Assessment of piRNA biogenesis and function in testicular germ cell tumors and their precursor germ cell neoplasia in situ

Abstract

Background: Aberrant overexpression of PIWI/piRNA pathway proteins is shown for many types of tumors. Interestingly, these proteins are downregulated in testicular germ cell tumors (TGCTs) compared to normal testis tissues. Here, we used germline and TGCT markers to assess the piRNA biogenesis and function in TGCTs and their precursor germ cell neoplasia in situ (GCNIS).

Methods: We used small RNA deep sequencing, qRT-PCR, and mining public RNAseq/small RNA-seq datasets to examine PIWI/piRNA gene expression and piRNA biogenesis at four stages of TGCT development: (i) germ cells in healthy testis tissues, (ii) germ cells in testis tissues adjacent to TGCTs, (iii) GCNIS cells and (iv) TGCT cells. To this end, we studied three types of samples: (a) healthy testis, (b) testis tissues adjacent to two types of TGCTs (seminomas and nonseminomas) and containing both germ cells and GCNIS cells, as well as (c) matching TGCT samples.

Results: Based on our analyses of small RNA-seq data as well as the presence/absence of expression correlation between PIWI/piRNA pathway genes and germline or TGCT markers, we can suggest that piRNA biogenesis is intact in germ cells present in healthy adult testes, and adjacent to TGCTs. Conversely, GCNIS and TGCT cells were found to lack PIWI/piRNA pathway gene expression and germline-like piRNA biogenesis. However, using an in vitro cell line model, we revealed a possible role for a short PIWIL2/HILI isoform expressed in TGCTs in posttranscriptional regulation of the youngest members of LINE and SINE classes of transposable elements. Importantly, this regulation is also implemented without involvement of germline-like biogenesis of piRNAs.

Conclusions: Though further studies are warranted, these findings suggest that the conventional germline-like PIWI/piRNA pathway is lost in transition from germ cells to GCNIS cells.

Keywords: GCNIS; Germ cell cancer; Germ cell neoplasia in situ; Nonseminoma; PIWI; Seminoma; Testicular germ cell tumor; piRNA.

Fig. 1

Length distribution of small RNA library reads in normal Adult testis (dataset N3), TGCTs (datasets SE3 for seminoma and NS3 for nonseminoma) and matched adjacent GCNIS-containing testis tissues (datasets GCNIS SE3 and GCNIS NS3, respectively) as well as somatic tissue (adult brain). The rest of the datasets are presented in Additional file 2: Figure S3

Fig. 2

Number of piRNA clusters predicted from small RNA library reads using probabilistic approach (a) and number of probabilistically predicted piRNA clusters overlapping previously annotated ones (b). P-values for two-tailed Mann-Whitney test are presented (n.s. – non-significant)

Fig. 3

Knockdown of PL2L60A in TERA1 cell line leads to transcriptional upregulation of transposable elements. a, Transcription level of PL2L60A gene and the following retrotransposons: full-length L1HS/L1PA2/L1PA3s, all Alu elements, AluYa5 subfamily. b, Fraction of TE-deriving small RNAs for full-length L1HS/L1PA2/L1PA3s, all Alu elements, AluYa5 subfamily. c, Chromatin modifications (H3K4me3 and H3K9me3) around all Alu elements, AluYa5 subfamily, and 5’UTR promoter of full-length L1HS/L1PA2/L1PA3. d, DNA methylation level around 5’UTR promoter of full-length L1HS/L1PA2/L1PA3 TEs. The graphs show mean and standard deviation across three biological replicates. P-value for two-tailed Mann-Whitney test is presented (n.s. – non-significant). scRNA – control scrambled RNA, siRNA – anti-PL2L60A siRNA

Fig. 4

L1 retrotransposition assay performed with pJM101 vector on Hela (positive control) and TERA1 cell lines. The latter was treated either with siRNA for PIWIL2 or with a scrambled RNA control