Biochemical and Biophysical Research Communications
An essential role for DNA methyltransferase 3a in melanoma tumorigenesis
Introduction
Melanoma, as the most lethal form of skin cancer, is responsible for 80 percent of deaths from skin cancers; only 14 percent of patients with metastatic melanoma survive for five or more years [1].
Abnormal DNA methylation and associated silencing of tumor suppressor genes are common to many types of cancers, including malignant melanoma [2], [3]. Tumor-specific hypermethylation can be a potential second hit [4], by preferentially targeting the wild-type allele of tumor suppressor genes, when the second allele is already mutated. Previous studies have revealed dozens of tumor-related genes, which are inactivated by DNA methylation in melanoma [5], including Rassf1a[6], [7], Rarb[5], [6], Mgmt[8], Cdkn1b[9], Cdkn2a[10], and Pten[11].
DNA methylation patterns are established and maintained by the coordinated action of three Dnmts, Dnmt1, Dnmt3a, and Dnmt3b. Dnmt1 functions exclusively as a maintenance Dnmt [12], [13]. In contrast, Dnmt3a and Dnmt3b are de novo Dnmts [14]. Previous studies have shown that both Dnmt1 and Dnmt3b play important roles in cancer cell survival and tumorigenesis [15], [16]. Overexpression of Dnmt1 and Dnmt3b has been reported in various cancer cells and tissues [17], [18]. Inhibition of the expression of Dnmt1 or Dnmt3b has been reported to significantly inhibit the proliferation of cancer cells and tumorigenesis [19], [20], [21]. However, the relationship between Dnmt3a and tumorigenesis is still largely unknown.
In the current study, Dnmt3a was specifically down-regulated in a mouse B16 melanoma cell line by stable transfection of a Dnmt3a-RNAi construct. Tumor growth and metastasis were evaluated in mice receiving B16 cell transplantation. Changes in gene expression in tumors were examined using microarray analysis and real-time RT-PCR. Additionally, promoter methylation was determined by bisulfite sequencing.
Section snippets
Materials and methods
Cells. B16 cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM, supplemented with 10% FBS.
Small hairpin RNA (shRNA) RNAi. The sequence of Dnmt3a shRNA was 5′ gtgcagaaacatcgaggacTTCAAGAGAgtcctcgatgtttctgcac 3′. A non-target shRNA with the sequence 5′ gcaagtctaaccaacgcgtTTCAAGAGAacgcgttggttagacttgc 3′ was used as negative control (NC). The shRNA was cloned into the pcDNA3 vector. The CMV promoter in the vector was replaced by a U6 promoter. The
The expression of Dnmt3a was specifically reduced in B16 cell clones
In order to examine the role of Dnmt3a in malignant melanoma, a shRNA, specifically targeted to Dnmt3a was ligated into an expression vector and stably transfected into B16 melanoma cells. A non-target shRNA was also stably transfected as a negative control (NC). Real-time RT-PCR and Western blot analysis were employed to test the efficiency and specificity of the Dnmt3a-RNAi. The results showed that two stably transfected B16 clones (Clone 10 and Clone 15) had the lowest level of Dnmt3a
Conclusion
Here, we report that the down-regulation of Dnmt3a dramatically inhibited the growth and metastasis of malignant melanoma, but did not affect in vitro cell growth. The most likely mechanisms for this inhibition of tumorigenesis include reactivation of MHC genes, chemokines and the T-cell mediated tumor immune response. Our results strongly suggest that Dnmt3a plays an essential role in melanoma tumorigenesis and in immune escape, and may be a promising therapeutic target for the treatment of
Acknowledgment
We thank Dr. Kehong Zhang for critical review of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (No. 30570971) and National Key Technology R&D Program (2006BAI23B02).
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