Browsing by Subject "Up-Regulation"

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Investigating the Role of DIS3L2 in Perlman Syndrome and Wilms Tumor
(2018-06-29) Hunter, Ryan Wayne; Amatruda, James F.; Mendell, Joshua T.; Abrams, John M.; Conrad, Nicholas
Wilms tumor, while the most common pediatric kidney tumor, has a poorly understood etiology. Several recent studies have uncovered a role for loss of let-7 in its pathogenesis. One crucial mechanism through which let-7 expression is controlled is via the activity of the RNA-binding protein LIN28, which binds the precursor of let-7 and mediates the addition of a series of uridines to the 3′ end. This oligouridylation marks pre-let-7 for degradation by the exoribonuclease DIS3L2. Loss-of-function mutations in DIS3L2 lead to the Perlman congenital overgrowth syndrome, characterized by high neonatal mortality and, interestingly, a strong predisposition to Wilms tumor. Furthermore, DIS3L2 has been found to be deleted or mutated in some cases of sporadic Wilms tumor. The importance of let-7 in Wilms tumorigenesis and a purported role for DIS3L2 in the LIN28-let-7 pathway have led to speculation that aberrant let-7 expression underlies Wilms tumor susceptibility in DIS3L2-deficient contexts. It is still unclear, though, how loss of DIS3L2 could lead to altered let-7 levels, as the uridylated pre-let-7 species that DIS3L2 degrades is believed to be a dead-end product that cannot be further processed by DICER into mature let-7. Thus far, regulation of mature let-7 levels by DIS3L2 has only been examined in a limited number of cell lines. To determine whether DIS3L2 regulation of let-7 differs in a broader set of cell types, we used genome-editing to knockout DIS3L2 in a wide-ranging panel of cell lines with differing levels of LIN28 expression. Consistent with prior reports, loss of DIS3L2 had no effect on mature let-7 expression in these cell lines. However, it remained possible that the regulation of let-7 by DIS3L2 differed in cell populations relevant to Wilms tumor pathogenesis in vivo. Thus, to examine DIS3L2 function in these contexts, we used CRISPR-Cas9 to generate mouse lines harboring either Dis3l2-null alleles or mutations commonly observed in Perlman syndrome. Interestingly, Dis3l2 mutants recapitulated some aspects of Perlman syndrome, including neonatal mortality and genitourinary abnormalities, but not overgrowth or Wilms tumor. Moreover, the phenotype that results from the most common Perlman mutation is the same as that seen in the Dis3l2 loss-of-function mouse model, suggesting that the DIS3L2 mutations reported in Perlman syndrome are indeed loss-of-function. Finally, we examined the molecular function of DIS3L2 in isolated primary nephron progenitor cells (NPCs), a likely cell of origin of Wilms tumors, from Dis3l2-null and wild-type embryos. Consistent with our findings in the cell lines, loss of DIS3L2 in NPCs does not affect let-7 expression or activity, yet leads to upregulation of Igf2, which is reported to be overexpressed in 70-80% of Wilms tumors. Therefore, Igf2 poses an attractive candidate for both overgrowth and oncogenesis associated with DIS3L2 loss.
Upregulation of Cytokines Midkine and Pleiotrophin in Keloids
(2020-01-21) Marella, Pooja; Tran, An; Glass, Donald A., II
Keloids are benign proliferative scars that are exaggerated responses to cutaneous wound healing. They can be painful and/or pruritic, and commonly affect the chest, upper back, shoulders, and earlobes. Keloids often affect skin of color; most keloid patients are of African, Hispanic, or Asian descent. Although keloids are heavily implicated in fibrosis, there remain gaps in our understanding of keloid pathogenesis and our elucidation of potential biomarkers. The goal of this project was to identify novel molecules upregulated in keloids in order to identify uncharacterized mechanisms underlying keloid pathogenesis. Matched sets of keloid tissue and perilesional normal tissue were obtained from three keloid patients recruited from the outpatient dermatology clinics of Parkland Hospital and UT Southwestern Medical Center. Whole transcriptome sequencing comparing keloid tissue to perilesional normal tissue showed 344 genes with differential expression across the three sample pairings. Of note were Midkine (MDK) and Pleiotrophin (PTN), which were upregulated 30 fold and 10 fold respectively. MDK and PTN are cytokine signaling molecules known to play a role in wound healing, mitogenicity, and inflammation. They are upregulated in several cancers, and higher expression generally indicates poor prognosis. Keloids have been implicated in cancer pathogenesis, and prior research has shown that human skin keloid fibroblasts display bioenergetics of cancer cells (Vincent AS, 2008). Because MDK and PTN were upregulated in whole keloid tissue, we hypothesized that MDK and PTN would be upregulated in primary keloid fibroblasts in vitro versus primary normal skin fibroblasts in vitro. We performed RT-PCR on RNA obtained from keloid fibroblasts and normal fibroblasts grown in vitro, using MDK, PTN, and GAPDH (positive control) primers, and water serving as a negative control. Results showed that keloid fibroblasts did not have significant increased expression of MDK and PTN at the RNA level compared to normal skin fibroblasts grown in vitro. It is possible that some other component of the keloid microenvironment, such as keratinocytes, endothelial cells, or inflammatory cells may be inducing upregulation of MDK and PTN in whole keloid tissue in vivo. Further work on this project involves performing Western blots on keloid fibroblasts and normal fibroblasts grown in vitro to determine whether MDK and PTN are upregulated at the protein level. In addition, in situ hybridization experiments for MDK and PTN on keloid sections will enable us to determine which cell type(s) are making MDK and PTN in keloid tissue.