Global changes of miRNA expression indicates an increased reprogramming efficiency of induced mammary epithelial cells by repression of miR-222-3p in fibroblasts

Sample collection

Goat ear fibroblasts were preserved by the College of Animal Science and Technology, Guangxi University. Based on the former research results, goat ear fibroblasts were seeded into a 60-mm cell culture dish at a density of 5 × 105 cells per dish. Following 24 h, the medium was changed to “VTFBR” (500 μg/mL VPA, 10 μM Tranylcypromine, 10 μM Forskolin, 1 μM TTNPB, 10 μM RepSox), a cocktail induction medium based on N2B27 and small molecule chemicals (Zhang et al., 2021). The induction culture was maintained for 8 days with medium changes every 2 days.

Creating small RNA libraries

Total RNA was isolated from reprogrammed iMECs cells using TRIzol reagent at the predetermined time periods (the initiating GEFs (0 day) and the day 8 post-induction iMECs), applying three replicates for each time point, and then pooled together for sequencing in a total of six libraries. A total of 1% gel electrophoresis was employed to detect total RNA whether degradation. Using 8% polyacrylamide gel electrophoresis, whole RNA was separated into fragments between 15 and 35 nt, and then the fragments were linked with proprietary adapters. Reverse transcription was used to create complementary DNA (cDNA) in order to create the libraries for sequencing. Using Illumina HiSeq to perform RNA sequencing with the SE50 sequencing strategy (Hafner et al., 2008).

Data analysis

Clean reads for subsequent analysis were obtained from raw bases corresponding to the sequenced reads by removing joints, removing low quality, and selecting fragments (Lindgreen, 2012). Clean reads in the genome were matched with miRNAs localization information (100% overlap) (Quinlan & Hall, 2010). Using miRDeep2 predicted novel miRNAs whose sequences have been discovered. Normalization was performed for each sample to obtain the trimmed mean of M-values (TMM). DESeq2 was used to identify differentially expressed miRNAs. When |log₂FC| > 1, padj < 0.05 and Cohen’s d > 1, the miRNAs were considered as differentially expressed (Quinlan & Hall, 2010).

MiRNAs enrichment analysis

GO and KEGG analyses of differentially expressed miRNAs were performed using the miRPath V4.0 database (Tastsoglou et al., 2023).

Cell transfection and induction

Goat ear fibroblasts were inoculated into 6-hole plate at a density of 2 × 105 cells per dish, and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal bovine serum at 37.5 °C in a humidified atmosphere of 5% CO₂ and 95% air. Cell transfection was performed when cell confluence reached 60%. The ransfected cells were divided into miR-222-3p mimic group, miR-222-3p mimic-NC group, miR-222-3p inhibitor group, and miR-222-3p inhibitor-NC group. Afterwards, lipofectamine TMRNAiMAX and each group of miRNA fragments were transfected into cells as described in the manufacturer’s protocol, and three biological replicates of each group were performed. After 12 h, the medium was replaced with N2B27 and small molecule compounds cocktail induction medium “VTFBR” (500 μg/mL VPA, 10 μM tranylcypromine, 10 μM Forskolin, 1 μM TTNPB, 10 μM RepSox) at 37.5 °C in a humidified atmosphere of 5% CO₂ and 95% air. The medium was changed every 2 days, and the induction culture was continued for 4 days. Three biological replicates were performed for each group. Cell induction was observed under a microscope every 2 days and photographed.

Oil red O staining and quantification

Four groups of iMECs were transfected for 4 days, and the medium in the petri dishes was aspirated medium. Cells were subsequently fixed with 4% paraformaldehyde for 10 min and then stained with oil red O staining solution for 15 min. The cells were then rinsed with 60% isopropanol for 15–30 s, washed three more times with PBS, and observed under a microscope. Finally for quantitative analysis of lipid accumulation, lipids were washed off using 100% isopropanol and then absorbance at 510 nm was measured using a spectrophotometer. Three biological replicates of each group were performed.

Immunofluorescence staining

The cells were permeabilised with 1% Triton X-100 after fixation by 4% paraformaldehyde for half an hour at room temperature. Subsequently, they were closed with 10% donkey serum for 1.5 h. Primary antibodies were incubated with the cells overnight at 4 °C at the appropriate dilution in containment buffer. The next day, the cells were incubated with the respective secondary antibodies for 1 h in the dark at room temperature (Zhang et al., 2021). Photographic documentation using a microscope. Quantification of iMEC using ImageJ software. Three biological replicates were performed for each group.

Statistical analysis

Reprogramming efficiency, the quantitation of Oil Red O staining and immunofluorescence were carried out by one-way ANOVA in Graphpad Prism 8.0 analyzer. Data are presented as mean ± SEM of independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. The experimental data all have three biologically independent samples.

Introduction to sequencing data

Based on our past experimental results, six small RNA-seq libraries were constructed for high-throughput sequencing, with three biological repetitions at each time-point (GEFs and iMECs) (Zhang et al., 2021). For the six small RNA libraries, the averages of 13,372,156.33 raw data were obtained, and the averages of 11,180,154.50 clean reads were obtained (Table S1). Lengths were ranged from 18 to 30 nt, and the peak appeared at 22 nt which conformed to the law of animal small RNA (Fig. 1A). The Q20 values of the miRNA libraries were all higher than 97%. The length distribution of small RNAs in GEFs and iMECs follows the typical read distribution of animal miRNAs, mostly in the range of 22 nt. The clean reads were contrasted to the goat reference sequence (Table S2). Principal component analysis (PCA) (Fig. 1B) was run to cluster the samples based on the expression values and showed a significant difference before and after induction between GEFs and iMECs, the main difference lay in pc1. We also showed the density distribution to visually demonstrate the expression level of all the genes that were detected in the two samples (Fig. 1C). In conclusion, the miRNA libraries used in the current study had high enough data quality to be used for subsequent statistical calculations.

Differentially expressed miRNA and GO and KEGG enrichment analysis

All six miRNA libraries detected a total of 457 miRNAs, including 116 novel miRNAs and 341 known miRNAs. The miRNAs showed were shown differential expression patterns (Fig. 2A). Compared with the GEF libraries, 33 known miRNAs were upregulated in the iMECs, and 46 known miRNAs were downregulated with |log₂FC| > 1, −log10(p value) < 0.05 and Cohen’s d > 1 (Figs. 2B, 2C, Table S3). From the graph above we can see that, chi-miR-34c-3p, chi-miR-34b-3p, chi-miR-34b-5p, chi-miR-34c-5p, and chi-miR-141 were the most obviously upregulated, but chi-miR-100-3p, chi-miR-296-3p, chi-miR-155-3p, chi-miR-665, and chi-miR-487b-3p were the most significantly downregulated. This suggested that these miRNAs may play a key role during mammary gland lineage formation. Interestingly, the expression of all 34 family members was observed to be significantly elevated in iMECs.

Both GO and KEGG analyses were carried out to mine cellular biological processes and signaling pathways, respectively, in order to highlight the relevance of differentially expressed miRNAs in the reprogramming of GEFs to iMECs. The KEGG pathway analysis results indicated the involvement of these DE-miRNAs mostly in adherens junction, TGF-β signaling pathway, GnRH secretion and the prolactin signaling pathway, etc. (Fig. 2D). According to the significance level of GO term enrichment, cells before and after induction were mainly enriched in the molecular functional classifications of protein interactions, enzyme activities, transcriptional regulation, as well as classification of cellular components of cell structure, organelles, protein complexes and cellular transport. In addition, biological processes involved cell cycle, cellular response to DNA damage stimuli and protein phosphorylation (Fig. 2E). Furthermore, we observed that downregulated miRNAs were associated with some hormone pathways (Table S4) while upregulated miRNAs were associated with some EMT pathways (Table S5). An explanation for this might be that upregulated miRNAs maintained epithelial cell characteristics by suppressing EMT, and downregulated miRNAs gave hormonal signals that allowed them to become a mammary gland spectrum.

MiR-221-3p plays a pivotal role in the generation of induced mammary epithelial cells, thus highlighting its significant contribution to this process

We found that among the differentially expressed miRNAs, miR-222-3p was significantly higher than other miRNAs expressed in fibroblasts in sequencing data. Meanwhile miR-222-3P not only regulates mesenchymal-epithelial transition but also plays an important role in adipogenesis. Therefore to investigate the impact of miRNAs on reprogramming efficiency of induced mammary epithelial cells, we focused on miR-221-3p as our target. Prior to induction with five small molecule compounds (VTFBR), goat ear fibroblasts were transfected with either a miR-221-3p mimic or inhibitor to upregulate or downregulate miR-221-3p expression, respectively. Our results demonstrated that cultures treated with the miR-221-3p inhibitor exhibited an increased number of independent and compact epithelial cell-like colonies, whereas fewer colonies were observed in cultures transfected with the miR-221-3p mimic compared to controls at 4 days (Fig. 3A). These findings suggest that upregulation of miR-221-3p by the mimic severely impairs small-molecule compound-induced colony formation and significantly reduces reprogramming efficiency (Fig. 3B). Thus, our observations indicate that elevated expression of miR-221-3p may hinder fibroblast-to-iMEC transformation.

MiR-221-3p mediates the biological properties of induced mammary epithelial cells

Subsequently, to gain deeper insights into the impact of miR-222-3p on iMEC functionality, we assessed the expression levels of MEC specific proteins and milk fat synthesis. The secretion of milk lipids represents a crucial characteristic of iMECs, and our findings revealed that treatment with an inhibitor targeting miR-221-3p resulted in enhanced cytoplasmic lipid droplet accumulation as evidenced by saturated oil red O staining. Conversely, iMECs treated with a mimic for miR-222-3p exhibited significantly reduced secretion of periplasmic lipid droplets (Figs. 4A, 4B). Furthermore, immunofluorescence staining demonstrated elevated expression levels of mammary epithelial cell-specific antigens CDH1, CK8, and CK14 in iMECs treated with the miR-221-3p inhibitor (Figs. 4C, 4D).