miR-141 is A Key Regulator of Renal Cell Carcinoma ... · 1 miR-141 is A Key Regulator of Renal...

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miR-141 is A Key Regulator of Renal Cell Carcinoma Proliferation and

Metastasis by Controlling EphA2 Expression

Xuanyu Chen1, Xuegang Wang1, Anming Ruan1, Weiwei Han1, Yan Zhao2, Xing Lu2, Pei Xiao2, Hangchuan Shi1,

Rong Wang1, Li Chen1, Shaoyong Chen3, Quansheng Du4, Hongmei Yang2, and Xiaoping Zhang1

1Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and

Technology, Wuhan 430022, China

2Department of Pathogen Biology, Tongji Medical College, Huazhong University of Science and Technology,

Wuhan 430030, China

3Cancer Biology Program, Hematology-Oncology Division, Department of Medicine, Beth Israel Deaconess

Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA

4Department of Neurology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia

Regents University, Augusta, GA 30912, USA

Corresponding Authors:

X. Zhang, Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science

and Technology, 1277 Jiefang Avenue, Wuhan 430022, Hubei Province, China. Phone: 86-27-85351625; Email:

[email protected].

H. Yang, Department of Pathogen Biology, Tongji Medical College, Huazhong University of Science and

Technology, 13 Hangkong Road, Wuhan 430030, Hubei Province, China. Phone: 86-27-83691053; Email:

[email protected].

Running Title: miR-141 inhibits proliferation and metastasis in RCC

Key Words: miR-141, renal cell carcinoma, proliferation, metastasis, EphA2

Grant Support

This study was supported by the National Natural Science Foundation of China (NSFC) (Grant No. 30872924,

81072095 & 81372760), Program for New Century Excellent Talents in University from Department of Education

of China (NCET-08-0223), the National High Technology Research and Development Program of China (863

Program) (2012AA021101) to X. Zhang, and the NSFC (Grant No. 31070142 & 81272560) to H. Yang.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Other Notes

Our manuscript contains 4,879 words, one table and seven figures. The supplemental information is composed

of supplemental experimental procedures, two tables and five figures.

Research. on September 24, 2020. © 2014 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 19, 2014; DOI: 10.1158/1078-0432.CCR-13-3224

http://clincancerres.aacrjournals.org/

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Translational Relevance

Renal cell carcinoma (RCC) is a particularly aggressive and chemoresistant malignancy.

Current targeted therapies based on the discovery of the VHL/HIF/VEGF pathway have changed

the treatment landscape for patients with metastatic RCC, whereas these therapies have not lived

up to their initial promise. A central role of microRNAs (miRNAs) in the initiation and

progression of cancers has begun to emerge. Here we screened and identified that miR-141 was

the most significantly downregulated in clear cell RCC (ccRCC) tissues and cell lines, and a

promising biomarker for discriminating ccRCC and normal renal tissues. Furthermore, miR-141

overexpression effectively suppressed tumor growth, local invasion and metastatic colonization in

human RCC orthotopic xenografts by decreasing a direct functional effector EphA2. Because our

findings are based on clinical RCC samples and a good animal model, miR-141’s ability to

suppress of tumorigenesis and metastasis may prove to be clinically useful.




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Abstract

Purpose: Although microRNAs (miRNAs) have been revealed as crucial modulators of

tumorigenesis, our understanding of their roles in renal cell carcinoma (RCC) is limited. Here we

sought to identify human miRNAs that act as key regulators of renal carcinogenesis.

Experimental Design: We performed microarray-based miRNA profiling of clear cell RCC

(ccRCC) and adjacent normal tissues and then explored the roles of miR-141 both in vitro and in

vivo, which was the most significantly downregulated in ccRCC tissues.

Results: 74 miRNAs were dysregulated in ccRCC compared with normal tissues. miR-141 was

remarkably downregulated in 92.6% (63/68) ccRCC tissues and would serve as a promising

biomarker for discriminating ccRCC from normal tissues with an AUC of 0.93. Overexpression of

miR-141 robustly impaired ccRCC cell migratory and invasive properties and suppressed cell

proliferation by arresting cells at G0/G1 phase in vitro and in human RCC orthotopic xenografts.

Significantly, the anti-tumor activities of miR-141 were mediated by its reversal regulation of

EphA2, which then relayed a signaling transduction cascade to attenuate the functions of focal

adhesion kinase (FAK), AKT, and MMP2/9. In addition, a specific and inverse correlation

between miR-141 and EphA2 expression was obtained in human ccRCC samples. Finally,

miR-141 could be secreted from the ccRCC donor cells, and be taken up and function moderately

in the ccRCC recipient cells.

Conclusion: miR-141 serves as a potential biomarker for discriminating ccRCC from normal

tissues and a crucial suppressor of ccRCC cell proliferation and metastasis by modulating the

EphA2/p-FAK/p-AKT/MMPs signaling cascade.




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Introduction

Renal cell carcinoma (RCC) accounting for 3% of adult malignancies is the most lethal

urological malignancy, with about 65,150 new cases and 13,680 deaths estimated for 2013 in the

United States (1). RCC is heterogeneous and comprises several histological subtypes according to

the differences in genetics, biology and behavior. The most common and aggressive RCC subtype

is clear cell RCC (ccRCC) with the highest rates of local invasion, metastasis, mortality and

refractory to current treatments. Recently, advancements in understanding of the VHL gene

pathway in ccRCC have produced pharmaceutic outcomes based on specific molecular targets that

have changed the treatment landscape for patients with metastatic RCC (mRCC) (2).

Unfortunately, the vast majority of treated patients with mRCC eventually develop progressive

disease due to acquired resistance or other reasons. Hence, a better understanding of the

mechanisms involved in the pathogenesis of ccRCC and more effective therapeutic approaches are

urgently required.

MicroRNAs (miRNAs), a group of small non-coding RNAs of about 22 nucleotides in length,

negatively regulate gene expression, primarily through interaction with the 3’untranslated region

(3′UTR) of target mRNAs (3). miRNAs are known to contribute to multiple tumorigenic steps in

human cancers, including RCC. Recent studies have identified regulatory activities of miRNAs in

ccRCC cell growth (4-8), apoptosis (5, 6, 8, 9), migration and invasion (5-8). A predominant and

systemic alteration in miRNA expression during renal carcinogenesis has been indicated by

present studies of miRNA expression profiling (10-17). Consistently, the miR-200 family

members (miR-200s) that comprise miR-141, 200c, 200a, 200b and 429 are among the most

markedly decreased miRNAs in ccRCC. These miRNAs were previously found to be closely




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associated with cell motility by modulating epithelial-mesenchymal transition (EMT), the

formation of cancer stem cells, sensitivity to chemotherapeutic agents, and apoptosis (9, 18-23).

However, how miR-200s function in ccRCC pathogenesis remains largely unknown. Growing

evidences reveal that extracellular miRNAs are chemically stable and can be detected in a broad

range of clinical samples and hence, have diagnostic and prognostic values (24-26). Furthermore,

certain secreted miRNAs from living cells (for example, miR-146a and miR-223), are transferable

and can affect surrounding and remote cells by regulating target mRNAs and proteins, implying

functions of miRNAs in intercellular communication (27-29). Given their small size and

substantial effects in a wide range of pathologies, miRNAs can be potentially utilized in ccRCC

therapy.

Here, we monitored miRNA expression profiles of ccRCC and identified miR-141 as one of

the most significantly downregulated miRNAs in ccRCC tissues and cells and a critical suppressor

of ccRCC cell growth and metastasis both in vitro and in vivo. We further demonstrated that the

attenuation of miR-141 in RCC is associated with the amplification of EphA2 and additional

downstream pathways. Moreover, our results based on clinical RCC samples showed that

miR-141 might be a potential biomarker for discriminating between ccRCC and normal tissues.

Finally, we found that miR-141 can be secreted from the ccRCC donor cells, and be taken up and

function moderately in the ccRCC recipient cells.

Materials and Methods

Human samples




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Surgical specimens (paired normal and cancerous tissues) were obtained from 78 patients with

kidney tumors in Department of Urology, Union Hospital, Tongji Medical College (Wuhan,

China), freshly frozen in liquid nitrogen and stored at -80°C for RNA and protein extraction.

Informed consent was obtained from patients and the study was approved by the Institutional

Review Board of Huazhong University of Science and Technology. Relevant clinical and

pathological information based on patient records was collected and listed in the Supplementary

Table S1.

miRNA microarray

The miRNA expression profiling was conducted using the commercially available G4471A

Human miRNA Microarray (Agilent Technologies, Palo Alto, CA), which consists of 961 probes

for 851 human miRNAs, based on Sanger miRBase release 12.0. The arrays were washed and

scanned with a laser confocal scanner (G2565BA, Agilent Technologies, Palo Alto, CA) according

to the manufacturer’s instructions. The intensities of fluorescence were calculated by Feature

extraction software (Agilent Technologies). Differentially expressed miRNAs were identified by

arbitrarily setting the threshold at a fold change of 2.0 or above combined with p < 0.05

(ANOVA ).

Cell culture, infection, transfection and preparation of conditioned media (CM)

Human RCC cell lines 786-O and A498 were obtained from the American Type Culture

Collection (ATCC, Manassas, VA). The RCC cell line SN12-PM6 was supplied by Dr. I.J. Fidler

(MD Anderson Cancer Center, Houston, TX). All cells were cultured in Dulbecco’s modified




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Eagle’s media plus 10% fetal bovine serum (FBS) with 1% penicillin-streptomycin at 37°C in 5%

CO2. Lentiviruses containing vector pGCSIL-GFP and pGCSIL-GFP-miR-141 were constructed

by GENECHEM (Shanghai, China) and used to infect 786-O or SN12-PM6 cells at a multiplicity

of infection (MOI) of 10 or 50, respectively, according to the manufacturer’s instructions.

miR-141 inhibitor (Inh-miR-141) and its negative control (Inh-NC) were designed and

synthesized by RiboBio (Guangzhou, China). Short interfering RNA (siRNA) against EphA2

(si-EphA2) and negative control (si-NC) with non-specific sequences was synthesized by

Genepharma (Shanghai, China). Sequence of EphA2 siRNA is as following:

5’-UGACAUGCCGAUCUACAUGdTdT-3’ (sense), 5’-CAUGUAGAUCGGCAUGUCAdTdT-3’

(antisense) (30). Cells were seeded in media without antibiotics approximately 24 hours before

transfections. Oligonucleotide transfection of a final concentration of 50 or 100 nM was

performed with Lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA) according to the

manufacturer’s protocol. Cells were used for experiments after siRNA transfection for 48 hours.

To prepare CM, cells infected with lentiviruses were cultured in fresh complete media. After

incubation for 0, 24, 48, 72 hours, respectively, media were collected and centrifuged at 2,000g for

15 minutes at 4°C to remove living cells and centrifuged again at 12,000g for 35 minutes at 4°C to

thoroughly remove cellular debris (27). Then the CM were transferred to an RNase/DNase-free

1.5 ml tube and used for RNA extraction. In addition, the media from miR-141 or miR-NC cells

after incubation for 48 hours were used to culture miR-NC cells for another 48 hours.

RNA extraction, quantitative real-time PCR (qRT-PCR) and semiquantitative

reverse-transcription PCR (RT-PCR)




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Total RNA of tissues and cells was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA).

Total RNA in CM was isolated by using TRI Reagent BD (Molecular Research Centre, Cincinnati,

OH) according to the manufacturer’s protocol with modification. In brief, 0.2 ml of CM was

added to 0.75 ml of TRI Reagent BD supplemented with 20 µl of acetic acid (5 mol/L). 25 fmol of

synthetic C.elegans miRNA cel-miR-39 (Qiagen, Hilden, Germany) was spiked-in as a normaliser

before chloroform extraction, and then the RNA was precipitated in isopropanol overnight at

-20°C. Finally the pellet was dissolved in 15 µl of DEPC-treated water. Reverse transcription of

miRNA and mRNA was done using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas,

Vilnius, Lithuania) and a reverse transcript primer from RiboBio (Guangzhou, China). qRT-PCR

analysis was performed with the Platinum SYBR Green qPCR Supermix UDG kit (Invitrogen,

Carlsbad, CA) using synthesized primers from RiboBio (Guangzhou, China) or

Sangon biotech (Shanghai, China). Mature miRNAs and mRNAs were measured in accordance

with the manufacturer's instructions (LightCycler® 480Ⅱ, Roche, Mannheim, Germany). RT-PCR

for EphA2 was performed with 2 x Taq PCR MasterMix (Tiangen Biotech, Beijing, China)

according to the manufacturer’s protocol. The primers were listed in the Supplemental

Experimental Procedures. Samples were normalized to RNU6B (U6) or GAPDH. Relative

expression was calculated using the power formula: 2-△Ct (△Ct=CtmiR-141-CtU6).

Cell viability, drug sensitivity, cell cycle, migratory and invasion assays

Cell viability was assessed at 24, 48, 72 and 96 hours upon treatments by the

3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) method (Sigma, USA) as

previously described (31). For drug sensitivity assay, cells were treated with various




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concentrations of cisplatin (DDP), 5-fluorouracil (5-FU) and tumor necrosis factor-related

apoptosis-inducing ligand (TRAIL) for 24 or 48 hours, then measured by above MTT assay.

Fluorescence -activated cell-sorting (FACS) (BD, USA) analysis was done using propidium iodide

(PI) stains for cell-cycle analysis according to the manufacturer's protocol. The 24-well transwell

plate with 8 µm pore polycarbonate membrane inserts (Corning, New York, USA) was used to

analyze the migration and invasive potential of cells according to manufacturer's protocol. For

invasion assay, the membrane was coated with the matrigel (200 ng/ml) (BD Biosciences, Bedford,

MA). After 24 hours of incubation, cells invading into the lower surface of the membrane insert

were fixed in 100% methanol, stained with 0.05% crystal violet, and quantified by counting in 10

random fields. To evaluate the effects of secreted miR-141 on cell migration and invasion, cells

overexpressing miR-141 or miR-NC were cultured in the lower chambers for 24 hours and then

control (miR-NC) cells were placed in the upper chamber for another 24 hours.

Xenograft orthotopic implantations

For in vivo studies, 1 x 106 SN12-PM6 cells stably expressing miR-141 or miR-NC were

injected into the left kidney of male BALB/c nude mice at 4-5 weeks of age as previously

described (32). The green fluorescence intensity of the xenografts was monitored periodically

using the Lumazone FA 2048 system (Roper Scientific, USA). 6 weeks and 8-9 weeks after the

implantation of the xenografts, animals were euthanized and xenografts were harvested, and

assessed for tumor weight, local invasion and distant metastasis. Renal tumors from xenografts

were flash frozen in liquid nitrogen and stored at -80°C for RNA extraction. Formalin-fixed,

paraffin-embedded RCC xenografts were assessed by hematoxylin and eosin (HE) staining and




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evaluated for EphA2 expression. All experiments were approved by the Animal Care and Use

Committee of Tongji Medical College of Huazhong University of Science and Technology.

Luciferase assays

Wild-type and mutant EphA2 3’UTR reporter and control construct were purchased from

GENECHEM (Shanghai, China). Tumor cells overexpressing miR-141 and miR-NC cultured in

48-well plates were cotransfected with 1.5 ug of firefly luciferase reporter and 0.35 ng Renilla

luciferase reporter with Lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA). 24 hours

posttransfection, firefly luciferase activities were measured using the Dual Luciferase Assay

(Promega, Madison, WI) and the results were normalized with Renilla luciferase according to the

manufacturer’s protocol.

Statistical analysis

Results are expressed as mean±SEM from at least three independent experiments. Using the

GraphPad Prism statistical program, data were analyzed using Student’s t- test unless otherwise

specified (Mann–Whitney test, ROC, Pearson’s correlation, etc.). The 2-tailed P values <0.05 were

considered significant.

Supplemental information

The supplemental information includes supplemental methods, two tables, and five figures.

Results




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Loss of miR-141 expression in ccRCC tissues and cells

Using a microarray platform which contains 851 human miRNAs to study miRNA profiles in

paired primary tumor and adjacent normal tissues (NTs) from 5 patients with the mean

tumor size of 4.7 cm (range, 2.3-6.0 cm), we identified 74 miRNAs dysregulated in ccRCCs

compared with NTs (fold change＞2.0 and p < 0.05), among which 44 were significantly

downregulated and the other 30 were upregulated (Supplementary Table S2). Notably, four

members (miR-141, 200b, 200c and 429) of miR-200s were all markedly reduced in ccRCC

tissues. Our miRNA expression pattern was similar with previous studies identified by microarray,

qRT-PCR or deep-sequencing, which covered less miRNAs (10-14, 17). Based on these findings,

we reasoned that miR-200s may contribute to the development of ccRCC. Among these

differentially expressed miRNAs in ccRCC, miR-141 was one of the most remarkably

downregulated miRNAs (104-fold) and its roles in RCC have not been fully documented yet

(10-14, 17). Thus, we chose miR-141 as a representative to substantiate the functions of miR-200s

in ccRCC.

Next, we validated the clinical significance of miR-141 by analyzing its expression in 68 pairs

of ccRCCs and NTs by qRT-PCR. In accordance with microarray results, qRT-PCR showed that

miR-141 was significantly downregulated in 92.6% (63/68) ccRCCs (p<0.0001) (Fig. 1A and B).

Receiver operating characteristics (ROC) analysis revealed that miR-141 might serve as a useful

biomarker for discriminating ccRCC from normal tissues with an area under the ROC curve (AUC)

of 0.93 (95% CI, 0.881 to 0.981) (Fig. 1C). At a threshold of 0.00005 for its relative expression,

the sensitivity was 86.76% and the specificity was 97.06%. As demonstrated, miR-141 expression




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was not associated with tumor stage, grade or size, while its expression gradually decreased during

tumor progression (Supplementary Fig. S1A-C). Notably, miR-141 was barely expressed in

ccRCC cell lines, including 786-O, SN12-PM6 and A-498 cells (Fig. 1D).

We then addressed whether miR-141 could be a potential marker to differentiate malignant

tumors from benign tumors. The levels of miR-141 in 2 chromophobe RCCs (chRCCs), 1 sarcoma

RCC, 7 renal angiomyolipomas (AMLs) and their NTs were analyzed by qRT-PCR. Unexpectedly

and significantly, miR-141 was also markedly decreased in these tumors, although there was no

difference between ccRCCs and AMLs (Fig. 1B and Supplementary Fig. S1D). The results imply

that miR-141 contributes to the development of tumors originated from kidney independent of

specific subtypes.

Overexpression of miR-141 attenuates ccRCC cell proliferation and motility

To explore the biological significance of miR-141, we stably overexpressed miR-141 in two

ccRCC cell lines 786-O and SN12-PM6 with lentiviruses carrying miR-141 and its control

(miR-NC) (Supplementary Fig. S2A and B). The efficacy of infection was tested by qRT-PCR

(Supplementary Fig. S2C and D). miR-141 expression was elevated up to 400- and 2400-fold in

786-O and SN12-PM6 cells, respectively. An increase in the control miR-16 expression was not

observed, suggesting that lentiviruses specifically increased miR-141 expression. There were no

marked cellular morphologic changes in the miR-141-overexpressed cells (Supplementary Fig.

S2A and B), however, the cellular proliferation (MTT and FACS) analyses showed that

overexpression of miR-141 suppressed ccRCC cell proliferation (p<0.05) (Fig. 2A), caused cell

cycle arrest at G0/G1 phase and decreased the S phase population (Fig. 2B and Supplementary Fig.




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S3A). More importantly, overexpression of miR-141 markedly impaired ccRCC cell migration

and invasiveness compared to miR-NC (p<0.001) (Fig. 2C and Supplementary Fig. S3B).

Additionally, miR-141 overexpression did not directly induce apoptosis in the two tested ccRCC

cell lines, with no pronounced alterations in the responsiveness to DDP, 5-FU and TRAIL (data

not shown). These findings indicate that miR-141 acts as a tumor-suppressor in RCC by inhibiting

cell proliferation and motility.

Extracelluar miR-141 modulates cell motility of recipient cells

Mounting evidence indicate certain miRNAs released from the donor tumor cells can be taken

up by the recipient tumor cells to function in cell-cell communications during cancer progression

(29). To determine whether miR-141 had similar features in the ccRCC cell lines, we first

prepared conditioned media (CM) from miR-141-overexpressed 786-O and SN12-PM6 cells, as

well as control cells (miR-NC). The levels of miR-141 in the CM from miR-141 and miR-NC

cells were measured at several time points. Similar to previous reports (27, 28), extracelluar

miR-141 in CM of miR-141 cells was significantly higher than that of miR-NC cells (Fig. 2D). As

a control, extracelluar miR-16 expression remained unchanged (Fig. 2D). In addition, the ratios of

extracelluar/intracellular miR-141 in the 786-O and SN12-PM6 cells were less than 2.2% and

0.01%, respectively (Supplementary Fig. S2E and F). To test whether extracelluar miR-141 was

taken up in the recipient ccRCC cells, CM from miR-141 or miR-NC cells were used to culture

miR-NC cells. As shown in Fig. 2E, levels of intracellular miR-141 in the miR-NC cells was

increased for about 10 or 7-fold after adding CM of the miR-141 cells. We next assessed whether

extracelluar miR-141 can make an impact on cell motility using transwell assay. The data showed




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that co-culture with CM of the miR-141 cells decreased migration and invasion of the recipient

miR-NC cells (Fig. 2F and Supplementary Fig. S3C). However, CM of miR-141 cells did not

impact cell proliferation and cell cycle of miR-NC cells (data not shown). The above data

demonstrated that miR-141 can be excreted into extracellular environment and plays active

biological functions in the recipient cells. However, the level of extracelluar miR-141 was

markedly lower than its intracellular counterpart in the parental cells, suggesting that more

predominant miRNA activities reside intracellularly. Accordingly, we next focused on the

functions of intracellular miR-141.

miR-141 suppresses tumorigenesis and metastasis in human RCC orthotopic xenografts

To identify antitumorigenic roles of miR-141 in RCC in vivo, SN12-PM6 cells overexpressing

miR-141 versus miR-NC were injected into the left kidney of nude mice and then tumor growth

and metastasis were evaluated. Our previous results have demonstrated that the renal orthotopic

xenografts can develop primary renal tumors and give rise to metastases in multiple organs (32).

Tumor growth was surveilled by detecting GFP expression using the automated fluorescence

imaging system. As shown in Fig. 3A, fluorescence imaging showed a significant reduction of

tumor growth in miR-141-overexpressed cells at as early as 3nd week. Meanwhile, no difference

of tumor incidence was observed between miR-141 and miR-NC tumors (Table 1). Over a period

of 6 weeks and 8-9 weeks postimplantation, there was a more significant decrease in tumor weight

and size upon expression of miR-141 (p<0.05) (Table 1 and Fig. 3B). More specifically, tumors

with miR-141 overexpression were commonly encapsulated and confined to kidney parenchyma,

whereas tumors of control cells presented more aggressive growth (Fig. 3B and C). In addition, no




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macroscopic metastases were found in tumors with miR-141 overexpression. In contrast, miR-NC

tumors extensively infiltrated the kidney fascia, especially at 8th-9th week after implantation

(Table 1 and Fig. 3B). Compared with miR-141 tumors, the miR-NC tumors were prone to local

invasion and metastasis. As shown in Fig. 3D, the miR-NC tumors metastasized to various sites,

including lung, liver, lymph nodes, cecum, musculus diaphragm and peritoneum ((Table 1 and Fig.

3D). These data further demonstrated that miR-141 functions as a critical tumor-suppressor in

RCCs by suppressing tumorigenesis, local invasion and metastatic colonization.

EphA2 is a novel direct target of miR-141

Our aforementioned work demonstrated that miR-141 plays a critical role in modulating

migration and invasion in ccRCC cells, which promoted us to identify specific miR-141 targets

with potential relevance in the regulation of metastasis. Recent work on miR-141 mostly focused

on the association with the process of EMT by downregulating ZEB2, TGFβ2 and upregulating

E-cadherin in different cancers (18-21). Several reports also showed miR-141 regulated Notch

signaling pathway by targeting JAG1 (33, 34). Consistent with previous observations, 786-O and

SN12-PM6 cells with overexpression of miR-141 showed decreased levels of ZEB2, TGFβ2,

JAG1, HES1 and increased E-cadherin expression (Supplementary Fig. S4). However, the

alterations just based on EMT cannot fully recapitulate its profound effects on tumor progression.

Next, we used different miRNA target-predicting algorithms such as TargetScan, Pictar, miRanda,

miRDB and miRwalk to identify potential effector (s) of miR-141 and found conserved miR-141

sites at the 3’UTR of EphA2. As EphA2 has been reported to be highly expressed in RCC cells

and tissues and associated with advanced stage disease and poor prognosis (35, 36), we reasoned




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that the loss of miR-141 might be an important factor of RCC malignancy by upregulating EphA2

expression.

To prove the above hypothesis, we examined the correlation between miR-141 and EphA2

expression in both in vitro and in vivo studies. As shown in Fig. 4A and B, enforced miR-141

expression led to a decrease in EphA2 mRNA and protein expression in both 786-O and

SN12-PM6 cells. Consistently, evaluation of primary tumors in the renal orthotopic xenografts

models showed an inversed correlation between miR-141 expression and EphA2 mRNA and

protein levels (Fig. 4C and D).

To further validate EphA2 as a direct target of miR-141, a 208 bp fragment from the EphA2

3’UTR containing the putative miR-141 target sites was cloned into a luciferase reporter construct.

As shown in Fig. 4E, the miR-141 binding site in EphA2 mRNA is a broadly conserved element

among vertebrates and locates in the vicinity of 753 to 759 bp of the EphA2 3’UTR.

Dual-luciferase reporter assays were performed in 786-O and SN12-PM6 cells with

overexpression of miR-141 versus miR-NC to assess the functions of this potential miR-141 target

site. Significantly, miR-141 overexpression substantially repressed activity of the reporter that

carried the wild-type but not mutant 3’UTR of EphA2 (Fig. 4F), suggesting the regulation is

mediated in sequence-specific manner and the tested region is a bona fide miR-141 targeting site.

miR-141-repressed tumor proliferation and aggressive behavior is mediated by EphA2

The above findings indicate that EphA2 is a candidate effector to mediate biological functions

of miR-141. We next examined whether knockdown of EphA2 could recapitulate the inhibitory

effects of miR-141 on ccRCC cell proliferation and progression. 786-O and SN12-PM6 cells were




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transfected with si-EphA2 versus si-NC. RT-PCR and WB analysis confirmed the EphA2 siRNA

markedly and specifically decreased EphA2 expression (Fig. 5A and B). Significantly, knockdown

of EphA2 in ccRCC cells attenuated cell growth, induced G0/G1 cell-cycle arrest, and suppressed

cell migration and invasion (Fig. 5C-E), similarly to the phenotypic alterations upon miR-141

overexpression. To further determine whether EphA2 is the direct and functional mediator of

miR-141-repressed cell migration and invasion, we performed a rescue experiment by

co-transfecting with EphA2 siRNA (versus the negative control) and miR-141 inhibitor (versus the

negative control) into 786-O and SN12-PM6 cells. Transient transfection of miR-141 inhibitor led

to downregulation of miR-141 and upregulation of EphA2 as determined by qRT-PCR and WB

(Fig. 5F). Importantly, the enhancement in ccRCC cell migration and invasion induced by

miR-141 inhibitor was effectively reversed by EphA2 attenuation (Fig. 5G and Supplementary Fig.

S3D). Collectively, these findings indicate that EphA2 is an essential functional effector of

miR-141 in ccRCC.

EphA2 protein is frequently upregulated in ccRCC tissues

We then further investigate based on clinical samples whether miR-141 is involved in the

pathogenesis of human ccRCC through EphA2. The expression of EphA2 mRNA and protein

from 20 pairs of ccRCC and their NTs were analyzed by qRT-PCR, WB and IHC, respectively.

These 20 pairs of samples have showed a statistically significant decrease in miR-141 in the

ccRCC tissues (Fig. 1A). Unexpectedly, no remarkable difference of EphA2 mRNA was observed

between ccRCC and NTs (Fig. 6A). Anyhow, Pearson’s correlation analysis showed a good

reverse correlation between levels of miR-141 and EphA2 mRNA in ccRCC tissues (R2=0.3661,




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p=0.0047) (Fig. 6B). Specifically, lower miR-141 levels were associated with higher EphA2

mRNA expression, and vice versa (Fig. 6C). Notably, compared to normal tissues, meaningful

overexpression of EphA2 protein was observed in 17 of 20 and decreased expression of EphA2

was showed in 1 of 20 ccRCC cases by WB analysis (Fig. 6D). A similar result was obtained in

IHC analysis (Supplementary Fig. S5), further supporting that the increased level of EphA2 is, at

least in part, attributed to loss of miR-141 in ccRCC. Collectively, these findings clearly

substantiate that EphA2 is a direct and functional target of miR-141.

miR-141-EphA2 modulates FAK and AKT phosphorylation

Next, we extended the studies on the miR141-EphA2 module to further downstream, based

on reported EphA2 signaling to the FAK and AKT pathways (28, 35-37). For this purpose, we

monitored whether p-FAK and p-AKT were the downstream effectors of the miR-141-EphA2

module. Expectedly, overexpression of miR-141 adequately led to compromised p-FAK and

p-AKT in 786-O and SN12-PM6 cells (Fig. 7A). Suppression of EphA2 also resulted in an

attenuation of p-FAK and p-AKT (Fig. 7B). Moreover, the rescue experiment showed that

upregulation of EphA2 expression following miR-141 inhibition led to increased phosphorylation

of FAK and AKT, while knockdown of EphA2 rescued the stimulatory effects of miR-141

inhibitor on p-FAK and p-AKT (Fig. 7C). These results suggest that miR-141 may act as a

tumor-suppressor through negatively regulating the EphA2/p-FAK/p-AKT pathway.

It has been reported that FAK and AKT promote cancer cell migration and invasion by

elevating MMP-2/9 expression, which have been implicated in the aggressiveness of RCC (37, 38).

Accordingly, we further pursued whether miR-141-EphA2 mediated tumorigenesis and




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progression in ccRCC was correlated with MMP-2/9. WB analysis showed that stable

overexpression of miR-141 or EphA2 knockdown in 786-O and SN12-PM6 cells led to a marked

reduction of MMP-2 (Fig. 7A and 7B). However, a significant downregulation of MMP-9 was

only observed in SN12-PM6 cells but not in 786-O cells. Together, these data imply that

miR-141-EphA2 may contribute to ccRCC metastasis through that p-FAK/p-AKT/MMPs cascade.

Discussion

Identification of additional and essential molecular determinant(s) is imminent to designate

alternative strategies to overcome resistance in RCC therapy. Recent advances have established

dysregulation of miRNAs as a common event in cancers. Others and our groups have identified

that global downregulation of miRNA expression is a rising feature in ccRCC (10-14, 16, 17).

Among these miRNAs, four members (miR-141, 200c, 200b and 429) of miR-200s were all

markedly reduced in ccRCC tissues. However, how miR-200s functions in ccRCC pathogenesis

are still not well understood. Despite of our globe miRNA screen in the small sample size (N=5),

miR-141 has been reported to be one of the most significantly down-regulated in ccRCC tissues

and cells (10-14, 16, 17). Thus, we chose miR-141 as a representative to study the roles of

miR-200s. We demonstrated that miR-141 serves as a potential biomarker for discriminating

ccRCC from normal tissues and a crucial suppressor of ccRCC cell proliferation and metastasis by

modulating the EphA2/p-FAK/p-AKT/MMPs signaling cascade. This is the first in vivo study on

the functional characterization and mechanistic investigation of miR-141 in RCC.




20

Our findings indicated that miR-141 can be a potential biomarker in RCC diagnosis. Similar to

Jung’s reports (11, 15), our study showed that miR-141 measurement yielded 92.6% accuracy in

discriminating ccRCC tissues from normal kidney tissues. In consistence with previous studies (15,

39), no significant correlation was identified between miR-141 expression and RCC tumor stage,

grade, or size. Recent reports have also showed the ability of miRNAs to distinguish between

RCC subtypes with an accuracy of about 90%, including ccRCC, papillary RCC (pRCC), chRCC

and the closely related benign tumor oncocytoma (40, 41). Here, miR-141 expression was equally

decreased in malignant (ccRCC, chRCC and sarcoma RCC) and benign renal tumors (AML).

Considering that these results come from the relatively small number of clinical samples and

SYBR Green dye-based assays, the clinical significance of miR-141 remains to be investigated by

TaqMan Assay with higher specificity in a large cohort of RCC patients. Another limitation of

present study is that benign renal lesion oncocytomas, which is histologically quite similar to

chRCC, have not been included in our study. The comparison among oncocytomas, AML, chRCC

and ccRCC may further determine the role of miR-141 as a potential biomarker.

It has been well documented that a reciprocal repression between ZEB1/2 and miR-200s

induces EMT, which confers invasive properties to tumors (18-21). However, the effects of

miR-141 on ZEB1/2 mediated EMT are less efficient compared with other miR-200s members

(18-21, 23), indicating the additional biological functions of miR-141 in malignancy. Consistently,

our in vitro data suggested that miR-141 overexpression dramatically attenuated ccRCC cell

migration and invasiveness, suppressed cell proliferation and induced cell cycle arrest at the G0/G1

phase. Moreover, our in vivo studies based on orthotopic xenograft model of human RCC cells

indicated that suppression of miR-141 led to a pronounced increase in renal tumor weight, local




21

invasion and metastasis rate. These data suggest that miR-141 is involved in RCC proliferation

and progression. However, similar correlation was not obtained in clinical ccRCC cohort (15, 39,

42). The inconsistency may be due to limited samples, improvement of early-diagnosis of clinical

RCC and genetic intratumor heterogeneity (variable levels of miR-141 expression within different

regions of one tumor sample) (39).

Most importantly, our results established EphA2 as a direct functional effector of miR-141 in

RCC. EphA2, a member of the erythropoietin-producing hepatocellular (Eph) tyrosine kinases

receptor family, is an emerging target for cancer therapeutics (43). High EphA2 expression has

been correlated with cancer progression and metastasis in many cancers, including RCC (35, 36).

Downregulation of EphA2 expression with various approaches has been shown to inhibit

malignant behavior in vitro and in vivo (43). Up to now, knowledge of functional roles and

regulatory mechanism of EphA2 in RCC is still missing. Here we showed that EphA2 protein was

significantly upregulated in the vast majority of clinical ccRCC tissues and EphA2 mRNA

expression was inversely correlated with miR-141 levels in ccRCC tissues and cells. The

restoration of miR-141 in ccRCC cells markedly downregulated EphA2 expression through direct

interaction with the 3′UTR of EphA2 mRNA, and vice versa. Our results also indicated that

EphA2 knockdown suppressed RCC cell growth by inducing G0/G1 cell-cycle arrest, migration

and invasion, which phenocopied the effects of miR-141 overexpression in vitro. Knockdown of

EphA2 rescued the promoting effects of miR-141 inhibitor on RCC cell migration and invasion.

These data clearly demonstrated that EphA2 contributes to cell growth and migration in RCC and

is a direct and functional target of miR-141.




22

Two prominent oncogenic pathways, FAK and AKT, have been associated with EphA2

overexpression (30, 44-46). FAK pathway has been reported to be inversely correlated with

miR-200a expression in ovarian tumors, the seed sequence of which is the same as that of the

miR-141 (23). In our study, either EphA2 knockdown or miR-141 overexpression suppressed the

phosphorylation of FAK and AKT in ccRCC cells. In contrast, inhibition of miR-141 led to

increased phosphorylation of FAK and AKT. Additional downstream mediators of FAK/AKT such

as MMPs (MMP-2/9) that have been implicated in the aggressiveness of RCC (37, 38) are known

to contribute to FAK/AKT-mediated cell proliferation and progression (47-50), which is further

supported by our study on the functional activities of miR-141 and EphA2. However, further

investigation of EphA2/p-FAK/p-AKT/MMP pathway regulation in RCC is mandatory.

In conclusion, our identification of the remarkable alterations of miR-141 in RCC and its

specific functional mediators may provide novel mechanism (s) in tumor progression and

additional diagnostic and/or therapeutic target(s). The specification of the

miR-141-EphA2-FAK/Akt-MMP (2/9) pathway also has fundamental importance in basic

research.

Acknowledgment

The authors thank Dr. I.J. Fidler (MD Anderson Cancer Center, Houston, TX) for providing

the SN12-PM6 cell line.

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Legends




26

Table 1. Incidence of renal tumor, tumor weight, invasion and metastasis in orthotopic

xenografts after 6 and 8-9 weeks.

Figure 1. miR-141 is frequently attenuated in kidney tumor tissues and ccRCC cell lines.

A, relative miR-141 expression was determined by qRT-PCR in paired normal and ccRCC tissues

and analyzed with Mann–Whitney test. Each dot represents one sample.

B, relative miR-141 expression levels in kidney tumors are presented as fold change=2(△Ct normal-△Ct

tumor) of tumor versus matched normal tissues. The 0.5-fold-change threshold was defined as

differentially expressed. The relative miR-141 expression levels in ccRCC samples were

summarized in the table.

C, receiver operator characteristic (ROC) analysis of the cohort (A) to assess the specificity and

sensitivity of miR-141 to differentiate between ccRCC and normal tissues.

D, qRT-PCR analysis of miR-141 expression levels in pools of normal and ccRCC tissues, and

ccRCC cell lines. miR-141 level of normal tissues was set as 1. Data were normalized to U6 and

represented as mean±SEM.

Figure 2. miR-141 acts as a tumor-suppressor in RCC.

A-C, overexpression of miR-141 inhibits ccRCC cell proliferation, migration and invasion, and

induces cell-cycle arrest at G0/G1 in vitro. 786-O and SN12-PM6 cells infected with miR-141

expression or control lentiviruses were monitored for cell proliferation (A), cell-cycle (B) and

transwell migration and invasion (C).




27

D-F, released miR-141 from the donor tumor cells functions in the recipient tumor cells. D,

time-course expression of miR-141 and miR-16 in the CM from ccRCC cells stably

overexpressing miR-141 versus miR-NC. miR-16 expression was used as a control for exported

miRNA. Samples were measured by qRT-PCR and data were normalized to cel-miR-39. E,

expression of miR-141 and miR-16 in the ccRCC cells stably expressing miR-NC. Cells were

cultured with the CM of miR-141 or miR-NC cells for 48 hours, followed by assessing miR-141

and miR-16 expression using qRT-PCR. Data were normalized to U6. F, migration and invasion of

ccRCC cells stably expressing miR-NC were analyzed by transwell assay upon pretreatment with

the CM of miR-141 versus miR-NC cells. The results were derived from at least three independent

experiments and analyzed with Student’s t-test. Data are represented as mean±SEM. *, p<0.05; **,

p<0.01; ***, p<0.001.

Figure 3. Overexpression of miR-141 attenuates the tumor growth, migratory and invasive

properties of SN12-PM6 cells in vivo.

A, representative fluorescence images with primary tumors in the left kidney of nude mice after

orthotopic injections of SN12-PM6 cells stably overexpressing miR-141 or miR-NC for three

weeks.

B, macroscopic appearance of the tumor xenograft (arrows) in nude mice from the 6th-week and

8th-9th-week group. L, left kidney; R, right kidney.

C, HE staining of the tumor xenograft. N, normal renal tissues; T, primary renal tumors. Original

magnification was ×100.

D, SN12-PM6 cells stably overexpressing miR-NC metastasized to multiple organs (arrows).




28

Figure 4. miR-141 downregulates EphA2 expression through specifically targeting its

3’UTR.

A and B, the mRNA (A) and protein (B) levels of EphA2 were examined by qRT-PCR and WB in

786-O and SN12-PM6 cells stably overexpressing miR-141 versus the control (miR-NC),

respectively. mRNA data were normalized to GAPDH and β-actin was used as loading control in

WB.

C, the expression of miR-141 and EphA2 mRNA was assessed by qRT-PCR in renal tumor

xenografts. Data were normalized to U6 and GAPDH, respectively.

D, analysis of EphA2 protein expression in orthotopic renal tumors by IHC. Original

magnification was ×400.

E, sequence alignment of the EphA2 3’UTR with wild-type (WT) versus mutant (mut) potential

miR-141 targeting sites.

F, luciferase reporter assays showing decreased reporter activity after transfection of wild-type

EphA2 3’UTR reporter construct in the 786-O and SN12-PM6 cells overexpressing miR-141. The

EphA2 3’UTR mutant and control constructs had no effect on reporter activity. A renilla luciferase

construct was co-transfected into cells as internal control. The normalized luciferase activity of

control construct in each experiment was set as 1. Data are represented as mean±SEM. *, p<0.05;

***, p<0.001.

Figure 5. Knockdown of EphA2 in ccRCC cells significantly inhibits cell growth, induced

G0/G1 cell-cycle arrest, and suppressed cell migration and invasion.




29

A and B, the levels of EphA2 mRNA (A) and protein (B) in 786-O and SN12-PM6 cells were

assessed by RT-PCR and WB, respectively. Cells were transfected with 100 nM EphA2 siRNA

versus non-specific control for 48 hours before detection. GAPDH and β-actin served as internal

controls for mRNA and protein loading, respectively.

C-E, cell proliferation (C), cell-cycle (D), transwell migration and invasion (E) assays of 786-O

and SN12-PM6 cells were performed after transfection with EphA2 siRNA (si-EphA2) versus

control (si-NC) for 48 hours.

F, after co-transfection with 50 nM siRNA duplexes (siRNA against EphA2 or negative control)

and 50 nM miRNA inhibitors (miR-141 or negative control), the levels of miR-141 and EphA2

mRNA were analyzed by qRT-PCR analysis (top) and the levels of EphA2 protein were measured

by WB (bottom) in both 786-O and SN12-PM6 cells.

G, cell migration and invasion in 786-O and SN12-PM6 cells were analyzed by transwell assays.

Cells were co-transfected with 50 nM siRNA duplexes (siRNA against EphA2 or negative control)

and 50 nM miRNA inhibitors (miR-141 or negative control). Data are represented as mean±SEM.

*, p<0.05; **, p<0.01; ***, p<0.001.

Figure 6. miR-141 expression is reversely correlated with expression of EphA2 mRNA and

EphA2 protein is frequently upregulated in clinical ccRCC tissues.

A, EphA2 mRNA expression was examined by qRT-PCR in 20 pairs of normal and ccRCC tissues

and analyzed with Mann–Whitney test. Each dot represents a sample. GAPDH served as internal

control.

B, correlation between levels of miR-141 and EphA2 mRNA in ccRCC tissues. Data was analyzed




30

with Pearson’s correlation analysis.

C, ccRCC tissues were divided into two groups according to their miR-141 expression. ccRCC

tissues with lower miR-141 expression displayed higher EphA2 level, and vice versa.

D, the expression of EphA2 protein was determined by WB in 20 pairs of normal (N) and ccRCC

(T) tissues. An increase in EphA2 protein was detected in 17 of 20 RCC cases, with the exception

of 3 pairs of ccRCCs (Italics).

Figure 7. Ectopic miR-141 expression and knockdown of EphA2 suppress activated focal

adhesion kinase (FAK) and AKT (p-FAK and p-AKT) and MMP2/9 expression.

A, WB showing decreased levels of p-FAK, p-AKT, MMP2/9 in miR-141-overexpressed 786-O

and SN12-PM6 cells, as compared to the miR-NC counterparts.

B, WB for FAK/p-FAK, AKT/p-AKT, MMP2/9 protein expression in 786-O and SN12-PM6 cells

following transfection with EphA2 siRNA (si-EphA2) versus control (si-NC).

C, the protein levels of FAK/p-FAK, AKT/p-AKT were determined by WB after co-transfection

with miR-141 inhibitor (versus control) and EphA2 siRNA (versus control) in 786-O and

SN12-PM6 cells. β-actin served as an internal control.




1

Table 1. Incidence of renal tumor, tumor weight, invasion and metastasis in orthotopic xenografts after 6 and 8-9 weeks.

Group No. of mice Tumor incidence (%)a Tumor weight (mg) (mean ± SEM)b Beyond renal fascia rate (%)a Metastasis rate (%)a

6th week 8th-9th week 6th week 8th-9th week 6th week 8th-9th week 6th week 8th-9th week

miR-NC 10 5/5 (100) 5/5 (100) 124.7 ± 29.6 368.6 ± 131.1 2/5 (40) 4/5 (80) 2/5 (40) 3/5 (60)

miR-141 10 5/5 (100) 5/5 (100) 38.7 ± 8.1 52.9 ± 6.2 0/5 (0) 0/5 (0) 0/5 (0) 0/5 (0)

P value NSc 0.023 0.043 0.011 0.033

Note: a P values were determined by Fisher’s exact test. b Tumor weight was calculated by subtracting the weight of the right kidney (normal) from the weight of the left kidney (implanted with tumor). If the

tumor xenograft weight was small and the left kidney was just slightly heavier than the right, the weight of the tumor was recorded as zero. c Not significant.

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Published OnlineFirst March 19, 2014.Clin Cancer Res Xuanyu Chen, Xuegang Wang, Anming Ruan, et al. Proliferation and Metastasis by Controlling EphA2 ExpressionmiR-141 is A Key Regulator of Renal Cell Carcinoma

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