Author's Accepted Manuscript MicroRNA-34a suppresses cell proliferation and metastasis by targeting CD44 in human renal carcinoma cell Gan Yu , Heng Li , Ji Wang , Kiranmai Gumireddy , Anping Li , Weimin Yao , Kun Tang , Wei Xiao , Junhui Hu , Haibing Xiao , Bin Lang , Zhangqun Ye , Qihong Huang , Hua Xu PII: DOI: Reference:

S0022-5347(14)03687-8 10.1016/j.juro.2014.05.094 JURO 11517

To appear in: The Journal of Urology Accepted Date: 19 May 2014 Please cite this article as: Yu G, Li H, Wang J, Gumireddy K, Li A, Yao W, Tang K, Xiao W, Hu J, Xiao H, Lang B, Ye Z, Huang Q, Xu H, MicroRNA-34a suppresses cell proliferation and metastasis by targeting CD44 in human renal carcinoma cell, The Journal of Urology® (2014), doi: 10.1016/ j.juro.2014.05.094. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain. All press releases and the articles they feature are under strict embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date.

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MicroRNA-34a suppresses cell proliferation and metastasis by targeting CD44 in human renal carcinoma cell Gan Yu1, 2 , Heng Li1, 2 , Ji Wang3, Kiranmai Gumireddy4, Anping Li4, Weimin Yao1, 2, Kun Tang1, 2, Wei Xiao1, 2, ﹟

﹟

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Junhui Hu1, 2, Haibing Xiao1, 2, Bin Lang5, Zhangqun Ye1, 2, Qihong Huang4, Hua Xu1,2*

Gan Yu and Heng Li contributed equally to this work.

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

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Technology, Wuhan 430030, China

2, Institute of Urology，Tongji Hospital , Tongji Medical College，Huazhong University of Science and Technology,

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Wuhan 430030, China

3, Department of Cell Death and Cancer Genetics, The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA

4, The Wistar Institute, Philadelphia, PA19104, USA

5, School of Health Sciences, Macao Polytechnic Institute, Macao, China

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*To whom correspondence should be addressed at: Dr. Hua Xu, Department of Urology, TongjiHospital, TongjiMedicalCollege, Huazhong University of Science and Technology, Wuhan 430030, China. Institute of Urology，Tongji Hospital , Tongji Medical College，Huazhong University of Science and Technology,

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Wuhan 430030, China

Runninghead: MiR-34a target CD44 in renal carcinoma.

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Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Keyword: Renal cell carcinoma, miR-34a, CD44

Total words: 2618

ACCEPTED MANUSCRIPT Abstract Purpose: To investigate the potential functions of miR-34a in CD44 transcriptional complexes in renal cell carcinoma.

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Materials and Methods: MiR-34a expression was detected by quantitative real-time PCR. Oligonucleotide was used to overexpress miR-34a. Cell proliferation assay, colony formation, flow cytometry and xenograft assay were used to examine the effect on cancer cell proliferation in

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vitro and in vivo. Luciferase assay was carried out to verify the precise target of miR-34a.

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Results: Promoter methylation contributed to loss of miR-34a in renal carcinoma cell lines (ACHN, 786-O, SN12PM6). Ectopic overexpression of miR-34a restrained cell growth, tube formation and migration/invasion, and could also significantly suppress the growth of renal carcinoma xenografts and metastasis in nude mice. Dual luciferase assay indicated that CD44 was

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a direct target of miR-34a in renal cancer cells, and knockdown of CD44 by RNAi in renal cancer cells suppressed tumor progress. Ectopic expression of CD44, conversely, could partially reverse the antitumor effects of miR-34a in renal cancer cells.

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Conclusions: Our findings indicate that miR-34a targets CD44 in renal cancer cells and suppresses

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renal cancer cell growth, tube formation and metastasis in vitro or in vivo, and miR-34 may thus be a potential molecular target for novel therapeutic strategies for renal clear cell carcinoma.

ACCEPTED MANUSCRIPT Introduction Renal cell carcinoma(RCC) is a common cancer that accounts for 2–3% of all cancerous diseases in adults, whose incidence ranked seventh in men and ninth in women, accounting 209

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000 new cases per year and 102 000 deaths per year worldwide.1 The incidence of this cancer was steadily increasing these years, studies aimed at mechanisms associated with renal carcinogenesis, identification of novel diagnostic biomarkers and development of effective therapeutic 3

The most common RCC type is clear cell RCC

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interventions for RCC are in great need.2,

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(ccRCC), which accounts for almost 75% of all RCCs characterized by loss of 3p in >90% of cases.4, 5

MicroRNAs (miRNAs) are a class of approximately 22 nucleotide noncoding RNA molecules that negatively regulate the expression of a wide variety of genes mainly through direct interaction

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with the 3’-untranslated regions (3’-UTR) of corresponding mRNA targets.6 It has been estimated that miRNAs regulate up to one third of the total human genes at the post-transcriptional level,7 indicating that miRNAs have pivotal roles in physiological and pathological processes. Increasing

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evidence shows that the deregulation of miRNAs is involved in a wide range of diseases,

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including human cancers. These data highlight the important roles of miRNAs in tumor development and provide new insights into the molecular mechanisms underlying carcinogenesis. 8, 9

However, despite that more and more evidences indicated miRNAs including miR-34a play a

great role in the pathway of RCC, the role of miR-34a in the physiological and pathological processes remain to be elucidated in renal carcinoma cells. A better understanding of the role of miR-34a as a potential therapeutic target in RCC genetic analyses suggested that RCC was a genetic disease involving multiple types of gene changes including miRNAs by the recent decades

ACCEPTED MANUSCRIPT of research.10 CD44 as a surface adhesion molecule has been identified as the prostate cancer and cystic cancer stem cell marker,11, 12 and emerged as an important gene in multiple aspects of cancer 13

and progression.14, 15 It is reported that CD44 was overexpressed in renal cancer

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development

and had a central role in regulating diverse aspects of RCC pathogenesis.16-19 However, the mechanism of up-regulation of CD44 in renal carcinoma cells has not yet been fully clarified.

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Interestingly, as predicated by TargetScan and reported by previous study, miR-34a could

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negatively regulate different kinds of mRNAs including CD44 in some human cancers.20-24 However, functions and mechanisms of miR-34a involved in RCC are still not fully elucidated and the therapeutic application of miR-34a has not been reported. In this study, we identified mir-34a was down-regulated in renal carcinoma cells, and explored its functions and mechanisms in renal

cell carcinoma. Materials and Methods

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carcinogenesis. Moreover, we verified CD44 as the direct functional target of miR-34a in renal

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Cell culture and transfection

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The study was approved by the Institutional Review Board of Huazhong University of Science and Technology, Tongji Medical College, Tongji Hospital. Human renal cancer cell lines (ACHN, 786-O, Caki-1, OS-RC-2 and SN12PM6) were obtained from the American Type Culture Collection (Manassas, VA), and a primary culture of human proximal convoluted tubule epithelial cell (HK-2) was obtained from the Shanghai cell bank of the Chinese Academy of Sciences. For cell proliferation assay, cells were cultured in DMEM (ACHN, 786-O, SN12PM6 and HK-2), RPMI1640 (Caki-1 and OS-RC-2), supplemented with 10% fetal bovine serum (FBS), 95%

ACCEPTED MANUSCRIPT air–5% CO2 at 37℃. Six synthetic, chemically modified short single or double stranded RNA oligonucleotides (miR-34a mimics, mimics NC, miR-34a inhibitor, inhibitor NC) were purchased from Ribo Biotech (Guangzhou, China). Prevalidated siRNA specific for CD44 and a nonsilencing

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siRNA control were purchased from Genepharma Biotech (Shanghai, China). Lenti-miR-34a, Lenti-NC and plasmid psiCheck-CD44 (wide-type 3’UTR and mutant 3’UTR) were purchased from Genechem Biotech (Shanghai, China) Primary antibody CD44 (1:1000) and GAPDH

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(1:10000) were purchased from Sigma-Aldrich, St. Louis, MO. Oligonucleotide and plasmid

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transfection was made by using X-treme GENE siRNA Transfection Reagent (Roche) and FuGene HD Transfection Reagent (Roche) respectively, according to the manufacturers’ protocol. The lentivirus mediating the overexpression of miR-34a and the control vector were supplied by JIKAI Company Shanghai China. Three days after infection, puromycin was added to the media at 2

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µg/ml, and cell populations were selected for 2 weeks. Renal cancer cell line ACHN and 786-O were infected at an MOI of 10-20 and harvested 48-72 h post-infection. In vitro tube formation assay and Methylation Specific PCR

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HUVECs were maintained in endothelial basic medium containing 2% FBS and 1% penicillin/

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streptomycin or the indicated conditioned media (CM) of ACHN, SN12PM6 and 786-O cells. HUVECs (3×104) were seeded into a 96-well culture plate pre-coated with Matrigel (BD Biosciences) overnight and then cultured in the indicated condition. After 4-6 h incubation, the formation of tubes was photographed with a phase contrast microscopy (100× magnification, Olympus Instruments, Inc.), and quantified by counting branch points in five randomly selected microscope fields per well. The experiments were conducted twice. Genomic DNA was isolated by QIAamp DNA Mini Kit (Qiagen) and bisulfite modification of the genomic DNA was carried

ACCEPTED MANUSCRIPT out using an Epitect Bisulfite Kit (Qiagen) according to the manufacturer’s instructions. Methylation

Specific

PCR

(MSP)

primers

were

designed

with

Methprimer

(http://www.urogene.org/methprimer/index1.html) and listed in Supplementary Table S1.

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Dual luciferase assay To verify the precise target of miRNAs, Dual luciferase assays were carried out in both ACHN and 786-O cells. Cells (1×105) were plated in 12-well plates, 24 h before transfection. 50 nmoles

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of miR-34a mimics/inhibitor or adjacent negative control and 50ng pmirGLO-30-UTR vector

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were co-transfected into cells using X-treme GENE transfection reagent (Roche Applied Science). Renilla and firefly luciferase activities were measured with the Dual-Luciferase Reporter System (Promega) 24 h after transfection. Firefly luciferase activity was normalized to Renilla luciferase expression for each sample. Each experiment was performed in triplicate.

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Statistical analysis

Data from all experiments were presented as means ± SEM. from at least three independent experiments and analyzed by Student’s t-test unless otherwise specified (Pearson’s correlation).

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The miR-34a expression in RCC samples and renal carcinoma cell lines were analyzed by

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Mann-Whitney test and Wilcoxon matched pairs test for paired data. A p-value＜0.05 was considered as statistically significant. Results

Promoter methylation contributed to loss of miR-34a in renal carcinoma cell lines To investigate the role of miR-34a in renal carcinoma, we firstly evaluated the miR-34a expressions in five renal carcinoma cell lines (ACHN, 786-O, SN12PM6, Caki-1 and OS-RC-2) and a non-tumorigenic renal cell line HK-2 by qPCR. Compared to HK-2 cells, three (ACHN,

ACCEPTED MANUSCRIPT 786-O and SN12PM6) of five renal carcinoma cell lines had a significantly lower level of miR-34a expression (Fig. 1A, p < 0.01). Moreover, we found the CD44 expressions were up-regulated in these five renal carcinoma cell lines (Fig. 1B, p < 0.01), suggesting a potential

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inverse correlation between the CD44 and miR-34a expressions in ACHN, 786-O and SN12PM6. To explore the contribution of promoter hypermethylation to the loss of miR-34a, we examined the miR-34a expressions in renal carcinoma cell lines treated with DNA methylation inhibitor,

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5-aza-dc. The treated ACHN, 786-O and SN12PM6 cell lines showed significant reactivation of

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miR-34a (Fig. 1D). In addition, genomic DNAs were extracted from six renal cell lines and miR-34a promoter methylation was determined by methylation-specific PCR (MSP). As shown in Fig. 1E, promoter hypermethylation was found in three (ACHN, 786-O, SN12PM6) of these five renal carcinoma cell lines. Taken together, these results suggested that miR-34a was

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down-regulated through promoter hypermethylation in renal cancer cell lines. miR-34a overexpression inhibits renal cancer cell proliferation, clonegenesis, and induces G1 arrest and apoptosis

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To further explore the roles of miR-34a in renal carcinoma, ACHN, 786-O and SN12PM6 were

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transfected with hsa-miR-34a mimics and further used for function analysis. For optimization of transfection, we used FAM-labeled pre-miR-NC as negative control, and found that 90% of transfection efficiency could be reached at the concentration of 100 nM without obvious toxicity. Unless noted otherwise, all miRNAs were transfected at the concentration of 100 nM. As shown in Fig.1C, miR-34a expression was greatly increased after hsa-miR-34a mimics transfection. Sustained cell growth is a distinctive hallmark of cancer. In ACHN, 786-O and SN12PM6 cells, overexpression of miR-34a could markedly suppress the proliferation of these cell lines by Cell

ACCEPTED MANUSCRIPT Counting Kit-8 (Dojindo) assay (Fig. 2A). Then, the ACHN cells stably expressing miR-34a or NC were used to generate the xenograft model in nude mice. As shown in Figure 2D, at day 32 post injection, there were still significant miR-34a expression in derived xenograft (Fig. 2Db). The

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average tumor in miR-34a overexpression group was 0.42 cm2, which was markedly smaller than NC group (Fig. 2Da).

Next, we checked whether miR-34a exerts an effect on the cell cycle distribution and apoptosis

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using flow cytometry assay in renal cancer cells. In cells transfected with hsa-miR-34a mimics for

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48h, percentage of G1/G0 phase population significant increased, and that of S and G2/M phases populations decreased when as compared with control (P ＜ 0.05). Interestingly, transfection with hsa-miR-34a mimics also led to an increase in cells with subdiploid (< 2C) DNA content, a marker for DNA fragmentation and apoptosis (Supplementary Fig. S2B). The effect of miR-34a

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overexpression on apoptosis induction were further confirmed in TUNEL and flow cytometry assays (Fig. 2C and Supplementary Fig. S2A). Additionally, the assessment of cell proliferation revealed an obvious loss of capability to form colonies after tranfected with miR-34a (Fig. 2B).

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We further determined the effects of the miR-34a overexpression on the expressions of

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downstream genes associated with cell-cycle and mitotic checkpoint in renal cancer cells. Our results showed that transfections with hsa-miR-34a mimics changed expression levels of these genes in a cell line-dependent manner.. Of them, we observed that manipulations of miR-34a expression increased mRNA levels of p15 and p21 in all three cell lines, and p27 mRNA level in ACHN and 786-O cells, but decreased p27 mRNA level in SN12PM6. It also caused significant decreases of mRNA levels of CyclinD1, CyclinD3 and CDK6 in two tested cell lines. The effects of miR-34a overexpression on protein levels of these genes were further identified by western blot

ACCEPTED MANUSCRIPT assays. As shown in Fig. 2E, transfection with hsa-miR-34a mimics significantly increased protein expressions of p15 and p21 in all three cell lines, and p27 protein expressionin ACHN and 786-O cells, but decreased protein expression of Cyclin D1 protein decreased in ACHN and 786-O cells

all tested cell lines, although the mRNA was little increased in 786-O.

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which were consistent to the mRNA changes. We also observed decreased CDK6 protein levels in

Mir-34a overexpression inhibits renal cancer cell migration and invasion by modulating

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EMT related proteins

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To assess changes in cell migration, ACHN and SN12PM6 cells transfected with hsa-miR-34a mimics or FAM-labeled pre-miR-NC (negative control) were allowed to migrate through a transwell membrane into complete media. Compared with the negative control, miR-34a overexpression led to 61%, 52% and 50% reduction of migratory cells in ACHN, 786-O and

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SN12PM6, respectively (Fig. 3A). To evaluate cell invasion capability, hsa-miR-34a transfected cells were plated on the membranes which were precoated with matrigel. As shown in Fig. 3A, mir-34a overexpression significantly reduced the invasive capabilities of ACHN, 786-O and

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SN12PM6 cells by approximately 54%, 32% and 28%. In intravenous injection assay,

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bioluminescence imaging revealed that fluorescence signals in lenti-miR-34a group were significantly weaker than lenti-NC group, indicating that less metastasis was formed in lung after miR-34a overexpression (Fig.3B). Tube formation assay in SN12PM6, ACHN and 786-O also showed an obvious loss of proangiogenic activity after has-miR-34a transfection (Fig.3D). Moreover, EMT-related factors like N-cadherin, E-cadherin, Vimentin and snail and proangiogenic factor VEGF were also examined by immunoblot and qPCR assay after mir-34a restoration. Our results indicated that miR-34a could promote E-cadherin and inhibited N-cadherin

ACCEPTED MANUSCRIPT and Vimentin mRNA in all three cell lines. Snail mRNA expression was selectively inhibited by miR-34a in ACHN and 786-O but was promoted in SN12PM6. Immunoblot showed that miR-34a consistently promoted E-cadherin protein expression and inhibited N-cadherin, Snail and Vimentin

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protein expression in all three cell lines. Although there was inconsistence in mRNA and protein expression, miR-34a mainly inhibited EMT in renal cancer cell (Fig. 3C and Supplementary Fig. S1B). In addition, miR-34a downregulated VEGF in all three renal cancer cell lines both in

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mRNA and protein levels. These data indicated that miR-34a functions as a tumor suppressor in

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these renal cancer cell lines.

MiR-34a functions as a tumor suppressor gene via directly targeting CD44 in renal cancer CD44 was overexpressed in renal cancer and had a central role in regulating diverse aspects of RCC pathogenesis. Interestingly, as predicated by TargetScan and previous studies, miR-34a could

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negatively regulate various of mRNAs including CD44 in some human cancers. In our study, restoration of miR-34a markedly decreased CD44 protein but not mRNA expression (Fig. 4A and B), indicating a potential post-transcriptional control of CD44 expression caused by miR-34a.

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According to the previous study in prostate cancer,12 we also performed a dual luciferase reporter

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assay and determined that CD44 was exactly the target of miR-34a in renal cancer cells (Fig. 4C and D). To confirm the inverse effect of CD44 and miR-34a in tumorigenesis, we blocked the CD44 expression by specific siRNA transfection, and found that CD44 silence resulted in decreased capability of cell proliferation, colony formation and invasion and migration (Fig. 5A-C), which is parallel with miR-34a overexpression in all the three renal cancer cell lines. Subsequently, To test whether restoration of CD44 could rescue the antitumor effects of miR-34a, we co-transfected miR-34a mimics and psiCheck-CD44 plasmids with wild type 3’UTR or mutant

ACCEPTED MANUSCRIPT 3’UTR into renal cancer cells. We found that CD44 with mutant 3’UTR could interfer the inhibitory effects of miR-34a, and overexpression of CD44 could partially reduce the tumor suppressor function caused by miR-34a restoration in RCC (Fig. 6A-C). Thus, our data presented

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here supported our hypothesis that the tumor-suppressive effect of miR-34a is mediated by directly targeting CD44. Discussion

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To better understand the molecular mechanism of renal tumorigenesis, the dysregulation of

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miRNAs is always an intriguing area.8 Of them, miR-34a is a star molecule which had been reported to be down-regulated in prostate cancer,12 cervical carcinoma,25 renal cancer

26

and may

play important roles in the occurrence and development of these human cancer by targeting certain targets. Previous studies reported epigenetic silencing of miR-34a in RCC.17, 27 However, little is

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known about the function or molecular mechanisms of miR-34a in renal cancer. In this study, we found miR-34a is frequently down-regulated in renal cancer cells, which, in consistence with previous reports25, 26, 27, was obviously associated with promoter hypermethylation. Our data also

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demonstrated that overexpression of miR-34a could inhibit the growth and migration/invasion

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ability of cancer cells.

Furthermore, we found that CD44 overexpression could interfere and reduce the pro-apoptotic effects of miR-34a which is similar to what’s observed in medulloblastoma.28 CD44 has been identified to be the direct target of miR-34a in prostate cancer. In this study we also confirmed CD44 as a direct functional target of miR-34a in renal cell carcinoma.28 Some reports suggested that CD44 protein might be involved in p-Chk1 and p-Chk2 activation and may be responsible for the dysregulation of genes modulating radiation sensitivity, metastasis and tumor development.

ACCEPTED MANUSCRIPT We proved that in renal cell carcinoma miR-34a could target CD44 by binding to its 3’ UTR and subsequently suppress CD44 which regulates transcription of a variety of genes in tumor cells.

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To our knowledge, this is the first report showing that up-regulation of CD44 may result from

miR-34a may be a novel candidate for therapeutic strategy against RCC. Conclusion

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down-regulation of miR-34a in human cancer cells. For the significant tumor suppressor function,

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Our findings indicate that miR-34a targets CD44 in renal cancer cells and suppresses renal

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cancer cell growth, tube formation and migration/invasion in vitro or in vivo, and provides a novel target miR-34a as a new therapeutic strategy against renal clear cell carcinoma.

Acknowledgments: This work was supported by National Natural Science Foundation of China (31072238,

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31172441, 31372562, 81170650); National Major Scientific and Technological Special Project for Significant New

Drugs Development (2012ZX09303018); NCIR01CA148759. The funders had no role in study design, data

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collection and analysis, decision to publish, or preparation of the manuscript.

Reference 1.

Gupta K, Miller JD, Li JZ et al: Epidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): a literature review. Cancer Treat Rev 2008; 34: 193.

2.

Sato Y, Yoshizato T, Shiraishi Y et al: Integrated molecular analysis of clear-cell renal cell

carcinoma. Nat Genet 2013; 45: 860. 3.

Oosterwijk E, Rathmell WK, Junker K et al.: Basic research in kidney cancer. Eur Urol 2011; 60: 622.

4.

van den Berg E and Dijkhuizen T: Classification of renal cell cancer based on (cyto)genetic analysis. Contrib Nephrol 1999; 128: 51.

5.

Junker K, Weirich G, Amin MB et al: Genetic subtyping of renal cell carcinoma by comparative genomic hybridization. Recent Results Cancer Res 2003; 162: 169.

ACCEPTED MANUSCRIPT 6.

Bartel DP: MicroRNAs: target recognition and regulatory functions. Cell 2009; 136: 215.

7.

Lewis BP, Burge CB, Bartel DP: Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120: 15.

8.

Calin GA and Croce CM: MicroRNA signatures in human cancers. Nat Rev Cancer 2006 6: 857.

9.

Bandyopadhyay S, Mitra R, Maulik U et al: Development of the human cancer microRNA network. Silence 2010; 1: 6. Catto JW, Alcaraz A, Bjartell AS et al: MicroRNA in prostate, bladder, and kidney cancer: a

11.

Chan KS, Espinosa I, Chao M et al: Identification, molecular characterization, clinical

systematic review. Eur Urol 2011; 59: 671.

RI PT

10.

prognosis, and therapeutic targeting of human bladder tumor-initiating cells. Proc Natl Acad Sci U S A 2009; 106: 14016. 12.

Liu C, Kelnar K, Liu B et al: The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nat Med 2011; 17: 211.

Jeong BJ, Liang ZL, Huang SM et al: CD44 is associated with tumor recurrence and is an

SC

13.

independent poor prognostic factor for patients with localized clear cell renal cell carcinoma after nephrectomy. Exp Ther Med 2012; 3: 811.

Bamias, A., Chorti, M., Deliveliotis, C. et al.: Prognostic significance of CA 125, CD44, and

15.

Costa WH, Rocha RM, Cunha IW et al: Immunohistochemical expression of CD44s in renal cell

M AN U

14.

epithelial membrane antigen in renal cell carcinoma. Urology, 62: 368, 2003 carcinoma lacks independent prognostic significance. Int Braz J Urol 2012; 38: 456. 16.

Altinel M, Arpali E, Turhan N et al: Increased expression of CD44s in conventional renal cell carcinomas with renal vein or vena cava thrombosis. a retrospective evaluation of the expression of CD44s by immunohistochemical analysis in conventional RCC patients and in

17.

TE D

conventional RCC patients with renal vein or vena cava thrombosis. Urol Int 2008; 81: 452. Lim SD, Young AN, Paner GP et al: Prognostic role of CD44 cell adhesion molecule expression in primary and metastatic renal cell carcinoma: a clinicopathologic study of 125 cases. Virchows Arch 2008; 452: 49. 18.

Tawfik OW, Kramer B, Shideler B et al: Prognostic significance of CD44, platelet-derived

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growth factor receptor alpha, and cyclooxygenase 2 expression in renal cell carcinoma. Arch Pathol Lab Med 2007; 131: 261. 19.

Yildiz E, Gokce G, Kilicarslan H et al: Prognostic value of the expression of Ki-67, CD44 and

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vascular endothelial growth factor, and microvessel invasion, in renal cell carcinoma. BJU Int 2004; 93: 1087.

20.

Basak SK, Veena MS, Oh S et al: Correction: The CD44 Tumorigenic Subsets in Lung Cancer Biospecimens Are Enriched for Low miR-34a Expression. PLoS One 2013; 8: e73195.

21.

Stankevicins L, Almeida da Silva AP, Ventura Dos Passos F et al: MiR-34a is up-regulated in response to low dose, low energy X-ray induced DNA damage in breast cells. Radiat Oncol 2013; 8: 231.

22.

Kang J, Kim E, Kim W et al: Rhamnetin and cirsiliol induce radiosensitization and inhibition of epithelial-mesenchymal transition (EMT) by miR-34a-mediated suppression of Notch-1 expression in non-small cell lung cancer cell lines. J Biol Chem 2013; 288: 27343.

23.

Garofalo M, Jeon YJ, Nuovo GJ et al: MiR-34a/c-Dependent PDGFR-alpha/beta Downregulation Inhibits Tumorigenesis and Enhances TRAIL-Induced Apoptosis in Lung Cancer. PLoS One 2013; 8: e67581.

ACCEPTED MANUSCRIPT 24.

Cao W, Yang W, Fan R et al: miR-34a regulates cisplatin-induce gastric cancer cell death by modulating PI3K/AKT/survivin pathway. Tumour Biol 2014; 35: 1287.

25.

Pang RT, Leung CO, Ye TM et al: MicroRNA-34a suppresses invasion through downregulation of Notch1 and Jagged1 in cervical carcinoma and choriocarcinoma cells. Carcinogenesis 2010; 31: 1037.

26.

Yamamura S, Saini S, Majid S et al: MicroRNA-34a suppresses malignant transformation by 2012; 33: 294.

27.

Lodygin D, Tarasov V, Epanchintsev A et al: Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle 2008; 7: 2591.

28.

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targeting c-Myc transcriptional complexes in human renal cell carcinoma. Carcinogenesis

de Antonellis P, Medaglia C, Cusanelli E et al: MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma. PLoS One 2011; 6: e24584.

Golshani R, Lopez L, Estrella V et al: Hyaluronic acid synthase-1 expression regulates bladder

SC

29.

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cancer growth, invasion, and angiogenesis through CD44. Cancer Res 2008; 68: 483.

Figure 1: Promoter methylation contributed to decrease of mir-34a in of renal carcinoma cell lines. A, mir-34a expression were evaluated by qPCR in renal carcinoma cell lines. U6 was internal control. B, CD44 expression

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levels were evaluated by qPCR in renal carcinoma cell lines. GAPDH was internal control. C, miR-34a expression after mimics transcription was detected by qPCR. D, mir-34a expression in five renal cancer cell lines after 5-AZA-dc treatment compared to mock-treated cells. E, Methylation-specific PCR was performed using primers

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specific for the unmethylated (U) or methylated (M) mir-34a promoter region. NC, unmethylated genomic DNA (Qiagen); PC, methylated genomic DNA (Qiagen). Data are plotted as the mean ± SEM of 3 independent

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experiments. **, P＜0.05.

Figure 2: Overexpression of mir-34a inhibits renal cancer cell growth in vivo and vitro. A, MTS kit used for cell viability assay at each time point (mean ± SEM; n=3). B (a), representative photographs of colony formation after miR-34a mimics transfection. B (b), number of colonies was quantified (mean ± SEM; n=3). C (a), apoptosis assay was performed using TUNEL. C (b) number of apoptotic cells was quantified (mean ± SEM; n=3). D (a), photographs of nude mice bearing subcutaneous tumors; D (b), qPCR assay to detect the mir-34a in tumors between the two groups. D (c), mean tumor volume measured by caliper on the indicated days. D (d), tumor weight of each nude mouse at the end of 32 days. E, cell-cycle and apoptosis related proteins and VEGF proteins

ACCEPTED MANUSCRIPT were determined by immunoblot analysis. GAPDH was used as control. *, P ＜ 0.05; **, P ＜ 0.01.

Figure 3: mir-34a inhibits renal carcinoma cell invasion, migration and tube formation. A (a), representative photographs of transwell assay (×200). A (b), number of migrated and invaded cells were quantified in 4 random

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images from each treatment group. Results are plotted as percent (%) relative to NC treatment. B (a), representative bioluminescent images of lungs of nude mice at the 30th days after IV. injection of treated ACHN. B (b), quantification analysis of fluorescence signal from captured bioluminescence images. C, EMT related proteins were determined by immunoblot analysis. GAPDH was used as control. D (a), representative images of tube

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formation HUVECs . D (b), branch nodes numbers was assayed after 6 h of culture under a phase contrast microscope. HUVECs were cultured in the following media: conditioned media of ACHN, 786-O and SN12PM6

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cells transfected with mir-34a or NC. NC, Negative Control. *, P ＜ 0.05; **, P ＜ 0.01.

Figure 4: mir-34a targets CD44 in renal carcinoma cell lines. A, CD44 mRNA expression was detected by qPCR after transfection of miR-34a mimics or inhibitor and corresponding negative control for 48h. B, CD44 protein expression was inhibited by miR-34a. C and D, The seed regions of the miR-34a target sites in CD44 and the

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luciferase activity assay. *, P ＜ 0.05; **, P ＜ 0.01.

Figure 5: The tumor-suppressive functions of miR-34a were mediated by reducing the production of CD44. A,

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MTS kit was utilized to quantify cell viability at each time point (mean ± SEM; n=3). B (a), representative photographs of transwell assay (×200). B (b), number of migrated and invaded cells were quantified in 4 random

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images from each treatment group (mean ± SEM; n=3). Results are plotted as percent (%) relative to NC group. C (a), representative photographs of cell culture plates following staining for colony formation of ACHN, 786-O and SN12PM6 cell after treatment. C (b), number of colonies was quantified. *, P ＜ 0.05; **, P ＜ 0.01.

Figure 6: Increased CD44 expression could efficiently reverse the effect of miR-34a on renal carcinoma cells. A, MTS kit was utilized to quantify cell viability at each time point (mean ± SEM; n=3). B (a), representative photographs of transwell assay (×200). B (b), number of migrated and invaded cells were quantified in 4 random images from each treatment group (mean ± SEM; n=3). Results are plotted as percent (%) relative to NC group. C (a), representative photographs of cell culture plates following staining for colony formation. C (b), number of colonies was quantified. *, P ＜ 0.05; **, P ＜ 0.01.

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ACCEPTED MANUSCRIPT Supplemental Materials and Methods RNA extraction and quantitative real-time RT-PCR Total RNA was extracted from renal cancer cell using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA integrity was evaluated by Nano Drop

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ND-1000 spectrophotometer. Total RNA was extracted from renal cancer cell using TRIzol reagent (Invitrogen Life Technologies) and then reverse-transcribed using Fermentas RT reagent Kit (Perfect Real Time) according to the manufacturer’s instructions. CD44 expression in renal

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cancer cell was measured by qPCR using SYBR Premix Ex Taq on MX3000 instrument. 2 µg of

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total RNA was converted to cDNA according to the manufacturer’s protocol. PCR was performed in a total reaction volume of 25.0 µl, including 10 µl SYBR Premix Ex Taq(2x), 1 µl of PCR Forward Primer (10 µM), 1 µl of PCR Reverse Primer (10 µM), 0.5 µl ROX Reference Dye (50x)*3, 2 µl of cDNA, 8 µl of double-distilled water. The quantitative real-time PCR reaction was

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set at an initial denaturation step of 10 min at 95°C; and 95°C(5 seconds),63°C(30 seconds),72°C(30 seconds) in a total 40 cycles with a final extension step at 72°C for 5 min. All experiments were done in triplicate. All samples normalized to GAPDH. Mir-34a expression in

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RCCC was measured by qPCR using SYBR Premix Ex Taq on MX3000 instrument. 2ug of total

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RNA was converted to cDNA according to the manufacturer’s protocol. PCR was performed in a total reaction volume of 20.0 µl, including 9 µl SYBR Premix Ex Taq(2x), 2 µl of PCR Forward Primer (10 µM), 2ul of PCR Reverse Primer (10 µM), 0.5 µl ROX Reference Dye

(50x)*3, 2µl

of cDNA, 5 µl of double-distilled water. The quantitative real-time PCR reaction was set at an initial denaturation step of 10 min at 95°C; and 95°C(5 seconds),60°C(20 seconds),72°C(20 seconds) in a total 40 cycles with a final extension step at 72°C for 5 min. All experiments were done in triplicate. All samples normalized to U6. The median in each triplicate was used to

ACCEPTED MANUSCRIPT calculate relative CD44/mir-34a concentrations (∆Ct = Ct median CD44/mir-34a-Ct median GAPDH/U6). Expression fold changes were calculated using 2-∆∆Ct methods. The CD44/mir-34a expression differences between cancer and control were analyzed using Student’s t test within

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SPSS (Version 17.0 SPSS Inc.). A value of p 0.05 was considered as statistically significant. Oligonucleotide transfection and efficiency test

Hsa-miR-34a mimics were synthesized by Genepharma, Shanghai, China. MiR-34a inhibitor

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(anti–miR-34a, chemically modified antisense oligonucleotides designed to target specifically

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against mature miR-34a). ACHN/SN12PM6/786-O Cells (2×105) were planted in 6-well plates the day before transfection. Transfection system was as follow, the cells were transfected with 100 nM of miR-34a precursor has-mir34a mimics, or anti-miR-34a inhibitor, or the negative control. The transfection using X-tremeGENE transfection reagent (Roche Applied Science,

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Indianapolis,IN, USA), according to the manufacture’s protocol. MiR-34a expression in the transfected cells that was transfected with hsa-miR-34a mimics or pre-miR-NC was determined by RT--qPCR at 48 h after transfection.

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Cell proliferation and colony formation assay

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ACHN, SN12PM6 and 786-O cells were infected with pre-miR-NC or has-miR-34a mimics for approximately 6 hours, then cells were digested and transferred to 96 well micro-plates, replanting at a density of approximately 2000(ACHN)/3000(SN12PM6)/1000(786-O) cells per well 24, 48 and 72h after transfection. All experiments were performed in triplicate. Cell viability was subsequently determined every 24 hours for 3 days by using the Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer's protocol and measure the absorbance by using Micro-plate reader (Thermo). 24 hours following transduction, cells were seeded in 6-well plates

ACCEPTED MANUSCRIPT at 1,000 cells/well, for colony formation, cells were maintained in complete medium for 10-12 days at which point crystal violet was used for visualizing colonies. The complete medium was changed every 3 days.

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Apoptosis Detection To detect apoptosis by flow cytometry cells were grown for the indicated duration after plasmid transfection and then treated with 0.1% Triton X-100 containing propidium iodide to stain nuclei

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or labeled using the FITC Annexin V Apoptosis Detection Kit 1 (BD Pharmingen™). DNA

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content and annexin V positive cells were measured using a FACSCalibur™ flow cytometer and CellQuest™ software. Apoptosis was evaluated by quantifying the percent of cells with hypodiploid DNA (sub-G1) as an indicator of DNA fragmentation or the percent positively stained with annexin V. For all assays 10,000 cells were counted.

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Transwell assay

Migration and invasion assays were performed as described as follows, renal cancer cell lines ACHN, SN12PM6 and 786-O were transfected with pre-miR-NC and has-miR-34a mimics for

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48h. Transwell inserts that have 6.5-mm polycarbonate membranes with pores 8.0 μm in size

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(Corning, New York, NY, and USA). Matrigel invasion assay was done using membranes coated with Matrigel matrix (BD Science, Sparks, MD, USA). A density about 1 × 105 of ACHN/SN12PM6 cells or 2 × 104 of 786-O was suspended and then seeded in the upper chambers of 24-well transwell plates with FBS-free medium. Culture medium containing 10% fetal bovine serum was deposited in the lower chambers. After 12-18 hours for ACHN/SN12PM6 and 8-12h for 786-O, cells that migrated were stained by 0.5% crystal violet solution for 15 min and counted. For invasion, Transwell membranes were prepared with matrigel for plating infected cells. After

ACCEPTED MANUSCRIPT 24 hours for ACHN/SN12PM6 or 12-14 hours for 786-O, cells that migrated were stained by 0.5% crystal violet solution for 15 min and counted. Each experiment was performed in duplicate. Western blot

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Briefly, protein was extracted from cells, Protein extraction with NP40 and were separated on 10% SDS-PAGE gel and then transferred to PVDF membranes (Bio-Rad). Nonspecific binding was blocked by incubating the PVDF membranes with 5% nonfat milk containing 0.1% Tween-20

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and then for 2 hr at room temperature. The membrane was incubated Primary antibodies included

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CD44 (1:1000 dilution; Abcam), E-cadherin vimentin, N-cadherin, snail, p15, p16, p27, cyclinD3, CDK4 and CDK6 (1:1000 dilution; Cell Signaling Technology), and GAPDH (1:2000 dilution; Cell Signaling Technology) in TBST +5% nonfat milk at 4

overnight. After washing with

TBST, the PVDF membranes were incubated with horseradish peroxidase-conjugated sheep

detection system (Pierce).

.At last the proteins were visualized using ECL-plus

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anti-mouse IgG(1:4,000) for 1 hr at 37

In vivo tumorigenicity assay

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Tumorigenicity in nude mice was determined as described previously 1, 2. Two groups of 5 mice

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each were injected subcutaneously with prepared ACHN at the same site. Tumor onset measured with calipers at the site of injection every three days by two trained laboratory staffs at different times on the same day 3 weeks after injection when appreciable tumor formed subcutaneously. Tumor volume was calculated using the formula, V = 0.5ab2, where a represents the larger and b represents the smaller of the 2 perpendicular indexes. Animals were sacrificed 32 days after injection and tumors were weighed. Nude mice were manipulated and cared according to NIH Animal Care and Use Committee guidelines in the ExperimentAnimalCenter of the Tongjimedical

ACCEPTED MANUSCRIPT collage of HuazhongUniversity of science and technology (Wuhan, Hubei, Province, P.R. China). Experimental lung metastasis model The antimetastatic activity of miR-34a was tested in nude mice ACHN lung metastasis model as

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described previously.3 ACHN cell was stably infected with Lenti-miR-34a or Lenti-NC containing GFP label. Treated cells (2 × 105) were suspended in 100µL of PBS and injected intravenously via the tail vein. Mice were sacrificed and lungs were resected 30 days later after injection. The

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incidence and volume of metastases were estimated by imaging of mice for bioluminescence using

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the Living Image software (Xenogen, Baltimore, MD). The photon emission level was used assess the relative tumor burden in the mice lungs. Nude mice were manipulated and cared according to NIH Animal Care and Use Committee guidelines in the Experiment Animal Center of the Tongji medical collage of Huazhong University of science and technology (Wuhan, Hubei, Province, P.R.

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China).

Supplementary Table S1: Primers list. Sequence (5' - 3')

p15

Sense: GGGCTTCCTGGACACGCTG

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Primer name

Antisense: CCAAGTCCACGGGCAGACG

p21

Sense: CCCGTGAGCGATGGAACT

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Antisense: CGAGGCACAAGGGTACAAGA

p27

Sense: GGCTCCGGCTAACTCTGA Antisense: TCTTCTGTTCTGTTGGCTCTTT

p57

Sense: CAGAACCGCTGGGATTACGACTT Antisense: AGTCGCTGTCCACTTCGGTCCACT

CyclinD1

Sense: GCTGCGAAGTGGAAACCATC Antisense:CCTCCTTCTGCACACATTTGAA

CyclinD3

Sense: GGAAGGCTGAGAGGGAACTAGGAG Antisense: GGCCAGGAACCAGAGAGTGAATGG

CDK6

Sense: GCTGACCAGCAGTACGAATG Antisense: GCACACATCAAACAACCTGACC

CDK4

Sense: ATGGCTACCTCTCGATATGAGC Antisense: CATTGGGGACTCTCACACTCT

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Sense: AAGCGGTCAGTGAGAAGGAAG Antisense: GGGGCCGTGTAGATAAACTCTAT

E-cadherin

Sense: ACCAGAATAAAGACCAAGTGACCA Antisense: AGCAAGAGCAGCAGAATCAGAAT

N-cadherin

Sense: ATGCCCAAGACAAAGAAACC Antisense: CTGTGCTTGGCAAGTTGTCT

MMP2

Sense: TGGCAAGGAGTACAACAGC

Vimentin

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Antisense: TGGAAGCGGAATGGAAAC Sense: GATGCGTGAGATGGAAGAGA Antisense: GGCCATGTTAACATTGAGCA MMP9

Sense: TGTACCGCTATGGTTACACTCG Antisense: GGCAGGGACAGTTGCTTCT Sense: ATTTCAACGCCTCCAAGAAG Antisense: CGAGGTGAGGATCTCTGGTT

Snail

Sense: GAAGATGCACATCCGAAGC

VEGF

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Antisense: GGAGAATGGCTTCTCACCAG

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Slug

Sense: AGGGCAGAATCATCACGAAGT

Antisense: AGGGTCTCGATTGGATGGCA GAPDH

Sense: TGGGTGTGAACCATGAGAAG

Antisense: GTGTCGCTGTTGAAGTCAGA MSP Methylation (M)

Sense: TAGGTTTGTTTTTCGAGTTTTTTTC

Unmethylation (U)

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Antisense: CCTCTTAAACCCCACAACGTA Sense: TGGGTGTGAACCATGAGAAG

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Antisense: GTGTCGCTGTTGAAGTCAGA

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Supplemental Figure legend

Supplementary Figure S1: the proliferation and cell cycle related (A), EMT related and VEGF (B) mRNA expression in ACHN, 786-O and SN12PM6 cells were determined by qPCR after has-miR-34a mimics transfection. GAPDH was used as control. Expression data were normalized to GAPDH level and fold change after transfection was shown in a form of log2 rate versus control group. Results are presented as mean ± SEM of 3 independent experiments. These differences were all significant.

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Supplementary Figure S2: A, flow cytometry apoptosis detection using Annexin- -APC after infection. Cells in the right-down quadrant were defined as early apoptosis cells. B, cell cycle

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distribution of ACHN, 786-O and SN12-PM6 were determined by flow cytometry assay after has-miR-34a mimics transfection.

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References

Saini S, Yamamura S, Majid S et al: MicroRNA-708 induces apoptosis and suppresses tumorigenicity in renal cancer cells. Cancer Res 2011; 71: 6208.

Liu C, Kelnar K, Liu B et al: The microRNA miR-34a inhibits prostate cancer stem cells and

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metastasis by directly repressing CD44. Nat Med 2011; 17: 211.

Parhar RS and Lala PK: Amelioration of B16F10 melanoma lung metastasis in mice by a

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combination therapy with indomethacin and interleukin 2. J Exp Med 1987; 165: 14.

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Key of Definitions for Abbreviations: RCC, Renal Cell Carcinoma; NC, Negative Control;

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