Data of expression status of miR- 29a and its putative target mitochondrial apoptosis regulatory gene DRP1 upon miR-15a and miR-214 inhibition

Data is about the mitochondrial apoptosis regulatory framework genes PUMA, DRP1 (apoptotic), and ARC (anti-apoptotic) analysis after the employment of their controlling miRNAs inhibitors. The data represents putative conserved targeting of seed regions of miR-15a, miR-29a, and miR-214 with respective target genes PUMA, DRP1, and ARC. Data is of cross interference in expression levels of one miRNA family, miR-29a and its putative target DRP1 upon the inhibitory treatment of other miRNAs 15a and 214.

DRP1 miR-15a Apoptosis miRNAs inhibition a b s t r a c t Data is about the mitochondrial apoptosis regulatory framework genes PUMA, DRP1 (apoptotic), and ARC (anti-apoptotic) analysis after the employment of their controlling miRNAs inhibitors. The data represents putative conserved targeting of seed regions of miR-15a, miR-29a, and miR-214 with respective target genes PUMA, DRP1, and ARC. Data is of cross interference in expression levels of one miRNA family, miR-29a and its putative target DRP1 upon the inhibitory treatment of other miRNAs 15a and 214.

Value of the data
Data comprising mitochondrial apoptosis regulatory proteins may be of value to the scientists working in different disorders including valvular heart diseases, heart failure, kidney diseases, and cancer.
Data showing microRNAs targeting mitochondrial apoptosis regulatory genes may be of potential value for researchers working in the field of translational research.

Data presenting microRNAs cross interference phenomenon may open some venues for researchers
working on miRNAs regulation and signaling.

Data
Regulation of Mitochondrial pathology associated genes may be of great merit for multiple disorders with damaged mitochondria as cardiac diseases, cancers and kidney diseases [1]. Present data is about the miRNAs regulation of mitochondrial apoptosis regulatory framework genes PUMA, DRP1 (apoptotic) and ARC (anti-apoptotic). Present data has been generated and confirmed by the multiple miRNAs targeting predictor sites involving bioinformatics tools in databases (Fig. 1). Further the data regarding miRNAs cross interference was performed in neonatal cultured cardiomyocytes by employing microRNA inhibitors and later by expression analysis of miRNAs and respective target genes.
Data involves miR-15a, miR-29a and miR-214 complementary sequence matching of targeting proapoptotic genes as PUMA, DRP1 and ARC (Fig. 1). Dynamin related protein 1 (DRP-1) get overexpressed as a result of cellular stress and apoptosis while playing important role in mitochondrial fission [2]. The p53 up-regulated modulator of apoptosis (PUMA) is involved in DRP1 accumulation in mitochondrial membrane [3]. Data presenting miR-214 seed matching with ARC which is an apoptosis inhibitory protein. Apoptosis repressor with caspase recruitment domain (ARC) attenuates mitochondrial membrane depolarization by directly binding with PUMA whenever highly expressed in cardiomyocytes [4].
Data of miR-15a inhibitor treatment leading to reduction in miR-29a expression and subsequent increase in its target DRP1 expression to certain extent may point towards cross interference phenomenon in miRNA inhibition (Fig. 2). Further, the data shows opposite response in the case of miR-214 inhibitor treatment that leads to the minute up regulation of miR-29a and subsequent reduction in the expression of its target gene DRP1 (Fig. 3). The data presented here showed non significant   alteration at expression level and this does not imply that specific miRNA inhibitor strongly attenuates the other miRNAs and respective targets. Our recent studies showed that miR-15a and miR-214 inhibitors treatment leads to significant inhibition of their target miRNAs and enhanced the expression of respective genes PUMA and ARC by many folds [1].

Culturing of cardiomyocytes
Neonatal Sprague Dawley rats were used for the current study and animal handling procedures were strictly under the guidelines and regulation of Bio-Ethics Committee of Quaid-i-Azam University Islamabad Pakistan. Neonatal rat's primary cardiomyocytes culturing was performed as described by Murtaza et al. [5]. Briefly, the cardiomyocytes were digested in 0.1% trypsin containing 0.02% EDTA in PBS and collagenase pancreatin mixture. After blocking the enzymatic activity, the harvested cells were re-suspended in complete medium containing 100 U/ml penicillin, 100 µg/ml streptomycin. Cardiomyocytes were separated from other cells of the heart tissues as fibroblasts and RBCs. The cardiomyocytes achieved in suspension were seeded on gelatin or polylysine coated plates. Total seeding density for cardiomyocytes was 1 × 10 6 cells per well of 6 well plates.

miRNA inhibitors treatment
Cardiomyocytes were transfected with inhibitors of miR-15a and miR-214 (IDT Lowa, USA) by Calcium chloride transfection and Glycerol shock procedure as described by Mortensen et al. [6]. Total concentration of miRNA inhibitors was 10 nM and incubation time was 48 h.

Quantitative reverse transcription PCR
miRNAs and target genes expression was performed by quantitative real-time polymerase chain reaction (qRT-PCR) [7]. Briefly, total RNA was extracted with Trizol (Invitrogen, UK), and expression of target genes was quantified using a SYBR Green qRT-PCR Kit (Thermo Fisher Scientific, USA). qRT-PCR primers were from Thermo Fisher Scientific. For miRNAs, stem-loop qRT-PCR was performed [8]. Following DNase treatment, RNA was reverse transcribed. qPCR was performed by syber green master mix on the real Time PCR (Thermo fisher, USA). Gene expression was normalized to that of GAPDH or U6 for mRNAs and miRNAs, respectively. The expression of target genes relative to the control was determined using the 2 −ΔΔCT method [7].

Statistical analysis
Present data were expressed as mean value 7 S.D by employing One-way analysis of variance (ANOVA) followed by the Tukey's. Data analysis was carried out by the SPSS (Chicago, IL, USA) 13.0 software. P value less than 0.05 was considered statistically significant.