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

ABSTRACT In the present study, mitral valve tissues from three mitral stenosis patients with RHD by valve replacement and two healthy donors were harvested and conducted DNA methylation signature on PRKCA by MeDIP-qPCR. The presence of hypomethylated CpG islands at promoter and 5ʹ terminal of PRKCA was observed in RHD accompanied with highly expressed PRKCA and down-regulated antisense long non-coding RNA (lncRNA) PRKCA-AS1 compared to health control. Furthermore, the enrichments of DNMT1/3A/3B on PRKCA were detected by ChIP-qPCR assay in vivo and in human cardiomyocyte AC16 and RL-14 cells exposed to TNF-α in vitro, and both demonstrated that DNMT1 substantially contributed to DNA methylation. Additionally, PRKCA-AS1 was further determined to bind with promoter of PRKCA via 5ʹ terminal and interact with DNMT1 via 3ʹ terminal. Taken together, our results illuminated a novel regulatory mechanism of DNA methylation on regulating PRKCA transcription through lncRNA PRKCA-AS1, and shed light on the molecular pathogenesis of RHD occurrence.


Introduction
Rheumatic heart disease (RHD) is an autoimmune inflammatory disease responding to group A streptococcal infection, which results in mitral valve damage and hemodynamic changes. RHD remains a health burden in developing countries [1]. Although the progression of valve surgery has improved the clinical treatment for RHD, frequent serious complications and irreversible valve dysfunction still challenges the living quality of patients owing to the failure of early detection [2].
DNA methylation as the most extensively epigenetic study is regulated by DNA methyltransferases (DNMTs) family, and controls gene expression without DNA sequence changes. In general, DNA methylation negatively governs transcription activity via impacting with polarity of promoter coordinated with multiple transcription factors and epigenetic regulators [3]. The mode of action of different DNMTs for DNA methylation catalysis is distinct. DNMT1 is responsible for hemimethylated modification in cell mitosis whereas DNMT3A/B are essential for de novo methylation in zygotic and embryonic development [4]. Nevertheless, the ability of CpG sites recognition and the efficiency of DNA methylation process by DNMTs seem to be less studied.
Increasing evidences have reported that abnormal DNA methylation plays an important role in RHD development and progression [5,6]. Previous study has characterized the phenotype of global differentially methylated regions in RHD in vivo and identified protein kinase C alpha (PRKCA) as one of the candidate genes whose DNA methylation is associated with the pathogenesis of RHD [7]. However, the underlying mechanism of DNA methylation on PRKCA transcription behind the RHD occurrence remains unclear.
In this study, we take PRKCA for example, and mainly focuses on the regulatory roles of DNMTs in PRKCA in RHD. Our results may provide a common research model of studying a single gene on DNA methylation, and advance the understanding on the epigenetic regulation for RHD pathogenesis.

Specimen collection
Three RHD patients (two males and one female) who received mitral valve replacement in Yantai Yuhuangding Hospital from April 2016 to May 2018 were enrolled in this study as the following criteria: I) Diagnosis of mitral valve stenosis; II) left ventricular ejection fraction volume >50% and left ventricular end-diastolic diameter <55 mm; III) Antistreptolysin O > 500 U, positive C reactive protein; IV) no history of cardiomyopathy, congenital heart disease, liver disease or renal disease. Healthy mitral valve tissues were obtained from two remains donation by traffic accidents. The average age of participants is 66.85 ± 7.33 year. All the participants and their families understood and signed the informed consent. The documents of signed informed consent and Ethics Committee of Yantai Yuhuangding Hospital were all provided to approve this study. The freshly isolated human tissues were washed by saline and stored in −80°C for the subsequent experiments.
DNA or RNA was mechanically sheared by Bioruptor Pico 60 KHz Sonicator (Diagenode, Seraing, Belgium). Ten percent of whole lysate was stored as input, whereas the rest of it was incubated with 1 μg primary antibodies of interest including 5-Methylcytosine (Cat. No. 61,479, Activemotif, Carlsbad, CA, USA), DNMT1 (NB100-56,519, Novus, Centennial, CO, USA) and SMAD2 (NBP2-67,376, Novus) at 4°C overnight. Additional 2-h pull down by protein A/G beads (Thermo Fisher Scientific) was performed at 4°C with gentle rotation. Beads were washed by the appropriated buffer for three times. Coprecipitated RNA was isolated by RNAiso (Takara, Tokyo, Japan). Coprecipitated DNA was purified by phenol chloroform methods. Primers designed to encompass approximately 150 bp around the target regions were used to detect the enrichment using qPCR.

RNA isolation
RNA was extracted using RNAiso reagent, quantified with a NanoDrop (Thermo Fisher Scientific). 100 ng RNA was conversed into cDNA using a reverse transcription kit (Roche, Basel, Switzerland). PRKCA and PRKCA-AS1 expressions were detected by qPCR.

Quantitative PCR (qPCR) assay
The DNA templates were detected using Fast Universal SYBR Green Real-time PCR Master Mix (Roche) under the following conditions: 95°C/2 min and 50 cycles of 95°C/5 s, 60°C/10 s, 72°C/ 30 s, and 72°C/10 min. GAPDH was used for the loading control. All primers used in this study are listed in Table S2.

Fluorescence in situ hybridization (FISH)
The FISH probes designed for PRKCA labeled with R-Phycoerythrin (excitation wavelength 565 nm) and for PRKCA-AS1 labeled with FITC (excitation wavelength 495 nm) were purchased from G&P Medical Enterprises (Beijing, China). Probes sequences can be found in Table S2. 40 μm mitral valve tissues sections or slides filled with AC16 cells washed by PBS were hypotension treatment in 0.075 M KCl at 37°C for 25 min, fixed by pure ethanol for 10 min and dropped on the glass slide, then aged at 56°C for 60 min. The slides were washed by PBS twice and dehydrated with the 70, 85, and 100% ethanol for 3 min in order, and naturally dried. 10 μl probes were added on and mounted immediately. Slides were next denatured 75°C for 5 min and 42°C for 16 h, then removed the cover glass and incubated in 2 × SSC for 5 min and 0.1% NP-40/2 × SSC for another 2 min at 46°C, followed by 70% ethanol at RT for 3 min and dried in dark. 15 μl DAPI were dropped and mounted again. After 10 min incubation in dark, the slides were observed under the fluorescence microscope with the appropriate filters.

Statistical analysis
All statistical analyses were conducted in SPSS 20 (IBM, Armonk, NY, USA). For qPCR data, the 2^delta delta method was used to calculate the expression and protein enrichment on DNA. Student's t-test was used to evaluate differences between groups. A p-value less than 0.05 was considered to indicate a significant difference.

Results
To investigate the epigenetic role of PRKCA in RHD, we characterized the DNA methylation pattern of PRKCA in RHD in vivo and in vitro, and further found that the abnormal DNA methylation was ascribed to DNMT1 binding affinity. Antisense lncRNA PRKCA-AS1 was determined to connect and recruit DNMT1 on the promoter of PRKCA. Finally, we further investigated the effect of p38/MAPK pathway on PRKCA-AS1 expression in RHD.

Characterization of DNA methylation on PRKCA in mitral valve of RHD
First, we investigated the DNA methylation signature of PRKCA in mitral valve tissues of RHD by MeDIP-qPCR. We observed that 'CG' loci with highest density were distributed at −590 to +648 containing promoter, first exon and partial first intron of PRKCA (consider 'A' of 'ATG' as +1) (Figure 1(a)), which were majorly concentrated on to investigate DNA methylation. The overall DNA methylation levels at these detected regions of PRKCA showed that PRKCA was hypomethylaed in RHD compared to health control (Fold change (FC) = 0.28, p = 0.021), where DNA methylation of promoter (FC = 0.23, p = 0.017) and the first exon (FC = 0.14, p = 0.009) of PRKCA was significantly lower in RHD compared to health control (Figure 1(b)). The mRNA (FC = 3.60, p = 0.022) (Figure 1(c)) and protein levels (FC = 3.14, p = 0.036) (Figure 1(d-e)) of PRKCA were both highly expressed in RHD compared to healthy control. Consistently, Pearson correlation analysis showed that DNA methylation at promoter (r = −0.892, p < 0.05) and exon (r = −0.895, p < 0.05) were strongly negatively correlated with PRKCA transcription (figure 1(f)). Taken together, we determined that the abnormally hypomethylated PRKCA especial at 5ʹ terminal was closely related to up-regulation of PRKCA in mitral valve tissues of RHD.

DNMT1 contributes to DNA methylation on promoter of PRKCA
Although DNMTs family contribute to DNA methylation, different catalytic means of DNMT1 (maintenance methylation) and DNMT3A/B (de novo methylation) have been declared. Next, we investigated which DNMTs were responsible for regulating gene body methylation of PRKCA. Two human cardiomyocyte cell lines AC16 and RL-14 were exposed to 100 ng/mL TNF-α for mimicking the in vitro inflammatory model or direct DNMTs knockdown via RNA interference or 20 ng/mL 5-Azacytidine (DNMTs inhibitor) treatment as a positive control. IL-1β and IL-4 levels in medium were detected by ELISA to confirm the inflammatory effect of TNF-α in cardiomyocyte cells ( Figure  S1). We observed that PRKCA was up-regulated after TNF-α treatment (AC16: FC = 3. Unexpectedly, protein levels of all DNMTs had almost unchanged induced by TNF-α compared to negative control either in AC16 or RL-14 cells (Figure 2(b-d)), which suggested that DNMT1 was likely a partial but not an essential factor for PRKCA induction by TNF-α. Results above indicated that DNMT1 contributed to DNA methylation on 5ʹ terminal negatively correlating with PRKCA transcription in RHD.

The interaction between 5ʹ terminal of PRKCA-AS1 and PRKCA
In general, DNMTs recognize unmethylated CpG loci and transfer methyl group for DNA methylation catalysis. Although DNMTs expression did not change, DNA methylation on PRKCA was weakened after TNF-α stimulation, suggesting an unknown epigenetic regulation coordinated with DNMT1 for DNA methylation on PRKCA. Previous studies have indicated that the antisense lncRNAs can link with DNA and transcription factors, and govern gene expression via chromatin organization [12,16,17], which enlighten us on the current system. To figure out the underlying mechanism of DNMT1 on PRKCA transcription, we focused on the antisense lncRNA PRKCA-AS1, and investigated their expression in RHD. PRKCA-AS1 was majorly expressed in nucleus and significantly down-regulated in RHD (FC = 0.113, p = 0.002) as well as in TNF-α treated AC16 (FC = 0.344, p = 0.035) and RL-14 cells (FC = 0.31, p = 0.028) compared to control (Figure 3(a)). Certainly, PRKCA-AS1 transcription As an antisense lncRNA transcribed from the second intron of PRKCA, the potential interaction between PRKCA-AS1 and genomic region of PRKCA was investigated by FISH. Multiple single-strand DNA probes spanning across PRKCA promoter and PRKCA-AS1 introns were designed for PRKCA-AS1 and PRKCA (Figure 3(b)). Although the substantial overlapping between PRKCA-AS1 and PRKCA was observed both in RHD and healthy control in vivo (Figure 3(c)), it is still hard to figure out the precise position of PRKCA-AS1 for PRKCA interaction. To this end, PRKCA-AS1 with respective truncation of four exons were transfected into AC16 cells, and the disappearance of affinity by the first exon absence compared to control and the other three truncated fragments of PRKCA-AS1 (Figure 3(d)) indicated that 5ʹ terminal of PRKCA-AS1 indeed bound with PRKCA. Interestingly, given the computational examination of PRKCA-AS1 by 'Triplex Domain Finder' tool (http://www. regulatory-genomics.org/tdf/basic-introduction/) [18], promoter of PRKCA was also included in the putative genomic targets of PRKCA-AS1 (Figure 3 (e)). The results above suggested that PRKCA-AS1 was proposed to bind with PRKCA.

Discussion
The pathogenesis of RHD is the result of valve damage caused by an abnormal immune response consisting of humoral and cellular components after exposure to group A streptococcal infection [1]. The role of autoimmune reactions in the pathogenesis of acute rheumatic fever was demonstrated when antibodies against group A streptococcus crossrecognize to a series of cardiac proteins such as cardiac myosin, tropomyosin, keratin, laminin and vimentin, and multiple cytokines including IL-1, IL-6, and TNF-α exerted inflammatory effects on cardiomyocytes. In our study, TNF-α is used to prepare an in vitro environment of inflammatory reaction. We observe that both NF-κB and JAK/STAT signaling pathways are all activated, which indicates that the ectopic cytokine stimulators can mimic rheumatism to induce auto inflammation in myocardial cells.
PRKCA has been associated with emotional memory formation, posttraumatic stress syndrome, cancer, and aging. PRKCA as a key regulator of cardiac contractility is reported to be implicated in heart failure risks and treatment outcomes [20]. The suppression of PRKCA may fit the criteria of a therapeutic target with milder systemic effects that still boosts contractility in HF patients. This increase in PKCα activity is perplexing because it is also accompanied by upregulation of a molecular braking mechanism. Previous studies revealed that differentially methylated PRKCA was both involved in development of fibromyalgia and RHD, which were usually caused by inflammatory cytokines and suffered from rheumatic symptoms [7,21]. Consistently, our data determines a hypomethylation pattern of PRKCA in RHD in vivo and in vitro. Moreover, we verify that transcription of PRKCA is up-regulated after DNMT1 knockdown, which suggests that the promoter and 5ʹ terminal methylated by DNMT1 contributes to regulating PRKCA transcription, and the other CpG loci on PRKCA showing no significant association with PRKCA transcription may not affected RNA polymerase.
In RHD, previous studies reported a controversial phenotype of DNMTs expression [22,23]. In this case, we announce that each DNMT manages DNA methylation pattern in different CpG loci, which suggests that the target strategy for the certain DNMT is superior to the classical DNMT inhibitors, which usually affect the global DNA methylation and cause more side reaction. Antisense lncRNA regulates the adjacent target gene via RNA-DNA interaction has been determined previously. X-chromosome inactivation by lncRNA Xist is one of the most classic case [24,25]. Besides, lncRNA can interact with transcription factors or enzymes in nucleus to modulate gene expression has also been reported [26][27][28]. Our results indicate that PRKCA-AS1 bridges PRKCA and DNMT1 through 5ʹ and 3ʹ terminals, respectively, and give one possible explanation for why DNMT1 can be recruited to promoter of PRKCA-AS1.
Although the sequences of PRKCA and PRKCA-AS1 are aligned but fail to find the identical sequence, the substantial interaction is confirmed by FISH assay, which implies that PRKCA-AS1 may recognize a certain DNA motif of PRKCA promoter through a complicate loop structure, and need to be clarified in future study. Moreover, FISH data shows the presence of PRKCA-AS1 transcript in nucleus and indicates that PRKCA-AS1 may bind multiple genomic regions via the similar DNA motif with PRKCA, which can be verified by highthroughput sequencing followed by pull down assay.
Finally, p38/MAPK/TGF-β1/Smad signaling pathway is determined to be the reason behind aberrant DNA methylation of PRKCA. The activated p38/MAPK/TGF-β1/Smad is able to suppress PRKCA-AS1, thereby retard DNMT1 recruitment to PRKCA promoter, and modulate DNA methylation and transcriptional repression similarly with a previous regulation model [29]. Interestingly, another study indicates an opposite effect that NF-κB/p65/MUC1-C complex occupy the promoters of DNMT1 and DNMT3B to drive their transcription, but not DNMT3A in carcinoma cells [30]. Taken together, the regulatory network bridging inflammatory signaling pathway and DNMTs seems to be complicate in different diseases.

Conclusion
Overall, our data suggests that PRKCA can be hypermethylated by PRKCA-AS1-meidated DNMT1 through p38/MAPK/TGF-β1/Smad signaling pathway against RHD, which advance the understanding of pathogenesis and progression of RHD, and provide the potential therapeutic targets for RHD.

RESEARCH HIGHLIGHTS
Regulatory mechanism of PRKCA in RHD DNA methylation by lncRNA Role of antisense lncRNA

Disclosure statement
No potential conflict of interest was reported by the author(s).

Availability of Data and Materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.