C-terminal domain small phosphatase 1 (CTDSP1) regulates growth factor expression and axonal regeneration in peripheral nerve tissue.

Peripheral Nerve Injury (PNI) represents a major clinical and economic burden. Despite the ability of peripheral neurons to regenerate their axons after an injury, patients are often left with motor and/or sensory disability and may develop chronic pain. Successful regeneration and target organ reinnervation require comprehensive transcriptional changes in both injured neurons and support cells located at the site of injury. The expression of most of the genes required for axon growth and guidance and for synapsis formation is repressed by a single master transcriptional regulator, the Repressor Element 1 Silencing Transcription factor (REST). Sustained increase of REST levels after injury inhibits axon regeneration and leads to chronic pain. As targeting of transcription factors is challenging, we tested whether modulation of REST activity could be achieved through knockdown of carboxy-terminal domain small phosphatase 1 (CTDSP1), the enzyme that stabilizes REST by preventing its targeting to the proteasome. To test whether knockdown of CTDSP1 promotes neurotrophic factor expression in both support cells located at the site of injury and in peripheral neurons, we transfected mesenchymal progenitor cells (MPCs), a type of support cells that are present at high concentrations at the site of injury, and dorsal root ganglion (DRG) neurons with REST or CTDSP1 specific siRNA. We quantified neurotrophic factor expression by RT-qPCR and Western blot, and brain-derived neurotrophic factor (BDNF) release in the cell culture medium by ELISA, and we measured neurite outgrowth of DRG neurons in culture. Our results show that CTDSP1 knockdown promotes neurotrophic factor expression in both DRG neurons and the support cells MPCs, and promotes DRG neuron regeneration. Therapeutics targeting CTDSP1 activity may, therefore, represent a novel epigenetic strategy to promote peripheral nerve regeneration after PNI by promoting the regenerative program repressed by injury-induced increased levels of REST in both neurons and support cells.

Characterization of traumatized muscle-derived multipotent progenitor cells from low-energy trauma.

BACKGROUND: Multipotent progenitor cells have been harvested from different human tissues, including the bone marrow, adipose tissue, and umbilical cord blood. Previously, we identified a population of mesenchymal progenitor cells (MPCs) isolated from the traumatized muscle of patients undergoing reconstructive surgery following a war-related blast injury. These cells demonstrated the ability to differentiate into multiple mesenchymal lineages. While distal radius fractures from a civilian setting have a much lower injury mechanism (low-energy trauma), we hypothesized that debrided traumatized muscle near the fracture site would contain multipotent progenitor cells with the ability to differentiate and regenerate the injured tissue. METHODS: The traumatized muscle was debrided from the pronator quadratus in patients undergoing open reduction and internal fixation for a distal radius fracture at the Walter Reed National Military Medical Center. Using a previously described protocol for the isolation of MPCs from war-related extremity injuries, cells were harvested from the low-energy traumatized muscle samples and expanded in culture. Isolated cells were characterized by flow cytometry and q-RT-PCRs and induced to adipogenic, osteogenic, and chondrogenic differentiation. Downstream analyses consisted of lineage-specific staining and q-RT-PCR. RESULTS: Cells isolated from low-energy traumatized muscle samples were CD73+, CD90+, and CD105+ that are the characteristic of adult human mesenchymal stem cells. These cells expressed high levels of the stem cell markers OCT4 and NANOG 1-day after isolation, which was dramatically reduced over-time in monolayer culture. Following induction, lineage-specific markers were demonstrated by each specific staining and confirmed by gene expression analysis, demonstrating the ability of these cells to differentiate into adipogenic, osteogenic, and chondrogenic lineages. CONCLUSIONS: Adult multipotent progenitor cells are an essential component for the success of regenerative medicine efforts. While MPCs have been isolated and characterized from severely traumatized muscle from high-energy injuries, here, we report that cells with similar characteristics and multipotential capacity have been isolated from the tissue that was exposed to low-energy, community trauma.

A microRNA Signature for Impaired Wound-Healing and Ectopic Bone Formation in Humans.

BACKGROUND: Heterotopic ossification (HO) is characterized by the abnormal growth of ectopic bone in soft tissues, frequently occurring within the military population because of extensive orthopaedic combat trauma. MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene expression. We hypothesized that a clinically relevant miRNA signature could be detected in patients following injury that progressed to form HO (HO+) or did not form HO (HO-). METHODS: Tissue samples were obtained from injured servicemembers during their initial surgical debridements, and miRNA profiling was performed using a real-time miRNA polymerase chain reaction (PCR) array. Primary mesenchymal progenitor cells (MPCs) were harvested from debrided traumatized human muscle tissue, and cells were isolated and cultured in vitro. Mimic miRNAs were transfected into MPCs, followed by downstream in vitro analyses. RESULTS: The investigation of the miRNA expression profile in the tissue of HO+ compared with HO- patients demonstrated a molecular signature that included the upregulation of miR-1, miR-133a, miR-133b, miR-206, miR-26a, and miR-125b. Transfection of each of these mature miRNAs into MPCs followed by osteogenic induction demonstrated that miR-1, miR-133a, miR-133b, and miR-206 enhanced osteogenic differentiation compared with control treatments. In silico and in vitro analyses identified the transcription factor SOX9 as a candidate downstream target of miR-1 and miR-206 miRNAs. CONCLUSIONS: Our data demonstrated a molecular signature of miRNAs in the soft tissue of wounded servicemembers that was associated with the development of HO, providing novel insights into the underlying molecular mechanisms associated with posttraumatic HO. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Circulatory miR-628-5p is downregulated in prostate cancer patients.

Prostate cancer (PCa) is a major health concern for men in the USA. Aberrant expression of microRNAs (miRNAs) has been associated with the pathogenesis of various cancers, including PCa. Circulatory forms of miRNAs have been detected in serum and hold promise as minimally invasive cancer biomarkers. This study aimed to identify potential circulatory miRNAs that can provide insights into new mechanisms for clinical diagnosis of PCa and can serve as potential biomarkers and/or therapeutic targets. Candidate serum miRNAs were detected by using PCR microarray in a learning set of six African American (AA) and six Caucasian American (CA) PCa patients. Discriminating performance of candidate miRNAs was validated by qRT-PCR in serum samples from 36 AA (24 PCa patients and 12 controls) and 36 CA (16 PCa patients and 20 controls). From the miRNA profiling experiments, three differentially expressed miRNAs (miR-25, miR-101, and miR-628-5p) were selected for future validation. In the validation set, there was an overall low expression of miR-25 (p < 0.01), miR-101 (p < 0.001), and miR-628-5p (p < 0.0001) in serum of PCa patients as compared with normal individuals. Subdivision on the basis of ethnicity showed that serum expression levels of miR-628-5p were significantly downregulated in both AA and CA PCa patients when compared with their respective controls. Our results demonstrate that the three miRNAs, particularly miR-628-5p, may be further developed as a biomarker, which can serve as novel noninvasive biomarker for PCa diagnosis and prognosis.

MicroRNA profiling in prostate cancer--the diagnostic potential of urinary miR-205 and miR-214.

Prostate cancer (PCa) is the most common type of cancer in men in the United States, which disproportionately affects African American descents. While metastasis is the most common cause of death among PCa patients, no specific markers have been assigned to severity and ethnic biasness of the disease. MicroRNAs represent a promising new class of biomarkers owing to their inherent stability and resilience. In the present study, we investigated potential miRNAs that can be used as biomarkers and/or therapeutic targets and can provide insight into the severity and ethnic biasness of PCa. PCR array was performed in FFPE PCa tissues (5 Caucasian American and 5 African American) and selected differentially expressed miRNAs were validated by qRT-PCR, in 40 (15 CA and 25 AA) paired PCa and adjacent normal tissues. Significantly deregulated miRNAs were also analyzed in urine samples to explore their potential as non-invasive biomarker for PCa. Out of 8 miRNAs selected for validation from PCR array data, miR-205 (p<0.0001), mir-214 (p<0.0001), miR-221(p<0.001) and miR-99b (p<0.0001) were significantly downregulated in PCa tissues. ROC curve shows that all four miRNAs successfully discriminated between PCa and adjacent normal tissues. MiR-99b showed significant down regulation (p<0.01) in AA PCa tissues as compared to CA PCa tissues and might be related to the aggressiveness associated with AA population. In urine, miR-205 (p<0.05) and miR-214 (p<0.05) were significantly downregulated in PCa patients and can discriminate PCa patients from healthy individuals with 89% sensitivity and 80% specificity. In conclusion, present study showed that miR-205 and miR-214 are downregulated in PCa and may serve as potential non-invasive molecular biomarker for PCa.