miR-106b regulates the reprogramming of spermatogonial stem cells into iPSC-like cells

Background Recent years have brought notable progress in raising the eciency of the reprogramming technique, so that approaches have evolved from known transgenic factors to only a few microRNAs. Nevertheless, there is a poor understanding of both the key factors and biological networks underlying this reprogramming. Therefore, the present study aimed to investigate the potential of miR-106b in regulating Spermatogonial stem cells (SSCs) to iPSC-like cells. We used SSCs because pluripotency can be induced in them under dened culture conditions with fewer issues compared to other adult stem cells. Methods As both signaling and post-transcriptional gene control are critical for the regulation of pluripotency, we traced the expression of Oct-4, Sox-2, Klf-4, c-Myc, and Nanog (OSKMN), and studied miR-106b targets using bioinformatic methods. Results Our results showed that transfected SSCs with miR-106b increased expression of the OSKMN factors, and this expression in iPSC and induced SSC groups was signicantly more than negative control groups. Moreover, using the functional miRNA enrichment analysis, online tools, and databases we predicted that miR-106b targeted a signaling pathway gene named MAPK1/ERK2, which regulates stem cell pluripotency. Conclusions Together, these data suggest that miR-106b regulates reprogramming of SSCs into iPSC-like cells by targeting the ERK2 gene as a part of the regulatory network that controls the pluripotency state and reprogramming process.


Abstract
Background Recent years have brought notable progress in raising the e ciency of the reprogramming technique, so that approaches have evolved from known transgenic factors to only a few microRNAs. Nevertheless, there is a poor understanding of both the key factors and biological networks underlying this reprogramming. Therefore, the present study aimed to investigate the potential of miR-106b in regulating Spermatogonial stem cells (SSCs) to iPSC-like cells. We used SSCs because pluripotency can be induced in them under de ned culture conditions with fewer issues compared to other adult stem cells.
Methods As both signaling and post-transcriptional gene control are critical for the regulation of pluripotency, we traced the expression of Oct-4, Sox-2, Klf-4, c-Myc, and Nanog (OSKMN), and studied miR-106b targets using bioinformatic methods. Results Our results showed that transfected SSCs with miR-106b increased expression of the OSKMN factors, and this expression in iPSC and induced SSC groups was signi cantly more than negative control groups. Moreover, using the functional miRNA enrichment analysis, online tools, and databases we predicted that miR-106b targeted a signaling pathway gene named MAPK1/ERK2, which regulates stem cell pluripotency. Conclusions Together, these data suggest that miR-106b regulates reprogramming of SSCs into iPSC-like cells by targeting the ERK2 gene as a part of the regulatory network that controls the pluripotency state and reprogramming process.

Background
Growing evidence suggests that stem cell differentiation and reprogramming techniques are quickly expanding. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons for reprogramming technology (1,2). Indeed, iPSCs and all iPSC-like cells are indispensable for generation and banking because of their aptitude to give rise to any kind of differentiated cells and tissues. They increase the potential for personalized cell therapies and introduce notable prospects for regenerative medicine, iPSC-based drug screening, disease modeling, and toxicity assessment (3,4). However, somatic cells can generally be reprogrammed to iPSCs with less than 1% e ciency, as well as the clinical application of iPSCs has been laden with some issues (5). In this regard, SSCs showed can be converted into pluripotent stem cells with fewer concerns in contrast to other adult stem cells. The SSCs are postnatal germline stem cells in the testis capable of differentiating into sperm cells and can also be reprogrammed under characterized culture conditions (6,7). Recently, SSCs have been demonstrated to be reprogrammed into multipotent SSCs (mSSCs) and pluripotency can be induced in homogeneous SSC populations without other cells. Unlike pluripotent stem cells (PSCs) with several issues, such as tumorigenicity and ethical concerns, these mSSCs can function as a pluripotent stem cell source free of the aforementioned problems (8,9).
On the other hand, various substantial functions have recently emerged for microRNAs (miRNAs), singlestranded noncoding small RNAs, in the regulation of pluripotency and lineage speci cation (10,11). miRNAs control protein synthesis by targeting mRNAs for translational repression or degradation at the posttranscriptional level. These molecules are phylogenetically conserved and have been validated to be in uential in a wide assortment of core biological procedures, including embryogenesis and support of "stemness" among others (12). Undoubtedly, the modulation of key pluripotency factors is a critical mechanism affecting the reprogramming e ciency. Several miRNAs are discovered to be important regulators of stem cells, which modulate the expression of the transcription factors OSKMN, leading somatic cells to a pluripotent state (13,14). Furthermore, the speci c identi cation of miRNA targets will help us to understand the functional role of miRNAs in PSCs. In this eld, Mei Y et al. showed the miR-21 expression pattern was highly correlated with MAPK/ERK activity during mesenchymal stem cell (MSC) differentiation (15,16).
According to the features of SSCs and current studies mentioned above, we hypothesized certain miRNAs in advance as post-transcriptional regulators of SSC reprogramming. In this investigation, we selected miR-106b, a member of the miR-106b-25 cluster, because our primary bioinformatic analysis revealed that this miRNA is one of the few which affects the genes of signaling pathways regulating pluripotency of stem cells. Besides, in iPSC, the miR-106b-25 cluster is induced in early reprogramming phases and restraint of this cluster decreases the reprogramming e ciency. TGFBR2 and CDKN1A (p21) are also targets of miR-106b which have already been related to iPSC induction (17). Based on these ndings, the present study was undertaken to evaluate the impact of miR-106b in cell reprogramming of SSCs to iPSClike cells by detecting the OSKMN expression. We also studied the miR-106b targets using bioinformatic methods to nd out more about parts of signaling involved in the SSCs reprogramming.

Animal housing
Adult male Wistar rats, 2-2.5 months old weighing 200-300g, were used in this study (n=6). These mice had free access to food and water ad libitum and were housed under a 12-h light-dark cycle at a stable temperature and humidity-controlled room.

Isolation and identi cation of SSCs
First of all, the testis tissue sections were excised from healthy groups, and isolation of SSCs was conducted as previously described (18). Secondly, the immunocytochemistry method was performed to anatomically visualize the localization of the promyelocytic leukemia zinc nger protein (PLZF) in the SSCs derived colonies after 7 days of culturing. The protocol of this technique was also explained earlier (18).

Preparation and transfection of hsa-mir-106b plasmids
The pLV-miRNA vector, comprising hsa-mir-106b lentivirus and co-expressing GFP protein in infected E. coli BL21 was purchased from Biosettia Inc. (mir-p081, Biosettia, San Diego, CA, USA). Transfection of SSCs was done by 2.5 μg of the pLV-miRNA vector. For this reason, mouse SSCs (1.0 ×10 6 ) were seeded in a 6-well plate before transfection so that the cell density was around 70%-90%. Gently, 500 μl of culture medium and 7.5 μl of Lipofectamine3000 (Invitrogen, USA) solution were mixed and incubated for 10-15 minutes at room temperature. Meanwhile, 2.5 μg of the pLV-miRNA vector was added to 500 μl of the medium. Then, 250 ml of the produced solution was added to each well and the cells were incubated at 37 ° C for 2-4 days. In order to con rm the transfection e ciency and observe the expression of the GFP gene under a uorescence microscope, the quantitative real-time reverse transcriptase PCR (qRT-PCR) technique was used. For this purpose, the sequences of forward and reverse primers, mir-106b, and U6 snRNA (as a reference gene) were downloaded from the www.ncbi.nlm.nih.gov/Gene website and designed using GeneRunner software ( Table 1). The cDNA was synthesized according to the manufacturer's protocols (Fermentas, USA) and the products were analyzed by electrophoresis in 1% agarose gel with a DNA ladder (1kb).

Characterization of SSCs
PLZF, also known as ZBTB16 (Zinc Finger And BTB Domain Containing 16) is a consensus marker for undifferentiated spermatogonia (20). Immunocytochemistry analysis illustrated the expression of this factor in the colonies derived from the cultured cell suspensions (Fig.1).

Con rmation of transfected cells
In order to corroborate the transfection of SSCs with Mir control and the expression vector containing miR106b, the cells were observed under a Phase-contrast microscope after 48 hours. The expression of GFP protein as a reporter gene was con rmed the transfection of the cells (Fig.2).

Gene expression signatures of pluripotency and miR-106b
Real-time PCR was utilized to determine the expression of miR-106b and OSKMN, a subset of pluripotency markers, in the all group study. The ndings con rmed that the SSCs remarkably were transfected with miR-106b (p-value < 0.01). The mean expression of the OSKMN genes in iPSC and induced SSC groups was signi cantly more than negative groups. Tukey's multiple comparison test of the results veri ed that the iPSCs expressed the aforementioned genes more than the SSC and Mir control groups (p-value < 0.01). Furthermore, the difference in expression of OSKMN genes between induced SSC and iPS groups was not signi cant (Fig.3).
miR-106b target MAPK1/ERK2 to regulate pluripotency The functional miRNA enrichment analysis revealed the miRNAs that target the genes of SPRPSCs (Target score: 50 % ≥). Among the miRNAs found via FunRich, miR-106b was selected based on its demonstrated potential. As the scalable Venn diagram illustrated, miR-106b targeted 7 common genes, and consequently comparing these common genes and the genes of SPRPSCs showed that miR-106b targeted ERK2 gene (Fig.4) (Table 3). In addition, the Heatmap image exhibited different expressions of ERK2 in the human proteome map, especially in the fetal and adult testis (Fig.5). Predicting PPI network of the common genes To gain a further understanding of the association among proteins of the common genes targeted by miR-106b, we constructed a PPI network using the STRING database. The interaction network presented no interaction between the proteins, but molecular function (GO) analysis in the database showed that PAK3 also enriched for Mitogen-Activated Protein (MAP) kinase activity (p-value: 9.50e-04) (Fig.6).

Discussion
Over the past years, much effort has been directed towards screening for small molecules to improve reprogramming e ciency and create new methods for iPSC derivation. miRNAs have played a key role in regulating pluripotency and lineage speci cation by modulating gene expression at the posttranscriptional level, resulting in iPSCs generation from various cell types (21)(22)(23)(24)(25). As a preliminary proofof-concept, the present investigation indicates that miR-106b is a major factor involved in reprogramming SSCs into iPSC-like cells. In light of the results, the SSCs infected with miR-106b expressed a subset of pluripotency markers (OSKMN) approximately in the same amount of iPSCs. In addition, the bioinformatic analysis in this study predicted that miR-106b targeted ERK2, a gene of signaling pathways that regulates stem cell pluripotency.
Transcriptional networks involving a set of pluripotent transcription factors control and sustain the pluripotency of stem cells. These pluripotent genes stimulate or suppress downstream gene expression, inducing the event of some signaling pathways and regulating the pluripotency of stem cells. As previously reported, signaling and post-transcriptional gene control are both important for pluripotency regulation, but it remains poorly known how they are incorporated to affect cell identity. In pluripotent cells, phosphorylation as a pervasive form of cell signaling plays a crucial role in controlling cell identity by relaying signaling of the growth factor via key pathways. The ERK2 is one of the best-characterized MAP kinase pathways that phosphorylates Klf4, OCT4, SOX2, and NANOG (26)(27)(28)(29)(30). For instance, The nuclear export of KLF4 requires ERK activation and the phosphorylation of KLF4 by ERK commences the interaction of KLF4 with the nuclear export factor XPO1, resulting in the export of KLF4. Mutation of ERK phosphorylation site in KLF4 prevents KLF4 nuclear export, decreases in mRNA of Nanog, Klf4, and Sox2, and differentiation (31). Based on these ndings and the results found in this investigation, it can be inferred that ERK2 activation controlled by miR-106b initiates reprogramming of SSCs to a pluripotent state.
Since Yamanaka and his colleagues rst generated iPSCs in 2006, their clinical applications have been laden with some issues such as tumorigenicity (32). The different level of c-Myc expression that has been linked to the risk of tumorigenesis is one of the variables that hinder the therapeutic application of iPSCs. Although c-Myc alone could induce miR-106b∼25 cluster, it has been reported that c-Myc activates as a proto-oncogene causing several cancers (33)(34)(35)(36). Here, we highlight that the induced SSCs express less c-Myc than iPSCs, so these iPSC-like cells have the potential to be a considerable alternative as a pluripotent stem cell source. Obviously, pathology experiments need to be conducted in the future to provide a remarkable understanding of the tumorigenicity effects of the induced SSCs.

Conclusions
Based on these ndings, the SSCs can be considered as a noticeable candidate for cellular reprogramming strategies. Moreover, miR-106b was also found to be a key molecule in the SSCs reprogramming that upregulates pluripotency-associated factors. Together, our results suggest that miR-106b regulates the reprogramming of SSCs into iPSC-like cells by targeting the ERK2 gene.   Molecular assessment. Analysis of miR-106b and OSKMN expression in the experimental groups. Signi cant differences: ***P ≤ 0.001; **P ≤ 0.01 (Error bars represent ±SD).