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
Data files
Jan 16, 2024 version files 1.84 MB
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EV_DESeq_miRNA_data.xlsx
77.57 KB
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miR-145_mimic_inhibitor_transfection_sequence_results.xlsx
1.34 MB
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PCR_Western_Luciferase_raw_data.xlsx
16.22 KB
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README.md
6.65 KB
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Supplemental_Data.pdf
300.22 KB
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VIC_DESeq_miRNA_data.xlsx
97.98 KB
Abstract
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.
- 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: https://doi.org/10.5061/dryad.dz08kps4h
- 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: vicky.yang@tufts.edu
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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.
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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)
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Was data derived from another source? No
miRNA and RNA sequencing analyzed by Qiagen Genomic Workbench CLC and DESeq2.
Western blot and PCR.