MicroRNA-19a and microRNA-19b promote the malignancy of clear cell renal cell carcinoma through targeting the tumor suppressor RhoB
MicroRNA-19a and microRNA-19b promote the malignancy of clear cell renal cell carcinoma through targeting the tumor suppressor RhoB
Shaoxi Niu 0 1
Xin Ma 0 1
Yu Zhang 0 1
Yen-Nien Liu 1
Xufeng Chen 1
Huijie Gong 0 1 2
Yuanxin Yao 0 1
Kan Liu 0 1
Xu Zhang 0 1
0 Department of Urology/State Key Laboratory of Kidney Diseases, Chinese PLA General Hospital/Chinese PLA Medical Academy , Beijing , People's Republic of China, 2 Department of Pathology, Duke University , Durham , North Carolina, United States of America, 3 Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University , Taipei , Taiwan
1 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute , UNITED STATES
2 Department of Urology, Dongzhimen Hospital, Beijing University of Chinese Medicine , Beijing , People's Republic of China
Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma, which shows high aggressiveness and lacks biomarkers. RhoB acts as a tumor suppressor that inhibits the progression of ccRCC. In the present study, we examined the effects of oncogenic microRNAs, miR-19a and miR-19b, on RhoB expression in ccRCC cells. The results showed that both miR-19a and miR-19b could directly target the 30untranslated region (3'UTR) of RhoB, resulting in the reduced expression of RhoB. With RT-PCR analysis, we detected the increased expression of miR-19a and miR-19b in ccRCC tissues compared to adjacent non-tumor renal tissues. These data also demonstrated an exclusive negative correlation between miR-19a/19b and RhoB expression in ccRCC specimens and cell lines. In addition, the knockdown of RhoB or overexpression of miR-19a and miR-19b in ccRCC cells could promote cell proliferation, migration and invasion. These data demonstrate the direct roles of miR-19a and miR-19b on the repression of RhoB and its consequences on tumorigenesis, cancer cell proliferation and invasiveness. These results suggest the potential clinical impact of miR-19a and miR-19b as molecular targets for ccRCC.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by the National
High Technology Research and Development
Program of China (2014AA020607).
Competing interests: The authors have declared
that no competing interests exist.
Renal cell carcinoma (RCC) is the second leading cause of cancer death in patients with
urological tumors, and accounts for approximately 3% of adult malignancies [
]. The overall
incidence and mortality of RCC have significantly increased over the past 20 years. Among all
RCC cases, approximately 70%~ 80% subtypes are clear cell renal cell carcinoma (ccRCC).
Surgery is the primary treatment for patients with localized ccRCC [
]. For patients with relapsed
or metastasized tumors, clinical treatment options are extremely limited because ccRCC is
often resistant to chemotherapy and radiotherapy [
]. In addition, there are no diagnostic and
therapeutic biomarkers currently available for this disease, and ccRCC patients are often
diagnosed at late stages with poor prognosis for clinical outcomes [
]. Thus, there is a clear need to
identify biomarkers for early diagnosis and molecular targets for establishing novel therapeutic
strategies for ccRCC.
The Rho protein family is a subgroup of small GTPases of the Ras superfamily, comprising
twenty members. Rho family proteins regulate a variety of cell functions, including actin
organization and cell shape, cell adhesion, cell motility, membrane trafficking and gene expression
]. A recent study has also indicated that Rho family proteins play an important role in
]. Interestingly, while other Rho proteins function as oncogenic proteins, RhoB acts
as a tumor suppressor in cancer cells [
]. Studies have shown that the decreased expression of
RhoB protein in solid tumors correlates with tumor staging , and the overexpression of
RhoB increases apoptosis and decreases the migration, invasion and metastasis of cancer cells
]. In addition, recent studies have shown that targeting RhoB could inhibit the tumor
growth of colorectal cancer and hepatocellular carcinoma in mouse xenograft models [
]. Consistent with these observations, in a previous study, we demonstrated that ccRCC
tumors have lower RhoB protein levels, and the overexpression of RhoB can inhibit cancer cell
proliferation and survival . These results thus indicate the potential of RhoB as a
therapeutic target for cancer treatment.
MiRNAs play vital roles in tumor progression and metastasis in many tumors, including
kidney cancer [
2, 3, 15, 16
]. MicroRNAs bind to the 3'-untranslated region (3'-UTR) of
target mRNAs and thus function as regulators for gene expression at the post-transcriptional
level . In colorectal cancer and hepatocellular carcinoma cells, studies have showed that
miR-21 can regulate RhoB protein expression [
]. To explore whether other miRNAs
also target and regulate the protein expression of RhoB mRNA, we used miRNA target
prediction algorithms with miRDB, TargetScan and PicTar to search for RhoB-targeting
miRNAs. The results showed that miR-21 is listed in the top miRNA candidates that may have
potential for RhoB-targeting. Moreover, we observed that miR-19a and miR-19b have the
highest scores for RhoB-targeting potential in this bioinformatics analysis. MiR-19a and
miR-19b belong to the miR-17±92 cluster located on chromosome 13q31.3 and have been
implicated as tumor-associated miRNAs involved in tumorigenesis. The miR-17±92
cluster has also been implicated in initiating carcinogenesis in B cell lymphoma and
targeting proapoptotic genes, such as PTEN, E2F1, and Bcl2l11/BIM [
studies have shown that miR-19a and miR-19b are involved in the carcinogenesis and cancer
development of different human cancers, including B-cell lymphomas , breast cancer
] and cervical cancer [
]. Other studies have also demonstrated that miR-19a and
miR19b directly target SOCS-1 (suppressor of cytokine signaling 1), a gene that shows loss of
function in multiple myeloma, and inhibits IL-6 growth signaling[
]. However, whether
miR-19a and miR-19b target and regulate the expression of RhoB in ccRCC remains
In the present study, we examined the potential regulatory effects of miR-19a and miR-19b
on RhoB protein expression and characterized their biological roles in ccRCC cells. We
examined the levels of RhoB mRNA, miR-19a and miR-19b in ccRCC in paired tumor and
nontumor specimens and ccRCC cell lines. These data demonstrated that miR-19a and miR-19b
can directly inhibit RhoB expression, resulting in consequent biological effects on the
tumorigenesis, proliferation, and invasiveness of ccRCC cells.
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Materials and methods
The protocol for the present study was approved by the Protection of Human Subjects
Committee, Chinese People's Liberation Army (PLA) General Hospital. All patients enrolled in the
present study provided signed informed consent. Specimens from human subjects were
collected from May to December 2013, and the experiments conducted with these human
specimens were initiated on January 2014.
Patients and samples
Seventy patients diagnosed with clear cell renal cell carcinoma and treated with surgery in the
Department of Urology of the Chinese PLA General Hospital in 2013 were enrolled in the
present study. Paired tumor tissues and adjacent non-tumor kidney tissues were collected and
added to the kidney cancer tissues bank after clinical and pathological confirmations. All tissue
samples were preserved with flash freezing in liquid nitrogen after resection and stored at −80
ÊC. Patients who received radical nephrectomy in our hospital, with pathologic diagnosis of
ccRCC, and both tumor and normal renal tissues available after surgery were included in the
present study. Patients receiving chemotherapy or radiotherapy prior to surgery, or patients
with multiple renal tumors or distant metastasis were excluded. TNM staging of ccRCC
samples was assessed according to the 7th edition of the AJCC Cancer Staging Manual [
the Fuhrman nuclear grading system was used to determined nuclear grades. The clinical
characteristics of the enrolled patients are shown in Table 1. All authors had access to information
on the identities of individual participants during data collection.
Cell culture and reagents
Human renal cancer cell lines 786-O, Caki-1, Caki-2, A498, SN12pm6, and ACHN, and
normal renal cell lines HKC and HK2 were purchased from the National Platform of
Experimental Cell Resources for Sci-Tech (Beijing, China). The cells were cultured in RPMI 1640/
DMEM/MEM medium (Thermo Fisher, NY, CA)supplemented with 10% fetal bovine serum
(Invitrogen, Carlsbad, CA), 100 U/ml of penicillin and 100 U/ml of streptomycin (Invitrogen,
Carlsbad, CA). All cell lines were maintained in a sterile incubator with a mixture of 95% air
and 5% CO2 at 37ÊC.
RNA isolation and quantitative real-time PCR
Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen, Carlsbad, CA)
according to the manufacturer's instructions. The TransScript Kit (TransGen Biotech Co.,
Beijing, China) was used for RT-PCR for RhoB. M-MLV (TIANGEN Biotech Co, Beijing, China)
was used to synthesize the complementary DNA templates for miRNA. The quantification of
both RhoB gene and miRNA expression was performed on an ABI PRISM 7500 Sequence
Detection System (Applied Bio systems, Foster City, CA) with SYBR Green (TransGen Biotech
Co., Ltd, Beijing, China). The relative expression of both mRNA and miRNA was determined
by using the ΔΔCt method. The expression of RhoB was normalized to the expression of
human peptidylprolyl isomerase A (PPIA), and the level of miRNA was normalized to U6. The
primers used in real-time RT-PCR are shown in Table 2.
Antibodies and western blotting
The anti-RhoB antibody was obtained from Proteintech (Chicago, USA), anti-cleaved
caspase9 antibody was purchased from Epitomics (Burlingame, CA), and anti-β-actin antibody was
obtained from ZSGB-BIO (Beijing, China). Preparation of the total cell lysate and Western
blot analysis were conducted with standard techniques as previously described [
Transfection and luciferase assay
The full-length RhoB 3'-UTR-luciferase reporter and its negative control vector psi-check2
were provided by Dr. Rossi (Department of Molecular Biology, Beckman Research Institute of
the City of Hope). The has-miR-19a and has-miR-19b mimics, control oligo (NC),
has-miR19a and has-miR-19b inhibitors, miRNA inhibitor control, and small interfering RNA
(siRNA) targeting RhoB (RhoB siRNA: Sense: 5’-ACGUCAUUCUCAUGUGCUUTT-3’;
Antisense: 5’-AAGCACAUGAGAAUGACGUTT) were all chemically synthesized by Shanghai
GenePharma Co. (Shanghai, China). For the mutagenesis of miRNA target sites in the 3'-UTR
region of the reporter, mutations were introduced into the seed sequence by using the
QuikChange Site-Directed Mutagenesis Kit II (Stratagene, USA) according to the manufacturer's
protocol. Mutations were confirmed by sequencing at Genewiz (Suzhou, China). The RNA
oligonucleotides transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad,
CA) according to the manufacturer's protocol. For Luciferase Assay, A498 or 786-O cells were
plated in 24-well plates. After 24 hours, the cells were cotransfected with oligos of miRNA
mimics (0.6 ug), miRNA inhibitor (0.6 ug) or negative control (0.6 μ g) together with the
appropriate reporter plasmid (0.2 ug). After 48 hours, the Dual-Glo Luciferase Assay System
(Promega, Madison USA) was used to measure the luciferase activity on a Centro XS3 LB960
luminometer (Berthold, USA), according to the manufacturer's instructions. The ratio of
Rluc/Fluc was used to measure the repression efficiency.
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Wound healing assay and plate colony formation assay
For wound healing assays, the cells were seeded onto six-well plates at 24 hours
post-transfection. Confluent monolayer cells were scratched by a 200-ul pipette tip, and then washed three
times with PBS. After fresh serum-free medium was replaced, wound closure was
photographed at 0, 12, and 24 hours at the same position. For the colony formation assay, cells
transfected with plasmid and mimics were seeded onto six-well plates (300 cells per well) and
cultured at 37ÊC for 14 days. The cells were fixed with 70% methanol for 15 minutes and
subsequently stained with 0.2% crystal violet. The number of positive colonies (consisting of more
than 50 cells) was counted after 4 weeks of growth.
Cell proliferation and migration analyses
Cell proliferation was measured by MTS assay using an ELx800 plate reader (BioTek, USA)
with the CellTiter 96 Aqueous One Solution (Promega, Madison, USA). Cell migration assay
was conducted with an 8 mm±pore size membrane Transwell apparatus (Corning Costar
Corp, Cambridge, USA). These assays were performed as previously described [
Flow cytometry analysis
Cell apoptosis were measured by flow cytometry analysis. Briefly, 1x106 cells were stained with
Annexin V-PE (Annexin V-PE kit, BD Biosciences, USA), and apoptotic cells were counted on
a FACSCalibur Flow Cytometer (BD Biosciences, USA). The data were analyzed using
CellQuest Pro software (BD Biosciences, USA). Up to 5x105 cells were analyzed for each sample.
All statistical analyses were performed with SPSS statistical software 13.0 (SPSS Inc., Chicago,
IL), and the statistical significance was set at P<0.05. ANOVA and Student's t-test were used
as appropriate for all results.
Negative collection between miR-19a/19b and RhoB in ccRCC specimens
and cell lines
To validate whether miR-19a and miR-19b functionally regulate the expression of RhoB, we
examined the expression of miR-19a, miR-19b and RhoB in patient tumor tissue samples (Fig
1A). The results showed that compared to the non-cancerous tissues ccRCC tumor tissues
have a higher level of miR-19a and miR-19b (p<0.05). Moreover, relatively lower amounts of
RhoB mRNA were detected in these tumor tissues. Notably, some tumor tissues had low
endogenous miR-19a and miR-19b expression when compared to paired non-tumorous
tissues (Fig 1B), and these tumor tissues expressed relatively high levels of RhoB protein (Fig 1C).
The relative expression of miR-19a,miR-19b and RhoB in paried specimens individually
showed in the supplementary information.(S1 Fig).The mRNA levels of miR-19a and miR-19b
in ccRCC cell lines were also dramatically inversely correlated to RhoB expression among
normal renal cells (HK2 and HKC) and ccRCC cell lines (A498, Caki-2, 786-O, ACHN, Caki-1,
and SN12) (Fig 1D). Moreover, the protein levels of RhoB were increased in normal renal cells
compared to those in ccRCC cell lines (Fig 1E). These results provide strong evidence to
support the hypothesis that RhoB is a tumor suppressor in ccRCC, and miR-19a and miR-19b
may act as oncomirs and down-regulate RhoB expression in ccRCC through a
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Fig 1. miR-19a/19b and RhoB expression levels are negatively correlated in patient tissues samples and cell lines. (A) QRT-PCR analysis for miR19a/b and
RhoB expression in paired patient specimens (n = 70). (B) QRT-PCR analysis for miR19a/b in patient specimens (n = 8). (C) Western blot analysis for RhoB
expression in the same specimens as panel B. N1-8 are normal tissues and A1-8 are paired tumor tissues. (D and E) QRT-PCR and Western blot analysis for
miR19a/b and RhoB expression in normal renal cells and ccRCC cell lines. β-actin was used as loading control for western blot. The data represent the average of
three independent experiments. Indicates statistical significance (P<0.05).
Correlations of miR-19a and miR-19b with clinical features
We next analyzed the correlations of miR-19a and miR-19b expression and the clinical
features of ccRCC. A total of 47 males and 20 females were included in this analysis. The data
showed that the expression of the these two miRNAs was significantly positively associated
with tumor size (P<0.001), TNM stage (P<0.001) and Fuhrman tumor grade (P<0.001,
Table 1). However, no significant correlation was observed between miRNA expression and
patients' age (p = 0.3667).
MiR-19a and miR-19b inhibit RhoB at both the mRNA and protein levels
To further confirm the relationship between miR-19a and miR-19b and RhoB, we examined
the expression of RhoB in cells after transfection with miR-19a and miR-19b mimics.
QRT-PCR and western blot analysis further supported the down-regulatory effect of miR-19a
and miR-19b on the protein levels of RhoB in ccRCC cells (Fig 2A). In contrast, cells
transfected with hsa-miR-19a and miR-19b inhibitors showed the increased expression of RhoB
(Fig 2B). The bioinformatics analyses showed a potential binding ability for both miR-19a and
miR-19b to the 3'-UTR region of RhoB (857±864 base pairs (bp), Fig 2C). Interestingly, the
miR-19a and miR-19b binding site in RhoB is a highly conserved sequence motif located
between 857 and 864 bp of the RhoB 3'UTR. Based on these findings, we performed a
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Fig 2. miR-19a and miR-19b regulate RhoB expression. (A and B) QRT-PCR analysis for miR19a/b (top) and Western -blot analysis of RhoB (bottom)
expression in A498 and 786-O cells after transfection with miR-19a and miR-19b mimics or inhibitors for 48 h. Control mimics or inhibitors were included as a
negative control. β-actin was included for equal protein loading. (C) The predicted binding motif sequence of miR-19a and miR-19b in the 3'-UTR region of RhoB.
(D) Reporter assay showing the effect of miR19a/b mimics on the luciferase activity of reporter plasmids containing RhoB-3'UTR-wt, RhoB-3'UTR-mut or empty
plasmid psi-check2 in A498 cells transfected with miR19a/b mimics. Control oligo was included as a negative control. (E) Reporter assay showing the effect of
miR19a and miR-19b inhibitors on luciferase activity of reporter plasmids containing RhoB-3'UTR-wt, RhoB-3'UTR-mut or empty plasmid psi-check2 in 786-O cells.
The data represent the average of three independent experiments. Indicates statistical significance (P<0.05).
luciferase reporter assay to examine the regulatory effects of miR-19a and miR-19b on the
gene post-transcription of RhoB. We co-transfected RhoB 3'-UTR-luciferase reporter and
miR-19a and miR-19b mimics into A498 cells. The transfection of miR-19a and miR-19b
mimics dramatically decreased the relative luciferase activity of the reporter containing the
wild-type RhoB 3'-UTR region (RhoB-3'UTR-wt). However, the co-transfection of miR-19a
and miR-19b mimics did not show obvious effects on the luciferase activity of the reporter
when the RhoB 3'-UTR region was mutated (RhoB-3'UTR-mut) (Fig 2D). In addition,
hasmiR-19a and miR-19b inhibitors and RhoB-3'UTR-wt or RhoB-3'UTR-mut were
co-transfected into 786-O cells, and the luciferase activity was determined. We found that the
co-transfection of has-miR-19a and miR-19b inhibitors increased the luciferase activity of
RhoB3'UTR-wt but had no effect on the luciferase activity of RhoB-3'UTR-mut (Fig 2E). These data
suggest that miR-19a and miR-19b suppress RhoB expression by direct binding to the 3'UTR
MiR-19a and miR-19b overexpression and RhoB knockdown show similar
phenotypes in promoting cell growth, invasion, and migration
We further tested whether the interaction of miR-19a and miR-19b with RhoB plays a role in
the proliferation and progression of ccRCC cells. We performed MTS assay to evaluate the
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Fig 3. Overexpression of miR-19a and miR-19b or RhoB knockdown show similar phenotypes in promoting the growth, migration and mobility of ccRCC
cells. (A) MTS results showing the promotion potential of miR-19a and miR-19b mimics overexpression or RhoB knockdown on the proliferation of A498 cells.
(B) Representative images and quantification show the clonogenic plating efficacy of A498 cells overexpressing miR-19a and miR-19b mimics. (C and D)
Representative images (right) and quantification (left) of wound healing assay (C) and transwell assay (D) showing the potential effects of the overexpression of
miR-19a and miR-19b mimics on the migration and invasiveness of the cells. Control oligo was included as a negative control. (E and F) Representative images
(right) and quantification (left) of wound healing assay (E) and transwell assay (F) showing the potential effects of RhoB knockdown on the migration and
invasiveness of the cells. Control siRNA was included as a negative control. The data are representative of three independent experiments performed in triplicate.
Significant differences from control oligo-transfected cells (P<0.05).
potential growth promoting effects of miR-19a and miR-19b on the cell growth of ccRCC. The
efficiency of RhoB knockdown was showed in S2 Fig. The results showed that the
overexpression of miR-19a and miR-19b mimics remarkably enhanced the proliferation potential of
A498 cells (Fig 3A). The expression of RhoB was lower with the transfection of si-RhoB both at
mRNA and protein level (S2 Fig). The clonogenic survival assay also showed that the
overexpression of miR-19a and miR-19b mimics increased the colony formation of A498 cells (Fig
3B). In addition, the transfection of miR-19a and miR-19b mimics in A498 cells enhanced the
migration and invasiveness of A498 cells (Fig 3C and 3D). These similar results were observed
in cells with RhoB knockdown by RhoB-siRNA transfection (Fig 3E). These data suggest that
miR-19a and miR-19b promote ccRCC cell proliferation, motility, and migration through
direct targeting of the RhoB tumor suppressor.
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Fig 4. MiR-19a and miR-19b inhibitors reduce cell migration and invasiveness, and inhibit cell proliferation. (A) Representative images showing that the
transfection of miR-19a and miR-19b inhibitors decreases the migration of 786-O cells by wound healing assay (Left); quantification of relative migration at 12
and 24 h (Right). (B) Representative images and quantification of relative migration, showing that the transfection of miR-19a and miR-19b inhibitors reduces the
invasiveness of 786-O cells by transwell assay. The migratory activities of 786-O cells transfected with control oligo were was included as a negative inhibitor
control. (C) The proliferation potential of 786-O cells transfected with miR-19a and miR-19b inhibitors or control oligo was determined by the MTS Assay. The
data are representative of three independent experiments. Significant differences from control oligo-transfected cells (P<0.05).
MiR-19a and miR-19b inhibitors induce cell growth, migration, and
invasiveness of 786-O cells
To investigate whether the miR-19a and miR-19b inhibitors could have potential inhibitory
effects on the migration and invasiveness of ccRCC cells, we transfected 786-O cells with
miR19a and miR-19b inhibitors or inhibitor control oligo. Interestingly, transfection with miR-19a
and miR-19b inhibitors resulted in the dramatically decreased migration and reduced
invasiveness of 786-O cells (Fig 4A and 4B). To determine the effects of miR-19a and miR-19b
inhibitors on cell proliferation features, we performed MTS assay of 786-O cells transfected
with miR-19a and miR-19b inhibitors or inhibitor control oligo. The results showed that
transfection with miR-19a and miR-19b inhibitors led to significantly reduced cell growth (Fig 4C).
These data are similar to those of a previous study showing the overexpression of RhoB in
786-O cells [
], which further supports the hypothesis that miR-19-RhoB signaling axis
regulates the migration, invasiveness, and proliferation of ccRCC cells.
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Fig 5. MiR-19a and miR-19b inhibitors induce cell apoptosis. (A) Flow cytometry results of Annexin-V5 analysis in 786-O cells transfected with miR-19a and
miR-19b inhibitors. An inhibitor control was included as a negative control. Graphs showing changes of the apoptotic percentage of the cells (left); quantification
of relative apoptosis (right). The data represent the means ± S.D. from three independent experiments performed in triplicate. Significant differences from control
oligo-transfected cells (P<0.05) (B) Western blot results showing the effect of miR-19a and miR-19b inhibitors on the expression of cleaved caspase-9 in 786-O
cells. (C) Potential mechanism showing the effect of the negative regulation of RhoB by miR-19a/b on the proliferation, migration, invasion and apoptosis of
Effect of miR-19a and miR-19b on cell apoptosis
We further determined the effect of miR-19a and miR-19b on apoptosis in ccRCC cells.
Apoptosis is a crucial event during malignant transformation [
], and we previously showed that
RhoB overexpression could influence the apoptosis of ccRCC cells [
]. In the present study,
we treated 786-O cells with miR-19a and miR-19b inhibitors and showed that transfection
with miR-19a and miR-19b inhibitors resulted in significant increases of apoptosis as
determined by Annexin V-PE staining (Fig 5A). We also examined changes in caspase-9, an
apoptotic protein in these cells, and found that the cleavage of caspase-9 was increased in 786-O
cells transfected with miR-19a and miR-19b inhibitors (Fig 5B). These results demonstrated
that levels of miR-19a and miR-19b affect the apoptosis of ccRCC cells.
In the present study, we investigated the potential regulatory effects of miR-19a and miR-19b
on RhoB expression and examined its consequences on the biological behavior of ccRCC cells
(Fig 5C). We observed the increased expression of miR-19a and miR-19b in tumor tissues
compared to that in paired and adjacent normal tissues, and this expression was inversely
correlation with RhoB expression. We also identified a similar inverse correlation between
miR10 / 13
19a and miR-19b and RhoB expression in established ccRCC cell lines. In addition, the
overexpression or inhibition of miR-19a and miR-19b respectively led to a decrease or increase of
RhoB. Luciferase reporter assay further showed that the transfection of miR-19a and miR-19b
inhibitors into ccRCC cells significantly suppressed RhoB 3'-UTR luciferase-reporter activity.
Thus, these results suggest that miR-19a and miR-19b can directly and inversely regulate RhoB
gene expression. Interestingly, these data further showed that transfection with miR-19a and
miR-19b mimics or RhoB-siRNA increased the viability and migration of A498 cells, and
transfection with miR-19a and miR-19b inhibitors showed inverse effects in 786-O cell lines.
Moreover, the down±regulation of miR-19a and miR-19b remarkably induces the apoptosis of
ccRCC cells and increases the levels of cleaved apoptotic casp9 protein. These data suggest the
potential effects of miR-19a and miR-19b on cancer cell invasiveness and metastasis via
targeting the RhoB tumor suppressor.
Rho GTPases have been implicated in tumor development and progression. Previous
studies have shown that the levels of both Rho GTPase mRNA and protein expression were
correlated with tumor progression [
]. Rho GTPase family proteins, such as Ras, Rac1 and Cdc42,
enhance oncogenesis, invasion and metastasis [
]. However, recent studies have indicated
that RhoB, unlike its other family proteins, RhoA and RhoC, whose expression is up-regulated
in different types of human cancers, may play a role as a tumor suppressor [
potential mechanism is that RhoB competes for binding to Rho effector proteins and interferes with
oncogenic signaling by Rho . Other studies have shown that RhoB expression changes the
apoptotic response of transformed cells to FTI (Farnesyltransferase inhibitor), DNA damaging
agents and paclitaxel [
]. Thus, RhoB could act as a death effector or modifier in
downstream or paralleled signaling to the DNA-damage-response pathway.
MiRNAs can act as ªoncomirsº or ªtumor suppressorsº in cancer progression [
have shown that miRNAs, such as miR-203a, miR-21, miR-17, miR-23 and miR-199a, are also
involved in the progression of ccRCC [
]. A previous study reported that the oncogenic
miR17-92 cluster is frequently up-regulated in human cancers and plays a role in tumorigenesis
]. MiR-19a and miR-19b are two important miRNAs in this cluster, and these molecules
could regulate the expression of tumor suppressor genes in lymphoma cells . Both
miR19a and miR-19b were also highly expressed in gliomas [
] and could regulate the cell
proliferation and invasion of cervical carcinoma through targeting CUL5 [
]. Notably, RhoB
activity has been identified by biochemical studies, suggesting that the
GTP-binding/GTPhydrolysis cycle is tightly regulated by RhoB and is essential for the regulation of Rho family
GTPase activities. Studies have further shown that the activities of classic regulators, such as
guanine nucleotide exchange factors, GTPase-activating proteins, and Rho GTPase, can be
controlled by miRNAs, such as miR-21[
In conclusion, the present study provides evidence for novel regulators, miR-19a and
miR19b, of RhoB expression, which plays important roles in the development of malignant ccRCC
via promoting cell proliferation, migration and invasiveness. These data also show the impact
of miR-19a and miR-19b as potential diagnostic biomarkers and therapeutic targets on clinical
management for ccRCC.
S1 Fig. The individual expression of the miR-19a, miR-19b and RhoB in specimens.
S2 Fig. The efficiency of RhoB knockdown.
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The authors would like to thank Dr. John Rossi at Beckman Research Institute of the City of
Hope for providing the plasmid DNA for the RhoB 3'-UTR-luciferase reporter and its negative
control vector psi-check2.
Conceptualization: Xufeng Chen, Xu Zhang.
Data curation: Shaoxi Niu.
Formal analysis: Shaoxi Niu.
Funding acquisition: Xin Ma.
Methodology: Yu Zhang.
Project administration: Xu Zhang.
Resources: Xu Zhang.
Software: Yen-Nien Liu, Kan Liu.
Supervision: Yu Zhang.
Validation: Huijie Gong, Yuanxin Yao.
Visualization: Kan Liu.
Writing ± original draft: Shaoxi Niu.
Writing ± review & editing: Yen-Nien Liu, Xufeng Chen.
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