Project description:MicroRNAs (miRNAs) are evolutionary conserved, non-coding, gene regulatory RNA molecules found in both plants and animals and amongst almost every cell and tissue type. They are about 22 nucleotides long and are involved in silencing of mRNA through sequence specific binding to the 3’ untranslated region (UTR) of the mRNA subsequently causing translational repression and/or will promote the degradation of protein-coding mRNA. Specifically, the miRNA family, mir-146 a/b, has been previously found to be involved in the regulation of the innate immune response by functioning as a negative regulator to help fine-tune the immune response. Microglial cells are the macrophage of the brain participating as major players of the innate immune response. During prion disease, no immune response is mounted against PrPSc possibly due to its similarity to host PrPc and thus, the host immune response would be suppressed and tightly regulated. Therefore, an increased expression of mir-146 by microglial cells during prion disease may function as one of these negative regulators. Our objective of this experiment is to use DNA microarrays to investigate the gene regulation of mir-146 previously found upregulated in our studies of mouse brain tissue specifically in microglial cells during prion disease with the aim of having a better understanding of prion pathobiology and a potential target for therapeutic intervention. Mir-146 expression was confirmed via in situ analysis of brain tissue and was further investigated in a microglial murine cell line (EOC 13.31). To mimic mir-146 upregulation during prion disease, mir-146 a & b were overexpressed in EOCs using a lipid based reverse transfection system. Furthermore, endogeneous mir-146 was knocked down as another method to help confirm the mRNA targets affected by mir-146. Simultaneously, another experiment was performed for the investigation of its involvement in the innate immune response by stimulating the EOCs with differing concentrations of LPS from E.coli (055:B5). Total RNA was collected and prepared at several timepoints and the levels of expression of both mir-146 a & b was tested via qRT-PCR. The RNA collected from the EOCs from each condition were used as target material for dual-color, competitive hybridization to Agilent whole mouse genome 4x44K oligo arrays. All the significant targets found on the mircorarrays were compared against the various conditions to find a consensus of affected mir-146 mRNA targets.

Project description:The staphylococcal accessory regulator A (sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. To assess the relative impact of these two functions, we previously used a proteomics approach that measures protein abundance as a function of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assess the impact of this on the accumulation of S. aureus exoproteins1. While this approach confirmed that protease-mediated degradation has a significant impact on the S. aureus exoproteome, it was potentially limited in that it did not take into account the possibility that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, we present an expanded proteomics approach that utilizes a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels. Specifically, proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases (sarA/protease) were resolved using one-dimensional gel electrophoresis. Using methods that focus on total proteoforms vs. methods that focus specifically on full-length proteins, quantitative proteomic comparisons of sarA vs sarA/protease mutants identified proteins that were degraded in a protease dependent manner owing to mutation of sarA, while comparisons of a sarA/protease mutant vs the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This approach provided for a more comprehensive and robust analysis of the impact of mutating sarA and protease-mediated degradation on the S. aureus exoproteome.

Project description:One of the major challenges for chemotherapy is appearance of resistance to compounds. Despite several singling pathways have been implicated in development of Adriamycin (ADM) resistance, mechanisms involved in ADM-resistant osteosarcoma progression remain largely unknown. The present study attempts to illustrate the role of long noncoding RNA ARSR in development of ADM-resistance. We found lncRNA ARSR overexpressed in the Adriamycin resistant cell lines U2OS/ADM and MG63/ADM, accompanied with acquired multidrug resistance against to paclitaxel and cisplatin. Overexpression of lncRNA ARSR triggered rhodamine 123 efflux and survival, as well as migration of ADM resistant cells. Conversely, depletion of ARSR promoted rhodamine 123 retention and apoptosis while reduced motility of ADM resistant cells. Further investigation revealed that upregulation of ARSR enhanced Akt phosphorylation on Ser473 and increment of multidrug resistance-associated protein-1, apoptosis inhibitor Survivin and matrix metalloproteinase-2. Reduction of ARSR overcame the resistance to ADM and magnified the suppression of Akt-mediated signaling pathways by MK-2206. The current study gained novel evidence for understanding the mechanisms underlying adaptive ADM resistance, and provided rational to improve Akt-targeted therapy towards refractory osteosarcoma. Methods: The expression of lncRNA was analyzed by RT2 lncRNA PCR Arrays. The differential expressed lncRNAs were further verified by qRT-PCR. The relationship between lncRNA expression and clinical features was evaluated with Spearman correlation test. Cell proliferation was measured by MTT assay. Cell apoptosis percentage was detected by ANNEXIN V-PI analysis. The promoter activity was illustrated by Luciferase assay, and the change of microRNA and protein was detected by qRT-PCR and western blot, separately. Results: The expression of HAND2-AS1 declined in bladder cancer and correlated with depth of invasion and grades negatively. Restoration of HAND2-AS1 hampered cell growth by provoking cell apoptosis. Furthermore, one of the HAND2-AS1 sponge, miR-146, was found overexpression in bladder cancer tissue and cell lines. Expression of miR-146 related to HAND2-AS1 expression negatively. One of the targeted genes of miR-146, retinoic acid receptor beta (RARB) was downregulated in bladder cancer. In addition, the expression of RARB related to miR-146 negatively. Lost-of-function and gain-of-function experiments were used to identify the mechanisms underlying association of lncRNA HAND2-AS1: miR-146: RARB. miR-146 targeted RARB directly and hindered RARB-mediate apoptosis. However, the hindrance was impaired by HAND2-AS1 notably. Conclusion: HAND2-AS1 diminished miR-146 expression, thereby releasing the suppression of miR-146 on RARB-mediated apoptosis, promoting bladder cancer regression. Long noncoding RNA profiling by array

			Action	DRS
	SRR24315447_1.fastq.gz	Fastqsanger.gz
	SRR24315447_2.fastq.gz	Fastqsanger.gz
	SRR24315448_1.fastq.gz	Fastqsanger.gz
	SRR24315448_2.fastq.gz	Fastqsanger.gz
	SRR24315449_1.fastq.gz	Fastqsanger.gz

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