MicroRNA-494 promotes cancer progression and targets adenomatous polyposis coli in colorectal cancer

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1 Zhang et al. Moleular Caner (8) 7: DOI.86/s RESEARCH Open Aess MiroRNA-494 promotes aner progression and targets adenomatous polyposis oli in oloretal aner Ying Zhang, Lu Guo,, Yuhuan Li,, Gui-Hai Feng, Fei Teng,, Wei Li, and Qi Zhou,* Astrat Bakground: Aerrant ativation of the Wnt/β-atenin signaling pathway is frequently oserved in oloretal aner (CRC). β-atenin is the major Wnt signaling pathway effetor and inativation of adenomatous polyposis oli (APC) results in nulear aumulation of β-atenin. It has een suggested that inativation of APC plays an important role in ativation of the Wnt/β-atenin pathway and in the progression of oloretal tumorigenesis. However, the mehanism through whih APC mediates oloretal tumorigenesis is not understood. Inreasing evidene suggests that the dysregulation of mirornas (mirnas) is involved in oloretal tumorigenesis. Although mir-494 has een reported as eing an upregulated mirna, the interplay etween mir-494 and APC-mediated oloretal tumorigenesis progression remains unlear. Methods: The expression of mir-494 in tissues from patients diagnosed with CRC was analyzed using a miroarray and real-time PCR. The effets of mir-494 on ell proliferation and tumorigenesis in CRC ells were analyzed y flow ytometry, olony formation assays, BrdU inorporation assays, and CCK8 assays. The orrelation etween mir-494 expression and APC expression, as well as the mehanisms y whih mir-494 regulates APC in CRC were also addressed. Results: mir-494 was signifiantly upregulated in CRC tissues, and this inrease was negatively assoiated with APC expression. APC was onfirmed to e a diret target of mir-494 in CRC. Furthermore, overexpression of mir- 494 indued Wnt/β-atenin signaling y targeting APC, thus promoting CRC ell growth. Conlusions: This study provides novel insights into the role of mir-494 in ontrolling CRC ell proliferation and tumorigenesis, and identifies mir-494 as a potential prognosti marker and therapeuti target. Keywords: Coloretal aner, mirorna, mir-494, Ap Bakground Coloretal aner (CRC) is one of the most ommon malignanies in the world, with over one million new patients diagnosed every year []. CRC progression is aompanied y the aumulation of mutations in tumor-suppressor genes and onogenes, inluding adenomatous polyposis oli (APC) []. Inativation of APC is a major initiating event in oloretal tumorigenesis []. Speifially, mutations in APC are a leading ause of CRC [4]. Mutations in APC have een found in * Correspondene: zhouqi@ioz.a.n Equal ontriutors State Key Laoratory of Stem Cell and Reprodutive Biology, Institute of Zoology, Chinese Aademy of Sienes, Beijing, China University of Chinese Aademy of Sienes, Beijing 49, China all patients diagnosed with familial adenomatous polyposis, as well as in almost 9% of patients diagnosed with CRC [5]. Most of the mutations in APC generate premature stop odons leading to trunated proteins that lak β-atenin inding sites. APC-free β-atenin stimulates the Wnt signaling pathway, leading to the ative transription of target genes suh as -My and ylin D, therey promoting tumorigenesis [6, 7]. APC and Axin serve as essential saffolds for glyogen synthase kinase eta (GSK-β) and β-atenin, and impaired assoiation of APC, Axin, with β-atenin leads to onstitutive ativation of the Wnt signaling pathway [8, 9]. In the intestine, the anonial Wnt pathway maintains the proliferative ell layer in the rypts []. Upon The Author(s). 8 Open Aess This artile is distriuted under the terms of the Creative Commons Attriution 4. International Liense ( whih permits unrestrited use, distriution, and reprodution in any medium, provided you give appropriate redit to the original author(s) and the soure, provide a link to the Creative Commons liense, and indiate if hanges were made. The Creative Commons Puli Domain Dediation waiver ( applies to the data made availale in this artile, unless otherwise stated.

2 Zhang et al. Moleular Caner (8) 7: Page of ativation of the Wnt pathway, β-atenin is released from the ytoplasmi omplex formed y APC, Axin, and GSK-β. Consequently, β-atenin is then ale to ind the T-ell fator/lymphoid enhaner fator inding fator (TCF/LEF) transription fators, resulting in inreased transription of downstream targets suh as -My or ylin D. In ontrast, in differentiated intestinal epithelial ells, APC ats as a negative regulator of the Wnt signaling pathway y inding to β-atenin in order to indue its degradation []. MiroRNAs (mirnas), are a lass of naturally ourring small, nonoding RNAs omprising of 9 to 5 nuleotides, that are an important lass of ellular regulators that modulate gene expression, and therey influene ell fate and funtion [ 6]. mirnas funtion y inding to target mrnas via sequene omplementarity, and repress translation or indue degradation of their target mrnas [7, 8]. So far, a numer of mirnas have een asried onogeni or tumor-suppressive funtions, and they are involved in almost every type of aner, inluding reast, lung, gastri arinoma, and CRC [9 ]. Some mirnas have een studied for their roles in oloretal arinogenesis [ 6]. For example, mir-494 has previously een reported to e upregulated in CRC, and it promotes ell migration and invasion in CRC y diretly targeting phosphatase and tensin homolog (PTEN) [7]. The roles and potential mehanisms of mirnas, mediated y APC, in CRC are still largely unknown. Here, we report that mir-494 ativates the Wnt/β-atenin signaling pathway y suppressing the expression of APC and onsequently plays an important role in the development and progression of CRC. Methods Tissue speimens and immunohistohemistry (IHC) staining The Ethis Committee of Institute of Zoology approved this study, and all patients gave their informed onsent prior to surgery. Colon arinoma tissues from human patients were otained from Beijing Military General Hospital (Beijing, China). The patients linial harateristis are shown in Tale. Detailed information inluding demography, linial harateristis, histopathology, APC mutation status, and survival status were olleted for all patients. Patients were followed after surgial treatment with a median follow-up of 6 months (range, 8 months). All patients were staged ased on the Tumor-Node-Metastasis (TNM) lassifiation. For mrna extration, samples were frozen in liquid nitrogen immediately after surgial removal and maintained at 8 C until use. Additional samples were fixed in % neutral-uffered formalin overnight, proessed, paraffin emedded, and setioned. A patient tissue with one synonymous mutation in the odon enoding amino aid 88 in APC, that did not ause a hange in amino aid Tale Clinial orrelation etween mir-494 expression and other liniopathologial harateristis in CRC Charateristis N of ases High mir-494 group P-value a Age (years) >6 9 Sex.887 Men 8 Women 8 8 TNM stage *.4 I + II 9 6 III + IV 7 4 Differentiation *.4 Well 5 4 Poorly 6 APC mutation status.567 Wild-type 5 Mutant 4 8 Survival status.476 Dead 9 Alive 4 a Chi-square test, *P <.5. CRC, oloretal arinoma sequene, was seleted for IHC analysis. IHC analysis was performed using μm setions whih were inuated with an anti-apc antiody (Aam atalog no. 5496) overnight at 4 C in a humidified hamer, followed y inuation with an HRP-onjugated seondary antiody for h. Staining was ompleted after 5 to min inuation with, -diaminoenzidine (DAB) as sustrate, whih resulted in a rown-olored preipitate at the antigen site. Cell lines and ell ulture Human olon aner ell lines were otained from Amerian Type Culture Colletion (Manassas, VA). HCT-6 and HT-9 ells were maintained in Duleo s modified Eagle s medium (DMEM) supplemented with % fetal ovine serum (FBS). Cells were maintained in a humidified inuator equilirated with 5% CO at 7 C. Miroarray analysis Total RNA was extrated from 7 tumor tissues and paired normal oloretal tissues using TRIzol reagent (Invitrogen). mirna miroarray profiling was performed as previously desried [8]. Data analysis was performed using GeneSpring GX software (Agilent). An mirna was designated as overexpressed if the expression in tumor tissues was greater than.-fold that in normal oloretal epithelial tissues.

3 Zhang et al. Moleular Caner (8) 7: Page of a R98 R7 R R R6 R96 R94 R R8 R5 R4 R8 R R R4 R9 R5 Relative RNA expression of mir-494 mir-665 mir-54 mir-496 mir-494 mir-8 mir-7 mir-45 mir-4-p mir-54 mir-4 mir Dlk-Dio mirna Adjaent Tumour * Sample pairs Fig. mir-494 is overexpressed in CRC. a Hierarhial lustering of mirna expression profiles from the Dlk-Dio region otained from 7 pairs of CRC tissues ompared with adjaent, non-tumor tissues. The mir-494 expression levels in CRC tissues and non-tumor tissues, evaluated y real-time PCR. The expression levels of mirna-494 in paired samples of CRC tissues and mathed adjaent non-tumor tissues. (*, P <.5;, P <., Student s t-test) Relative mrna level of mir Normal Caner Numer 46 Median.66.6 S.D * Real-time PCR detetion of mature mirnas Total RNA was isolated using TRIzol reagent (Invitrogen) aording to the manufaturer s protool. All-in-One mirna First-Strand DNA Synthesis Kit (Geneopoeia) was used to transrie μl ofpurifiedrna.real-time PCR, using mir-speifi primers and universal adaptor PCR primers (Geneopoeia), was performed with a Stratagene MxP QPCR System (Genetimes Tehnology, Shanghai, China). The reations were inuated in a 96 well plate at 95 C for min, followed y 4 yles at 95 C for 5 s, and 6 C for min. All reations were run in tripliate. RNA extration and real-time PCR Total RNA was isolated as desried aove. For reverse transription into DNA, μg of RNA from eah sample was inuated with random primers and then sujeted to real-time PCR. Real-time quantifiation was performed using a Stratagene MxP quantitative PCR system (Genetimes Tehnology, Shanghai, China). The reation solutions were inuated in a 96-well plate at 95 C for min, followed y 4 yles at 95 C for 5 s, and 6 C for min. All reations were run in tripliate. The primer pairs used were as follows: APC, 5 -CTGCGGACCG AGGTTGGCTC- (forward) and 5 -CTTCCTGCCAGA CGCTCGCC- (reverse); and GAPDH, 5 -CTCTGCTC CTCCTGTTCGAC- (forward) and 5 -CGACCAAATC CGTTGACTCC- (reverse). Oligonuleotide transfetions All ommerial mirnas and the APC targeted small interfering RNAs (sirnas) were synthesized and purified y RioBio Co. (Guangzhou, China). The APC sirna sequenes used were APC-siRNA, 5 -GGATCA GCCTATTGATTAT- and APC-siRNA, 5 -GTACGC CAGTCAACTTTCA-. Transfetions of the aforementioned mirnas and sirnas were performed using Lipofetamine (Invitrogen), aording to the manufaturer s instrutions.

4 Zhang et al. Moleular Caner (8) 7: Page 4 of a Numer 6 9 Numer 4 8 mir-control mir-494 G/G: 7.8 % G/M:.56 % S: 6.64 % 6 9 Channels (FL-A) Anti-Control G/G: % G/M:. % S: 8.7 % 6 9 Channels (FL-A) Numer 6 9 Numer 4 8 mir-control mir-494 G/G: 6.8 % G/M:.97 % S: 4.65 % 6 9 Channels (FL-A) G/G: 76.7 % G/M:.6 % S:. % 6 9 Channels (FL-A) Perentage Perentage 5 5 mir-control mir-494 Anti-Control G S G/M G S G/M.5 * * Anti-Control Relative olony numer..5. d Relative BrdU inorporation.5 * *..5. e Cell proliferation (A55nm)..5. mir-control mir-494 Anti-Control # ## ### D D D D4 Fig. (See legend on next page.)

5 Zhang et al. Moleular Caner (8) 7: Page 5 of (See figure on previous page.) Fig. mir-494 promotes the proliferation of CRC ells. a The effet of mir-494 overexpression on ell yle progression. HCT-6 ells were transfeted with a ontrol mirna or a mir-494 mimi for 4 h and then swithed to onditioned medium without serum for 4 h. The ells were ultured in medium ontaining % FBS for 4 h and olleted for ell yle analysis y flow ytometry. The data shown in the right-hand panel are representative of three independent experiments. Representative profiles are shown in the left-hand panel. The effet of mir-494 knokdown on ell yle progression. After HCT-6 ells were transfeted with the ontrol mirna inhiitor or the mir-494 inhiitor for 4 h, ells were swithed to onditioned medium without serum for 4 h. The ells were then ultured in medium ontaining % FBS for 4 h and olleted for ell yle analysis y flow ytometry. The data shown in the right-hand panel are representative of three independent experiments. Representative profiles are shown in the left-hand panel. Colony formation assay. HCT-6 ells staly expressing the indiated mirnas were maintained in ulture media for 7 days and stained with rystal violet. The numer of olonies in eah ondition was ounted, and is expressed as a mean ± S.D. from tripliate experiments. *, P <.5(Student s t-test). d The effet of mir-494 on ell proliferation. HCT-6 ells were transfeted with the indiated onstruts, and ell proliferation was measured y BrdU inorporation. Data are mean ± S.D. from tripliate experiments. *, P <.5(Student s t-test). e The effet of mir-494 on ell proliferation. HCT-6 ells were transfeted with a mir-494 mimi or with a mir-494 inhiitor. CCK8 assays were performed at,,, and 4 days post-transfetion. Data are mean ± S.D. from tripliate experiments., P <.;, P <., ompared to Anti-Control. #, P <.5; ##, P <.; ###, P <., ompared to mir-control, Student s t-test Western lot analysis Western lotting was performed as previously desried (). The ommerial antiodies used were anti-apc (Aam atalog no. 5496), anti-β-atenin (Santa Cruz atalog no. 796), anti-ylin D (Cell Signaling Tehnology atalog no. 978S), and anti-firillarin (Aam atalog no. a58). Colony formation assays HCT-6 ells were transfeted with the indiated longlasting syntheti mirnas. The ommerial mirnas used were a negative mir-ontrol (Rioio atalog no. mir4 -), a mir-494 mimi (Rioio atalog no. mir486 -), a negative anti-mir-ontrol (Rioio atalog no. mir -), or an anti-mir- 494 (Rioio atalog no. mir86 -). At 48 h post-transfetion, ells were plated in tripliate at 5 ells per 6 mm dish. After 7 days, the olonies were stained with.% rystal violet solution and ounted using a light mirosope. Flow ytometry Cells were harvested y trypsinization, washed with ieold PBS, and fixed in 7% ie-old ethanol in PBS. Before staining, ells were sedimented in a hilled entrifuge and resuspended in old PBS. Bovine panreati RNase (Sigma Aldrih) was added to a final onentration of μg/ml, and the ells were inuated at 7 C for min, followed y inuation with μg/ml propidium iodide (Sigma Aldrih) for min at room temperature. The ell yle profiles of 4 ells were analyzed using a FACSCaliur flow ytometer (BD Biosienes). Statistial analysis All experiments were repeated at least three times. Data shown are presented as mean ± SD of three or more independent experiments. A Student s t-test was used for statistial analysis of data. Differenes in liniopathologial harateristis etween the two groups were examined y Fisher s and hi-square tests. All P-values were determined using two-sided tests and statistial signifiane was ased on a P-value of.5. Correlation analysis was performed using satter plots and Pearson s orrelation analysis. The orrelation oeffiients and the orresponding P-values were alulated y the or.test funtion of R. Results mir-494 is overexpressed in oloretal aner The Dlk-Dio region is loated on human hromosome 4q, and onstitutes one of the largest mirna lusters in the human genome [9]. Many of these mirnas are differentially expressed in various aners, as well as several other disease states []. The expression and roles of Dlk-Dio region mirnas in onogenesis are very ontroversial and differ in different tissues []. We examined several mirnas from the Dlk-Dio region using a miroarray-ased expression analysis of 7 CRC tissues and mathed non-tumor adjaent tissues. As shown in Fig. a, a omparative analysis indiated that mir-494 was differentially overexpressed in CRC tissue when ompared to that in adjaent, non-tumor tissue from the same patient. We further onduted real-time PCR to analyze the expression of mir-494 in nontumor adjaent tissues and 46 arinoma tissues from CRC patients (Fig. ), as well as in the 7 pairs of arinoma tissues and mathed non-tumor adjaent tissues (Fig. ). The real-time PCR data was onsistent with the miroarray data. Taken together, these results indiate that mir-494 is upregulated in CRC. Upregulation of mir-494 is assoiated with advaned liniopathologial features of CRC To determine the linial signifiane of mir-494 in CRC, we analyzed the assoiation etween mir-494 CRC tissue expression levels and various CRC liniopathologial parameters. The median mir-494 expression in all 46 CRC patients was.6. Using the median expression level as a utoff, the patients were

6 Zhang et al. Moleular Caner (8) 7: Page 6 of divided into two groups, a high mir-494 expression group (n = ), and a low mir-494 expression group (n = 6). As shown in Tale, mir-494 was signifiantly upregulated in CRC patients with a higher TNM stage (P =.4). We also found that there were inreased mir- 494 levels in poorly differentiated CRC tissue ompared to well-differentiated CRC tissue (P =.4). However, mir- 494 expression was not signifiantly orrelated with age, gender,mutationinapc,orsurvivalstatus. mir-494 promotes CRC ell proliferation To explore the role of mir-494 upregulation in the development and progression of CRC, we examined its effets on ellular proliferation and tumorigenesis. The HCT-6 line was hosen as it expresses wild-type APC, and it is widely used in CRC researh. Flow ytometry analysis revealed that overexpression of a mir-494 mimi aused an inrease in the perentage of ells in S phase, and a derease in the perentage of ells in the G/G phase. Conversely, suppression of mir-494 expression using a mir-494 inhiitor inreased the perentage of ells in the G/G phase (Fig. a and ). To investigate the role of mir-494 in olon tumorigenesis, a olony formation assay was performed, whih revealed that mir-494 overexpression signifiantly inreased the proliferation rate of CRC ells (Fig. ). We further a Luiferase APC WT 5 mir APC mutant 5 Relative mrna levels of APC HCT-6 * *.5 * *..5. mir-control Relative luiferase mir pgl Relative mrna levels of APC HT-9 * * d APC -Tuulin APC -Tuulin HCT-6 Fig. Luiferase reporter assays demonstrating the target relationship etween mir-494 and APC mrna. a Predited mirna inding sites within the -UTR of APC mrna. The pairing etween mir-494 and the putative inding sites in the -UTR of APC mrna are shown. Mutations in the APC -UTR are underlined. Luiferase, normalized to renilla luiferase, was measured in homogenates of HEK9T ells transfeted with the wild-type -UTR and mutant -UTR APC luiferase onstruts and a mir-494 mimi or a sramled mirna ontrol. APC mrna expression is redued y the mir-494 mimi and inreased y the mir-494 inhiitor. d Expression of APC protein is redued y the mir-494 mimi and inreased y the mir-494 inhiitor. (*, P <.5, Student s t-test, n = independent experiments) HT-9

7 Zhang et al. Moleular Caner (8) 7: Page 7 of examined the effets of suppressing mir-494 expression on CRC ell proliferation. Consistent with the aovementioned results, a olony formation assay showed that suppression of mir-494 expression dramatially inhiited the growth rate of HCT-6 CRC ells, as ompared to ontrol ells transfeted with a ontrol mirna (Fig. ). To further demonstrate the importane of mir-494 in ell proliferation, we performed a BrdU inorporation assay, whih is a more sensitive assay to measure ell proliferation. As a result, the numer of BrdU-positive ells inreased when mir-494 was overexpressed, whereas the numer dereased when mir-494 was downregulated (Fig. d). A seond ell proliferation study, onduted using a CCK8 assay, also indiated a role for mir-494 in positively regulating ell proliferation (Fig. e). Colletively, our data suggest that mir-494 may mediate CRC ell proliferation y regulating the G/S transition. mir-494 diretly targets APC in CRC ells Using a ioinformatis analysis, we identified putative inding sites for mir-494 within the -UTR of the human APC mrna (Fig. a). To validate APC as a mir- 494 target, we onstruted a luiferase expression vetor ontaining the -UTR segment of APC along with the putative mir-494 inding sites. Co-transfetion of a mir-494 mimi and the APC -UTR expression vetor into HEK9T ells resulted in a signifiant suppression of APC luiferase. In addition, mutating the putative mir-494 inding sites ompletely eliminated this inhiitory effet (Fig. ). These data strongly suggest that APC is indeed the diret target of mir-494. To examine whether mir-494 an diretly target the APC mrna in HCT-6 and HT-9 CRC ell lines, we transiently transfeted the mir-494 mimi into HCT-6 and HT-9 ells. Endogenous APC mrna and APC protein levels were then measured 48 h after transfetion. As a result, the expression of oth APC mrna and APC protein dereased in the presene of the mir-494 mimi (Fig. and d). In ontrast, when HCT-6 and HT-9 ells were transfeted with the mir-494 inhiitor, the APC mrna and APC protein levels inreased (Fig. and d). Downregulation of APC is inversely orrelated with mir-494 expression in CRC To further evaluate the relationship etween mir-494 and APC in human CRC, we analyzed the expression of APC and mir-494 in paired CRC and non-tumor adjaent tissue using immunohistohemistry and real-time a Normal Carinoma Overexpression 6% Unhanged 5% µm Underexpression 79% Relative mrna level of APC 8 4 5µm Relative mirna level of mir-494 Fig. 4 Expression of APC is often dereased and inversely orrelated with mir-494 expression in human CRC tissues. a Immunohistohemial staining of APC in tumor tissue and orresponding normal oloni epithelium. Normal epithelium shows vesiular rown ytoplasmi APC staining. This staining is nearly asent in the tumor tissue. APC mrna expression is frequently dereased in tumor tissues when ompared to the mathed non-anerous tissues, as evaluated y real-time PCR. The relative levels of mir-494 expression were plotted against the relative levels of APC mrna expression

8 Zhang et al. Moleular Caner (8) 7: Page 8 of PCR. Using oth immunohistohemistry (Fig. 4a) and real-time PCR (Fig. 4), tumors showed dereased APC expression when ompared with expression in tumoradjaent tissues. Furthermore, using real-time PCR, APC mrna expression levels in tumor tissues were found to e inversely orrelated with mir-494 levels (Fig 4). This relationship had a Pearson s orrelation oeffiient of.5679 (P <.), indiating a strong negative orrelation etween mir-494 and APC expression levels in olon arinomas. Taken together, these data suggest that dereased APC expression may result from overexpression of mir-494 in human CRC. mir-494 ativates the Wnt/β-atenin pathway Next, we investigated the funtional relevane of the interation etween mir-494 and APC y determining the effets of hanges in their expression levels on the of the Wnt pathway. First, we examined the effets of hanges in mir-494 expression levels on the transriptional of the TCF transription fator. HCT-6 ells were transfeted with a luiferase reporter onstrut haroring either three optimal TCF-inding sites (Topflash) or three mutated TCF-inding sites (Fopflash). Transfetion with the mir-494 mimi signifiantly inreased Top/Fop transriptional, whereas transfetion with the mir-494 inhiitor signifiantly dereased Top/Fop transriptional (Fig. 5a). Next, we examined the indution of -My and ylin D, important downstream target genes in the Wnt/β-atenin pathway (5). We used luiferase reporter onstruts under the ontrol of promoters ontaining either of four My or ylin D responsive elements. We oserved an inrease in oth -My and ylin D reporter ativities in ells o-transfeted with the mir-494 mimi, and a orresponding derease in -My and ylin D reporter in ells o-transfeted with the mir-494 inhiitor (Fig. 5 and ). Furthermore, ellular frationation showed that mir-494 mimi overexpression promoted the nulear aumulation of β-atenin (Fig. 5d), indiating that mir-494 ativates the Wnt/β-atenin signaling pathway y promoting nulear β-atenin aumulation. Moreover, we oserved an inrease in the ylin D protein, a Wnt/β-atenin signaling pathway target gene, in ells overexpressing the mir-494 mimi (Fig. 5e). Thus, overexpression of mir-494 ativates the Wnt/ β-atenin pathway. a Relative Top/Fop luiferase * * * * * * Relative -My luiferase Relative Cylin D luiferase d Control mir-494 e C N C N C N -Catenin -Catenin -Tuulin Cylin D Firillarin -Tuulin Fig. 5 mir-494 indues β-atenin signaling (Top/Fop, -My, ylin D). a Dual luiferase assay demonstrating the effet on Top/Fop reporter in HCT-6 ells. Values were normalized to a renilla transfetion ontrol. The figure shown is a representative of at least three independent experiments. Dual luiferase assay showing the effet of expression of a mir-494 mimi on a -My reporter onstrut in HCT-6 ells. Dual luiferase assay showing the effet of expression of a mir-494 mimi on a ylin D reporter onstrut in HCT-6 ells. d Western lot analysis of β-atenin expression in the ytoplasm (C) and nuleus (N) of the indiated ells. α-tuulin and firillarin were measured y western lot to monitor the effiieny in the preparation of ytosoli and nulear protein extrats, respetively. e Western lot analysis of β-atenin and ylin D in the indiated ells. (*, P <.5, Student s t-test, n = independent experiments)

9 Zhang et al. Moleular Caner (8) 7: Page 9 of Suppression of APC is funtionally important for the iologial effets of mir-494 To explore the funtional signifiane of APC depletion in upregulating the Wnt/β-atenin pathway in olon aner ell lines, as well as in the ativation of β-atenin indued y mir-494, we studied the effets of APC depletion using speifi sirnas. As shown in Fig. 6a,, and, sirna silening of APC expression enhaned the Top/Fop, -My, and ylin D transriptional ativities. In addition, silening APC also inreased Top/Fop, -My and ylin D transriptional ativities in HCT-6 ells whose mir-494 expression levels were suppressed (Fig. 6d, e, and f). These results demonstrate that APC lies downstream of mir-494 and is funtionally important for mir-494 indued Wnt/β-atenin signaling in olon aner ell lines. Disussion The key finding in this study is that mir-494 expression is markedly upregulated in CRC tissues ompared to normal oloretal tissues. Furthermore, etopi expression of mir-494 enhanes the proliferation of CRC ells, whereas inhiition of mir-494 expression has the opposite effet. Moreover, we demonstrate that mir-494 upregulation in CRC ells ativates the Wnt/β-atenin pathway y diretly targeting APC. These findings suggest that this dysregulation of mir-494 levels may play a Relative Top/Fop luiferase Relative -My luiferase Relative Cylin D luiferase d Relative Top/Fop luiferase e Relative -My luiferase f Relative Cylin D luiferase g APC -Tuulin Fig. 6 APC is funtionally involved in mir-494 indued β-atenin signaling of CRC ells. a Dual luiferase experiment demonstrating the effets on Top/Fop reporter in the indiated ells. Values were normalized to a renilla transfetion ontrol. Thefigureshownisrepresentativeofat least three independent experiments. Dual luiferase assay showing the effets on a -My reporter onstrut in the indiated ells. Dual luiferase assay showing the effets on a ylin D reporter onstrut in the indiated ells. d Dual luiferase experiment demonstrating the effets on Top/Fop reporter in the indiated ells following mir-494 downregulation. e Dual luiferase assay showing the effets on a -My reporter onstrut in the indiated ells following mir-494 downregulation. f Dual luiferase assay showing the effets on a ylin D reporter onstrut in the indiated ells following mir-494 downregulation. g APC western lot of the indiated ells. (, P <., Student s t-test, n = independent experiments)

10 Zhang et al. Moleular Caner (8) 7: Page of an important role in promoting proliferation and tumorigenesis in CRC. Aerrant Wnt/β-atenin signaling has een linked to the pathogenesis of various diseases, inluding aner, and is thought to promote tumor progression [ 8]. Multiple key negative regulators, suh as APC, GSK-β, and Axin, are suppressed in aner, ontriuting to the promotion of tumor progression through regulation of the Wnt/β-atenin pathway. For instane, inativation of GSK-β via kinases promotes mammary tumorigenesis y ativation of the anonial Wnt pathway []. In mouse models, loss of APC funtion has een shown to lead to oloretal tumorigenesis through hyperativation of Wnt/β-atenin signaling []. Herein, we demonstrate that mir-494 suppresses APC expression y diretly targeting the -UTR of the APC mrna. Consistent with the tumor-suppressive effets of APC, mir-494 was upregulated in CRC, and its overexpression dramatially promoted CRC ell proliferation. The Wnt/β-atenin signaling pathway is ontrolled at multiple levels. β-atenin is the major ellular effetor of Wnt signaling and is normally retained y a degradation omplex ontaining Axin, APC, GSK-β, and asein kinase a. In the asene of Wnt, the omplexed β-atenin is targeted for proteasome-mediated degradation following its phosphorylation and uiquitination [ 5]. APC is ruial for the apture of β-atenin y the degradation omplex, and inds β-atenin diretly via its entral armadillo repeats [6]. Upon Wnt ativation, Axin is removed from the destrution omplex allowing the release of β-atenin. The aumulated β-atenin enters the nuleus and inds to the TCF/LEF family of transription fators to indue target gene expression [7, 8]. A key event therefore in oth Wnt signal transdution and aner ell proliferation is the regulation of β-atenin staility and. In this study, we have unovered a mehanism for ontrolling APC expression, and therey the of the Wnt pathway, through the ation of mir-494, and suggest that mir-494 ontriutes to the pathogenesis of CRC. mir-494 is a produt of the Dlk-Dio region, whih is loated on human hromosome 4q, and onstitutes one of the largest mirna lusters in the human genome. The expression patterns of mir-494 are not onsistent aross different types of tumors, suggesting that there is tissue-dependent regulation of expression of this mirna, among other potential mehanisms [9 44]. In this study, we demonstrated that mir-494 is upregulated in CRC tissues ompared to adjaent, non-anerous tissues from the same patient. Furthermore, we demonstrated that etopi mir-494 expression inreases the growth rate of HCT-6 ells ompared to a ontrol mirna, whereas suppression of mir-494 expression inhiited ell proliferation and olony formation. The data strongly suggest that upregulation of mir-494 orrelates with linial CRC progression and that mir-494 may funtion as an ono-mirna. Conlusions Our study has shown that elevated expression of mir- 494 promotes ell proliferation and tumorigenesis in CRC y suppressing the expression of APC, an inhiitor of β-atenin signaling. These findings unover a novel moleular mehanism for the hyperativation of the Wnt/β-atenin signaling pathway in CRC, suggesting that mir-494 may serve as a potential therapeuti target for the treatment of CRC. Areviations APC: Adenomatous polyposis oli; CRC: Coloretal aner; DAB: Diaminoenzidine; DMEM: Duleo s modified Eagle s medium; FBS: Fetal ovine serum; GSK-β: Glyogen synthase kinase eta; IHC: Immunohistohemistry; mirnas: MiroRNAs; PTEN: Phosphatase and tensin homolog; TCF/LEF: T-ell fator/lymphoid enhaner inding fator; TNM: Tumor-Node-Metastasis Aknowledgements The authors wish to thank; the patients involved in the study and their guardians; Xin Tong for providing CRC tissue; Meng Wang for providing the APC -UTR vetor onstrut; and Haixi Sun for ioinformati analysis. We also thank all memers of our group for disussions and assistane with this study. Funding This work was supported y the National Natural Siene Foundation of China (4795 to Q.Z. and 64) and the Key Researh Projets of the Frontier Siene of the Chinese Aademy of Sienes (QYZDY-SSW-SMC to Q.Z.). Availaility of data and materials The dataset (s) supporting the findings of this study are inluded in the artile. Authors ontriutions QZ, WL, and YZ oneived the projet idea and designed the experiments; YZ, LG, YL, FT, performed the experiments; GF and YZ analyzed the data; QZ, WL, and YZ wrote the manusript. All authors read and approved the final manusript. Ethis approval and onsent to partiipate The proedures of this study were approved y the Ethis Committee of Institute of Zoology. Consent for puliation Not appliale. Competing interests The authors delare that they have no ompeting interests. Pulisher s Note Springer Nature remains neutral with regard to jurisditional laims in pulished maps and institutional affiliations. Reeived: 6 June 7 Aepted: 6 Deemer 7 Referenes. Harrison S, Benziger H. The moleular iology of oloretal arinoma and its impliations: a review. Surgeon. ;9(4):.. Pandurangan AK. Potential targets for prevention of oloretal aner: a fous on PIK/Akt/mTOR and Wnt pathways. Asian Pa J Caner Prev. ; 4(4): 5.. Miyaki M, et al. 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