Epigenetic Regulation of Claudin-1 in the Development of Ovarian Cancer Recurrence and Drug Resistance.

Over 21,000 women are diagnosed with ovarian cancer (OC) in the United States each year and over half that number succumb to this disease annually, often due to recurrent disease. A deeper understanding of the molecular events associated with recurrent disease is needed to identify potential targets. Using genome-scale DNA methylation and gene expression data for 16 matched primary-recurrent advanced stage serous epithelial OCs, we discovered that Claudin-1 (CLDN1), a tight junction protein, shows a stronger correlation between expression and methylation in recurrent versus primary OC at multiple CpG sites (R= -0.47 to -0.64 versus R= -0.32 to -0.57, respectively). An independent dataset showed that this correlation is stronger in tumors from short-term (<3y) survivors than in tumors from long-term (>7y) survivors (R= -0.41 to -0.46 versus R= 0.06 to -0.19, respectively). The presence of this inverse correlation in short-term survivors and recurrent tumors suggests an important role for this relationship and potential predictive value for disease prognosis. CLDN1 expression increased following pharmacologic inhibition of DNA methyltransferase activity (p< 0.001), thus validating the role of methylation in CLDN1 gene inhibition. CLDN1 knockdown enhanced chemosensitivity and suppressed cell proliferation, migration, and wound healing (p< 0.05). Stable CLDN1 knockdown in vivo resulted in reduced xenograft tumor growth but did not reach significance. Our results indicate that the relationship between CLDN1 methylation and expression plays an important role in OC aggressiveness and recurrence.

Urine RNA Processing in a Clinical Setting: Comparison of 3 Protocols.

OBJECTIVE: The objective of this study was to compare quantitative and qualitative RNA extraction results from clinical voided urine samples between 3 commercially available extraction protocols. METHODS: For phase 1, fresh voided urine samples from 10 female subjects were collected and processed in clinic and transported to the laboratory with cold packs. RNA was purified with 1 of 3 RNA extraction protocols: (1) TRI Reagent Protocol; (2) Absolutely RNA Nanoprep Kit; and (3) ZR Urine RNA Isolation Kit. Real-time polymerase chain reactions (RT-PCR) were performed. As the ZR Urine RNA Isolation Kit provided the highest quality RNA in phase 1, for phase 2, RNA was extracted from 9 additional voided urine specimens using this kit to perform additional qualitative analyses. RESULTS: Median RNA yield was significantly higher with the TRI Reagent Protocol as compared with the other protocols (P = 0.007). However, there was a significantly lower median threshold cycle value from polymerase chain reaction (indicating improved downstream application performance) with the ZR Urine RNA Isolation Kit as compared with the other methods (P = 0.005). In phase 2, the median RNA integrity number of urine RNA was 2.5 (range, 1.6-5.9). CONCLUSIONS: Although other methods may provide a higher quantity of RNA, when using clinical urine samples, the ZR Urine RNA Isolation Kit provided the highest quality of extracted RNA. This kit is especially attractive for the clinical setting because it does not require an initial centrifugation step. The urine RNA obtained with this kit may be useful for polymerase chain reaction but is not likely to be of high enough integrity for RNA sequencing.

Cannabinoid exposure and altered DNA methylation in rat and human sperm.

Little is known about the reproductive effects of paternal cannabis exposure. We evaluated associations between cannabis or tetrahydrocannabinol (THC) exposure and altered DNA methylation in sperm from humans and rats, respectively. DNA methylation, measured by reduced representation bisulfite sequencing, differed in the sperm of human users from non-users by at least 10% at 3,979 CpG sites. Pathway analyses indicated Hippo Signaling and Pathways in Cancer as enriched with altered genes (Bonferroni p < 0.02). These same two pathways were also enriched with genes having altered methylation in sperm from THC-exposed versus vehicle-exposed rats (p < 0.01). Data validity is supported by significant correlations between THC exposure levels in humans and methylation for 177 genes, and substantial overlap in THC target genes in rat sperm (this study) and genes previously reported as having altered methylation in the brain of rat offspring born to parents both exposed to THC during adolescence. In humans, cannabis use was also associated with significantly lower sperm concentration. Findings point to possible pre-conception paternal reproductive risks associated with cannabis use.