Suppresses Colorectal Carcinoma Aggressiveness by ... · colorectal carcinoma tissues without metastasis and those with metastasis. We found that lncRNA antisense transcript of SATB2

Genome and Epigenome

SATB2-AS1 Suppresses Colorectal CarcinomaAggressiveness by Inhibiting SATB2-DependentSnail Transcription and Epithelial–MesenchymalTransitionYi-QingWang1,2, Dong-Mei Jiang1,2, Sha-Sha Hu1,2, Li Zhao1,2, LanWang2, Min-Hui Yang1,2,Mei-Ling Ai2, Hui-Juan Jiang2, Yue Han2, Yan-Qing Ding1,2, and Shuang Wang1,2

Abstract

Accumulating evidence suggests that long noncoding RNA(lncRNA) plays important regulatory roles in cancer biology.However, the involvement of lncRNA in colorectal carcinomaprogression remains largely unknown, especially in colorectalcarcinomametastasis. In this study, we investigated the changesin lncRNA expression in colorectal carcinoma and identified anew lncRNA, the antisense transcript of SATB2 (SATB2-AS1), as akey regulator of colorectal carcinoma progression. SATB2-AS1was frequently downregulated in colorectal carcinoma cells andtissues, and patients whose tumors expressed SATB2-AS1 at lowlevels had a shorter overall survival and poorer prognosis.Downregulation of SATB2-AS1 significantly promoted cell pro-liferation, migration, and invasion in vitro and in vivo, demon-strating that it acts as a tumor suppressor in colorectal carcinoma.

SATB2-AS1 suppressed colorectal carcinoma progression byserving as a scaffold to recruit p300, whose acetylation ofH3K27 and H3K9 at the SATB2 promoter upregulated expres-sion of SATB2, a suppressor of colorectal carcinoma growth andmetastasis. SATB2 subsequently recruited HDAC1 to the Snailpromoter, repressing Snail transcription and inhibiting epithe-lial-to-mesenchymal transition. Taken together, these data revealSATB2-AS1 as a novel regulator of the SATB2-Snail axis whoseloss facilitates progression of colorectal carcinoma.

Significance: These data show that the lncRNA SATB2-AS1mediates epigenetic regulation of SATB2 and Snail expressionto suppress colorectal cancer progression.

See related commentary by Li, p. 3536

IntroductionColorectal carcinoma is the third most common cancer, with

approximately 1.3millionnewcancer cases and690,000mortalitiesworldwide each year (1). Although the initial events in colorectalcarcinoma have been relatively well-studied, and treatments forearly-stage disease have significantly improved over the past dec-ades, themechanisms ofmetastasis and relapse, which are themaincauses of death, remain poorly characterized (2). Thus, gaining abetter understanding of tumorigenesis and developing new diag-nosis and treatment strategies for colorectal carcinoma is stillurgently needed to improve colorectal carcinoma clinical outcome.

Long noncoding RNAs (lncRNA), important new members ofthe ncRNA family, are functionally defined as transcripts >200

nucleotides in length with no protein-coding potential. LncRNAshave crucial functional importance in various biological process-es, ranging from epigenetic gene regulation and transcriptionalcontrol to posttranscriptional regulation (3, 4). Increasing evi-dence has also established that the deregulation of lncRNAsexpressionmay be important in cancer biology, typically resultingin the aberrant expression of gene products that contribute to theprogression of human tumors (5–8). For example, LncRNA-ATB,which activated by the TGFb, competitively binds miR-200s toupregulate ZEB1 and ZEB2 during the epithelial-to-mesenchymaltransition (EMT) and promote the invasion-metastasis cascadein hepatocellular carcinoma (5). Thus, lncRNAs have beenhighlighted as new players in cancer progression by functioningas tumor suppressors, oncogenes or both, depending on thecircumstance. However, there are only preliminary studies on thefunction of lncRNAs in colorectal carcinoma, and the overallbiological mechanisms and clinical significance of lncRNAs incolorectal carcinoma remain largely unknown.

We speculated that there are still a large number of previ-ously unexplored lncRNA alterations in colorectal carcinoma,especially colorectal carcinoma metastasis-related lncRNAs. Weperformed lncRNA differential expression profiling betweencolorectal carcinoma tissues without metastasis and those withmetastasis. We found that lncRNA antisense transcript ofSATB2 (SATB2-AS1) was significantly downregulated in colo-rectal carcinoma tissues with metastasis, which was associatedwith advanced stage and poor prognosis of patients withcolorectal carcinoma. The effects of lncRNA SATB2-AS1 oncolorectal carcinoma cell growth and metastasis were assessed

1Department of Pathology, Nanfang Hospital, Southern Medical University,Guangzhou, China. 2Department of Pathology, School of Basic Medical Sciences,Southern Medical University, Guangzhou, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Y.-Q. Wang and D.-M. Jiang contributed equally to this article.

Corresponding Authors: Shuang Wang, Nanfang Hospital, Southern MedicalUniversity, #1838 Guangzhou North Road, Guangzhou, Guangdong 510515,China. Phone: 0086-020-62789364; Fax: 0086-020-61642148;E-mail: [email protected]; and Yan-Qing Ding, [email protected]

Cancer Res 2019;79:3542–56

doi: 10.1158/0008-5472.CAN-18-2900

�2019 American Association for Cancer Research.

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by gain- and loss-of-function experiments in vitro and in vivo.We also unveiled the mechanisms underlying the inhibitoryeffects of SATB2-AS1 on colorectal carcinoma progression viaactivating SATB2 transcription and then inhibiting EMT. Thisstudy provides a more detailed understanding of SATB2-AS1and its functions as a novel therapeutic target in colorectalcarcinoma pathogenesis.

Materials and MethodsEthics statement

The use of tissues for this study has been approved by the ethicscommittee of Nanfang Hospital, Southern Medical University(Guangzhou, China). All of the patients signed an informedconsent, and the investigators obtained the written informedconsent, before the use of these clinical materials for researchpurposes. This study was performed in strict accordance with therecommendations in theGuide for theCare andUseof LaboratoryAnimals of the National Institutes of Health. The protocol wasapproved by the Committee on the Ethics of Animal Experimentsof Southern Medical University.

Microarray and computational analysisThree colorectal carcinoma tissues with metastasis and three

colorectal carcinoma tissues without metastasis were used forthe lncRNA expression profiling study. Briefly, total RNA wasextracted from tissues and used to synthesize double-strandedcDNA. cDNA was labeled and hybridized to the LncRNA Expres-sion Microarray (Arraystar). After hybridization and washing,slides were scanned with the Axon GenePix 4000B microarrayscanner (Molecular Devices). The data were extracted using theNimbleScan software (Roche NimbleGen, Inc.). A P value wascalculated using the t test. The threshold for differentiallyexpressed genes was set as a fold change �2.0 and a P value<0.05. The microarray data in this article have been deposited inNCBI Gene Expression Omnibus (GEO) and are accessiblethrough (GEO) Series accession number GSE109910.

Tissue specimens and cell linesFresh and formalin-fixed, paraffin-embedded colorectal tumor

tissue samples were obtained from patients with a diagnosis ofprimary colorectal carcinoma and thenunderwent elective surgeryin Nanfang Hospital, Southern Medical University. A total of 40cases of fresh colorectal carcinoma and paired nontumormucosaltissues were freshly frozen in liquid nitrogen and stored at�80�Cuntil further use. And 176 cases of archived-colorectal carcinomatissue samples were collected and used to investigate the expres-sion of SATB2-AS1 using in situ hybridization (ISH). Completefollow-up, ranging from 1 to 118 months, was available for thecohort of 176 patients, and the median survival was 56 months.No patient received any pre-operative chemotherapy andradiotherapy.

The human colorectal carcinoma cell lines DLD1, HCT116,SW480, SW620, LoVo, LS174t, and HT29 were obtained from theATCC. A subclone named M5 with enhanced metastatic abilitiesin liver was isolated by in vivo selection of SW480 cells in ourprevious studies (9, 10). NCM460, a normal colon mucosaepithelial cell line (11), was obtained from the cell bank at theChinese Academy of Sciences (Shanghai, China). Details of cellculture are summarized in the Supplementary Materials andMethods.

RNA isolation and qRT-PCRTotal RNA was extracted using TRIzol Reagent (Ambion by life

Technologies). cDNA was synthesized using the Prime-Script RTReagent Kit (Promega). Two-step qRT-PCR was performed asdescribed previously (10). The results were normalized to theexpression of b-actin. The assay was performed in triplicatefor each case to allow for the assessment of technical variability.The primer sequences used for PCR are listed in SupplementaryTable S1.

ISH and evaluation of staining of SATB2-AS1ISH was performed according to the manufacturer's protocol

(Exiqon). The ISH stained tissue sections were reviewed andscored separately by two blinded pathologists. Staining forSATB2-AS1 was assessed using a relatively simple, reproduciblescoring method (10). Additional details are described in theSupplementary Materials and Methods.

Construction of cell lines with stably overexpressed SATB2-AS1The full-length human SATB2-AS1 with 3316-bp DNA frag-

ment amplified and cloned into GV303 lentiviral vector (Gene-chem). Virus particles were harvested 48 hours after GV303-SATB2-AS1 transfection cells using lipofectamine 2000 reagent(ThermoFisher Scientific). Colorectal carcinoma cells wereinfected with the recombinant lentivirus-transducing units plus8 mg/mL Polybrene (Sigma) and then subjected to fluorescence-activated cell sorting (FACS) analysis for GFP expression to gaincolorectal carcinoma cells with stable overexpression of SATB2-AS1. The empty lentiviral vector GV303 was used as the control.

Oligonucleotide transfectionThe siRNAs SATB2-AS1, siRNA p300, siRNA SATB2, siRNA

Snail, and negative control siRNA (silencer negative controlsiRNA) were synthesized by GenePharma (Shanghai, China).Oligonucleotides transfection was performedwith Lipofectamine2000 following the manufacturer's protocol. Target sequences forsiRNAs were shown in Supplementary Table S2.

Functional assays in vitroCell proliferation, colony formation, wound-healing, and inva-

sion assays in vitro were performed according to standard proto-cols as previously described (12, 13). The details are described inthe Supplementary Materials and Methods. All experiments wereperformed in triplicate.

In vivo tumorigenic and metastasis assaysFour-week-old female athyic Balb/c-nu/nu nude mice were

obtained from the Laboratory Animal Centre of SouthernMedicalUniversity andmaintained in laminarflow cabinets under specificpathogen-free conditions. For in vivo tumorigenicity, SATB2-AS1overexpressing LoVo cells and empty vector stably transfectedcells were trypsinized, counted, and resuspended in sterile PBS. Atotal of 5 � 106 SATB2-AS1 overexpressing LoVo cells or controlcells were subcutaneously injected into the right and left bilateralupper limbs ofmice, respectively. The tumor volumes and overallhealth of the mice were monitored. The size of the tumor wasdetermined by caliper measurements. Tumor volume was calcu-latedwith the following the formula: 0.5� length�wildth2. Eachexperimental group contained six mice.

For developing the in vivometastatic model, mice were injectedintravenously via the lateral tail vein with 2 � 106 cells. After 4

SATB2-AS1/SATB2/Snail Axis Inhibits CRC Aggressiveness

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weeks of monitoring, the mice were sacrificed by cervical dislo-cation. The lungs were removed from the adjacent organs bydissection, and fixed using 10% neutral-buffered formalin. Sub-sequently, the consecutive tissue sections were obtained andstained with hematoxylin and eosin to observe the metastaticnodules in the lungs under the microscope.

RNA immunoprecipitationRNA immunoprecipitation (RIP) assays were performed

using the Magna RIP RNA-Binding Protein Immunoprecipita-tion Kit (Millipore) as described previously (7). Briefly, cellswere cross-linked with 1% (w/v) formaldehyde and suspendedin lysis buffer containing a protease inhibitor cocktail and anRNase inhibitor. Magnetic beads were preincubated with ananti-rabbit immunoglobulin G (IgG) or anti-rabbit p300 anti-body (ab14984; Abcam) for 30minutes at room temperature,and lysates were then immunoprecipitated with the beads at4�C overnight. RNA was purified from the RNA–protein com-plexes that bound to the beads and then was analyzed by real-time RT-PCR.

Chromatin immunoprecipitationChromatin Immunoprecipitation (ChIP) was performed using

the EZ ChIP Chromatin immunoprecipitation Kit (Millipore),according to its manual. Briefly, cross-linked chromatin wassonicated into 200- to 1000-bp fragments. The chromatinwas immunoprecipitated using anti-p300, anti-SATB2, or anti-HDAC1 antibody. Normal mouse IgG was used as a negativecontrol. Real-time PCR was conducted using SYBR Green Mix(Takara). Primer sequences are listed in Supplementary Table S1.

RNA pull-down assayRNA pull-down assay was performed as described previous-

ly (14, 15). Briefly, Biotin-labeled SATB2-AS1 and its antisenseRNAs were in vitro transcribed with the Biotin RNA Labeling Mix(Roche Diagnostics) and T7 RNA polymerase (Ambion by lifeTechnologies). The cell lysates were freshly prepared usingWhole Cell Lysis Assay (KGP2100; Keygen BioTECH). The Strep-tavidin Magnetic Beads (Life Technologies) were first preparedaccording to manufacturer's instructions and then immediatelysubjected to labeled RNA (50 pmol) capture in RNA capturebuffer [20mmol/L Tris-HCl (pH7.5), 1MNaCl, 1mmol/L EDTA]for 30 minutes at room temperature with agitation. The RNA-captured beads were washed once with 20 mmol/L Tris (pH 7.5)and incubated with 1mg cell lysates diluted in 1X Protein-RNABinding Buffer [20 mmol/L Tris (pH 7.5), 50 mmol/L NaCl,2 mmol/L MgCl2, 0.1% Tween-20 detergent] supplemented with50% glycerol for 2 hours at 4�C with rotation. The RNA-bindingprotein complexes were washed sequentially with Wash buffer[20 mmol/L Tris (pH 7.5), 10 mmol/L NaCl, 0.1% Tween-20Detergent] for three times, and eluted by Elution buffer. Theeluted protein complexes were heated for 10 minutes at 100�C,separated by SDS-PAGE, and then analyzed by Western blottingassay. RNA inputwas detected using streptavidin-HRPby dot-blotassay.

Statistical analysisAll the statistical analyses were performed using the SPSS

version 16.0 software (SPSS). Differences between groups wereidentified using a two-tailed Student t test. Associations betweenSATB2-AS1 expression and clinicopathologic characteristics

were determined by the x2 test. Survival curves were plottedby the Kaplan–Meier method and compared by the log-rank test.The significance of various variables for survival was analyzed bythe Cox proportional hazards model for multivariate analyses. Aprobability value of 0.05 or less was considered to be significant.

ResultsMicroarray analysis identifies deregulated lncRNAs related tocolorectal carcinoma metastasis

To identify novel functional lncRNAs in colorectal carcinomametastasis, we performed lncRNAmicroarray analysis to comparelncRNA expression levels between colorectal carcinoma tissueswith and without metastasis. We found 140 upregulated and 168downregulated lncRNAs in colorectal carcinoma tissues withmetastasis compared with those without metastasis (Fig. 1A).Among them, we paid close attention to the top 20 lncRNAs ineach group. Of interest, the expression of lncRNA SATB2-AS1(AK056625) was downregulated in the colorectal carcinomatissues with metastasis. SATB2-AS1 is an antisense cognate geneof SATB2, a colorectal carcinoma metastasis suppressor genedemonstrated in our previous studies (10, 16, 17). There isevidence that antisense transcript lncRNAs can directly or indi-rectly regulate the expression of the sense genes. Therefore, wefocused on SATB2-AS1 and investigated its clinical significanceand biological function in colorectal carcinoma.

LncRNA SATB2-AS1 expression was significantlydownregulated in colorectal carcinoma tissues and cell lines

To validate the microarray analysis findings and investigate theroles of SATB2-AS1 in colorectal carcinoma, we first measuredSATB2-AS1 expression in 40 colorectal carcinoma tissues and theirpair-matched noncancerous mucosa tissues. We found thatSATB2-AS1 transcript was expressed at lower levels in colorectalcarcinoma tissues compared with nontumor tissues of the samedonor (P ¼ 0.005, Fig. 1B). Significantly, downregulation ofSATB2-AS1 in tumor samples was associated with lymph-nodemetastasis (P ¼ 0.013, Fig. 1B). A similar trend was observed inunpaired colorectal carcinoma and adjacent normal samples fromThe Cancer Genome Atlas (TCGA) cohort (P < 0.001, Fig. 1C).Moreover, we also measured SATB2-AS1 levels in a panel ofcolorectal carcinoma cell lines and a human colon mucosaepithelial cell line (NCM460). Compared with NCM460,SATB2-AS1 expression levels were significantly decreased in alleight colorectal carcinoma cell lines (P < 0.001, Fig. 1D).

Downregulation of SATB2-AS1 is associated with advancedstage and poor prognosis of patients with colorectal carcinoma

To further explore the clinicopathologic and prognosticsignificance of SATB2-AS1 expression, we investigated SATB2-AS1expression in an independent panel of 176 primary colorectalcarcinoma tissues with clinical follow-up information using ISH.SATB2-AS1-specific staining was localized in the nucleus ofbenign and tumor epithelial cells (Fig. 1E). According to thereclassification as described above, we divided the 176 patientswith colorectal carcinoma into a high SATB2-AS1 expressiongroup (n ¼ 72) and a low expression group (n ¼ 104). The lowSATB2-AS1 expression group showed an advanced T stage (P ¼0.048), lymph node metastasis (P ¼ 0.004), and distant metas-tasis (P ¼ 0.025) compared with the high SATB2-AS1 expressiongroup (Supplementary Table S3).

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In addition, patients with low SATB2-AS1 expression had asignificantly poorer prognosis than those with high SATB2-AS1expression (P ¼ 0.010, Fig. 1F). We also validated the prognosticsignificance of SATB2-AS1 in the TCGA cohort (n ¼ 525). Asshown in Fig. 1G, Kaplan–Meier curve showed that low SATB2-AS1 expression was correlated with poor survival in patients withcolorectal carcinoma (P¼ 0.035). Logistic multivariate regressionrevealed that low levels of SATB2-AS1 were independently asso-ciated with colorectal carcinoma overall survival [P ¼ 0.002;hazard ratio (HR), 0.537; 95% confidence interval (CI), 0.361–0.801). Taken together, we identified SATB2-AS1 expression levelas an independent prognostic factor of disease outcome inpatients with colorectal carcinoma.

SATB2-AS1 overexpression represses colorectal carcinoma cellproliferation

To elucidate causal roles of SATB2-AS1 in colorectal carcinoma,we applied lentivirus delivery of SATB2-AS1 to ectopicallyoverexpress SATB2-AS1 in two colorectal carcinoma cell lines,M5 and LoVo cells. Increased expression of SATB2-AS1 afterinfection of lentivirus was confirmed in the two cell lines byreal-time RT-PCR (P < 0.05, Supplementary Fig. S1). The CCK8assays revealed a significantly slower proliferation rate in SATB2-AS1-overexpressing M5 and LoVo cells when compared withcontrol cells (P < 0.001, Fig. 2A). Meanwhile, SATB2-AS1 over-expression suppressed colony formation in M5 (P < 0.001) andLoVo cells (P ¼ 0.023, Fig. 2B).

Figure 1.

LncRNA SATB2-AS1 is downregulated in colorectal carcinoma, especially in colorectal carcinomawith metastasis, and this lncRNA could be an independentprognostic factor for the prediction of the overall survival of patients with colorectal carcinoma. A, Hierarchical clustering analysis of lncRNAs that weredifferentially expressed in colorectal carcinoma tissues with and without metastasis. C, colorectal carcinoma tissues without metastases; CM, colorectalcarcinoma tissues with lymph nodemetastases. B, The level of SATB2-AS1 in paired colorectal carcinoma and adjacent noncancerous tissues. nmCRC, colorectalcarcinoma tissues without metastases; mCRC, colorectal carcinoma tissues with lymph nodemetastases. C, The level of SATB2-AS1 in unpaired colorectalcarcinoma and noncancerous tissues samples from TCGA cohort. D, The level of SATB2-AS1 in colorectal carcinoma and colon mucosa epithelial (NCM460) celllines. E, Expression analysis of SATB2-AS1 in normal colorectal mucosa and colorectal carcinoma tissues by ISH. a, Positive expression of SATB2-AS1 in normalcolorectal mucosa. b–d, Negative, medium, and high expression of SATB2-AS1 in colorectal carcinoma tissue, respectively. Scale bars are shown in the right leftcorner of each picture. F and G, Kaplan–Meier analysis of overall survival in all patients with colorectal carcinoma according to SATB2-AS1 expression accordingto our data (F) and TCGA cohort (G).






Figure 2.

SATB2-AS1 overexpression inhibits colorectal carcinoma cell growth andmetastasis in vitro and in vivo.A, SATB2-AS1 overexpression suppressed cellproliferation in colorectal carcinoma cell lines as determined by CCK-8 assay. B, SATB2-AS1 overexpression inhibited colony formation in colorectal carcinomacells. Representative images (left) and quantitative analyses (right) are shown. C, SATB2-AS1 overexpression induced cell-cycle arrest in G1 phase in colorectalcarcinoma cells. D, SATB2-AS1 overexpression inhibited colorectal carcinoma cell invasion in Matrigel invasion assay. E, SATB2-AS1 overexpression suppressedcell migration in the wound-healing assay in M5 and LoVo cells. The experiments were performed at least in triplicate, and the data are expressed as themean� SD. F, SATB2-AS1 overexpression inhibited subcutaneous tumor formation in nudemice. LoVo cells with ectopic overexpression of SATB2-AS1 andcontrol cells were inoculated into nudemice (n¼ 6 per group). These graphs show the tumor xenografts 3 weeks after ectopic-subcutaneous implantation innude mice. The effect of SATB2-AS1 on colorectal carcinoma tumor growth was evaluated based on tumor volume in the two groups. G, Representativephotographs of hematoxylin and eosin (H&E) and IHC staining for Ki-67 and SATB2 antibody of primary cancer tissues. H, Representative pictures of lungmetastasis by hematoxylin and eosin staining in nude mice 4 weeks after tail vein injection with SATB2-AS1 overexpression LoVo cells or control cells (n¼ 8 pergroup). I, Statistical comparisons of lung metastasis and pulmonary tumor colonies in the two groups of mice after tail vein injection. � , P < 0.05; �� , P < 0.001.

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We next probed the potential mechanisms underlying thegrowth-inhibitory effects of SATB2-AS1 overexpression. Flowcytometry cell-cycle assays in these cell lines demonstrated thatSATB2-AS1 overexpression induced G1 phase cell-cycle arrest inM5 (P ¼ 0.035) and LoVo cells (P < 0.001, Fig. 2C). However,the proportion of apoptotic cells remained similar betweenSATB2-AS1-overexpressing cells and controls cells (P < 0.05,Supplementary Fig. S2). These data suggest that the SATB2-AS1-mediated decrease in colorectal carcinoma cell proliferationis modulated by the G1–S checkpoint, rather than by apoptosis.

SATB2-AS1 overexpression inhibits colorectal carcinoma cellmigration and invasion

We also examined the effects of SATB2-AS1 overexpression oncolorectal carcinoma cell migration and invasion. Cell invasionanalysis demonstrated that enforced expression of SATB2-AS1in two different colorectal carcinoma cell lines significantlyinhibited cancer cell invasion through Matrigel, a basement-membrane-like extracellular matrix (P < 0.001, Fig. 2D). Thewound-healing assay also illustrated that the exogenous expres-sion of SATB2-AS1 in colorectal carcinoma cells caused a signif-icant decrease in cell migration (M5 cells, P ¼ 0.013 and LoVocells, P < 0.001, Fig. 2E). These findings indicated that SATB2-AS1was sufficient to repress both colorectal carcinoma cell invasionand migration in vitro.

SATB2-AS1 overexpression suppresses tumorigenicity andmetastasis of colorectal carcinoma cells in vivo

In light of our in vitro findings, we tested the effects of SATB2-AS1 overexpression in vivo. LoVo cells stably overexpressingSATB2-AS1 and control cells were subcutaneously inoculated intonude mice (n ¼ 6 per group). Mice injected with cells over-expressing of SATB2-AS1 showed a measurably suppressed effecton tumor growth compared with those injected with control cells(Fig. 2F). SATB2-AS1 overexpression significantly inhibited cellproliferation in LoVo cells as determined by Ki-67 staining(Fig. 2G). We further explored the role of SATB2-AS1 in lungcolonization by inoculating cells directly into the tail veins ofnude mice (n¼ 8 per group). The results showed that SATB2-AS1overexpression decreased the number of definite pulmonarycolonization (oneof eightmice) comparedwith the control group(five of eight mice; P ¼ 0.039, Fig. 2H and 2I). Moreover,compared with mice injected with control cells, the number ofpulmonary tumor colonies in the SATB2-AS1-overexpressing cell-injected mice was significantly decreased (P ¼ 0.041, Fig. 2I).Consistent with the in vitro results, these data also indicated animportant inhibitory role for SATB2-AS1 in tumor growth andmetastasis in vivo.

siRNA-mediated knockdown of SATB2-AS1 expressionpromotes colorectal carcinoma cell proliferation, invasion, andmigration

To further confirm the effects of SATB2-AS1 on suppressing themalignant phenotypes in colorectal carcinoma cells, we alsoknocked down SATB2-AS1 expression by siRNA transfection inSW480 and DLD1 cells, which have relatively high SATB2-AS1expression. SW480 cells with depleted SATB2-AS1 expressionexhibited enhanced cell growth by more than 40% at day 4(P ¼ 0.002) and DLD1 cells showed 20% enhanced cell growthat day 6 (P ¼ 0.049), compared with negative control scrambledsiRNA transfection (Supplementary Fig. S3A). Furthermore,

colony formation assays revealed that SATB2-AS1 siRNA-treatedcells exhibited significantly increased cell growth comparedwith the negative control siRNA (SW480, P ¼ 0.026 and DLD1,P ¼ 0.001, Supplementary Fig. S3B). SATB2-AS1 knockdownreduced the G1 phase and increased the S-phase cell populationin SW480 (P ¼ 0.013) and in DLD1 cells (P < 0.001, Supple-mentary Fig. S3C). Moreover, we also found that depletionof SATB2-AS1 increased the invasion and migration potential ofcolorectal carcinoma cells, compared with the control cells(SW480, P < 0.001 and P ¼ 0.041 and DLD1, P < 0.001 andP¼ 0.007, Supplementary Fig. S3D and S3E). Consistent with thegain-of-function results, these data also suggest that SATB2-AS1 isa suppressor in colorectal carcinoma cells.

SATB2, the sense-cognate gene for SATB2-AS1, is a keydownstream target of SATB2-AS1

Given the close proximity of SATB2-AS1 to SATB2 (Fig. 3A), wehypothesized that SATB2-AS1 could exert its biological effects viaSATB2 modulation. To examine whether SATB2-AS1 is co-expressed with SATB2 in human colorectal carcinoma samples,we measured SATB2mRNA levels in the same set of 40 colorectalcarcinoma tissues shown in Fig. 1B. Similar to SATB2-AS1, SATB2mRNA was significantly downregulated in the majority of colo-rectal carcinoma samples (P < 0.001, Supplementary Fig. S4),and SATB2-AS1 and SATB2 RNA levels in these tissues werepositively correlated (R2 ¼ 0.1698, P ¼ 0.008, Fig. 3B left). Asshown in Fig. 3B (right), a significantly positive correlationbetween SATB2-AS1 and SATB2 was found in the TCGA cohort(n¼ 623, R2¼ 0.6222, P < 0.001). Furthermore, wemeasured thelevels of SATB2 in colorectal carcinoma cells with overexpressingSATB2-AS1. SATB2-AS1 overexpression significantly increasedSATB2 mRNA levels in M5 and LoVo cells (M5 cells, P < 0.001and LoVo cells, P ¼ 0.033, Fig. 3C, top left). When measured bywestern blotting, the expression of SATB2proteinwas increased inboth M5 and LoVo cells with overexpressing of SATB2-AS1(Fig. 3C, top right).Meanwhile, SATB2-AS1 knockdownmediatedby siRNA significantly reduced SATB2expression comparedwith anegative control siRNA (Fig. 3C, bottom). These observationsconfirmed the regulation of SATB2 expression by SATB2-AS1in vitro at the mRNA and protein level. Furthermore, theresults from mouse models showed that the overexpression ofSATB2-AS1 could significantly increase SATB2 protein levelsin vivo (Fig. 2G, bottom). Of note, the depletion of SATB2 alsoaffected SATB2-AS1 expression levels to some extent (Supplemen-tary Fig. S5). Taken together, these results suggest an importantrole for SATB2-AS1 in modulating the expression of SATB2, thesense-cognate gene for SATB2-AS1, and vice versa.

SATB2-AS1 promotes SATB2 expression by recruiting p300 toaccelerate histone H3 acetylation

We propose that the overlapping parts of the SATB2-AS1 andSATB2 transcripts could form an RNA duplex to further alter thesecondary or tertiary structure of SATB2, which increases thestability of SATB2. As a result, we assessed the stability of theSATB2 transcript by blocking new RNA synthesis with Dactino-mycin D. We did not observe increased SATB2 mRNA stability incolorectal carcinoma cells stably overexpressing SATB2-AS1 com-pared with control cells (P > 0.05, Supplementary Fig. S6).

Nuclear and cytoplasmic total RNA fractions were preparedfrom colorectal carcinoma cells. As shown in Fig. 4A, SATB2-AS1was 377-fold enriched in the nuclear fraction relative to the






cytoplasm, suggesting that the SATB2-AS1 transcript was mainlylocated in the nucleus. A similar subcellular location for SATB2-AS1 in colorectal carcinoma cells was confirmed by fluorescence

ISH (FISH, Fig. 4A, right). Because nuclear-enriched lncRNAsare known to be functionally involved in epigenetic and tran-scriptional regulation, we wondered whether the mechanism of

Figure 3.

SATB2, the sense-cognate gene for SATB2-AS1, is a key downstream target of SATB2-AS1.A, Genomic location of SATB2 and SATB2-AS1 from ENCODEcollection. Higher levels of epigenetic modification marks on histone 3 at lysines 4 (H3K4me3), 9 (H3K9ac), and 27 (H3K27ac) and several transcription factorbinding sites uniform peaks of p300 were observed within the SATB2 promoter region. B, The correlation between SATB2-AS1 transcript levels and SATB2 mRNAlevels in colorectal carcinoma tissues was measured according to our data (n¼ 40; left) and the TCGA cohort (n¼ 623; right). C, SATB2-AS1 regulated theexpression of SATB2 onmRNA and protein levels. �, P < 0.05; �� , P < 0.01; �� , P < 0.001.

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Figure 4.

SATB2-AS1 promotes the expression of SATB2 by recruiting p300 to accelerate histone H3 acetylation. A, Nuclear-enriched SATB2-AS1was determined by RNAfraction assays (left) and fluorescence in situ hybridization (right). B, RIP experiments were performed in LoVo cells using a p300 or nonspecific IgG antibody todetermine the amount of SATB2-AS1 RNA associated with p300 or IgG relative to the input control. C, RNA pull-down assays were used to identify proteinsassociated with SATB2-AS1. Biotinylated SATB2-AS1 and antisense RNAwere incubated with cell extracts and the associated proteins were resolved bySDS-PAGE.Western blotting was performed to analyze the specific interaction between p300 and SATB2-AS1. Dot-blot of RNA-protein binding samplesindicates equal RNA transcript present in the assay.D, RNAs corresponding to different fragments of SATB2-AS1 or its antisense sequence (dotted line) werebiotinylated and incubated with LoVo cell extracts, targeted with streptavidin beads. p300 protein was detected byWestern blotting. E, SATB2-AS1overexpression significantly increased the protein level of H3K9ac, H3K27ac, and SATB2, and did not affect the protein level of H3k4me3 and p300. F and G,p300 could bind to SATB2 promoter. Schematic illustration of SATB2-AS1 and SATB2 structures is shown (F, top). Arrows show the direction of transcription.SATB2-AS1 exons, red bars marked as E1–E3. SATB2 exons, as blue bars. There are 164 nucleotide overlapping and complementary regions between the secondexon of SATB2-AS1 and SATB2 exon1. The numbered sites denote the promoter fragments of SATB2 gene. The ability of p300 to bind to SATB2 promoter regionswas assessed by ChIP (F) and was increased as a result of SATB2-AS1 overexpression (G). H, SATB2 protein was upregulated by the histone deacetylase inhibitorTSA in colorectal carcinoma cells, whose effect was equal to that of SATB2-AS1 overexpression. The induction of SATB2 expression by SATB2-AS1 could bereversed by the acetyltransferase inhibitor C646. I, Depletion of p300mediated by siRNA significantly increased H3K9 and H3K27 acetylation and SATB2 proteinlevels in colorectal carcinoma cell lines with overexpressing SATB2-AS1. J, H3K9ac, H3K27ac, and SATB2 levels were upregulated with the increasing p300recruited by SATB2-AS1 overexpression, whereas H3K9ac, H3K27ac, and SATB2 were downregulated when SATB2-AS1was lowly expressed in tissues fromthe same donor gland in human colorectal carcinoma. �� , P < 0.01; �� , P < 0.001.






SATB2-AS1 promotes SATB2 expression through modulation oftranscription factor recruitment and chromatin modification. Weperformed a computational screen. ENCODE data in the UCSCGenome Browser showed that higher levels of epigenetic modi-fication marks on histon 3 at lysines 4 (H3K4), 9 (H3K9), and27 (H3K27) and several transcription factor binding sites uniformpeaks of p300, a histone acetyltransferase, within the SATB2promoter region in K562 cells (Fig. 3A). Then, we performed RIPwith an antibody against p300 using colorectal carcinoma cellsextracts, and observed significant enrichment of SATB2-AS1 withthe p300 antibody compared with the nonspecific IgG controlantibody (P ¼ 0.005, Fig. 4B). The previous study confirmed thatlncRNAZEB1-AS1 recruitedp300 toZEB1promoter, upregulatinghistonemarkers at theZEB1promoter (18). As expected, p300wasable to interact with ZEB1-AS1 in colorectal carcinoma cellsextracts with p300 antibody. Meanwhile, the p300 binding toSATB2-AS1was specific, because of failing todetect thepresenceofPACER, a lncRNA reported that could not interact with p300 (19),in p300 RIP samples (Supplementary Fig. S7A–S7B). Further-more, RNA pull-down was performed to validate the associationbetween SATB2-AS1 and p300 in colorectal carcinoma cells,which confirmed the physical association between SATB2-AS1and p300 in vitro (Fig. 4C). Deletion-mapping analyses identifiedthe 361�720 nt region at the 50 end of SATB2-AS1 that is requiredfor its association with p300 (Fig. 4D). However, SATB2-AS1could not affect p300 mRNA and protein expression levels(Fig. 4E, top; Supplementary Fig. S8). Subsequently, we per-formed ChIP using an anti-p300 antibody to determine whetherp300 was bound to the SATB2 promoter region and SATB2-AS1overexpression affects its binding capability. We observed a sig-nificant enrichment of SATB2 promoter fragments with the p300antibody (Fig. 4F). Moreover, a 23.3- and 3992.1-fold increase inSATB2 promoter DNA bound to p300 was identified in cells withoverexpressing SATB2-AS1 comparedwith control cells expressingendogenous levels of SATB2-AS1 (P < 0.05, Fig. 4G). Takentogether, these results demonstrate that SATB2-AS1 served as ascaffold to recruit p300 to the SATB2 promoter.

We next addressed whether SATB2-AS1 overexpression, whichincreases p300 occupancy, also affected the levels of H3K9and H3K27 acetylation and H3K4 tri-methylation (H3K4me3)in the SATB2 promoter region. As shown in Fig. 4E, there was asignificant increase in the protein level of acetylated H3K9(H3K9ac) and H3K27 (H3K27ac), and SATB2 as a result ofSATB2-AS1 overexpression. However, no significant differencewas observed in the protein level of H3K4me3. We also treatedthe colorectal carcinoma cells with the histone deacetylase inhib-itor trichostatin A (TSA; 100 nmol/L) and acetyltransferase inhib-itor C646 (10 mmol/L). Interestingly, SATB2 was upregulated byTSA in both LoVo and M5 cells, and that the effect was equal tothat of SATB2-AS1 overexpression. Meanwhile, the induction ofSATB2 expression by SATB2-AS1 could be reversed by C646, asshown in Fig. 4H.

Accordingly, we addressed whether the enhancement ofH3K9ac and H3K27ac by SATB2-AS1 was mediated by p300. Incolorectal carcinoma cells with overexpressing SATB2-AS1 trans-fected with specific p300 siRNAs, the protein levels of H3K9ac,H3K27ac, and SATB2 were significantly decreased compared tothe cells transfected with negative control siRNA (Fig. 4I). Con-sistent with the in vitro results, p300, H3K9ac, H3K27ac, andSATB2 levels were upregulated with SATB2-AS1 overexpression inhuman colorectal carcinoma tissues. However, with SATB2-AS1

low-expression, p300, H3K9ac, H3K27ac, and SATB2 levels weredownregulated from the same donor gland (Fig. 4J). These resultssupport the hypothesis that the transcript activation of SATB2 isdependent on the level of SATB2-AS1 recruiting p300, whichinduces H3K9 and H3K27 acetylation in the SATB2 promoterregion.

SATB2-AS1 requires p300/SATB2 to suppress colorectalcarcinoma growth, invasion, and migration

To test the contribution of p300 and SATB2 on the importantrole of SATB2-AS1, we knocked down the expression level of p300or SATB2 in vector- or SATB2-AS1-expressing colorectal carcino-ma cells with siRNAs targeting p300 or SATB2, respectively.SATB2-AS1 overexpression significantly reduced LoVo cell prolif-eration, cell-cycle progression, invasion, andmigration.However,this potential was completely abolished to the levels similar to thecontrol cells when knockdown of p300 mediated by siRNA(Fig. 5A–E). These results suggest that p300 is specifically requiredfor SATB2-AS1 to affect LoVo cells behaviors. Moreover, weobserved that depletion of SATB2 impairs LoVo cell proliferationefficiency and diminishes the suppressive effects of SATB2-AS1overexpression in a similar manner (Fig. 5A–E). Similar resultswere obtained using M5 cells (Supplementary Fig. S9A–S9E).Taken together, these rescue effects obtained by knocking downp300or SATB2 indicated that the ability of SATB2-AS1 to suppressgrowth, invasion, and migration is in large part attributed to itsability to recruit p300, which subsequently enhances H3K9 andH3K27 acetylation levels in the SATB2 promoter and activatesSATB2 gene transcription.

Suppression of EMT by SATB2-AS1 depends on SATB2-mediated recruitment of HDAC1 to repress Snail transcription

Our previous studies showed that SATB2 was associated withcell invasion and migration in colorectal carcinoma (10, 16, 17).Gene set enrichment analysis (GSEA) revealed that lower expres-sion of SATB2 was positively correlated with an enrichment ofmetastasis signatures (Fig. 6A, GSE17536, GSE13067, andGDS4718). Indeed, SATB2 downregulation, directly regulated bymiR-182, induced colorectal carcinoma cells EMT (16), whichindicated that SATB2 downregulation-induced EMT might be amechanism that SATB2-AS1 depletion facilitated colorectal car-cinoma progression. Interestingly, both SATB2-AS1- and SATB2-overexpression resulted in significantly decreased mRNA andprotein levels of Snail, a central regulator of EMT (Fig. 6B and C).

To further explore the underlying mechanism of how SATB2regulates Snail expression, we searched Snail promoter sequencewith BLAST software and identified that three potential bindingregions with AT-rich sequence (42.5%, 29.3%, and 30.8%) werepredicted to be embedded in Snail gene promoter (Fig. 6D, top).ChIP assays revealed that endogenous SATB2 protein was boundto the most AT-rich region (P1, �1069 to �710 bp) of Snailpromoter (Fig. 6D).

Recruitment of histone deacetylase (HDAC) has been shown astypical functional characteristics of MAR-binding proteins (20).To investigate whether HDACs is involved in the regulation ofSnail, we immunoprecipitated protein complex from colorectalcarcinoma cells extracts using anti-SATB2 antibody with normalmouse IgG as the negative control and detected HDAC1 andHDAC2 protein in the precipitates, and found that SATB2 co-precipitated with HDAC1 but not with HDAC2. The interactionbetween SATB2 and HDAC1 was further confirmed by detecting

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SATB2 in the immunoprecipitate of HDAC1 in DLD1 cells(Fig. 6E).

As histone deacetylation is tightly coupled to HDAC-medi-ated gene repression, we therefore examined HDAC1 occupan-cy at the Snail promoter by ChIP assays. We observed a signif-icant enrichment of Snail promoter (P1 and P2) DNA with anti-HDAC1 antibody. The ability of HDAC1 binding to the Snailpromoter (P1) was significantly increased in colorectal carci-noma cells overexpressing SATB2-AS1 compared with controlcells (Fig. 6F), which was consistent with the above results thatthe promoter region of Snail interacted with SATB2. Meanwhile,the SATB2 downregulation significantly increased Snail pro-moter luciferase activity (Fig. 6G). These results indicated thatSATB2 recruit HDAC1 to Snail promoter to repress transcriptionby reducing levels of acetylated histone. In addition, we alsoobserved that p300 occupied at the Snail promoter throughChIP assays. However, SATB2-AS1-overexpression could notsignificantly increase this occupancy ability (SupplementaryFig. S10), suggesting that p300 recruitment by SATB2-AS1cannot enhance the acetylation levels of Snail promoter. Inter-estingly, the depletion of Snail affected SATB2-AS1 expressionlevels to some extent (Supplementary Fig. S11).

To examine the contribution of Snail to the phenotype causedby SATB2-AS1 overexpression, we performed rescue experiments.We increased Snail level in SATB2-AS1-overexpressing andcontrol cells by transfecting Snail overexpression plasmid. Snailoverexpression significantly repaired the inhibiting effects causedby overexpression of SATB2-AS1 on colorectal carcinoma cellproliferation, invasion, and migration to the levels similar to the

control cells (Figure 7A–E). These results suggest that Snail isspecifically required for SATB2-AS1 to affect LoVo cells behaviors.Moreover, we also found that Snail overexpression rescued thesuppressive effects of SATB2 overexpression in a similar manner(Fig. 7A–E).

Snail has been proposed as a central regulator of EMT. There-fore, we next further investigated the expression changes of classicEMTmarkers. As expected, a significant gain of epithelial markers(E-cadherin and ZO-1) and loss of the mesenchymal markervimentin was observed both in SATB2-AS1- and SATB2-over-expression colorectal carcinoma cells. However, these changeswere completely attenuated to the levels similar to the controlcells by HDAC1 depletion (Fig. 7F). Taken together, these resultsindicate that suppression of colorectal carcinoma progression bySATB2-AS1 depends on SATB2-mediated recruiting HDAC1 torepress Snail transcription and EMT (Fig. 7G).

DiscussionIn this study, we uncovered a long noncoding antisense tran-

script for SATB2, called SATB2-AS1, which functions as a regulatorof SATB2 gene expression. We present data showing that theSATB2-AS1 expression levels were downregulated in colorectalcarcinoma tissues and that the downregulation of SATB2-AS1expression was intimately linked to the TNM classification andpoor outcome of patients with colorectal carcinoma. We alsovalidated that SATB2-AS1 played an important suppressed roleduring colorectal carcinoma tumorigenesis and progression.Additionally, we demonstrated that SATB2-AS1 recruited p300

Figure 5.

SATB2-AS1 requires p300/SATB2 to suppress colorectal carcinoma cell growth, invasion, and migration. SATB2-AS1 overexpression significantly reduced thecolorectal carcinoma cell proliferation (A), colony formation (B), cell-cycle progression (C), migration (D), and invasion (E). The potential effects of SATB2-AS1 oncolorectal carcinoma cells were completely abolished similar to the control cells by the p300 knockdown by siRNA. The depletion of SATB2 impairs colorectalcarcinoma tumor cell proliferation efficiency and diminishes the suppression effect of SATB2-AS1 overexpression in a similar manner. �� , P < 0.01; �� , P < 0.001.






Figure 6.

SATB2-AS1 repressed Snail transcription depending on SATB2-mediated recruitment of HDAC1. A, GSEA showed the enrichment of metastasis signatures incolorectal carcinoma cells with SATB2 downregulation. B and C, Both SATB2-AS1 and SATB2 inhibited the expression of Snail on mRNA and protein levels.D, ChIP analysis of SATB2 occupancy on the Snail promoter. Immunoprecipitated DNAwas compared with input and is shown as percentage. Data arerepresented as mean� SD. E, Coimmunoprecipitation of endogenous HDAC1 with anti SATB2 antibodies (top) and endogenous SATB2 with anti-HDAC1antibodies (bottom) in colorectal carcinoma cells. F, ChIP analysis of HDAC1 occupancy on the Snail gene. Significantly increased recruitment of HDAC1 to Snailpromoter was observed upon SATB2-AS1 overexpression. G, Dual luciferase reporter assay was performed in colorectal carcinoma cells to verify the effect ofSATB2 on Snail promoter activity. � , P < 0.05; �� , P < 0.01; ��, P < 0.001; ns, nonsignificant.

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Figure 7.

SATB2-AS1 requires SATB2/Snail to suppress colorectal carcinoma cell growth, invasion, migration, and EMT. SATB2-AS1 overexpression significantly reducedthe colorectal carcinoma cell proliferation (A), colony formation (B), cell-cycle progression (C), invasion (D), and migration (E). The potential effects ofSATB2-AS1 on colorectal carcinoma cells were completely abolished similar to the control cells by Snail overexpressing. Snail overexpression also rescued thesuppressive effects of SATB2 overexpression in a similar manner (A–E). F,Western blot analysis showed that epithelial markers were reduced and vimentin as amesencymal marker was enhanced both by SATB2-AS1 and SATB2-overexpression. The changes were completely attenuated to the levels similar to the controlcells by HDAC1 depletion in colorectal carcinoma cells. G, Schematic diagram showing the mechanism of action of SATB2-AS1 in colorectal carcinoma. SATB2-AS1was downregulated in colorectal carcinoma and consequently decreased p300 recruitment. In turn, the reduction in p300 recruitment suppressed theaccumulation of the active marks H3K27ac and H3K9ac and repressed SATB2 levels. Subsequently, the decreasing of HDAC1 recruited by SATB2 increasedactivating histone acetylation marks and promoted Snail expression, which induced EMT and colorectal carcinoma progression. � , P < 0.05; ��, P < 0.01;�� , P < 0.001.






to the SATB2promoter and increasedH3K9ac andH3K27ac levelsat the SATB2 promoter region, resulting inmarked transcriptionalactivation of SATB2 gene. Then, SATB2 inhibited EMT throughrecruiting HDAC1 to repress SATB2 transcription. Thus, ourresults reveal a novel mechanism of SATB2-AS1-SATB2-Snailregulatory axis involved in epigenetic regulation and theSATB2-AS1-SATB2-Snail pathway constitutes a previously unrec-ognized carcinogenesis and progression regulator in colorectalcarcinoma.

Recent advances in genomics technology have led to the explo-sive discovery that mammalian genomes encode numerouslncRNAs (3), which advancing our comprehensive understandingof biological processes in human health and disease. It has beenidentified that thousands of lncRNAs that are differentially tran-scribedbetweennormal tissues and tumors (21, 22). Validationofthese expression-deregulated lncRNAs may provide biomarkersfor diagnosis, determining prognosis and as a therapeutic target.We applied high-throughput lncRNA expression profiling todetect colorectal carcinoma metastasis–related lncRNAs, whichmight play critical roles in colorectal carcinoma progressionprocesses. Among them, we focused on an antisense lncRNA,SATB2-AS1, and then demonstrated that SATB2-AS1 was down-regulated in colorectal carcinoma tissues and cell lines and thatthe low SATB2-AS1 expression levels were significantly associatedwith aggressive stages andpoor survival of patientswith colorectalcancer. These data indicate that the expression of SATB2-AS1mayhave considerable potential in predicting the prognosis of colo-rectal carcinoma.

Accumulating data are revealing the extensive functional rolesof lncRNAs in tumorigenesis. Gupta and colleagues reported thatHOTAIR induced genome-wide retargeting of PRC2, leading toH3K27me3, which promoted breast cancer metastasis by silenc-ing multiple metastasis suppressor genes (8). Subsequent studiesshowed that HOTAIR deregulation is associated with patientprogression in 26 human tumor types (23). LncRNA CCALenhances proliferation, cell-cycle progression, invasion, andmigration, which are in large part attributed to its ability todegrade AP-2a and subsequently activate the Wnt/b-cateninsignaling pathway (24). Very recently, one study showed thatelevated expression of SATB2-AS1 increases cell proliferation andgrowth in osteosarcoma (25). In this study, we comprehensivelyconfirmed the effects of SATB2-AS1 on colorectal carcinomacarcinogenesis through gain- and loss-of-function experiments.We identified that SATB2-AS1 plays a key role in colorectalcarcinoma carcinogenesis by acting as a tumor suppressor, whichinhibits colorectal carcinoma growth and metastasis in vitro andin vivo. The results also indicate that the enhanced expression ofSATB2-AS1 by gene transfer can reverse the malignant phenotypeof colorectal carcinoma, suggesting SATB2-AS1 as a potentialtherapeutic target for colorectal carcinoma.

Many new functions have been ascribed to antisense lncRNAsfrom data generated over the last year. However, the mechanismsinvolved in mediating the progression of colorectal carcinoma bySATB2-AS1 remain unknown. The sense-cognate gene of SATB2-AS1, SATB2, one of matrix attachment region (MAR)-bindingproteins, binds to AT-rich MARs of DNA and induces a localchromatin-loop remodeling, controllinggeneexpression(20,26).In our previous studies, we found that SATB2 is ubiquitouslyexpressed in noncancerous tissues; in contrast, its expression isfrequently low in tumors and tumor cell lines, leading to increasedcolorectal carcinoma cell proliferation and metastasis (10, 16,

17).However, themechanisms involved in deregulating of SATB2expressionhave not been clearly defined. Remarkably, SATB2-AS1abundance through ectopic overexpression or siRNA-mediatedknockdown specifically affects SATB2 mRNA and protein expres-sion. Moreover, SATB2-AS1 and SATB2 RNA levels in colorectalcarcinoma tissues were significantly correlated. Thus, we specu-lated that SATB2-AS1 could exert its biological effects viaSATB2 modulation. Previously, studies have revealed that theantisense might form sense-antisense pairs to regulate epigeneticsilencing, transcription, mRNA stability, and gene translation onthe opposite strand (27, 28). For example, lncRNA BACE1-anti-sense transcript (BACE1-AS) increases BACE1 mRNA stability bygenerating additional Ab1-42 in Alzheimer's disease (27). How-ever, SATB2-AS1 failed to significantly alter the SATB2 mRNAdegradation rate. Thus, SATB2-AS1 does not appear to affect thestability of SATB2 by forming form sense-antisense gene pairs.

Apart from proteins, the functionality of lncRNAs begins withtheir cellular localization. These so-called nuclear-enrichedlncRNAs are shown to be involved in key cellular processesassociated with gene expression including but not limited toepigenetic regulation, chromosomal interaction, and transcrip-tional regulation (29).Here, SATB2-AS1wasdetermined to enrichin nucleus of colorectal carcinoma cells by ISH, FISH, andRNA fraction assays, which is consistent with a potential role inepigenetic regulation. Indeed, SATB2-AS1 could bind with p300protein, thereby inducing the accumulation of the active marksH3K9ac and H3K27ac, which activate SATB2 transcription.Importantly, we also observed that changes in SATB2-AS1 abun-dance specifically affected SATB2 expression through p300recruitment in vivo. These data suggest SATB2-AS1 epigeneticallyregulate the SATB2 promoter at the histone level. MutiplelncRNAs have previously been described to regulate genes expres-sion via epigenetic regulation, such as the recently identifiedlncRNA ANRASSF1 (28) andNEAT1 (30). Our results, along withrecent studies, indicate that lncRNAs have the potential to influ-ence the epigenetic environment of genes in a location-specificmanner. Furthermore, the most important effect exerted bySATB2-AS1 on cell proliferation, invasion, and migration is par-tially reversed by the siRNA-mediated knockdown of p300 orSATB2 after transfection with siRNAs. Thus, our results reveal anovelmechanism for epigenetic regulation at SATB2 that involvesthe antisense lncRNA SATB2-AS1 and demonstrates that theSATB2-AS1/SATB2 pathway constitutes an important role incolorectal carcinoma progression.

EMT is an evolutionary conserved trans-differentiation processproposed to play a key role in cancer metastasis, therapeuticresistance and cancer stem cell-like features (31–33), which areessential features for colorectal carcinoma progression (34–36).During EMT, epithelial cells lose their epithelial adherence, losetheir polarity, attain front-to-real polarity, become spindleshaped, and gain migratory and invasive capacity. EMT and thereverse of this program, termed mesenchymal-to-epithelial tran-sition (MET), induce multiple fundamental changes in cell phys-iology in addition to the morphologic differences. EMT can betriggered by a set of pleiotropically acting transcription factors(TF), including Snail1/2, Twist1/2, and Zeb1/2 (37). Snail(Snail1), as one of the most important EMT-TFs, is frequentlyoverexpressed in different types of metastatic cancers, includingcolorectal carcinoma (38). Epigenetic modification, transcrip-tional, and posttranscriptional regulatory mechanisms areinvolved tightly in Snail expression (39). Our previous study

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showed that SATB2 could reduce Snail protein level, induce MET,and suppress colorectal carcinoma metastasis (16). However, themechanism involved in the repression of Snail by SATB2 has notbeen clearly defined.

As a MAR-bind protein, SATB2 has been reported to bind AT-rich regulatory elements for transcriptional regulation in pre-Bcells, osteoblasts, brain, and embryonic stem cells (26, 40, 41).Here, we further identified that SATB2 recruited corepressorHDAC1 to Snail promoter and removed activating histone acet-ylation marks, thereby decreasing Snail expression. Consistentwith our results, HDAC inhibitor treatment enhances Snail acet-ylation and induces EMT via inducing Snail transcription (42). Inthe first half of present study, we found SATB2-AS1 could recruitp300. Although, p300 occupancy at Snail promoter was observed,SATB2-AS1-overexpression could not significantly increase thisoccupancy ability. Thus, the Snail expression did not appear to bedirectly regulated by SATB2-AS1-recruited p300. It is likely thatthere are other factors that recruitment p300 to regulate Snailexpression. To our knowledge, we demonstrated a novel mech-anism responsible for the transcriptional regulation of Snailmediated by SATB2, a MAR-bind protein. Our study furthermorerevealed that both upregulation of SATB2-AS1 and SATB2repressed Snail expression, thereby increasing epithelial markersand decreasing mesenchymal markers, and induced MET, whichexplained the mechanism of SATB2-AS1 inhibited colorectalcarcinoma progression. Strikingly, Snail could decrease theexpression of SATB2-AS1. These data hinted the probable exis-tence of a negative feedback loopbetween SATB2-AS1 and Snail incolorectal carcinoma cells. SATB2-AS1 transcriptional inactiva-tion and the associationbetween the SATB2-AS1 andSnail presentan interesting issue for further investigation.

The published study showed that Snail impairs the transitionfrom early to late G1 by maintaining low levels of cyclin D andblocks the G1–S transition by maintaining high levels of p21 inMDCK and MCA3D cells after 72 hours in culture with basalconditions (43). However, Snail-overexpressing cells can respondto mitogenic signals by transiently decreasing p21 expression,which favors the transition to S phase (43). The study alsoreported that Snail repressed p21 expression in infected B cellscontributing to KSHV induced B-cell oncogenesis (44). Snailknockdown was reported to led to the upregulation of p21 anddownregulation of Cdc2, causing a decrease of G1 and S periodsand an increase of the G2–M population (45). In our rescueexperiments, we observed that Snail overexpressing promotedG1–S transition and blocked the phenotype caused by the over-expression of SATB2-AS1. These data indicated that Snail hascomplicated roles on regulating cell-cycle progression dependingon cell lines, cell culture conditions, and its target genes in

different cancer cells. In addition, SATB2-AS1 might directly/indirectly regulate a number of genes contributing globally tocancer progression, including cell-cycle regulation. Snail is onlyone of many genes regulated by SATB2-AS1. Further characteri-zation of these genes will provide new insights in SATB2-AS1-mediated cell-cycle regulation.

In summary, we determined that SATB2-AS1 was downregu-lated in colorectal carcinoma and consequently decreased p300recruitment. In turn, the reduction in p300 recruitment sup-pressed the accumulation of the active marks H3K27ac andH3K9ac and repressed SATB2 levels, then activating Snail expres-sion and inducing EMT to promote colorectal carcinoma cellproliferation, invasion and migration in vivo and in vitro. Here,we reported the involvement of SATB2-AS1 as an epigeneticregulator that modulates colorectal cancer aggressiveness by reg-ulating of SATB2 expression and EMT. Overall, we highlight anovel mechanism of regulation by the SATB2-AS1-SATB2-Snailaxis, which is mediated by epigenetic regulation. Our resultssuggest that SATB2-AS1 deregulation contributes to the develop-ment and progression of colorectal carcinoma, suggesting thatSATB2-AS1 may represent an effective target for blocking orreversing colorectal carcinoma progression.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y.-Q. Ding, S. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.-Q. Wang, D.-M. Jiang, S.-S. Hu, Y. HanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.-Q. Wang, D.-M. Jiang, S.-S. Hu, L. Zhao, L. Wang,M.-L. Ai, H.-J. JiangWriting, review, and/or revision of the manuscript: Y.-Q. Wang, D.-M. Jiang,S. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Zhao, M.-H. Yang,Study supervision: Y.-Q. Ding, S. Wang

AcknowledgmentsThe National Basic Research Program of China (973 Program,

2015CB554002), the National Natural Science Foundation of China(81472318 and81502483), and theNatural Science Foundation ofGuangdongProvince (2014A030313308 and 2014A030310134) supported this work.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 16, 2018; revised January 17, 2019; accepted March 4,2019; published first March 11, 2019.

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Wang et al.




2019;79:3542-3556. Published OnlineFirst March 11, 2019.Cancer Res Yi-Qing Wang, Dong-Mei Jiang, Sha-Sha Hu, et al. Mesenchymal Transition

− Transcription and EpithelialSnailInhibiting SATB2-Dependent Suppresses Colorectal Carcinoma Aggressiveness bySATB2-AS1

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Suppresses Colorectal Carcinoma Aggressiveness by ... · colorectal carcinoma tissues without metastasis and those with metastasis. We found that lncRNA antisense transcript of SATB2