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MiR-29c regulates the expression of miR-34c and miR-449a by targeting DNA methyltransferase 3a and 3b in nasopharyngeal carcinoma
- Man Niu†1, 2,
- Dan Gao†2,
- Qiuyuan Wen3,
- Pingpin Wei†2,
- Suming Pan2, 4,
- Cijun Shuai5,
- Huiling Ma2,
- Juanjuan Xiang1, 2,
- Zheng Li1, 2,
- Songqing Fan3,
- Guiyuan Li1, 2 and
- Shuping Peng1, 2Email author
© Niu et al. 2016
Received: 18 May 2015
Accepted: 8 March 2016
Published: 15 March 2016
Nasopharyngeal carcinoma (NPC) is prevalent in South East Asia and Southern China particularly, despite the reported 5-year survival ratio is relative higher than other deadly cancers such as liver, renal, pancreas cancer, the lethality is characterized by high metastatic potential in the early stage and high recurrence rate after radiation treatment. MicroRNA-29c was found to be down-regulated in the serum as well as in the tissue of nasopharyngeal carcinoma tissue.
In this study, we found accidentally that the transfection of pre-miR-29c or miR-29c mimics significantly increases the expression level of miR-34c and miR-449a but doesn’t affect that of miR-222 using real-time quantitative PCR in nasopharyngeal carcinoma cell lines. To explore the molecular mechanism of the regulatory role, the cells are treated with 5-Aza-2-deoxycytidine (5-Aza-CdR) treatment and the level of miR-34c and miR-449a but not miR-222 accumulated by the treatment. DNA methyltransferase 3a, 3b were down-regulated by the 5-Aza-CdR treatment with western blot and real-time quantitative PCR.
We found that pre-miR-29c or miR-29c mimics significantly increases the expression level of miR-34c and miR-449a. We further found DNA methyltransferase 3a and 3b are the target gene of miR-29c. Restoration of miR-29c in NPC cells down-regulated DNA methyltransferase 3a, 3b, but not DNA methyltransferase T1.
The regulation of miR-29c/DNMTs/miR-34c\449a is an important molecular axis of NPC development and targeting DNMTs or restoring of miR-29c might be a promising therapy strategy for the prevention of NPC.
Nasopharyngeal carcinoma (NPC) is prevalent in South East Asia and Southern China particularly. Despite the reported 5-year survival ratio is relative higher than other deadly cancer such as liver, renal, pancreas cancer, the lethality is characterized by high metastasis in the early stage and high recurrence rate after radiation treatment. Due to the secluded anatomical sites, early symptom of patients is not typical, 80 - 90 % patients with NPC are diagnosed until the late advanced stage. EB virus infection, genetic factors, environmental and diet factor are widely recognized to be associated with the etiology of NPC carcinogenesis [1, 2]. However, recent studies have found that genome-wide epigenetic modifications in tumor associated gene are also involved in this process [3–5].
Epigenetic modification refers to the changes in gene expression, but not a genetic change in the DNA sequence, and can be stably transmitted through meiosis in the process of growth and cell proliferation. Epigenetic factor has been proved to play an important role in the carcinogenesis and development of nasopharyngeal carcinoma (NPC). Detection of epigenetic modifications can serve as molecular context of NPC and it is advantageous in the prognosis of NPC. The regulation of the epigenetic modification is reversible so that different intervention measures in epigenetic aspect may be used as a novel strategy to treat NPC, as well as the development of novel NPC radiotherapy sensitizing agent and novel drugs.
MicroRNAs (miRNAs), small non-coding RNA, exist in many organisms and play a important role in the regulation of protein expression by binding the 3′-untranslated region (3-UTR) of their target mRNAs through completely or incompletely complementary seed sequences and assembled in RNA-induced silencing complex(RISC), mediating the degradation of mRNA or the blockade of the translation of encoded protein. Abnormal expression of miRNAs has been demonstrated in most tumor types including NPC [6–8]. In previous studies of our laboratory as well as other research groups, miR-29c was found to be down-regulated in the serum of NPC patients [9–12], while, the effect of miR-29c and the pathways in which miR-29c works during the development and progression of NPC are not well defined. Therefore, in this study, we investigated the biological functions and molecular mechanism of miR-29c in NPC, which may help to further elucidate the roles of miRNAs in the development of NPC and provide a novel candidate target for therapeutic strategies for NPC.
In this study, we accidently found that pre-miR-29c transfection in nasopharyngeal carcinoma increased the expression of miR-34c and miR-449a. In order to seek for the molecular mechanism of this event, we hypothesized that miR-29c down-regulated DNA methytranferases (DNMTs), which catalyze the addition of a methyl group to the cytosine residue of CpG nucleotides. In NPC tissue, the down-regulation of miR-29c leads to the high level of DNMTs, which further promote the methylation of the CpG islands of tumor suppressors such as miR-34c and miR-449a. Our experimental data showed that epigenetic modifications of miR-34c and miR-449a are affected by the DNMTs, especially DNMT3a and DNMT3b.
Cells and cell culture
Human nasopharyngeal carcinoma cell lines, HNE-1,CNE-2,C666-1 and the immortalized human nasopharyngeal epithelial cell, NP69 were described previously [13, 14]. The NPC cell lines were maintained in 1640 (Gibco, Grand Island, NY, USA), supplemented with 10 % fetal bovine serum (FBS) (Gibco, Grand Island, NY,USA) and 1 % penicillin-streptomycin-glutamine (Gibco, Grand Island, NY,USA) at 37 °C and 5 % CO2. The nasopharyngeal epithelial cell line NP69, which is immortalized with an SV40 T-antigen, was a kind gift from Professor Sai Wah Tsao of the Department of Anatomy, University of Hong Kong, China, and was maintained in keratinocyte-serum free medium (Invitrogen, Carlsbad, CA, USA) with the addition of growth factor supplements (Life Technologies, Gaithersburg, MD, USA) .
Cells were incubated with the 5-Aza-2′-deoxycytidine(5-Aza-CdR) (10 μM) (Sigma, MO, USA) for 96 h, with or without Trichostatin A(TSA) (10 μM) (Sigma, MO, USA) or TSA alone for the last 24 h.5-Aza-CdR is methylation methytranferase inhibitor, an epigenetic modifier that inhibits DNA methyltransferase activity which results in DNA demethylation (hypomethylation) and gene activation by remodeling “opening” chromatin. Genes are synergistically reactivated when the demethylation is combined with histone hyperacetylation. Trichostatin A is a histone deacetylase inhibitor.
Pre-miRNA constructs and miRNA mimics transfection
Pre-miR-29c or scramble cDNA together with restriction enzyme sites were inserted into pSuper vector (OligoEngine,WA,USA) and transformed into Ecoli JM109. The clones with positive inserts were subjected to the plasmids extraction and confirmed to be correct by DNA sequencing. Cells were seeded in 6-well dish (4*106cells/well) the day before and were transfected with scramble pSuper or pre-miR-29c/pSuper with Lipofectamine™ 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Forty-eight hours after the transfection, the expression of miR-29c, miR-34b, miR-449a was detected by real-time PCR, and the expression of DNMT3a, 3b, T1 was tested by real-time PCR and Western blotting.
Quantitative real time PCR (qRT-PCR)
Total RNA was extracted using miRNeasy Mini kit (Qiagen, Germany) according to the manufacturer’s instructions. For miRNA expression analysis, cDNA was synthesized using miScript II RT Kit (Qiagen, Germany). A PCR analysis was performed using miScript SYBR Green PCR Kit (Qiagen, Germany). Hsa-miR-29c-1 miScript Primer, Hsa-miR-34c-1 miScript Primer, Hsa-miR-222-1, Hsa-miR-449a-1 miScript Primer (Qiagen, Germany) were used and RNU6 (Qiagen, Germany) acted as an internal control. The PCR cycle parameters were as follows: 95 °C for 15 min, 39 cycles of denaturation at 95 °C for 15 s, annealing at 50 °C for 30s, and extension at 70 °C for 30s. For mRNA expression analysis, cDNA was synthesized using cDNA reverse transcription kit (Thermo Fisher Scientific, MA, USA) and a PCR analysis was performed using QuantiFast SYBR Green PCR Kit following the manufacturer’s instructions. The PCR cycle parameters were as follows: denaturation at 95 °C for 5 min, 39 cycles of denaturation at 95 °C for 10s, annealing at 60 °C for 30s, and extension at 72 °C for 30s. DNMT3a, 5′ primer (5′-CCGGA ACATT GAGA CATCT-3′) and 3′ Primer (5′-CAGCAGATGGTGCAGTAGGA-3′); DNMT3b, 5′ primer (5′-GGAGA CTCAT TGGAG GACCA; and 3′ Primer (CTCGG CTCTG ATCTT CATCC-3′); DNMT1, 5′ primer (5′-GAGCCACAGATGCTGACAAA-3′) and 3′ primer (5′-TGCCA TTAACACCACCTTCA-3′). β-actin, 5′ primer(5′-CCTATCGAGCATGGAGTGGT-3′) and 3′ Primer (5′-CTGAGGCATAGAGGGACAGC -3′), β-actin acted as internal control. These experiments were performed according to the manufacturer’s protocol of Bio-Rad CFX96 System.
Western blot analysis
Cells were harvested at the indicated time and rinsed tweic with cold PBS. Cell extracts were prepared with lysis buffer containing 50 mM Tris–HCl, pH7.5, 150 mM NaCl, 2 mM EDTA, 1%Triton, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture(Roche, USA) for 20 min on ice. Lysates were cleared by centrifugation at 14,000 rpm at 4 °C for 10 min. Supernatants were collected, and protein concentrations were determined by Pierce BCA Protein Assay (Pierce, USA). The proteins samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 10 % (wt/vol) polyacrylamide gels and transferred to nitrocellulose membrane (Millipore, USA). After blocking with 5 % non-fat dry milk for 1 h at room temperature, the membrane was incubated with the primary antibodies in 5 % non-fat dry milk overnight at 4 - 8 °C. The following antibodies were utilized: anti-DNMT3a mouse polyclonalantibody (Santa Cruz, USA), anti-DNMT3b rabbit polyclonal antibody (Anbo, USA), anti-DNMT1 rabbit polyclonal antibody (Santa Cruz, USA), anti-β-actin mouse polyclonal antibody (Abclonal, USA). Membranes were washed and incubated with horseradish peroxidase-conjugated secondary anti-mouse antibody or anti-rabbit antibody (CST, USA). After additional washes with phosphate-buffered saline, the band signals were visualized and quantified with chemiluminescence kit (AidLab, China).
Immunohistochemical staining and evaluation
The paraffin sections of NPC tissue microarray were collected from the patients of the Pathology Department of the Second Xiangya Hospital of Central South University between 2007 - 2014. The tissue slides were heated 65 °C for 1 h, and deparaffinized in xylene and rehydrated through graded alcohols (100, 90, 70 and 50 % alcohol; 5 min for each). For antigen retrieval, tissue slides were incubated in sodium citrate buffer (0.01 M, pH 6.0) for 20 min in a household Pressure cooker. After cooling to room temperature, the slides were washed in PBS (150 mM sodium chloride, 150 mM sodium phosphate, pH 7.2). The endogenous peroxidase activity was removed by incubating with 3 % hydrogen peroxide for 10 min and was blocked in normal goat serum (Maixin, China) for 30 min. The primary antibodies (anti-DNMT1, anti-DNMT3a and anti-DNMT3b) were applied at 4 °C overnight. Polymerized HRP and anti-rabbit IgG (Maixin, China) were added according to the manufacturer’s instructions. A color reaction was developed using DAB Color Developing Reagent Kit (Boster, China), and all of the slides were counterstained with hematoxylin staining kit. Negative control slides were included in the experiment. The immune histochemical staining of these sections was scored microscopically (Olympus, Japan) at × 400 magnification in all of the available tumor cells or epithelial cells meeting the typical morphological criteria by 3 pathologists using the qualitative scale that is described in the literature. The number of cells staining was scored as 0 (no staining), 1 + (<1/3 positive cells), 2 + (>1/3 and < 2/3 positive cells) and 3 + (>2/3 positive cells). The intensity of staining was scored from1 + (weak) to 3 + (strong). The immune reactive score was categorized into three groups by comprehensive evaluation of the percentage of positive cells and staining intensity.
Hsa-miR-29c is down-regulated in NPC cell lines and NPC tissues, correlated with clinical stage of NPC
MiR-29c increases the expression level of miR-34b/c and miR-449a significantly
DNMT3a and 3b are direct targets of miR-29c
Expression of DNMT3a, 3b, T1 associated with prognosis of nasopharyngeal carcinoma
Correlation between DNMT3a expression and clinicopathological characteristics of nasopharyngeal carcinoma
No. of patients (n, %)
Low Exp (n, %)
High Exp (n, %)
Correlation between DNMT3b expression and clinicopathological characteristics of nasopharyngeal carcinoma
No. of patients (n, %)
Low Exp (n, %)
High Exp (n, %)
Correlation between DNMT1 expression and clinicopathological characteristics of nasopharyngeal carcinoma
No. of patients (n, %)
Low Exp (n, %)
High Exp (n, %)
As well known, miRNAs play an important role in various cellular activities by regulating gene expression of their targets. Recent studies have shown that the expression of miRNA is regulated by epigenetic modifications by DNA methylation or histone modification. MiRNA also can be the key factor to regulate the levels of DNA methylation or histone modification which affect the expression level of other molecules. All these factors (extracellular signals, miRNAs, transcription factor, targeted gene) are the members of the vast gene expression regulatory networks. In cancer cells, the epigenetic modifications of miRNAs have been reported. Those miRNAs acting as tumor suppressor often were silenced by frequent hypermethylation or histone deacetylation. Furthermore, it shows tumor specialty. When treated with demethylating agent 5-aza-deoxycytidine (5-Aza-CdR) and histone deacetylase (4-Phenylbutyrie acid, PBA), the expression of 5 % miRNAs in bladder carcinoma cell line T24 increased by 3 folder than that of untreated. MiR-34c acts as a suppressor in many tumors. It’s down-regulated and the target genes DCBLD2, FOXN3, IKZF1, NPTN PAFAH1B1, USP10, YY1, ARHGAP1, ARHGEF3, BCL11B, C16orf5, CNTNAP1, FOXN3, FUT8, IL6R, ITGB8, ITSN1, JAG1, MLL2, NDST1,NOTCH2, NPNT, PPFIA1, PTPRM, PVRL1, SERPINE1, VCL were up-regulated in NPC . Single hyper methylation of CpG island in the promoter region of miR-34c gene repressed miR-34c expression by reducing DNA binding activities of Sp1 and promoted self-renewal and epithelial-mesenchymal transition of breast tumor-initiating cells . Differential methylation of CpG islands neighboring the miR-34c promoter inhibited the expression of miR-34c in gastric cancer cell lines and in paclitaxel-resistant gastric cancer samples. MiR-34c was down-regulated and its target microtubule-associated protein tau (MAPT) protein expression was high. Over expression of miR-34c significantly down-regulated MAPT protein expression and increased the chemo sensitivity of paclitaxel-resistant gastric cancer cells . Aberrant DNA methylation of miR-34c was correlated with a high probability of recurrence and associated with poor overall survival and disease-free survival in non-small cell lung cancer [35, 36]. MiR-449a was also found to be down-regulated in NPC . MiR-449a can directly target HDAC1 in primary lung cancer and inhibit cell growth and anchorage-independent growth . Trichostatin A (TSA) could strongly increase miR-449a levels in testicular cancer cell lines and miR-449a down-regulated the histone deacetylase Sirt1 . These studies manifest that miR-34c and miR-449a were regulated by the epigenetic factors. According to our previous data, miR-29c, miR-34c, miR-449a were down-regulated in NPC. In this study, we treated the NPC cell line HNE-1 and CNE-2 by 5-AzadC for 96 h and then found that miR-34c and miR-449a increased. MiR-34c and miR-449a were associated with cell proliferation, apoptosis, anti-tumor drug resistance and serum biomarkers of recurrence in other cancers, this new miRNA-miRNA pathway may provide a new sight on the diagnosis, treatment and prognosis of NPC.
In a summary, we found that miR-29c was further confirmed to be down-regulated in NPC cell lines HNE-1, CNE2, C666-1 and tissues, and firstly reported that restoration of miR-29c increases the expression miR-34c and miR-449a which were regulated by DNA methytranferases through epigenetic factors. Our experiments verified that DNMT-3a and -3b are the targets of miR-29c. As epigenetic regulation is reversible, the effects can be available through specific drugs such as DNMT inhibitors (5-Aza-CdR) with or without HDAC inhibitor (TSA). Another kind of strategy, oligonucleotides (synthetic miRNA oligonucleotides) can be used directly in vivo to correct the disorders in miRNA expression levels, which is expected as a new therapeutic tool for the nasopharyngeal carcinoma patients.
This work was supported by Natural Science Foundation of China(81572577, 81372366, 81000882, 81472773), Program for New Century Excellent Talents in University (NCET-12-0544), overseas, Hong Kong & Macao Scholars Collaborated Researching Fund of National Natural Science Foundation of China (81428018), 111 project(111-2-12), national 863 plan (2012AA02A206), the Fundamental Research Funds for the Central Universities (2013JSJJ046), The Hunan Province Natural Science Foundation of China(10JJ7003), the Open-End Fund for the Valuable and Precision Instruments of Central South University, and Innovation project for postgraduate of Central South University(2015zzts278).
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