Exogenous miR-29b Reduces DNA Methylation and Apoptosis in Transgenic Cells

MicroRNAs (miRNAs/miR) are a class of short, non-coding, single-stranded, 22-24 nucleotides long RNAs, that affect transcriptional and post-transcriptional regulation of gene expression (Rashmi et al., 2019). Emerging evidence shows that more than hundred miRNAs are regulated by epigenetic mechanisms and about one-half of them are modulated by DNA methylation (O’Brien et al., 2018). Epigenetic regulates gene activity that does not involve a change in the DNA sequence however, there are modifications to the chromatin (Humphries et al., 2019). DNA methylation does not always take place alone but often occurs in the presence of other epigenetic modifications, such as histone modification, which constitutes the second major epigenetic regulatory system of miRNAs. DNA methylation at the fifth carbon of cytosine residues in cytosine phosphate guanine (CpG) and non CpG dinucleotide sites is a key mechanism for epigenetic modification in eukaryotes. During gametogenesis and early embryogenesis, global DNA methylation patterns are dynamically reprogrammed (Gruber and Zavolan, 2013). This process is established and maintained by DNA methyl transferases. DNMT 3A/3B enzymes are accountable for de novo methylationand DNMT1 is responsible for the maintenance of methylation (Garzon et al., 2009). Abnormal activities of these enzymes can be detrimental to the fetal development (Song et al., 2017).
       
miRNA expression is tissue-specific and depends on the cellular context, histone modification might regulate distinct subpopulations of miRNAs in different of cell types. Expression of miRNAs is tightly regulated in a developmental-stage-dependent, as well as in an organ-dependent manner (Kloosterman and Pasterk, 2006; Takada et al., 2006). These observations suggest the existence of a regulatory circuit between epigenetic modulation and miRNAs, which could have a significant effect on transcription. Mature miRNAs form part of an active RNA-induced silencing complex (RISC) containing dicer and many associated proteins. This complex acts as a regulator of numerous biological processes by either triggering the degradation of the target mRNAs or suppressing their translation through incomplete base-pairing to the 3’ untranslated region (O’Brien et al., 2018). Although the functioning patterns and target genes of most miRNAs are still unknown. Diverse miRNAs have been reported to participate in processes such as embryogenesis, embryonic development, stem cell pluripotency, differentiation, organogenesis, growth, cell proliferation and apoptosis (Gilchrist et al., 2016). Recently, there is an increasing interest in miRNAs and their role in gene expression patterns. Several miRNAs presumably influence pre-implantation embryo development through DNA methylation (Sah et al., 2020) and suggested that miRNA 29b participates in DNA methylation in different cells by regulating DNMT3A/3B expressions (Liang et al., 2018). miR-29b is also involved in the regulation of DNA methylation during reprogramming and embryogenesis (Singh et al., 2019). However, to the best of our knowledge, there are no reports available on the effects of miR-29b (mimics and inhibitors) on transgenic cells containing the human insulin gene. Therefore, the present study aims to investigate the effect of this miRNA on cultured buffalo fibroblast cells on the expression of some genes related to -epigenetics and -apoptosis.

All the media and chemicals utilized in this study were procured from Sigma Chemical Co. (USA) and the plasticware was obtained from Nunc (Denmark), unless otherwise mentioned. Fetal bovine serum (FBS) was acquired from Gibco Life Technologies (USA).
 
Establishment of buffalo fetal fibroblast (BuFF) cell line
 
Female buffalo fetus obtained from slaughterhouse-based animal, was washed twice with normal saline fortified with antibiotics (gentamicin sulfate and penicillin-streptomycin). Then surface of the fetus was washed with 70% ethanol followed by several washings with antibiotic-fortified normal saline. Ear pinna tissue was washed 4-6 times with DPBS containing 50 μg/mL gentamicin sulfate. The biopsies were then cut with the help of a surgical blade into small pieces (~1 mm³) which were then again washed 3-4 times with DPBS followed by the cell culture medium (DMEM supplemented with 2.0 mM L-glutamine,1% non-essential amino acids, 20% FBS and 50 μg/mL gentamicin sulfate). The explants were cultured into tissue culture flasks in a CO₂ incubator (5% CO₂ in air) at 37°C. The media was then replaced with a fresh medium every 3^rd day until the fibroblast monolayer attained 30-40% confluence. Then cell monolayer was washed with DPBS and partially detached by trypsin-EDTA (0.25%) for 2 min to ensure fractional detachment of the fibroblasts while other cells, especially epithelial cells, remain attached to the culture flask. The sub-cultured cells were seeded in a new culture flask and anchored cells were allowed to grow till confluency. For characterization, passage- 3 cells were fixed for 1 h in 4% paraformaldehyde, permeabilized with 0.5%. Triton-X-100 for 20 min and blocked in 3% bovine serum albumin for 1 h. After blocking, cells were incubated for 1 h with primary antibody (mouse anti-cytokeratin, 1: 500 or anti-vimentin 1:500) diluted in blocking solution then with secondary antibody (goat anti-mouse IgG, 1:1000) conjugated with fluorescein isothiocyanate (FITC) for 1 h. Positive control was performed by incubating cells with mouse β-tubulin (1: 500) whereas for negative control the cells were incubated with secondary antibody only. Finally, cells were counterstained with Hoechst-33342. Fluorescence signals were detected with a fluorescence microscope (Nikon, Japan).
 
Transfection of buFF cells with pAcISUBC vector and their enrichment
 
An expression vector “pAcISUBC” was earlier constructed, had human insulin (hINS) gene beta-lactoglobulin (buBLG) promotor and buBLG 3’ UTR into pAcGFP-N1 (Clontech Laboratories Inc, USA) vector backbone (Kaushik et al., 2014). Buffalo fetal fibroblast cells were transfected by nucleofection (AMAXA Biosystem, Germany) with pAcISUBC plasmid containing hINS gene. Transfected cells were cultured for the first 24 h in the culture medium containing 800 µg/mL G418 in 4 well culture plates with a change of medium every 48 h for 2-3 weeks to obtain transfected cell colonies. After 2-3 weeks of enrichment of transfected cells, transgene integration was confirmed by PCR amplification of hINS gene fragment as well as GFP expression was observed under the fluorescence microscope (Mehta et al., 2019).
 
Exogenous miR-29b treatment to transgenic BuFF cells
 
Transgenic buFF cells (10⁵/well) were seeded in a 6-well plate 24 h before transfection. Transfection of miRNAs was done with lipofectamine-3000 (Invitrogen, USA), according to the manufacturer’s protocol. Briefly, miR-29 b mimics, inhibitorand scramble sequence (Ambion, USA), individually diluted to 40 nM in serum-free Opti-MEM and lipofectamine 3000 (10 µL/250 μL Opti-MEM) were incubated at room temperature for 15 min. After incubation, the two solutions were mixed thoroughly, incubated for 20 min and added to the cultured transgenic BuFF cells and kept in CO₂ incubator (at 37°C, >95% RH and 5% CO₂ in air). Thereafter, fresh complete medium was added following 4 h of incubation.
 
Expression of epigenetic and apoptosis-related genes
 
The miR-29b treated transgenic cells were harvested 48 h later from all three groups (miR-29b mimic-treated, miR-29b inhibitor-treatedand untreated control) and the effect of miR-29b was studied on some epigenetic-related (DNMT1, DNMT3A, DNMT3B, HDAC1) and apoptosis-related (MCL-1, BCL-XL) genes by qPCR. For this, total RNA isolation was performed using the Single Cell RNA Purification Kit (NORGEN, Canada) following the manufacturer’s protocol and cDNA was synthesized using SuperScript III Kit (Invitrogen, USA). Briefly, the reaction mixture consisted of 100 ng RNA, 1 µL oligo dT, 1 µL 10 mM dNTP mix, 1 µL random primers and 10 µL DNase-/RNase-free water. The mixture was incubated at 65°C for 5 min, followed by a cooling step on ice for 3 min. A master mix containing 4.5 µL of 5X First Strand Buffer, 1 µL of 0.1M DTT and 0.25 µL (50 U) of SuperScript III RT was added. The reaction was performed using the following program: 25°C for 5 min, 50°C for 60 minand 70°C for 15 min. Subsequently, the cDNA was diluted 1:4 (v:v) with nuclease-free waterand gene amplification was carried out using Maxima SYBR Green Master Mix (Fermentas, USA) along with primer sets (Table 1). The thermal cycling conditions consisted of an initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s annealing at 60°C for 30 sand extension at 72°C for 30 s. The expression levels of the target genes were normalized using internal control genes, with GAPDH and Β-TUBULIN (Shyam et al., 2020).
 

 
Statistical analysis
 
Statistical analysis was performed using Graph Pad Prism 7 software. One-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s test was used to analyze the data and Student’s t-test was employed for comparing the means of different groups. The presented data represent the mean value along with the standard error of the mean.

Establishment and characterization of buffalo fetal fibroblast (BuFF) cells
 
The fibroblast cells from cultured ear tissues started to migrate after 3-5 days of culture (Fig 1A). When these cells were reached 30-50% confluency, then sub-cultured by partial trypsinization and cells were transferred to a new culture flask (Fig 1B). At passage-3, these cells showed the expression of vimentin (marker of fibroblast) and β-tubulin (positive control) while no expression was observed for cytokeratin-8 or cytokeratin-18 (epithelial cell marker), which revealed that cultured cells were fibroblast cells and had no cross contamination with epithelial cells (Fig 2).
 

 

 
Transfection of BuFF cells with pAcISUBC vector and their enrichment
 
Buffalo fetal fibroblast cells were transfected with pAcISUBC vector containing hINS gene and transfection efficiency was 5.15±0.49% and 13.31±0.48%, respectively observed by lipofectamine-3000 and by nucleofection using Lonza buffer (Fig 3). After the nucleofection, transfected cells were grown in selection media containing geneticin (800 µg/ml), only transfected cells survived in selection media. After 15-20 days of selection, a pure population of transgenic cells was obtained (Fig 4). Transfected cells showed normal morphology and GFP expression. RT-PCR of these cells showed a 275 bp human insulin gene fragment amplification, indicating transgene integration in cells in genome (Fig 5).
 

 

 

 
Exogenous miR-29b treatment to transgenic BuFF cells
 
After geneticin selection, transgenic cells were transfected with miR-29b mimics, inhibitor and scramble sequences. On culture, all three groups of cells showed normal morphology and growth patterns. Transfection of miR was confirmed by red fluorescence produced through TAMARA (5-carboxytetramethylrhodamine) dye tagged with scramble sequences (Fig 6).
 

 
Effect of miR-29b on gene expression in transgenic cells
 
Transgenic cells treated with miR-29b mimic, inhibitor and control were harvested after 48 h of treatment and total RNA was isolated and c-DNA was synthesized. The expression of DNMT1, DNMT3A and DNMT3B were significantly decreased (P<0.05) in miR-29b mimic treated transgenic cells, while in inhibitor-treated cells, the expression was significantly increased (P<0.05). The expression HDAC1 gene had no significant difference (P<0.05) when treated with miR-29b mimic, whereas miR-29b inhibitor treatment showed a significant increased expression (P<0.05) as compared to control (Fig 7). The expression of apoptosis-related genes BCL-XL and MCL-1 were increased significantly (P<0.05) in miR-29b mimic-treated cells, while in miR-29b inhibitor-treated cells showed significantly reduced (P<0.05) MCL-1 expression, whereas, no significant change (P<0.05) in BCL-XL expression as compared to their control (Fig 8).
 

 

       
The present study aimed to investigate the role of miR-29b mimic and inhibitor on transgenic buffalo fetal fibroblast cells (containing human insulin gene). miR-29b has been reported to reduce DNA methylation by regulating DNMTs in several types of cells (Zhang et al., 2015; Wu et al., 2022). Fabbri et al., (2007) reported that miR-29b targets both DNMT3A and DNMT3B in lung cancer leading to a reversal of aberrant DNA methylation. It also targets DNMT3A and DNMT3B directly and DNMT1 indirectly to induce tumour suppressor gene repression and global DNA hypomethylation in acute myeloid leukemia (Garzon et al., 2009). Takada et al., (2009) reported their role in the regulation of genomic DNA methylation in mouse primordial germ cells by targeting DNMT3A and DNMT3B. Treatment with miR-29b mimic and inhibitor modifies the epigenetics status of cloned embryos by decreasing the DNA methylation in different farm animals i.e. bovine (Liang et al., 2018), buffalo (Singh et al., 2019) and pig IVF embryo (Zhang et al., 2018). Singh et al., (2019) reported that significantly lower expression of DNMT3A and DNMT3B, but not that of DNMT1 were observed in buffalo blastocysts produced through SCNT in the mimic treatment group, compared with untreated controls. Similar results have been reported in another study in bovine SCNT blastocyst (Liang et al., 2018). In mouse embryos also reported that miR-29b reduces the DNMT3A and DNMT3B expression and was the direct target gene of miR-29b (Movahed et al., 2019). Our results agreed with these studies and suggest that treatment of transgenic cells with miR-29b mimic an effective approach for reducing DNA hypermethylation in transgenic cells. Due to global hypomethylation, plentiful gene expression is affected and cells may proliferate and maintain viability.
       
Apoptosis is also an important parameter for cellular health, proliferation and quality of the transgenic cell line. Flavin et al., (2009) reported that miR-29b treated hepatocellular carcinoma cells significantly reduced the expression of the anti-apoptotic genes BCL-2 and MCL-1, while in non-cancerous cells miR-29b plays the opposite role, inhibiting apoptosis in mammary epithelial cells (Yang et al., 2016). In the present study, we found that the expression level of anti-apoptotic genes BCL-XL and MCL-1 was significantly increased as compared to control when transgenic cells were treated with miR-29b. The expression level MCL-1 was significantly increased as compared to the control while BCL-XL expression had no significant changes. This showed that miR-29b might inhibit apoptosis during cell proliferation and development.

The present study indicates that miR-29b mimic reduces both DNA methylation and apoptosis by altering the gene expression profile in transgenic cells. Since transgenic cells are used in animal cloning as donor cells, thus the use of miR-29b mimic may improve nuclear reprogramming and cloning efficiency.

The authors declare no conflict of interest.