DOI: 10.5061/dryad.dz08kps4h
title: Defining the role of the miR-145 – KLF4 – αSMA axis in mitral valvular interstitial cell activation in myxomatous mitral valve prolapse using the canine model

description: Mitral valve prolapse (MVP) is a common valvular disease, affecting 2-3%
 of the adult human population and is a degenerative condition. 5-10% of
 the afflicted will develop severe mitral regurgitation, cardiac
 dysfunction, congestive heart failure, and sudden cardiac death. Naturally
 occurring myxomatous MVP in dogs closely resembles MVP in humans
 structurally, and functional consequences are similar. In both species,
 valvular interstitial cells (VICs) in affected valves exhibit phenotype
 consistent with activated myofibroblasts with increased αSMA expression.
 Using VICs collected from normal and MVP-affected valves of dogs, we
 analyzed the miRNA expression profile of the cells and their associated
 small extracellular vesicles (sEV) using RNA sequencing to understand the
 role of non-coding RNAs and sEV in MVP pathogenesis. miR-145 was shown to
 be upregulated in both the affected VICs and sEV, and overexpression of
 miR-145 by mimic transfection in quiescent VIC recapitulates the activated
 myofibroblastic phenotype. Concurrently, KLF4 expression was suppressed by
 miR-145, and KLF4 overexpression resulted in a decrease in αSMA
 expression, and its suppression resulted in an increase in αSMA
 expression, thus confirming the miR-145 – KLF4 – αSMA axis. Targeting this
 axis may serve as potential therapy in controlling pathologic
 abnormalities found in MVP valves.
description: miRNA and RNA sequencing analyzed by Qiagen Genomic Workbench CLC
 and DESeq2. Western blot and PCR.
description: * Title of Dataset: Data for the article "Defining the role of the
 miR-145 – KLF4 – αSMA axis in mitral valvular interstitial cell activation
 in myxomatous mitral valve prolapse using the canine model" *
 Persistent Identifier: *
 Dataset Contributors: Vicky K. Yang, Nicole Moyer, Runzi Zhou, Sally
 Carnevale, Dawn M. Meola, Sally R. Robinson, Guoping Li, Saumya Das *
 Contact Information: Name: Vicky K. Yang Affiliation: Department of
 Clinics Sciences, Cummings School of Veterinary Medicine, Tufts University
 Email: --- * This data includes four files
 with information on canine mitral valve miRNA, mRNA, PCR, and protein
 information in association with the developement of mitral valve prolapse.
 * Description of File 1: File name: PCR Western Luciferase raw data.xlsx
 Data structure: Data are organized based on figures. Each dataset is
 associated with a particular figure (worksheet name). General data
 description: This file contains data associated with PCR, immunoblotting,
 and luciferase assay results. Methodology is described in the article.
 Worksheet 1: Worksheet name: Fig 1 qPCR Ct values Description of data:
 Normalized qPCR Ct values of p21, aSMA, TGFbR2, and TGFb2 of VICs isolated
 from normal and MVP-affected canine mitral valves. Each data point
 represents a different dog cell line (normal valve = 17, diseased valve =
 20). Ct numbers are normalized against average of 2 housekeeping genes
 (HPRT and RPS19) for each of the individual cell lines. Worksheet 2:
 Worksheet name: Fig 1 western results Description of data: Protein
 expression for aSMA and KLF4 normalized to b-actin for VICs isolated from
 normal and MVP-affcted canine mitral valves. Each data point represents a
 different dog (normal valve = 4 for aSMA, 8 for KLF4; diseased valve = 5
 for aSMA, 6 for KLF4). Ct numbers are normalized against average of 2
 housekeeping genes (HPRT and RPS19) for each of the individual cell lines.
 Worksheet 3: Worksheet name: Fig 5 Luciferase luminescence Description of
 data: Luminescence signal for miR-145 transfected and negative control
 stransfected cells co-transfected with KLF4 3'UTR sequence plasmid or
 KLF4-mutant 3'UTR sequence plasmid into HEK cells. 3 different mutant
 or miR-145 3'UTR plasmids were used and transfected into HEK cells.
 Luminescence signal was normalized within each cell line to the signal
 from miR-145 3'UTR plasmid transfected cells that were also
 transfected with miR-145 for comparison. Worksheet 4: Worksheet name: Fig
 5 KLF4 stimulation Description of data: aSMA protein expression of
 valvular interstitial cells by Western blot normalized to b-actin as a
 function of KLF4 recombinant protein stimulation (0 ng/ml vs. 0.1 ng/ml)
 during cell culture. 7 cells lines were tested, and the expression level
 were normalized within each cell line to the control (0 ng/ml of KLF
 protein) condition for comparison. Worksheet 5: Worksheet name: Fig 5 KLF4
 siRNA Description of data: aSMA protein expression of valvular
 interstitial cells by Western blot normalized to b-actin for control
 sample (vehicle only added), cells transfected with scramble siRNA (NC),
 and cells transfected with the 3 DICER KLF4 siRNA. 3 cells lines were
 tested, and expression level were normalized within each cell line to the
 control condition for comparison. Worksheet 6:\ Worksheet name: Fig 6 EV
 co-culture Description of data: aSMA protein expression by Western blot
 normalized to b-actin. 9 valvular interstitial cell lines were used, and 3
 conditions were tested: control sample (vehicle only), cells co-cultured
 with EV harvested from MVP/Diseased VICs, and cells co-cultured with
 normal valve VICs EV (10X9 particles/ml). Expression levels were
 normalized within each cell line to the control condition for comparison.
 * Description of File 2: File name: EV DESeq miRNA data.xlxs Data
 structure: Sheet 1 shows miRNA sequence count for each of the miRNA
 species collected from EVs of culture supernatant of VICs of each dog
 study subject. General data description: See above. * Description of File
 3: File name: VIC DESeq miRNA data.xlxs Data structure: Sheet 1 shows
 miRNA sequence count for each of the miRNA species collected from VICs of
 each dog study subject. General data description: See above. * Description
 of File 4: File name: miR-145 mimic inhibitor transfection sequence
 results.xlxs Data structure: Sheet 1 shows mRNA sequence count for each of
 the mRNA species collected from VICs from 6 dogs (D#) transfected with
 control scramble miRNA, miR-145 mimic, and miR-145 inhibitor. General data
 description: See above. * Description of File 5: File name: Supplemental
 Data.pdf Data structure: Each western blot image showing the original
 uncropped results for each Figure General data description: Figures are
 ordered sequentially from each figure number. Image name: Figure 1c
 Uncropped Gel Description: Respresentative western blots of aSMA, KLF4,
 and b-actin for individual canine VIC cell lines (some from normal valves,
 some from MVP-affected valves). Red boxes show sections displayed in the
 manuscript figure. Image name: Figure 2a Uncropped Gel Description:
 Representative western blots of CD9, TSG101, Alix, and Calnexin signals
 from canine VIC EVs harvested by Izon size exclusion chromatograph. MDCK
 EVs were used as positive control, and Cell protein (VIC) were used as
 negative control. Red boxes show sections displayed in the manuscript
 figure. Image name: Figure 5b Uncropped Gel Description: Representative
 western blots of aSMA and b-actin for canine VICs treated with KLF4 at
 0-10ng/ml. Results for a separted TGF-beta experiment not part of this
 paper is also shown. Red boxes show sections displayed in the manuscript
 figure. Image name: Figure 5c Uncropped Gel Description: Representative
 western blots of aSMA and b-actin for canine VICs treated with
 control/vehicle only (C), scramble siRNA (NC), and KLF4 DICER siRNA. Red
 boxes show sections displayed in the manuscript figure. Image name: Figure
 6b Uncropped Gel Description: Representative western blots of aSMA and
 b-actin for canine VICs co-cultured with EVs harvested from normal
 (fibroblastic) valve associated VICs and MVP (myofibroblastic) associated
 VICs. Red boxes show sections displayed in the manuscript figure. *
 Submitted to IJMS for publication (under review) * Was data derived from
 another source? No