Biochemical and Biophysical Research Communications 406 (2011) 534–538

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier .com/locate /ybbrc

Down-regulation of NDRG2 gene expression in human colorectal cancerinvolves promoter methylation and microRNA-650

Li Feng a, Yun Xie b, Hao Zhang a, Yunlin Wu c,⇑a Department of Gastroenterology, Minhang District Central Hospital, Shanghai, Chinab Department of Pathology, Minhang District Central Hospital, Shanghai, Chinac Department of Gastroenterology, Rui-jin Hospital, Shanghai Jiao Tong University, Shanghai, China

a r t i c l e i n f o

Article history:Received 10 February 2011Available online 23 February 2011

Keywords:NDRG2HypermethylationColorectal cancermiR-650

0006-291X/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.bbrc.2011.02.081

⇑ Corresponding author. Address: Rui-jin Hospital AUniversity School of Medicine, 197 Rui-jin Road II, S+86 21 64150773.

E-mail address: wuyunlin195136@126.com (Y. Wu

a b s t r a c t

The expression of N-myc downstream-regulated gene 2 (NDRG2) is present in normal tissues but low orundetectable in various cancers and thus poses a potential tumor suppressor gene. However, the expres-sion of NDRG2 in colorectal tissues remains unknown. Here, our results showed that NDRG2 was down-regulated in colorectal cancer compared to benign colorectal tissues by using immunohistochemicalstaining and semi-quantitative RT-PCR analyses. Bisulfite sequencing analysis showed that the reducedNDRG2 expression was accompanied by de novo DNA methylation at the NDRG2 promoter. We alsofound that microRNA-650 (miR-650) targets a homologous DNA region in the promoter region of theNDRG2 gene and represses its expression at the transcriptional level. Reporter assay with 30untranslatedregion of NDRG2 cloned downstream of the luciferase gene showed reduced luciferase activity in thepresence of miR-650, providing strong evidence that miR-650 is a direct regulator of NDRG2. In conclu-sion, these results suggest that NDRG2 expression is regulated by promoter methylation and miR-650 inhuman colorectal cancer cells, and endogenous small noncoding RNA induced control of transcriptionmay be a potential system for expressional regulation in human colorectal cancer cells.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Human NDRG2 gene locates at chromosome 14q11.2 and en-codes a 41 k Da protein of the N-myc downstream-regulated genefamily. NDRG2 is normally expressed in brain, heart and is one ofthe four members of the NDRG family [1]. NDRG2 is regarded as tu-mor suppressor gene (TSG) transcriptionally repressed by c-Myc[2]. Previous studies have suggested that NDRG2 is down-regu-lated or undetectable in many human cancers, such as, pancreatic,breast and liver cancers as well as colorectal cancer and high-riskadenomas [3,4]. NDRG2 is tumor suppressor gene associated withcell growth, differentiation and apoptosis [5,6]. NDRG2 has beenfound over-expressed in the brain of Alzheimer’ patients, this indi-cates that it may play role in both cell growth and neurodegener-ation [7]. In meningiomas, NDRG2 expression levels weresignificantly reduced in high-grade compared to low-grade tumors[8]. Consistent with its function as tumor suppressor gene, over-expressed NDRG2 represses glioblastoma cell proliferationin vitro and NDRG2 expression correlates positively with survivalin high-grade glioma [9].

ll rights reserved.

ffiliated to Shanghai Jiao Tonghanghai 200025, China. Fax:

).

Transcriptional inactivation by cytosine methylation at pro-moter CpG islands of tumor suppressor genes is believed to be amechanism involved in human carcinogenesis [10,11]. Here, wefound that NDRG2 was down-regulated in colorectal cancer com-pared to the control tissues, and hypermethylation of the promoterof NDRG2 was responsible for it. We also show that miR-650 tar-gets a homologous DNA region in the 3’UTR region of the NDRG2gene and represses its expression at the transcriptional level.

2. Material and methods

2.1. Patients and cell line

Human colorectal cancer samples were collected from surgicalspecimens from 70 patients with colorectal cancer at Departmentof Pathology, Minhang District Central Hospital, Shanghai, China.Non-tumor samples from the macroscopic tumor margin were iso-lated at the same time and used as the matched adjacent non-neo-plastic tissues (>5 cm). All the samples were divided into two parts.Tissue samples were collected, immediately snap frozen in liquidnitrogen, and stored at �80 �C until RNA extraction, all sampleswere obtained their informed consent and with institutional re-view board approval of the hospital. All patients obtained a con-firmed diagnosis of colorectal carcinoma after resection. Four

L. Feng et al. / Biochemical and Biophysical Research Communications 406 (2011) 534–538 535

colorectal cancer cell lines (SW480, HT29, SW620, LOVO) were allpreserved in our laboratory and maintained in RPMI 1640 with 10%FBS.

2.2. Tissue microarray and immunohistochemistry

After screening H and E-stained slides for optimal tumor tissueand tissue adjacent to tumor (TAT) with a distance of 5 cm fromthe tumor, we constructed TMA slides (Shanghai Biochip, Shanghai,China). Two cores were taken from each formalin-fixed, paraffin-embedded HCC and TAT sample by using punch cores that mea-sured 1.0 mm in greatest dimension from the non-necrotic areaof tumor foci and TAT. Immunohistochemistry was performed bya 2-step method using primary antibody including heat-inducedantigen-retrieval procedures. Sections were incubated overnightat 37 �C with primary antibody; after the primary antibody waswashed off, the components of the Envision detection system wereapplied with an anti-mouse polymer (EnVision1/HRP/Mo, Dako,Glostrup, Denmark). The primary antibodies used were allmouse-antihuman monoclonal antibodies against NDRG2 (1:100dilution; abcam, USA). Negative controls were treated identicallybut with the primary antibody omitted.

2.3. Scoring of expression of NDRG2

Immunoreactivity was evaluated independently by threeresearchers who were blinded to patient outcome. The percentageof positive tumor cells was determined by each observer, and theaverage of three scores was calculated. We randomly selected tenhigh-power fields; and counted 1000 cells in each core. Whenthe mean of percentage of positive cells is close to 0% or 100%,the standard deviation (SD) is close to 0, and when the mean isapproximately 50% the SD is approximately 5%. Thus, the SD doesnot increase with the mean. The following categories were used forscoring: intensity of staining, none (0), mild (1), moderate (2),strong (3); percentage of the positive staining,<5% (0), 5–25% (1),25–50% (2),>50% (3). Combining intensity and percentage stainingresulted in the following score: 0–1, negative (�); 2–4, moderate(+); 5–6 strong (++)[12].

2.4. DNA methylation analysis of the NDRG2 gene

Genomic DNA (2 lg) was modified with sodium bisulfite usingEpiTect Bisulfite kit (Qiagen). Methylation status was analyzed bybisulfite genomic sequencing of the CpG islands. The fragment cov-ering 16 CpG sites from NDRG2 promoter region was amplifiedfrom bisulfite-modified DNA. The primers used were 50-TTTTCGAGGGGTATAAGGAGAGTTTATTTT-30 (sense) and 50-CCAA AACTCTAACTCCTAAATAAACA-30 (antisense). Amplified bisulfite-sequencing PCR products were cloned into pMD18-T simple vector(Takara). Methylation status of human colorectal normal tissuesand tumor samples was examined by methylation-specific PCR(MSP) analysis. Primers for methylated reaction were 50-ATTCGGGTATCG AGAGGGACGC-30 (sense), and 50-AAAAAACCCTATAACTTCGCCG-30 (antisense), and for the unmethylated reactionwere 50-ATTTGGGTATTGAGAGGGATGT-30 (sense), and 50-AAAAAACCCTATAACTTCACCA-30 (antisense).

2.5. 0-aza-20-deoxycytidine treatment

We investigated the effect of a demethylating agent, 50-aza-20-deoxycytidine (Sigma) on the expression of NDRG2 in SW480,HT29, SW620, LOVO cells. Cells were plated at a density of2 � 105 per well in 6-well plates for 18 h, then treated with50-aza-20-deoxycytidine (DAC) at concentrations of 50 lM/L induplicate. After treatment for 48 h, the cells were harvested.

2.6. Real time RT-PCR

To test NDRG2 expression in colorectal cancer tissues, real timeRT-PCR was carried out. Primers used in the RT-PCR: NDRG-F: 50-CGATCCTTACCT ACCTACCACGATGTG-30; NDRG-R: 50-GCATGTCCTCGAACTGAACAGT-30. GAPDH-F, 50-AACTTTGGCATTGTGGAAGG-30; GAPDH-R, 50-ACACATTGGGG GTAGGAACA-30. A 7900HT FastReal-Time PCR System (Applied Biosystems, Foster City, CA) wasused for testing. For detecting mature miR-650, reverse transcrip-tion was done following the applied Biosystems Taqman MicroRNAAssay protocol, and the specific primers was designed according tothe reference. All the experiments were performed triplicate. Theexpression of NDRG2 and miR-650 were normalized to GAPDHand U6, respectively and were given by: 2�dCt. dCt was calculatedas Ct (NDRG2)-Ct (GAPDH) or Ct (miR-650)-Ct (U6).

2.7. Western blot analysis

Protein of treated cell lines was extracted by mammalian pro-tein extraction reagent (Pierce, USA) supplemented with proteaseinhibitors cocktail (Sigma, USA). Fifty micro grams protein sampleswere resolved by 10% SDS–PAGE and then transferred to PVDFmembranes. Autoradiograms were quantified by densitometry(Quantity One software; Bio-Rad, Hercules, CA). Actin-specific anti-body was used for loading control. Mouse monoclonal anti-NDRG2(1:1000, abcam, USA) and Mouse monoclonal anti-Actin (1:1000,abcam, USA) were used.

2.8. Cell transfection

The pre-miR-650 precursor molecule (miR-650 mimics), anti-miR-650 and negative control RNA-oligonucleotides were gainedfrom Ambion corporation (Ambion, Austin, USA). The day beforetransfection, cells were seeded in antibiotic free medium. Transfec-tion of miRNAs was carried out using Lipofectamine 2000 in accor-dance with the manufacturer’s procedure (Invitrogen, California,USA). The level of miR-650 mimics expression in the cells was as-sayed by real-time PCR.

2.9. Luciferase activity assay

A fragment of the wild-type (WT) NDRG2 30UTR containingthe predicted miR-650 binding site was amplified by RT-PCR.The primers used were 50-CATACTAGCTAACCTTGACCTTTAACCCGTGAT-30 (sense) and 50-CTTAA GCTTCCTGACACACATTCACGTAGGT-30 (antisense). Restriction sites are bolded and underlined.Site-directed mutagenesis of the miR-650 target site was carriedout using Stratagene Quik-Change site-directed mutagenesis kit(Stratagene, Heidelberg, Germany). The construct was sequencedand named NDRG-UTR-Mut. The pMIR-report luciferase vectorwas used for the construction of NDRG-UTR or NDRG-UTR-Mutplasmids (Ambion, USA). SW480 and LOVO cells were culturedin 24-well plates. In each well, 10 ng of phRL-TK renilla luciferasevector (Promega, USA) was co-transfected to normalize transfec-tion efficiency. Five hundred nano grams of NDRG-UTR or NDRG-UTR-Mut plasmids together with 10 nM miR-650 mimics ornegative control was also co-transfected. Transfection was doneusing Lipofectamine 2000 and Opti-MEM I reduced serummedium (Life Technologies, California, USA). Firefly luciferaseactivity was measured using the Dual luciferase assay kit(Promega). Normalized relative luciferase activity (RLA) was cal-culated as the following formula: RLA = [firefly luciferase]/[renillaluciferase].

536 L. Feng et al. / Biochemical and Biophysical Research Communications 406 (2011) 534–538

2.10. Statistical analysis

Pearson Chi-Square test and one-ANOVA were used for statisti-cal analysis of group differences. With regard to survival analysis,we analyzed 70 patients with colorectal carcinoma using Kaplan–Meier analyses. Univariate and multivariate survival analyses werethen conducted using the Cox regression model. P values less than0.05 was considered significant.

Fig. 2. (A)representative MSP results of NDRG2 hypermethylation in primarycolorectal cancer tumors. Case numbers are shown on top. M: methylated primers;U: unmethylated primers. (B) A map of the CpG islands in relation to the promoterof the NDRG2. The locations of sense and antisence primers used for bisulfite-sequencing PCR were indicated by underlining. The translation start site is shownby a horizontal arrow. (C) Demonstration of NDRG2 promoter methylation bysequencing of sodium bisulfite–modified DNA from the indicated colorectal cancercell lines. Black and white areas represent respectively the percentage of methyl-ated and unmethylated CpG sites out of the colonies sequenced for each case.

Table 1

3. Results

3.1. Expression profile of NDRG2 in colorectal lesions

Tissues from patients with colorectal cancer and other colorec-tal lesions were retrospectively identified from the Department ofPathology, Minhang District Central Hospital, Shanghai, China. The70 colorectal cancers comprised 40 early cases and 30 advancedcases, the clinicopathologic characteristics were analyzed accord-ing to tumor size, histological grading and presence of nodalmetastasis. NDRG2 expression in normal colorectal epitheliumand carcinomas detected by immunohistochemistry were semi-quantitated. Overall, NDRG2 was absent in 43 of 70 carcinomas(61.43%) and 6 of 70 (8.56%) in normal colorectal samples. Repre-sentative examples of NDRG2 protein expression in colorectal can-cer samples are shown in Fig. 1.

Clinical characteristics of colorectal cancer patients according to hypermethylationstatus of NDRG2.

Group NDRG2 methylation p value

U M

Normal tissues 59 11Cancer tissues 25 45 p < 0.001Differentiation Well 16 10

Moderate 3 15Poor 6 20 p = 0.002Yes 19 21

Metastasis No 6 24 p = 0.017<4 11 25

Size (cm) P4 14 20 p = 0.354NDRG2 expression Yes 20 7

3.2. Promoter methylation status in colorectal carcinomas

To elucidate the mechanism of NDRG2 down-regulation in colo-rectal carcinoma, we examined the methylation status of the pro-moter region using MSP (see Fig. 2A). We found that in 45 of the 70(64.28%) colorectal carcinomas analyzed, the NDRG2 promoter washypermethylated. 5 of the 25 unmethylated carcinoma samples(20%) demonstrated positive cytoplasmic staining and 38 of 45methylated carcinoma samples (84.44%) showed loss of expressionof NDRG2. Thus, the immunostaining results were strongly corre-lated (p < 0.001) with NDRG2 methylation status (Table 1).

No 5 38 p < 0.001

aStatistically significant when compared with the normal tissues.

3.3. NDRG2 expression could be restored with 5’-Aza-dC treatment incolorectal cancer cell lines

The area of the CpG-rich region around the transcription initia-tion site of NDRG2 gene between the nucleotides �426 and �100which spanned 16 CpG site was sequenced (Fig. 2B). As shown inFig. 2C, most CpG dinucleotides were methylated in colorectal can-cer cell lines. To confirm that CpG methylation is indeed responsi-ble for the silencing of NDRG2, we treated these heavilymethylated and silenced cell lines with DAC, a methyltransferaseinhibitor. NDRG2 expression was markedly induced after the treat-ment in all the cell lines (Fig. 3A). Bisulfite DNA sequencing of the

Fig. 1. Immunohistochemical staining for NDRG2 with anti-NDRG2 in the cancerous andNDRG2 expression graded according to the number of stained cells and the staining int

colorectal carcinoma cell lines confirmed the promoter methyla-tion status with or without treatment of DAC (Fig. 3B and C.).

3.4. Hypermethylation of NDRG2 is associated with colorectal cancerdifferentiation and metastasis

To characterize the correlation between hypermethylation ofNDRG2 promoter and clinical features of colorectal cancer, several

normal tissues. The nuclei were countered stained with hematoxylin. The scores ofensity of staining were shown on the top of the pictures.

Fig. 3. (A) NDRG2 mRNA expression levels in four colorectal cancer cell lines weredetected by semi-quantitative RT-PCR. Four colorectal cancer cell lines (SW480,HT29, SW620, LOVO) were treated with DAC for 48 h. GADPH was co amplified asan internal control. (B) Demethylation function of DAC was confirmed by MSPassay. (C) An illustrative fragment of the sequencing electropherogram is shown forLOVO cells treated with or without DAC, the CpG sites are underlined.

Fig. 4. Inverse relationship between expression of NDRG2 and miR-650. (A)Representative nucleotide sequence matches between possible target genes andmiRNAs. Putative binding site of miR-650 in NDRG2 30UTR region (as detected byTargetScan). Only matched nucleotides with miRNA seed sequences are indicatedwith vertical lines, the arrows indicate mutant sites. (B) Comparison of miR-650expression (x axis) to NDRG2 mRNA expression (y axis) in the colorectal cancersamples. Inverse correlation was also obtained by Spearman’s correlation, r = �0.69,P < 0.001. (C) Relative levels of NDRG2 protein expression in SW480 after transient

L. Feng et al. / Biochemical and Biophysical Research Communications 406 (2011) 534–538 537

clinicopathological characteristics including age, gender, grosstype, cell differentiation, tumor size and nodal metastases werecompared between patients with normal and hypermethylationof NDRG2 promoter (Table 1). The result found that hypermethyla-tion NDRG2 promoter was not associated with tumor size(p = 0.354), gross type of colorectal cancer (p = 0.719), patient’sage and gender (data not shown). However, hypermethylation ofNDRG2 was significantly correlated with nodal metastasis(p = 0.017) and poor differentiation (p = 0.002).

transfection with the miR-650 precursor molecule, negative control (mock), anti-miR-650 and DAC plus anti-miR-650. Actin was used as internal control. (D) Thefirefly luciferase reporter activity is significantly reduced in NDRG-UTR vectorcompared with NDRG-UTR-Mut (P < 0.001). The data were normalized to Renillaluciferase activities. Values are expressed as the mean ± SD. of three replicateexperiments.

3.5. Inverse relationship between expression of NDRG2 and miR-650

MicroRNAs (miRNAs) are noncoding RNAs that can regulate theexpression of protein-coding genes at post-transcriptional levelthrough imperfect base-pairing with the 30UTR of target mRNAs.Growing evidence now suggests that miRNAs regulate a widerange of biological processes, including those linked to cancer.Using computational and expression analyses, NDRG2 was identi-fied as a candidate target of miR-650 (Fig. 4A). To test this hypoth-esis, we performed semi-quantitative PCR using 30 paired samplesabove-mentioned. As shown in Fig. 4B, downregulation of NDRG2was inversely associated with the up-regulation of miR-650 incolorectal cancer. To confirm the inverse relationship betweenNDRG2 and miR-650, we transiently trasfected the miR-650 mim-ics, negative control (mock), anti-miR-650 and DAC plus anti-miR-650 into the SW480 cells. As expected, the exogenousexpression of miR-650 results in reduction of NDRG2 protein. Cellstransfected with the negative control did not exhibit any change inNDRG2 levels. Down-regulation of endogenous miR-650 with anti-miR-650 or DAC plus anti-miR-650 led to a significant increase inNDRG2 protein expression in SW480 cells (Fig. 4C).

To validate whether miR-650 directly recognizes the 30UTRs ofNDRG2 mRNA or not, we cloned a sequence with the predicted tar-get sites of miR-650 or a mutated sequence with the predicted tar-get sites to downstream of the pMIR luciferase reporter gene.When the wild-type or mutation-type vector was transfected withmiR-650, the luciferase activity of wild-type vector was signifi-cantly decreased (P < 0.05) compared with mutation-type vector(Fig. 4D). While the wild-type or mutation-type vector was trans-fected with negative control miRNA, there was no significant dif-ference between the wild-type or mutation-type vector. Thesedata suggest that miR-650 may play a major role in the regulationof NDRG2.

4. Discussion

NDRG2 is down-regulated or undetectable in many humancancers, such as, pancreatic, breast and liver cancers as well as

538 L. Feng et al. / Biochemical and Biophysical Research Communications 406 (2011) 534–538

colorectal cancer and high-risk adnomas [3,4]. To determinewhether NDRG2 is aberrantly expressed in colorectal cancer, weconducted tissue microarray analysis. In concert with the findingsobtained by other groups, our results showed that NDRG2 wasdown-regulated in colorectal cancer compared to benign colorectaltissues. In colorectal cancer, a growing number of genes have beenidentified as undergoing aberrant promoter hypermethylation,suggesting that promoter hypermethylation is an important mech-anism involved in colorectal cancer [13]. It has been demonstratedthat hypermethylation in CpG-rich promoter region is stronglyassociated with transcriptional silencing [14]. To further elucidatethe molecular mechanisms underlying up-regulation of NDRG2 incolorectal cancers, we analyzed the methylation status of NDRG2promoter. We first analyzed NDRG2 CpG island hypermethylationin 70 patients with primary colorectal cancer by use of MSP. Weobserved that NDRG2 CpG island hypermethylation was a commonevent in colorectal cancer tissues.

Sequencing of the CpG islands of the sodium bisulfite-treatedDNA revealed that most of these CpG dinucleotides in the NDRG2promoter were methylated in colorectal cancer cell lines. Treat-ment of colorectal cancer cells with a demethylating agent 5-aza-dC leads to reexpression of NDRG2 gene in colorectal cancercells, as assessed by semi-quantitative RT-PCR. Demethylation treat-ment with 5-aza-dC up-regulated the gene expression in those celllines with low NDRG2 gene expression, confirming that hyperme-thylation is important for the down-regulation of NDRG2 geneexpression, which has also been reported by other investigators.

Promoter hypermethylation of some genes is significantlylinked to pathological or clinical parameters. There was a signifi-cant correlation between hypermethylation of NDRG2 and unfa-vorable variables, including nodal metastasis, advanced colorectalcancer. However, there was no significant correlation with otherparameter, such as tumor differentiation, patient age.

MicroRNAs (miRNAs) are highly conserved, small noncodingRNAs that can downregulate various gene products by transla-tional repression when partially complementary sequences arepresent in the 30 untranslated regions (3’-UTR) of the target mRNAsor by directing mRNA degradation. Growing evidence now sug-gests that miRNAs regulate a wide range of biological processes,including those linked to cancer [15–17]. Using computationaland expression analyses, NDRG2 was identified as a candidate tar-get of miR-650. It is reported that miR-650 is involved in lymphaticand distant metastasis in human colorectal cancer, and ectopicexpression of miR-650 promotes tumorigenesis and proliferationof colorectal cancer cells, at least partially through directly target-ing ING4 [18]. To test whether the NDRG2 is a direct target of miR-650, we performed semi-quantitative PCR using 30 paired samplesabove-mentioned. We found that downregulation of NDRG2 wasinversely associated with the up-regulation of miR-650 in colorec-tal cancer. Transfection of miR-650 resulted in down-regulation ofNDRG2 in SW480 cells. Reporter assay with 30untranslated regionof NDRG2 cloned downstream of the luciferase gene showed re-duced luciferase activity in the presence of miR-650, providingstrong evidence that miR-650 is a direct regulator of NDRG2.

In conclusion, our data presented here clearly demonstrate thathypermethylation of promoter CpG island of NDRG2 gene and miR-650 are two of the important mechanisms by which NDRG2 geneexpression can be downregulated, although these are not the only

mechanisms of NDRG2 regulation. Although additional studies arerequired to characterize the biological significance of NDRG2 inac-tivation in colorectal tumorigenesis, our study suggests that aber-rant hypermethylation of the NDRG2 gene could be a molecularmarker for detection and treatment of human colorectal cancers.

Acknowledgment

This work was supported by The Shanghai Municipal NaturalScience Foundation (Grand no: 10ZR1426300).

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