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Epigenetic loss of the PIWI/piRNA machinery in human testicular tumorigenesis.

Ferreira HJ, Heyn H, Garcia del Muro X, Vidal A, Larriba S, Muñoz C, Villanueva A, Esteller M - Epigenetics (2013)

Bottom Line: Although most cancer research has focused in mRNA, non-coding RNAs are also an essential player in tumorigenesis.Herein we have studied the putative existence of epigenetic aberrations in the activity of PIWI proteins, an Argonaute family protein subclass, and the small regulatory PIWI-interacting RNAs (piRNAs) in testicular cancer, as the PIWI/piRNA pathway plays a critical role in male germline development.We have observed the existence of promoter CpG island hypermethylation-associated silencing of PIWIL1, PIWIL2, PIWIL4, and TDRD1 in primary seminoma and non-seminoma testicular tumors, in addition to testicular germ cell tumor cell lines.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics and Biology Program (PEBC); Bellvitge Biomedical Research Institute (IDIBELL); Barcelona, Spain; Programme in Experimental Biology and Biomedicine; Centre for Neurosciences and Cell Biology; University of Coimbra; Coimbra, Portugal.

ABSTRACT
Although most cancer research has focused in mRNA, non-coding RNAs are also an essential player in tumorigenesis. In addition to the well-recognized microRNAs, recent studies have also shown that epigenetic silencing by CpG island hypermethylation of other classes of non-coding RNAs, such as transcribed ultraconserved regions (T-UCRs) or small nucleolar RNAs (snoRNAs), also occur in human neoplasia. Herein we have studied the putative existence of epigenetic aberrations in the activity of PIWI proteins, an Argonaute family protein subclass, and the small regulatory PIWI-interacting RNAs (piRNAs) in testicular cancer, as the PIWI/piRNA pathway plays a critical role in male germline development. We have observed the existence of promoter CpG island hypermethylation-associated silencing of PIWIL1, PIWIL2, PIWIL4, and TDRD1 in primary seminoma and non-seminoma testicular tumors, in addition to testicular germ cell tumor cell lines. Most importantly, these epigenetic lesions occur in a context of piRNA downregulation and loss of DNA methylation of the LINE-1 repetitive sequences, one of the target genomic loci where the PIWI/piRNA machinery acts as a caretaker in non-transformed cells.

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Figure 3. Molecular environment of the epigenetic loss of PIWI-protein genes in primary testicular tumors: diminished piRNA expression and LINE1 hypomethylation. (A) Expression levels of the piRNAs DQ598918, DQ589977 and DQ601609 determined by quantitative reverse transcription PCR. (B) DNA methylation levels at the LINE1 sequence determined by sodium bisulfite modification coupled to pyrosequencing.
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Figure 3: Figure 3. Molecular environment of the epigenetic loss of PIWI-protein genes in primary testicular tumors: diminished piRNA expression and LINE1 hypomethylation. (A) Expression levels of the piRNAs DQ598918, DQ589977 and DQ601609 determined by quantitative reverse transcription PCR. (B) DNA methylation levels at the LINE1 sequence determined by sodium bisulfite modification coupled to pyrosequencing.

Mentions: Once we had determined the existence of promoter CpG island hypermethylation events in the described PIWI-class protein genes and the diminished expression of their corresponding transcripts in testicular tumorigenesis, both in primary tumors and cultured transformed cells, we wondered about the downstream impact of the described epigenetic inactivation. We first analyzed the expression levels of piRNAs in the same samples studied for PIWIL1, PIWIL2, PIWIL4, and TDRD1 CpG island methylation and transcription. piRNAs show a high diversity in their genomic sequences and are transcribed from a relatively small number of genomic regions called piRNAs clusters.15 After a primary RNA is generated, piRNA accumulation requires amplification by the above mentioned ping-pong mechanism involving at least two distinct Piwi proteins, a process that occurs in the cytoplasm.1,2 Herein, we randomly selected three piRNAs transcribed from different genomic loci to sample the global levels of expression of these small regulatory ncRNAs: DQ598918 (chr7:99 691 656–99 691 686), DQ589977(chr17:79 479 330–79 479 358) and DQ601609 (chr1:179 557 005–179 557 036). Using quantitative RT-PCR to study the expression levels of the three piRNAs, we found diminished expression of all of them in primary testicular tumors in comparison to normal testis (Fig. 3). The diminished expression of the piRNAs DQ598918, DQ589977 and DQ601609 occurred both in seminoma and non-seminoma tumors (Fig. 3).


Epigenetic loss of the PIWI/piRNA machinery in human testicular tumorigenesis.

Ferreira HJ, Heyn H, Garcia del Muro X, Vidal A, Larriba S, Muñoz C, Villanueva A, Esteller M - Epigenetics (2013)

Figure 3. Molecular environment of the epigenetic loss of PIWI-protein genes in primary testicular tumors: diminished piRNA expression and LINE1 hypomethylation. (A) Expression levels of the piRNAs DQ598918, DQ589977 and DQ601609 determined by quantitative reverse transcription PCR. (B) DNA methylation levels at the LINE1 sequence determined by sodium bisulfite modification coupled to pyrosequencing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928173&req=5

Figure 3: Figure 3. Molecular environment of the epigenetic loss of PIWI-protein genes in primary testicular tumors: diminished piRNA expression and LINE1 hypomethylation. (A) Expression levels of the piRNAs DQ598918, DQ589977 and DQ601609 determined by quantitative reverse transcription PCR. (B) DNA methylation levels at the LINE1 sequence determined by sodium bisulfite modification coupled to pyrosequencing.
Mentions: Once we had determined the existence of promoter CpG island hypermethylation events in the described PIWI-class protein genes and the diminished expression of their corresponding transcripts in testicular tumorigenesis, both in primary tumors and cultured transformed cells, we wondered about the downstream impact of the described epigenetic inactivation. We first analyzed the expression levels of piRNAs in the same samples studied for PIWIL1, PIWIL2, PIWIL4, and TDRD1 CpG island methylation and transcription. piRNAs show a high diversity in their genomic sequences and are transcribed from a relatively small number of genomic regions called piRNAs clusters.15 After a primary RNA is generated, piRNA accumulation requires amplification by the above mentioned ping-pong mechanism involving at least two distinct Piwi proteins, a process that occurs in the cytoplasm.1,2 Herein, we randomly selected three piRNAs transcribed from different genomic loci to sample the global levels of expression of these small regulatory ncRNAs: DQ598918 (chr7:99 691 656–99 691 686), DQ589977(chr17:79 479 330–79 479 358) and DQ601609 (chr1:179 557 005–179 557 036). Using quantitative RT-PCR to study the expression levels of the three piRNAs, we found diminished expression of all of them in primary testicular tumors in comparison to normal testis (Fig. 3). The diminished expression of the piRNAs DQ598918, DQ589977 and DQ601609 occurred both in seminoma and non-seminoma tumors (Fig. 3).

Bottom Line: Although most cancer research has focused in mRNA, non-coding RNAs are also an essential player in tumorigenesis.Herein we have studied the putative existence of epigenetic aberrations in the activity of PIWI proteins, an Argonaute family protein subclass, and the small regulatory PIWI-interacting RNAs (piRNAs) in testicular cancer, as the PIWI/piRNA pathway plays a critical role in male germline development.We have observed the existence of promoter CpG island hypermethylation-associated silencing of PIWIL1, PIWIL2, PIWIL4, and TDRD1 in primary seminoma and non-seminoma testicular tumors, in addition to testicular germ cell tumor cell lines.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics and Biology Program (PEBC); Bellvitge Biomedical Research Institute (IDIBELL); Barcelona, Spain; Programme in Experimental Biology and Biomedicine; Centre for Neurosciences and Cell Biology; University of Coimbra; Coimbra, Portugal.

ABSTRACT
Although most cancer research has focused in mRNA, non-coding RNAs are also an essential player in tumorigenesis. In addition to the well-recognized microRNAs, recent studies have also shown that epigenetic silencing by CpG island hypermethylation of other classes of non-coding RNAs, such as transcribed ultraconserved regions (T-UCRs) or small nucleolar RNAs (snoRNAs), also occur in human neoplasia. Herein we have studied the putative existence of epigenetic aberrations in the activity of PIWI proteins, an Argonaute family protein subclass, and the small regulatory PIWI-interacting RNAs (piRNAs) in testicular cancer, as the PIWI/piRNA pathway plays a critical role in male germline development. We have observed the existence of promoter CpG island hypermethylation-associated silencing of PIWIL1, PIWIL2, PIWIL4, and TDRD1 in primary seminoma and non-seminoma testicular tumors, in addition to testicular germ cell tumor cell lines. Most importantly, these epigenetic lesions occur in a context of piRNA downregulation and loss of DNA methylation of the LINE-1 repetitive sequences, one of the target genomic loci where the PIWI/piRNA machinery acts as a caretaker in non-transformed cells.

Show MeSH
Related in: MedlinePlus