Examination of Methylation Sites for Forensic Age Determination from Semen

Methylation Sensitive High-Resolution Melt (MS-HRM) is based on quantitating the melt curve from an experimental sample against a standard of known methylation levels. Whereas most applications of age prediction using methylation markers are based upon pyrosequencing or SNaPshot technologies, these analysis methods are both cost and instrumentation prohibitive. This study sought to use to the varied methylation status of the ELOVL2 and FHL2 alleles, both having known correlation with age (Hamano et. al.), in a labor and time efficient manner to develop an age prediction model. A non-linear regression and standard curve was compiled from the methylation status in a sample (n=7) of extracted semen samples and compared to chronological age. The methylation status of ELVOL2 and FHL2 from each sample was obtained, with the conclusion that no correlation in methylation percentage and biological age existed for this sample of individuals aged 20-33. The principal objective of this study, to expand the application of MS-HRM age prediction from blood to other body fluids, will need further testing using larger sample sizes and broader age ranges prior to application in forensic casework.https://scholarscompass.vcu.edu/uresposters/1281/thumbnail.jp

miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis

Identification of dysregulated microRNAs (miRNAs) in prostate cancer is critical not only for diagnosis, but also differentiation between the aggressive and indolent forms of the disease. miR-9 was identified as an oncomiR through both miRNA panel RT-qPCR as well as high-throughput sequencing analysis of the human P69 prostate cell line as compared to its highly tumorigenic and metastatic subline M12, and found to be consistently upregulated in other prostate cell lines including DU-145 and PC3. While miR-9 has been characterized as dysregulated either as an oncomiR or tumour suppressor in a variety of other cancers including breast, ovarian, and nasopharyngeal carcinomas, it has not been previously evaluated and proven as an oncomiR in prostate cancer. miR-9 was confirmed an oncomiR when found to be overexpressed in tumour tissue as compared to adjacent benign glandular epithelium through laser-capture microdissection of radical prostatectomy biopsies. Inhibition of miR-9 resulted in reduced migratory and invasive potential of the M12 cell line, and reduced tumour growth and metastases in male athymic nude mice. Analysis showed that miR-9 targets e-cadherin and suppressor of cytokine signalling 5 (SOCS5), but not NF-ĸB mRNA. Expression of these proteins was shown to be affected by modulation in expression of miR-9

Optical Tweezers as an Effective Tool for Spermatozoa Isolation from Mixed Forensic Samples

A single focus optical tweezer is formed when a laser beam is launched through a high numerical aperture immersion objective. This objective focuses the beam down to a diffraction-limited spot, which creates an optical trap where cells suspended in aqueous solutions can be held fixed. Spermatozoa, an often probative cell type in forensic investigations, can be captured inside this optical trap and dragged one by one across millimeter-length distances in order to create a cluster of cells which can be subsequently drawn up into a capillary for collection. Sperm cells are then ejected onto a sterile cover slip, counted, and transferred to a tube for DNA analysis workflow. The objective of this research was to optimize sperm cell collection for maximum DNA yield, and to determine the number of trapped sperm cells necessary to produce a full STR profile. A varying number of sperm cells from both a single-source semen sample and a mock sexual assault sample were isolated utilizing optical tweezers and processed using conventional STR analysis methods. Results demonstrated that approximately 50 trapped spermatozoa were required to obtain a consistently full DNA profile. A complete, single-source DNA profile was also achieved by isolating sperm cells via optical trapping from a mixture of sperm and vaginal epithelial cells. Based on these results, optical tweezers are a viable option for forensic applications such as separation of mixed populations of cells in forensic evidence

Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3’-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression

The stability and persistence of blood and semen mRNA and miRNA targets for body fluid identification in environmentally challenged and laundered samples

The identification of body fluids in evidentiary stains may provide investigators with probative information during an investigation. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to detect the presence of mRNA and miRNA in fresh and environmentally challenged samples. Blood, semen, and reference markers were chosen for both mRNA/miRNA testing. Samples of blood and semen were exposed to heat, humidity, and sunlight, and controlled conditions (room temperature, low humidity, and darkness) for 6 months. All mRNA targets were observed through six months under controlled conditions, but were undetected after 30 days in experimental conditions. However, miRNA targets persisted under all test conditions for the duration of the study. Additionally, cotton stained with blood or semen was laundered using a liquid detergent in various washing and drying conditions. An unstained cutting was evaluated for potential transfer. Both miRNA targets were observed in all stained samples regardless of the wash protocol used. Of the mRNA markers, HBB was detected in all bloodstained samples and PRM1 persisted in all but one semen stained sample. The unstained samples showed transfer of at least one body fluid specific miRNA marker in all samples and at least one body fluid specific mRNA in approximately half of the samples. These results support that RNA markers can be used for body fluid identification in challenging samples, and that miRNA markers may be more persistent than mRNA for blood and semen stains. However, some caution is warranted with laundered items due to possible transfer

Infrared Laser Heating Applied to Nanopore Sensing for DNA Duplex Analysis

Temperature studies coupled with resistive-pulse nanopore sensing enable the quantification of a variety of important thermodynamic properties at the single-molecule limit. Previous demonstrations of nanopore sensing with temperature control have utilized bulk chamber heating methodologies. This approach makes it difficult to rapidly change temperatures and enable optical access for other analytical techniques (i.e., single-molecule fluorescence). To address these issues, researchers have explored laser-based methodologies through either direct infrared (IR) absorption or plasmonic assisted heating. In this paper, we demonstrate the use of IR-based direct absorption heating with the DNA sensing capabilities of a biological nanopore. The IR heating enables rapid changes of the temperature in and around an α-hemolysin pore, and we use this to explore melting properties for short (≤50 bp) double-stranded DNA homopolymers. We also demonstrate that the IR heating enables one to measure the percentage of different-sized DNA molecules in a binary mixture

While protein levels of SOCS5 are increased upon miR-9 inhibition, messenger RNA levels are not significantly different.

<p>SOCS5 expression in the P69, M12, and M12 stably transformed with vector expressing miR-9 inhibitor. A: messenger RNA relative quantitation shows that mRNA levels are not significantly impacted by miR-9. mRNA is normalized to GAPDH and reported as relative to the M12 cell line; results are compiled data from three independent lysates, each performed in triplicate. Regardless of similar SOCS5 mRNA levels, (B) Western blot analysis and (C) quantitation show that miR-9 inhibition results in increased levels of SOCS5. Blot is representative of 6 independent experiments, Quantitation the average of 3 independent experiments normalized to β-Actin and reported as relative to the M12 cell line (p<0.05). (D) The proven SOCS5 mRNA:miR-9 hybrid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159601#pone.0159601.ref021" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159601#pone.0159601.ref022" target="_blank">22</a>]. miR-9 is the top sequence, and hybridized below is the complementary region of the 3’-UTR of SOCS5. (E) SOCS5 expression is suppressed by miR-9. M12 cells were transiently transfected with a dual luciferase reporter construct containing a portion of the SOCS5 3’-UTR, with the wild type or mutated miR-9 binding seed region. Firefly luciferase expression is reported as normalized to renilla luciferase activity and relative to mutated seed region expression. Results are the mean of 2 independent experiments, each performed in triplicate. (p = .016).</p

Tumour growth and metastasis is reduced upon miR-9 inhibition.

<p>A: Subcutaneous injections into the flank of the respective cell lines into nude athymic mice. Tumour reduction was significantly reduced in mice injected with M12 cells transformed with miR-9 inhibitor (p <.0001). Results are reported as the average ratio of tumour volume to the final average M12 tumour volume. N = 5 mice for each treatment. B: Intraprostatic (IP) injection into nude athymic mice resulted in 0 metastatic lesions when miR-9 was inhibited, as compared to 7 lesions in the mouse injected with M12 cells alone.</p

messenger RNA and protein levels of CDH1 are increased upon miR-9 inhibition.

<p>A: RT-qPCR analysis shows that mRNA levels are impacted by miR-9, and miR-9 inhibition relieves mRNA and protein levels (p<0.03 for P69 and miR-9 inhibited lines vs. M12). mRNA is normalized to GAPDH and reported as relative to the M12 cell line using the comparative C_T method. Results are compiled data from two biological replicates, each performed in triplicate. B: Proven binding site for miR-9 in e-cadherin. Adapted from RNAHybrid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159601#pone.0159601.ref021" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159601#pone.0159601.ref022" target="_blank">22</a>] analysis of CDH1 mRNA (NM_004360.3). miR-9 is the left sequence, and hybridized on the right is the complementary region of the 3’-UTR of CDH1. C: Western blot analysis and D: quantitation. Blot is representative of 5 independent experiments, Quantitation the average of 3 independent experiments normalized to β-Actin and reported as relative to the M12 cell line (p<0.05). (E) e-cadherin expression is suppressed by miR-9. M12 cells were transiently transfected with a firefly luciferase reporter construct containing a portion of the CDH1 3’-UTR, with the wild type or mutated miR-9 binding seed region along with a renilla luciferase plasmid. Firefly luciferase expression is reported as normalized to renilla luciferase activity and relative to mutated seed region expression. Results are the mean of 2 independent experiments, each performed in triplicate. (p <0.01). Western blot analysis shows that NF-kB levels do not change significantly between M12 or M12 cells stably transfected with miR-9 inhibitor. Blot (F) is representative of 3 independent experiments and quantitation (G) is from one representative experiment.</p