Hypomethylation at H19DMR in penile squamous cell carcinoma is not related to HPV infection.

Penile squamous cell carcinoma (SCC) is a rare and aggressive tumour mainly related to lifestyle behaviour and human papillomavirus (HPV) infection. Environmentally induced loss of imprinting (LOI) at the H19 differentially methylated region (H19DMR) is associated with many cancers in the early events of tumorigenesis and may be involved in the pathogenesis of penile SCC. We sought to evaluate the DNA methylation pattern at H19DMR and its association with HPV infection in men with penile SCC by bisulfite sequencing (bis-seq). We observed an average methylation of 32.2% ± 11.6% at the H19DMR of penile SCC and did not observe an association between the p16INK4a+ (p = 0.59) and high-risk HPV+ (p = 0.338) markers with methylation level. The average methylation did not change according to HPV positive for p16INK4a+ or hrHPV+ (35.4% ± 10%) and negative for both markers (32.4% ± 10.1%) groups. As the region analysed has a binding site for the CTCF protein, the hypomethylation at the surrounding CpG sites might alter its insulator function. In addition, there was a positive correlation between intense polymorphonuclear cell infiltration and hypomethylation at H19DMR (p = 0.035). Here, we report that hypomethylation at H19DMR in penile SCC might contribute to tumour progression and aggressiveness regardless of HPV infection.

Long Non-coding RNAs and CRISPR-Cas Edition in Tumorigenesis.

Long non-coding RNAs (lncRNAs) are one of the most abundant and heterogeneous transcripts with key roles in chromatin remodeling and gene regulation at the transcriptional and post-transcriptional levels. Due to their role in cell growth and differentiation, lncRNAs have emerged as an important biomarker in cancer diagnosis, prognosis, and targeted treatment. Recent studies have focused on elucidating lncRNA function during malignant transformation, tumor progression and drug resistance. The advent of the CRISPR system has made it possible to precisely edit complex genomic loci such as lncRNAs. Thus, we summarized the advances in CRISPR-Cas approaches for functional studies of lncRNAs including gene knockout, knockdown, overexpression and RNA targeting in tumorigenesis and drug resistance. Additionally, we highlighted the perspectives and potential applications of CRISPR approaches to treat cancer, as an emerging and promising target therapy.

CRISPR/Cas9 small promoter deletion in H19 lncRNA is associated with altered cell morphology and proliferation.

The imprinted H19 long non-coding RNA, a knowing oncofetal gene, presents a controversial role during the carcinogenesis process since its tumor suppressor or oncogenic activity is not completely elucidated. Since H19 lncRNA is involved in many biological pathways related to tumorigenesis, we sought to develop a non-cancer lineage with CRISPR-Cas9-mediated H19 knockdown (H19-) and observe the changes in a cellular context. To edit the promoter region of H19, two RNA guides were designed, and the murine C2C12 myoblast cells were transfected. H19 deletion was determined by DNA sequencing and gene expression by qPCR. We observed a small deletion (~ 60 bp) in the promoter region that presented four predicted transcription binding sites. The deletion reduced H19 expression (30%) and resulted in increased proliferative activity, altered morphological patterns including cell size and intracellular granularity, without changes in viability. The increased proliferation rate in the H19- cell seems to facilitate chromosomal abnormalities. The H19- myoblast presented characteristics similar to cancer cells, therefore the H19 lncRNA may be an important gene during the initiation of the tumorigenic process. Due to CRISPR/Cas9 permanent edition, the C2C12 H19- knockdown cells allows functional studies of H19 roles in tumorigenesis, prognosis, metastases, as well as drug resistance and targeted therapy.