DNA methylation directly downregulates human cathelicidin antimicrobial peptide gene (CAMP) promoter activity

LL-37, the active product of human cathelicidin antimicrobial peptide (CAMP) has a broad spectrum of antibacterial activity. LL-37 also has important physiological functions in immune regulation, angiogenesis and in modulating apoptosis. The roles of LL-37 in oral squamous cell carcinoma (OSCC) are still not clear. The correlation between DNA methylation and human CAMP expression is also unknown. Here human CAMP/LL-37 expression was assessed by immunohistochemistry in normal and OSCC tissues. The results indicated that low expression of CAMP/LL-37 correlated with histological differentiation and lymph node metastasis and also promoted tumor progression. A cell-specific methylation pattern in the promoter region of human CAMP was detected. Treatment with 5-aza-2′-deoxycytidine, a DNA demethylation reagent can increase human CAMP expression in epithelial cancer cells. The reporter assay showed that unmethylated human CAMP promoter activity was significantly higher than methylated promoter activity. Taken together, these results suggested that human CAMP/LL-37 might act as a tumor-suppressor in OSCC and DNA methylation might play roles during carcinogenesis via directly downregulating human CAMP promoter activity.


INTRODUCTION
Keratinocytes are the first mucosal epithelial cells to provide barrier protection against both oral and ingested enteric pathogens [1].Antimicrobial peptides (AMPs) within keratinocytes also appear to increase resistance to bacterial invasion [1,2].Human cationic antibacterial peptide (CAMP, also called hCAP18) is the only cathelicidin in humans, and is primarily found in the secondary granules of neutrophils [3,4].The C-terminal end of this protein also contains a 37-amino acid-long peptide (LL-37) with a broad-spectrum antibacterial activity [4,5].Besides its broad antimicrobial activity, LL-37 also plays roles in stimulation and modulation of cytokine release from different immune cells [3,4].Recently, Overexpression of LL-37 was found to promote development and progression of ovarian, lung and breast cancers, and to suppress tumorigenesis in colon and gastric cancer [6,7].However, the roles of LL-37 in oral squamous cell carcinoma (OSCC) are not clear.
Gene expression can be regulated epigenetically by DNA methylation, histone variants and modifications and nucleosome positioning [12].DNA methylation at CpG islands is associated with gene silencing [13,14].In recent years it has been shown that DNA methylation at CpG sites in non-CpG island promoters can also contribute to regulate gene expression [12,[15][16][17][18]. Studies on transcriptional regulation of human CAMP have been widely reported [3][4][5][6][7][8][9][10][11].However, detailed mechanism about how DNA methylation regulates human CAMP expression remains unclear.The purpose of this study is to explore roles of CAMP/LL-37 in OSCC and regulatory roles of DNA methylation in human CAMP promoter region.

Expression of CAMP/LL-37 and Ki-67 in OSCC tissues
To determine the cell proliferation activity and its association with CAMP expression we first compared the expression of CAMP/LL-37 and cellular proliferative activity marker Ki-67 in 70 OSCC and 20 normal tissues by immunohistochemistry.The results showed that CAMP/LL-37 is strongly expressed in normal oral mucosa (Figure 1A).However, it is weakly expressed in OSCC, especially in poorly-differentiated OSCC (Figure 1A).Ki-67 was only sparsely distributed in basal and parabasal layers in normal oral mucosa.However, Ki-67 is strongly expressed in OSCC (Figure 1A), while the low or high CAMP expression cases were 52/70 (74.3%) and 18/70 (25.7%), the low or high Ki-67 expression cases were 23/70 (32.8%) and 47/70 (67.2%), respectively (Figure 1A and Table 1).Moreover, among 52 OSCC with low CAMP/ LL-37 expression, 43 cases showed high expression of Ki67, whereas 18 cases showed high expression of CAMP/LL-37, and 14 cases showed low expression of Ki67 (Table 1).These results showed that CAMP/ LL-37 expression was negatively correlated with Ki-67 (Table 1).Subsequently, we examined the correlation between clinicopathological features and CAMP/LL-37 expression in OSCC.The low expression of CAMP/LL-37 was related to histological differentiation (p < 0.01) and lymph node metastasis (p < 0.01) in OSCC tissues, but was unrelated to age, gender and tumor size (Table 2).

Low expression of CAMP in epithelial cancer cells
To determine the expression levels of human CAMP in the epithelial cancer cell, HaCaT cell, TR146 cell and human oral cancer cell HSC-3, HSC-4, we then performed qRT-PCR and western blot analysis.The qRT-RCR analysis results showed that the mRNA copies of human CAMP in these epithelial cancer cell lines are barely detectable (Figure 1B).The expression levels of human CAMP are extremely low in these epithelial cancer cells compared with the positive control [TR146 cells treated with1,25(OH) 2 D 3 ].However, the expression of human CAMP proteins in these epithelial cancer cells was also detectable (Figure 1C).Among these epithelial cancer cells, the expression levels of human CAMP in HaCaT cells are relatively high (Figure 1C).

Hypermethylation of CpG sites in human CAMP promoter
The transcriptional regulation by human CAMP promoter has widely been reported [8][9][10][11].Our bioinformatics analysis using CpGplot program indicated that the human CAMP promoter is not located within CpG islands (http://www.ebi.ac.uk/Tools/seqstats/ emboss_cpgplot/).However, several CpG sites can be found at its promoter region (Figure 2A).To determine the methylation status of these CpG sites in human CAMP promoter, bisulfite sequencing was performed.Because a product size larger than 300 bp is very difficult to amplify [19], three short fragments containing CpG sites in human CAMP promoter were designed.The CAMP-F1 fragment (185 bp) has 2 CpG sites, CAMP-F2 fragment (172 bp) and CAMP-F3 fragment (179 bp) contains 5 and 2 CpG sites, respectively.These fragments were then used for amplification.As shown in Figure 2, the methylation levels of 5 CpG sites at CAMP-F2 in HaCaT and HSC-4 cells were relatively high.In contrast, the methylation levels in TR146 and HSC-3 cells were relatively low.For TR146 and HSC-3 cells, the first and the fifth CpG sites with respect to the transcriptional direction at CAMP-F2 are fully unmethylated (Figure 2E).For HSC-4 cells, the first CpG sites at CAMP-F2 are fully unmethylated and most of the fifth CpG sites are methylated (Figure 2F).In contrast, the methylation levels for the CpG sites at CAMP-F1 and CAMP-F3 of all cells were low (Figure 2).The results indicated that the cell-specific methylation pattern in the promoter region of the human CAMP may contribute to controlling its cell-specific expression.The results also suggested that the hypermethylation of CpG sites at CAMP-F2 in human CAMP promoter may participate into the inhibitory effects on the expression of human CAMP in these epithelial cancer cells.

Induction of human CAMP expression by 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment
To induce DNA demethylation, cells were incubated with 5-Aza-CdR (an inhibitor of DNA methylation) continuously for 6 days.After incubation, cells were collected for further analysis.Bisulfite sequencing (BSP) was also performed to confirm the demethylation effects of 5-Aza-CdR treatment.The results showed that 5-Aza-CdR treatment induced an obvious level of demethylation (Figure 3A).The 5-Aza-CdR treatment can significantly increase the expression of human CAMP while the induction effects are moderate in these cell lines (Figure 3B and 3C).The induction effects for mRNA copies detected by qRT-PCR were not significant which might be caused by low levels of mRNA copies of CAMP which are hard to accurately quantify in these cell lines (data not shown).These results indicated that other transcriptional regulators are also needed in these cell lines, and that DNA methylation in the promoter region of human CAMP might downregulate expression of human CAMP.

Direct downregulation of human CAMP gene promoter activity by DNA methylation
Treatment of 5-Aza-CdR can induce genomewide demethylation, therefore the induction effects observed by 5-Aza-CdR treatment might also be indirect (Figure 3).To clarify whether DNA methylation can directly downregulate human CAMP promoter activity, a pCpGfree-basic-Lucia construct devoid of CpG dinucleotides was used for luciferase reporter assay.pCpGfree-CAMP (-828/-1) was treated with M. SssI and M. HpaII methyltransferase.S-adenosylmethionine (SAM) was used as the methyl group donor for the enzymatic reaction.Plasmids of no treatment, M.SssI or SAM treatment were used as control.Methylated or unmethylated plasmids were then transfected into cells and luciferase activities were measured.The results indicated that a higher promoter activity was detected in these epithelial cancer cells transfected by unmethylated pCpGfree-CAMP (-828/-1) constructs.About 20-35% lower promoter activity was found when pCpGfree-CAMP (-828/-1) constructs treated with M.SssI (methylated constructs) were transfected into HaCaT, TR146, HSC-3 or HSC-4 cells compared with unmethylated constructs (Figure 4).In addition, cells transfected with pCpGfree-CAMP (-828/-1) constructs that were only methylated by M. HpaII methyltransferase treatment also indicated a similar downregulation of promoter activity (Figure 4), which suggests that the CpG sites for M. HpaII in human CAMP promoter are important for the regulation while scattered methylation on CpG sites may not be effective to attenuate the gene promoter activity.The results obtained here also implied that the closest CpG site (-233/-232) to the major transcriptional start site of human CAMP is important for the transcriptional regulation (Figure 4).These observations are consistent with the results that the induction of human CAMP expression by 5-Aza-CdR treatment (Figure 3).Taken together, these results strongly suggest that DNA methylation directly downregulates human CAMP promoter activity in these epithelial cancer cells.

DISCUSSION
Recently, LL-37 has been associated with tumor surveillance and anti-tumor effects [3,6,7].Meanwhile, tumor-promoting effects have also been described [3,6,7].The role of LL-37 in tumor development is tissue specific [6].The expression levels of LL-37 are increased in ovarian, lung, breast cancer and malignant melanoma cells [3,6].In contrast, the expression levels of LL-37 are decreased in cells from colon or gastric cancers [3,6].Mechanistically, LL-37 contributes to   immunity, pathophysiology and cell signaling involved in malignant tumor growth [6,20].Oral cancer is the sixth most common cause of death from cancer worldwide and the most common form of oral cancer is OSCC [21].
Little is known about the roles of LL-37 in OSCC.The immunohistochemistry results reported here indicated that the low expression cases of CAMP/LL-37 were 52/70 (74.3%) in OSCC compared with normal oral mucosa (Figure 1A).The results also showed that the low expression of CAMP/LL-37 was related to histological differentiation, lymph node metastasis, and promoted tumor progression in OSCC tissues (Table 2).These results suggested that CAMP/LL-37 might act as a tumor suppressor in OSCC.Though the mechanism of CAMP/ LL-37 downregulation in OSCC is still not fully clear, for the first time the results reported here implied that CAMP/LL-37 might be useful for the development of a novel diagnostic marker and therapeutic target for OSCC patients.
Due to the important roles of human CAMP/LL-37 in infectious diseases and tumors [1][2][3][4][5][6][7], the mechanism of transcriptional regulation of human CAMP has attracted wide attention of researchers.DNA methylation has been functionally linked to gene repression [13].It is known that the level of cytosine methylation of promoters is negatively correlated with gene expression [14,27].
Here we explored the possible roles of DNA methylation in the promoter region for human CAMP expression.The results observed suggested that DNA methylation contributed to regulation of human CAMP expression and other transcription regulators also existed (Supplementary Figure 1).It was proposed that cytosine methylation might change the spatial structure of DNA and thus might affect transcriptional regulation by changes in the affinity of transcription factors binding to DNA [27][28][29].Bioinformatics analysis using the TRANSFAC program indicated that methylation of the important CpG site at -233/-232 could affect binding of transcription factors including GR-alpha, TFII-I, c-Jun and ER-alpha to human CAMP promoter.Though we couldn't detect methylation of CpG site at -233/-232 of human CAMP promoter in selected epithelial cancer cell lines, we did detect full methylation of CpG site at -233/-232 human CAMP promoter in a colonic cancer cell line, Caco-2 cell (Supplementary Figure 2).According to results of reporter assay, we expected that the CpG site at -233/-232 would play important roles for transcription regulation of human CAMP in some specific cells, such as colonic cancer Caco-2 cells.Because hypermethylation of CpG sites at the CAMP-F2 fragment in human CAMP promoter could be detected in these oral epithelial cancer cells, TR146, HSC-3 and HSC-4 cells (Figure 2), we suggested that the transcription factors binding to these CpG sites at CAMP-F2 in human CAMP promoter might involve into the transcriptional regulation process in cancer progression for OSCC.Based on these results, we speculated that DNA methylation of these CpG sites in the promoter region might play important roles during carcinogenesis via directly downregulating human CAMP promoter activity in cancer progression for OSCC.These investigations could be further confirmed using more oral cancer cell lines to have better understanding roles of DNA methylation of these CpG sites in cancer progression for OSCC.Further studies would also be necessary to clarify functional role of CAMP/LL-37 in oral cancer cells.
The investigation represents novel mechanistic insights into the epigenetic regulation of human CAMP in oral epithelial cancer cells.Undoubtedly, the correlation established here between DNA methylation in the promoter region and human CAMP expression will aid future elucidation of the regulatory mechanism of human CAMP expression in physiological and pathological conditions.

Patients and tissue samples
A total of 70 patients (ages 28 to 80 years, 48 men and 22 women) who had undergone surgery at the Affiliated Hospital of Guilin Medical University were enrolled in this study.All patients underwent complete surgical resection between 2009 and 2015.This study was approved by the Ethical Committees of Guilin Medical University and informed consent was obtained from all patients.Histologically, 59 cases were classified as well or moderately differentiated, with 11 classified as poorly differentiated OSCC.Clinical details including age, gender, tumor size, tumor differentiation and lymph node metastasis were gathered from surgical records of the patients.Tumors from each patient were formalinized and cut into parallel 4-5 μm sections.

Immunohistochemical staining
The sections were incubated with primary monoclonal anti-LL-37 antibody (1:200; Santa Cruz Biotechnology, sc-166770, Dallas, TX).In addition, to determine the proliferative cell activity and its correlate with CAMP/LL-37 expression, we examined Ki-67 expression using anti-Ki-67 monoclonal antibody (MIB-1, Dako, Carpinteria, CA).The sections were incubated with primary antibodies at 4˚C overnight after antigen retrieval by microwave treatment in citrate buffer (pH 6.0), detection by the avidin-biotin peroxidase complex system using an LSAB kit (Doko, Kyoto, Japan).A labeling index, percentage of CAMP/LL-37 or Ki-67 positive cells was determined by examining of at least 1000 tumor cells at 200× magnification.The expression of CAMP/LL-37 and Ki-67 were divided into high expression (more than 20% positive cells) and low expression (less than 20% positive cells).

Cell culture
Human HaCaT and TR146 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life technologies, Grand Island, NY) supplemented with 10% Fetal Bovine Serum (FBS).HSC-3 and HSC-4 cells (human oral squamous cell carcinoma) were maintained in RPMI-1640 ((Life technologies, Grand Island, NY) supplemented with 10% Fetal Bovine Serum (FBS).All cells were incubated at 37°C in a 5% CO 2 incubator.HaCaT cells were obtained from the American Type Culture Collection (ATCC, Manassas, USA).TR146 cells were provided by Dr. Xia Liu, Shandong Academy of Pharmaceutical Sciences, Jinan, P. R. China.HSC-3 and HSC-4 cells were obtained from Japanese Collection of Research Bioresources Cell Bank.

Plasmids construction
The PCR fragments of -828/-1 of human CAMP were amplified from human blood genomic DNA (Promega, Madison, WI) using primer pairs P1/P2 (Table 3) and Herculase ® II Fusion DNA Polymerase (Agilent technologies, Santa Clara, CA).Amplified fragments were inserted into the pGEM-T vector (Promega, Madison, WI) to generate plasmid pGEM-T-828, which was verified by DNA sequencing.A 845 bp fragment amplified with primer pair P3/ P4 (Table 3) was ligated into a Lucia reporter plasmid pCpGfree-basic-Lucia (Invivogen, San Diego, CA) without a promoter and devoid of CpG dinucleotides via the AvrII and BamHI restriction sites to generate vector pCpGfree-CAMP (-828/-1).Constructs were confirmed by automated sequencing (Sangon, Shanghai, China).

5-Aza-CdR treatment
5-Aza-CdR (Sigma) was dissolved in 99% ethanol to 10 mM, stored at -20°C and used at a final concentration of 0.5 μM or 1 μM [30].Treatment with 5-Aza-CdR was performed as described previously [30].Cells were seeded at a density of 2 to 3 × 10 5 cells/ml.The medium was replaced every 24 h for 6 days with fresh medium containing 0.5 μM or 1 μM 5-Aza-CdR, and then cells were collected for further analysis.

CpG methyltransferase treatment and luciferase reporter assay
The plasmid pCpGfree-CAMP (-828/-1) was treated with methyltransferase M. SssI or M. HpaII.Methylation was confirmed by treatment of restriction enzyme HpaII.Cells were seeded in 6-well plates.At approximately 60-90% confluence, cells were transfected with plasmids methylated or unmethylated using Lipofectamine 3000 (Life technologies) according to the manufacturer's recommendations.Briefly, 2.5 μg of plasmid DNA and 5 μl P3000 reagent was diluted in 125 μl Opti-MEM and then were added into 125 μl Opti-MEM with 5 μl Liofectamine 3000 reagent.After 5 min of incubation, the mixture was added into each well.After 24 h, medium was aspirated and replaced with 800μl fresh medium.48 h after transfection, medium was collected and 20 μl aliquots were added into 96 well plates.100 μl Quanti-Luc TM (Invivogen) was injected into each well and luciferase activities were read.

Western blot analysis
Cells were washed with Dulbecco's phosphatebuffered saline (DPBS, Gibco BRL, Life Technologies) and proteins were extracted using mammalian cell lysate buffer (Biyuntian, Beijing, China).Samples were then centrifuged

Statistical analysis
Generally, statistical analysis was performed with SPSS 19.0 (IBM, Armonk, NY).Comparisons between two groups were done with Student's t test, and multiple comparisons were done with one-way ANOVA followed by Bonferroni's post hoc test.For immunohistochemical staining analysis, the χ2 ant T test was used for comparison of the data between two groups.A p value less than 0.05 was considered to indicate statistical significance.

Figure 1 :
Figure 1: Expression analysis of human CAMP/LL-37 in normal oral mucosa, OSCC tissues and epithelial cancer cells.(A) Expression of human CAMP/LL-37 was examined by immunohistochemistry. Representative images of CAMP/LL-37 and Ki-67 in normal oral mucosa, well differentiated adenocarcinoma and poorly differentiated adenocarcinoma cases.(B) Human CAMP mRNA copies in cancer cells were detected by qRT-PCR, normalized to β-actin (ACTB) mRNA.TR146 cells treated with 1,25(OH) 2 D 3 as described in materials and methods were used as positive control.+VD3, 1,25(OH) 2 D 3 treatment.(C) Human CAMP protein expression in epithelial cancer cells was detected using western blot analysis.Intensity levels of each band are normalized to β-actin.The error bars show the means ± SE of three to six independent experiments.

Figure 2 :
Figure 2: Cell specific methylation pattern for human CAMP.Bisulfite sequencing of CAMP-F1 fragment with 2 CpG sites (185 bp), CAMP-F2 fragment with 5 CpG sites (172 bp) and CAMP-F3 fragment with 2 CpG sites (179 bp) in human blood cells and epithelial cancer cells.At least eight independent clones were sequenced.Circles represent different CpG sites.Open and closed circles indicate unmethylated and methylated CpGs, respectively.(A) A schematic illustration of the human CAMP promoter.The major transcriptional start site is indicated by the arrow at position +1.Upper tick marks indicate individual CpG sites in human CAMP promoter.(B) Methylation pattern in human peripheral blood cells.(C) Methylation pattern in HaCaT cells.(D) Methylation pattern in TR146 cells.(E) Methylation pattern in HSC-3 cells.(F) Methylation pattern in HSC-4 cells.

Figure 3 :
Figure 3: Induction of human CAMP expression by 5-Aza-CdR treatment.The expression levels of human CAMP protein were determined by western blot analysis.(A) The DNA methylation levels of CpG sites at CAMP-F2 in human CAMP promoter for the untreated cells and cells subjected to 5-Aza-CdR treatment were determined by bisulfite sequencing.At least five independent clones were sequenced each time.(B) Human CAMP protein was detected after 5-Aza-CdR treatment in epithelial cancer cells.(C) The expression levels of human CAMP protein were quantified after 5-Aza-CdR treatment.Intensity levels of each band are normalized to β-actin.The error bars show the means ± SE of three to six independent experiments.*Significantly increased compared to vehicle control (p < 0.05).

Figure 4 :
Figure 4: Direct downregulation of human CAMP promoter activity by DNA methylation.(A) A schematic illustration of the human CAMP promoter.The major transcriptional start site is indicated by the arrow at position +1.Upper tick marks indicate individual CpG sites in human CAMP promoter.The symbols of '#' represent HpaII sites.(B) Luciferase activities of unmethylated and methylated human CAMP promoter constructs in epithelial cancer cells.The error bars show the means ± SE of three to six independent experiments.SAM, S-adenosylmethionine. NS, not significant.*Significantly decreased compared to untreated construct control (p < 0.05).

Table 2 : Human CAMP/LL-37 expression and its correlation with clinicopathological features in OSCC
a W/M, Well or Moderately-differentiated OSCC.
a Regions of oligonucleotide not derived from human CAMP are underlined.b