Microrna Regulation On The Expression Of CD38 And Other Asthma Related Genes In Human Airway Smooth Muscle Cells

University of Minnesota Ph.D. dissertation. April-2015. Major: Veterinary Medicine. Advisor: Mathur Kannan. 1 computer file (PDF); x, 200 pages.CD38 is a multifunctional enzyme that regulates intracellular calcium ([Ca++]i ) homeostasis. It is expressed in airway smooth muscle (ASM) cells where it elevates [Ca++]i through its enzymatic product cyclic ADPribose (cADPR) and increases ASM contractility. Increased expression of CD38 in the ASM cells derived from the asthmatic patients (AS-HASM) and attenuated airway hyperresponsiveness to contractile stimuli shown by CD38-/- mice implicate the importance of CD38 in asthma. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is considered to be an important mediator for airway pathology in asthma. The reason for the differential expression of TNF-alpha-induced-CD38 in AS-HASM cells, does not involve transcriptional regulation of CD38 which is through signaling pathways mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K), and transcription factors nuclear factor kappa-B (NF-kB) and AP-I. Thus I hypothesized that post-transcriptional regulation of CD38 by microRNAs account for the differential expression of TNF-alpha induced -CD38 in AS-HASM cells. Among the several potential microRNAs predicted for CD38 by microRNA target-predicting algorithms, I selected miR-140-3p and miR-708 for further studies, as these showed differential expression in the AS-HASM cells compared to those from healthy subjects. Overexpression of these either microRNAs in ASM cells inhibited the TNF-alpha induced expression of CD38 at messenger RNA (mRNA) and protein levels. Luciferase-reporter assays with a mutated 3'UTR of the CD38 transcript confirmed the specific target sites for both microRNAs. Transcript stability assays revealed that mRNA degradation is not the mechanism underlying regulation by microRNAs. Examination of the expression and activation levels of proteins in the upstream signaling pathways of CD38 revealed that miR-140-3p, by inactivating p38 MAPK and NF-kB, and miR-708, by inactivating c-Jun N-terminal kinase (JNK) MAPK and Akt by elevating the expression of their phosphatases MKP-1 and PTEN respectively, control the expression of CD38 indirectly. Further, we found that miR-708 downregulates the expression of many chemokines and inhibits the serum induced proliferation of human ASM cells. We conclude that both microRNAs have therapeutic potential in controlling asthma related symptoms through regulating the expression of CD38 and chemokines and controlling the proliferation of human ASM cells

Seroprevalence of SARS-CoV-2 (COVID-19) exposure in pet cats and dogs in Minnesota, USA

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is continuing to spread globally. SARS-CoV-2 infections of feline and canine species have also been reported. However, it is not entirely clear to what extent natural SARS-CoV-2 infection of pet dogs and cats is in households. We have developed enzyme-linked immunosorbent assays (ELISAs) using recombinant SARS-CoV-2 nucleocapsid (N) protein and the receptor-binding-domain (RBD) of the spike protein, and the SARS-CoV-2 spike-pseudotyped vesicular stomatitis virus (VSV)-based neutralization assay to screen serum samples of 239 pet cats and 510 pet dogs in Minnesota in the early phase of the COVID-19 pandemic from mid-April to early June 2020 for evidence of SARS-CoV-2 exposures. A cutoff value was used to identify the seropositive samples in each experiment. The average seroprevalence of N- and RBD-specific antibodies in pet cats were 8% and 3%, respectively. Among nineteen (19) N-seropositive cat sera, fifteen (15) exhibited neutralizing activity and seven (7) were also RBD-seropositive. The N-based ELISA is also specific and does not cross react with antigens of common feline coronaviruses. In contrast, SARS-CoV-2 antibodies were detected at a very low percentage in pet dogs (~ 1%) and were limited to IgG antibodies against SARS-CoV-2 N protein with no neutralizing activities. Our results demonstrate that SARS-CoV-2 seropositive rates are higher in pet cats than in pet dogs in MN early in the pandemic and that SARS-CoV-2 N-specific IgG antibodies can detect SARS-CoV-2 infections in companion animals with higher levels of specificity and sensitivity than RBD-specific IgG antibodies in ELISA-based assays

MicroRNA Mediated Chemokine Responses in Human Airway Smooth Muscle Cells.

Airway smooth muscle (ASM) cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs) are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM) cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR)-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma

Principal Component Analysis.

<p>There is a clear primary separation of samples based on miR-708 mimic versus scrambled control. Secondary separation was by donor ID.</p

Chemokine release from HASM cells following miR-708 transfection.

<p>HASM cells from 3–6 donors were transfected with mimic or scrambled sequence mimic of miR-708 and treated with TNF-α (10ng/ml) following growth arrest of cells. Untransfected cells treated with TNF-α served as an additional control. Twenty hours later cell culture supernatants were collected for the measurement of chemokines. Note the release of CCL11 was significantly inhibited at every time point following miR-708 transfection when compared to scrambled sequence mimic transfection. Data represents mean±SEM.</p

Differentially expressed genes in TNF-α-stimulated HASM cells following miR-708 mimic transfection.

<p>Differentially expressed genes in TNF-α-stimulated HASM cells following miR-708 mimic transfection.</p

Primer Sequences.

<p>Primer Sequences.</p