
shown in Fig. 6b. Subsequent cell proliferation and
colony formation assays revealed that ectopic expression of
circLARP4 inhibited cell growth and colony-forming
capacity of GC cells, while knockdown of circLARP4
reversed these effects (Additional file 4: Figure S3c, d).
EdU incorporation assay showed that the proliferation of
HGC-27 cells was impaired by transfection with
circLARP4 overexpression vector, but was strengthened by
knockdown of circLARP4 (Fig. 6c). Transwell invasion
assay demonstrated that cell invasive potential was
weakened by circLARP4 overexpression, but was
enhanced by knockdown of circLARP4 (Fig. 6d). Taken
together, these results imply that that circLARP4 may be a
tumor suppressive factor in GC cells.
circLARP4 Acts as a miRNA sponge for miR-424 in GC
Given that circRNAs can act as miRNAs sponge and
circLARP4 is stable in the cytoplasm, we first applied the
ComiR prediction tool as well as the circLARP4 genomic
sequence to predict 5 miRNAs that have the potential to
bind to circLARP4 (Fig. 7a and Additional file 5: Figure
S4a). We constructed a circLARP4 fragment and
incorporated it into downstream of the luciferase reporter gene,
and hypothesized that circLARP4 related 5 miRNAs could
reduce the luciferase activity of circLARP4. We then
carried out a luciferase assay using these 5 miRNAs. The
luciferase reporter vector for pMIR-Luc-circLARP4 was
co-transfected with each miRNA mimic into HEK-293 T
cells. Compared with the negative control (NC), miR-424
lowered the luciferase reporter activity by approximately
75% (Fig. 7b), indicating that miR-424 might have the
greater potential to bind to circLARP4 compared with
other 4 miRNAs. Afterwards, we examined the mutual
regulation between circLARP4 and miR-424 by qRT-PCR
analysis, which showed that circLARP4 overexpression
reduced the expression level of miR-424 in HCG-27 cells,
while circLARP4 knockdown increased the expression
level of miR-424 in MKN-28 cells (Fig. 7c1), but miR-424
mimic or inhibitor had no influence on the expression of
circLARP4 (Additional file 5: Figure S4b). Luciferase
reporter vectors containing the wild type or mutant
circLARP4 (Additional file 5: Figure S4c) and miR-424
mimic were co-transfected into HGC-27 and MKN-28
cells. Interestingly, the luciferase activity of wild type
circLARP4 significantly decreased in miR-424 mimic group
in HGC-27 cells and increased in miR-424 inhibitor group
in MKN-28 cells, compared with their NC group (Fig. 7c2).
However, the luciferase activity of mutant circLARP4 had
no difference between miR-424 group and NC group in
these two cell lines. Moreover, the luciferase activity of
wild type LATS1 3’UTR was decreased by miR-424 mimic
but increased by co-transfection with miR-424 mimic and
circLARP4 in HGC-27 cells (Additional file 5: Figure S4e),
while the luciferase activity of wild type LATS1 3’UTR
was increased by miR-424 inhibitor but decreased by
cotransfection with miR-424 inhibitor and si-circLARP4 in
MKN-28 cells (Additional file 5: Figure S4f ). However, the
luciferase activity of mutant LATS1 had no difference
between NC, miR-424 and co-transfection groups
(Additional file 5: Figure S4e, f ).
Furthermore, we used the online circular RNA
interactome to reveal a high degree of AGO2 occupancy in the
region of circLARP4 (Additional file 1: Table S8). To
confirm this result, we performed RNA
immunoprecipitation (RIP) for AGO2 in HGC-27 and MKN-28 cells
and investigated the expression level of endogenous
circLARP4 and miR-424 pulled-down from
AGO2expressed cells by qRT-PCR analysis. The results
indicated that Ago2 antibody precipitated the AGO2 protein
from the cell lysates (Fig. 7d, up panel), and circLARP4
and miR-424 were highly expressed in the AGO2 pellet
compared with those in the input control (Fig. 7d, down
panel). We also observed the expression of LATS1, a
target of miR-424 and its downstream gene YAP after
co-transfection with circLARP4 and miR-424 mimic or
si-circLARP4 and miR-424 inhibitor by qRT-PCR
(Additional file 5: Figure S4d) and western blot analysis
(Fig. 7e), indicating that circLARP4 revived LATS1 and
p-YAP expression and decreased YAP expression and
counteracted the effects of miR-424 on their expression
in HGC-27 cells. Reversely, knockdown of circLARP4
reduced LATS1 and p-YAP expression and increased
YAP expression and reversed the effect of miR-424
inhibitor on their expression in MKN-28 cells.
Moreover, ectopic expression of circLARP4 attenuated the
proliferative effect caused by miR-424 in HGC-27
cells, and knockdown of circLARP4 weakened the
anti-proliferative effect induced by miR-424 inhibitor
in MKN-28 cells (Fig. 7f ). Altogether, these results
inferred that circLARP4 could sponge miR-424 and
inhibit its activity in GC cells.
circLARP4 Acts as an independent prognostic factor for
OS of GC patients
FISH analysis showed that the expression level of
circLARP4 was markedly downregulated in GC tissues
compared with the pair-matched normal tissues (Fig. 8a).
In addition, we also found that circLARP4 expression
level was decreased in GC patients with tumor size
(TS) > 3 cm or stage N2 + N3 compared with those with
TS ≤ 3 cm or stage N0 + N1 (Fig. 8b). We further
analysed the correlation of circLARP4 expression level with
various clinicopathological characteristics of 80 GC
patients, who were divided into two groups-circLARP4
high expression and circLARP4 low expression
according to the cut-off value (Additional file 6: Figure S5a).
High expression of circLARP4 was found negatively
associated with the tumor size and lymphatic metastasis,
but had no correlation with other clinical and
pathological parameters (Additional file 1: Table S9).
Therefore, circLARP4 expression might be related with early
stages of this disease. Kaplan-Meier analysis showed that
GC patients or early stage patients (stageI + II) (log-rank
test, Fig. 8c) rather than late stage ones (stage II + III or
stage III) (log-rank test, Additional file 6: Figure S5b)
with circLARP4 high expression had a significantly
longer OS than those with circLARP4 low expression.
We then analysed the associations between circLARP4
expression level and the therapeutic outcomes in 72 GC
patients treated with adjuvant chemotherapy of
oxaliplatin and 5-Fu. These patients or early stage ones (Fig. 8d)
rather than late stage ones (stage II + III or stage III)
(Additional file 6: Figure S5c) with circLARP4 high
expression had more favorable therapeutic outcome than
those with circLARP4 low expression. As for the
patients without chemotherapy, circLARP4 high or low
expression had no impact on the survival of these patients
(Additional file 6: Figure S5d).
To define whether the ability of circLARP4 to predict
survival was independent of other clinicopathological
factors of GC patients, univariate and multivariate Cox
proportional hazards analyses revealed that circLARP4
level and lymphatic metastasis were independent
prognostic factors for OS of GC patients (Additional file 1:
We have previously reported that LATS1 expression was
downregulated in GC tissues [
]. Here, we verified the
decreased expression of LATS1 in GC tissues by using
the large sample size of TCGA sequencing data. Despite
individual study having shown that miR-21 enhances
radio-resistance of cervical cancer by targeting LATS1
], we here filtered out 5 miRNAs (miR-16, miR-15a,
miR-15b, miR-590 and miR-424), which possessed very
high stringency with LATS1 gene 3’UTR. The
expressions levels of these miRNAs were upregulated in GC
tissues, and except for miR-16, other miRNAs had
negative correlation with LATS1 expression. Taken into
account the strongest correlation of miR-424 with LATS1
expression in GC tissues, we analyzed the correlation of
miR-424 and LATS1 expression with clinicopathological
characteristic and prognosis of GC patients. We found
that both of miR-424 high expression and LATS1 low
expression were associated with pathological stage, OS
and recurrence of GC patients, and miR-424 but not
LATS1 gene was an independent prognostic factor for
tumor recurrence of GC, suggesting a potential
diagnostic marker for GC patients.
In view of the tissue diversity, aberrant expressions of
miR-424 have been investigated in a variety of cancers. On
the one hand, miR-424, downregulated in hepatocellular
carcinoma (HCC) [
], cervical cancer [
esophageal carcinoma [
], inhibits cell growth and invasion
], reverses epithelial-mesenchymal transition 
and chemo-resistance [
], and strengthens the sensitivity
of chemotherapy and radiotherapy [
a tumor suppressive role in cancers. On the other
hand, miR-424 promotes tumorigenesis and
progression of prostate cancer  and reduces chemotherapy
sensitivity by inhibiting apoptosis in breast cancer [
Our present studies showed that, miR-424 mimic
stimulated cell growth and invasion, while miR-424
inhibitor reversed these effects by targeted regulation of
LATS1 gene. Additionally, elevated miR-424
expression is also associated with metastasis and poor
prognosis of non-small cell lung cancer [
accelerates gastric cancer proliferation [
]. These data
support our findings that miR-424 may harbor an
oncogenic role in GC.
Increasing evident shows that circRNAs are not simply
by-products of splicing errors, rather they can modulate
gene expression and act as miRNA sponge involved in
cancer pathogenesis [
]. CircRNAs can function
as tumor suppressors or oncogenes in cancers. For
example, circCCDC66, circ_0067934 and circHIAT1
promote tumor growth and metastasis [
circZKSCAN1 and circZNF292 suppress tumor
progression by multiple signaling pathways [
]. Here, we
identified a circRNA derived from LARP4 gene locus,
termed as circLARP4, which had the potential to sponge
miR-424. Recent studies have shown that LARP4 as a
La-related RNA-binding protein inhibits cancer cell
migration and invasion [
]. We found that circLARP4 was
differentially-expressed between GC and adjacent
normal tissues, and was derived from Exon 9, 10 of the
LARP4 gene and intermediate long intron. Compared
with the linear LARP4, circLARP4 exhibited stable
expression in GC cells in a time-dependent manner, and
was mainly localized in the cytoplasm. Further
functional experiments revealed that overexpression of
circLARP4 inhibited DNA synthesis, cell proliferation and
invasion by sponging miR-424 and regulating the
expression of LATS1 and YAP genes, but knockdown of
circLARP4 reversed these effects, suggesting that
circLARP4 may function as a tumor suppressive factor in
GC via regulation of miR-424/LATS1/YAP signaling
circRNAs can act as promising potential biomarkers
for cancer diagnosis and prognosis due to their high
stability and specific loop structure [
]. It has been
reported that circPVT1, circ_0000190is and
fourcircRNA-based classifier are independent prognostic
markers for survival and recurrence of patients with GC
49, 50, 51
]. In this study, we also found that circLARP4
expression was downregulated in GC tissues, and was
correlated with tumor size and lymphatic metastasis,
and could act an independent prognostic marker for OS
of GC patients as well as the patients with
chemotherapy. Moreover, patients with circLARP4 high expression
had a significantly better survival than those with
circLARP4 low expression.
In summary, we identified an oncogenic miR-424, which
was negatively correlated with LATS1 expression. High
expression of miR-424 or low expression of LATS1 was
positively associated with pathological stage, OS and
recurrence of patients with GC, and miR-424 promoted cell
growth and invasion by targeting LATS1 gene. We further
characterized a circLARP4 derived from LARP4 gene and
demonstrated that circLARP4 was a tumor suppressive
factor in GC by sponging miR-424 and regulating LATS1
expression (Fig. 9). The regulatory network involving
circLARP4/miR-424/LATS1 axis may highlight a better
understanding of gastric tumorigenesis and progression.
Additional file 1: Table S1. Clinicopathological data of GC patients
from TCGA database. Table S2 Clinicopathological data of GC patients
from Tissue Microarray. Table S3 List of primers of the genes. Table S4
Correlation of LATS1 and miR-424 expression with clinicopathologic
features of GC patients. Table S5 Summary of univariate and multivariate
Cox regression analysis of recurrence duration. Table S6 Summary of
univariate and multivariate Cox regression analysis of recurrence duration.
Table S7 Identification of circRNAs sponging miR-424 in gastric cancer.
Table S8 AGO2 binding sites in circLARP4 genomic region. Table S9
Correlation of circLARP4 expression with clinicopathologic characteristics
of GC patients. Table S10 Summary of univariate and multivariate Cox
regression analysis of overall survival duration. (DOCX 49 kb)
Additional file 2: Figure S1. The genetic alteration frequency and
methylation levels of LATS1 in GC patients. a The genetic alteration
frequency of LATS1 amplification, deletion and mutation in different
pathological subtypes of GC. b The correlation of LATS1 gene expression
with its putative copy number alterations in GC. c The correlation of
LATS1 gene expression with its methylation level in GC. d The correlation
of LATS1 gene expression with miR-15b-5p in GC. (PDF 2166 kb)
Additional file 3: Figure S2. The correlation of LATS1 and miR-424
expression with OS and recurrence of GC patients. a and b Kaplan Meier
analysis of the correlation of LATS1 and miR-424 with OS of GC patients in
TCTA RNA sequencing database. c Kaplan Meier analysis of the correlation
of LATS1 expression with the recurrence of early stage patients (stage I + II)
or late stage ones (stage III + IV). d Kaplan-Meier plotter analysis of the
correlation of LATS1 expression with OS of GC patients with stage II or stage
IV. (E) Kaplan-Meier plotter analysis of the correlation of LATS1 expression
with recurrence of GC patients with stage II or stage III. (PDF 2418 kb)
Additional file 4: Figure S3. The effects of circLARP4 on GC cell
proliferation. a The expression level of LATS1 was examined after transfection
with miR-424 mimic and (or) LATS1 in HGC-27 cells, and miR-424 inhibitor and
(or) sh-LATS1 in MKN-28 cells indicated by qRT-PCR. b The expression level of
circLARP4 was detected in GC cell lines and GES-1 cells by qRT-PCR and
spearman correlation analysis of the correlation of circLARP4 with miR-424 and
LATS1 expression in GC cells. c Detection of cell proliferation of HGC-27 or
MKN-28 cells transfected with circLARP4 overexpression or si-circLARP4 vectors
by MTT assay. d Assessment of cell colony formation of HGC-27 or MKN-28
cells transfected with circLARP4 overexpression or si-circLARP4 vectors.
*P < 0.05; **P < 0.01. (PDF 3665 kb)
Additional file 5: Figure S4. The binding sites of circLARP4 with
miRNAs. a Schematic representation of potential binding sites of miRNAs
with circLARP4. b The effects of miR-424 mimic or inhibitor on the
expression level of circLARP4 in HCG-27 or MKN-28 cell line indicated by
qRT-PCR. c The binding sites of wild type or mutant circLARP4 3’UTR with
miR-424.-5p. d qRT-PCR analysis of the expression levels of LATS1 and
YAP after transfection with circLARP4 + miR-424 in HGC-27 cells or
sicircLARP4 + miR-424 inhibitor in MKN-28 cells. e the luciferase activity of
wild type LATS1 3’UTR was examined by co-transfection with miR-424
mimic + circLARP4 in HGC-27 cells. f the luciferase activity of wild type
LATS1 3’UTR was detected by co-transfection with miR-424 inhibitor +
sicircLARP4 in MKN-28 cells. *P < 0.05; **P < 0.01. (PDF 2681 kb)
Additional file 6: Figure S5. Correlation of circLARP4 expression level
with OS of GC patients. a Receiver operating characteristic (ROC) curve
analysis of the cutoff value, sensitivity, specificity and AUC of circLARP4 in GC
patients. b Kaplan-Meier analysis of the correlation of circLARP4 expression
with OS of GC patients with stage II + III or stage III. c Kaplan-Meier analysis of
the correlation of circLARP4 expression level with therapeutic outcomes of GC
patients with stage II + III or stage III treated with adjuvant chemotherapy of
oxaliplatin and 5-Fu. d Kaplan-Meier analysis of the correlation of circLARP4
expression level with therapeutic outcomes of GC patients without adjuvant
chemotherapy. (PDF 3527 kb)
We thank Shanghai KANGCHEN (Shanghai, PR, China) for providing the
technical support for our study.
This study was supported by grants from the National Natural Science
Foundation of China (No. 81573747), Hong Kong Scholars Program (No.
XJ2015033), Shanghai Science and Technology Commission Western
Medicine Guide project (No. 17411966500) and Shanghai Jiao Tong
University School of Medicine doctoral innovation fund (No. BXJ201737).
JSZ and JZ conceived of the study and carried out its design and JZ drafted
the manuscript. HL, LDH, RZ, YXH and XYC performed the experiments. JZ
and GW conducted the statistical analysis. JZ wrote the paper and JSZ
revised the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Hospital’s Protection of Human
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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