Coordination of PRKCA/PRKCA-AS1 interplay facilitates DNA methyltransferase 1 recruitment on DNA methylation to affect protein kinase C alpha transcription in mitral valve of rheumatic heart disease

Figures

• 

  Figure 1.. DNA methylation of PRKCA in mitral valve of RHD. (a) the overview of CpG loci distribution within human PRKCA gene. (b) DNA methylation on 5ʹ terminal of PRKCA (promoter, first exon and first intron) in mitral valve of RHD by MeDIP-qPCR assay. (c) PRKCA transcription levels in mitral valve of RHD by qPCR assay. (d) PRKCA protein levels in mitral valve of RHD by WB assay. (e) PRKCA protein levels calculated by gray intensity analysis. (f) the correlation between DNA methylation and PRKCA transcription by pearson correlation analysis. the given data was processed as mean ± standard error and compared between RHD and HC groups by student’s t-test. ‘RHD’: rheumatic heart disease; ‘HC’: healthy control; ‘FC’: fold change

• 

  Figure 2.. The effect of DNMTs on DNA methylation of PRKCA in human cardiomyocyte cells. (a) the mRNA and (b) protein of PRKCA in AC16 cells induced by TNF-α, DNMTs RNA interference and 5-azacytidine. (c) the mRNA and (d) protein of PRKCA in RL-14 cells induced by TNF-α, DNMTs RNA interference and 5-azacytidine. DNA methylation on 5ʹ terminal of PRKCA (promoter, first exon and first intron) in (e) AC16 and (f) RL-14 cells induced by TNF-α, DNMTs RNA interference and 5-azacytidine. the given data from triplicate experiments was processed as mean ± standard error and compared by student’s t-test. No. 1 to 6 indicate six comparison with control group. ‘FC’: fold change; ‘si’: siRNA; ‘AZA’: 5-azacytidine

• 

  Figure 3.. Interaction between PRKCA-AS1 and PRKCA in RHD. (a) the RNA levels of PRKCA-AS1 in mitral valve of RHD tissues, TNF-α-induced AC16 and RL-14 cells. the given data was processed as mean ± standard error and compared between RHD and HC groups by student’s t-test. (b) the overview of PRKCA and PRKCA-AS1 location. blue: promoter; orange: exon of PRKCA; green: intron of PRKCA; yellow: exon of PRKCA-AS1; red arrows: the probes for FISH assay; orange and yellow arrows: the transcription orientation. (c) the staining for PRKCA-AS1 transcripts and PRKCA genomic region in RHD with 400 x magnification by FISH assay. (d) the staining for PRKCA-AS1 transcripts and PRKCA genomic region in AC16 cells with different truncations of exogenous PRKCA-AS1 with 400 x magnification by FISH assay. red: the staining for allele PRKCA in genomic DNA; green: the staining for PRKCA-AS1 transcripts; yellow: the overlap of two kinds of probes. (e) the computational examination of PRKCA-AS1 for target promoter of genes by ‘triplex domain finder’ tool. ‘FC’: fold change; ‘Δ’: deletion

• 

  Figure 4.. The role of PRKCA-AS1 in regulating PRKCA expression in RHD. (a) the RNA levels of PRKCA-AS1 in TNF-α-induced AC16 and RL-14 cells. (b) DNA methylation and (c) DNMT1 occupancy on 5ʹ terminal of PRKCA (promoter, first exon and first intron) in TNF-α-induced AC16 and RL-14 cells. (d) DNMT1 occupancy and (e) DNA methylation on 5ʹ terminal of PRKCA (promoter, first exon and first intron) in TNF-α-induced AC16 and RL-14 cells treated with different truncations of exogenous PRKCA-AS1. (f) interaction between DNMT1 and different fragments of PRKCA-AS1 by RIP-qPCR assay. the given data from triplicate experiments was processed as mean ± standard error and compared by student’s t-test. ‘FC’: fold change; ‘Δ’: deletion

• 

  Figure 5.. The role of p38/MAPK in regulating PRKCA-AS1 expression in RHD. (a) the RNA levels of PRKCA-AS1 and PRKCA in TNF-α-induced AC16 cells treated with inhibition of different signaling pathways. (b) Smad2 occupancy on PRKCA-AS1 in TNF-α-induced AC16 cells treated with inhibition of different signaling pathways. (c) Smad2 occupancy on PRKCA-AS1 in mitral valve of RHD. the given data from triplicate experiments was processed as mean ± standard error and compared by student’s t-test. ‘FC’: fold change; ‘BK’: blocking