miR-22-3p Suppresses Adipocyte Formation of Telomerase Transformed Mesenchymal Stromal Cells (iMSC3) by Regulating Non- targeted MAPK Signaling Pathway

Sara Sultan Alomran1, Muhammad Nasir Khan Khattak1,2, Amir Ali Khan1,2*, Sallam Hasan Abdallah2, Abeer Maher Fayyad1 and Khalid Bajou1,2 1Department of Applied Biology, University of Sharjah, Sharjah, United Arab Emirates 2Human Genetics and Stem Cells Research Group, Research Institute of Sciences and Engineering, University of Sharjah, Sharjah, United Arab Emirates Article Information Received 27 November 2019 Revised 04 February 2020 Accepted 20 February 2020 Available online 09 September 2020


INTRODUCTION
O besity is a chronic disorder with increasing worldwide prevalence due to lack of physical activity and imbalanced diets. It leads to other diseases including type 2 diabetes mellitus, insulin resistance and cancer (Noriyoki et al., 2011;Garg et al., 2014;Yang et al., 2016). It is characterized by increase in fat mass either by enhanced adipocyte size or number or a combination of both (Wang et al., 2014). There are two types of adipose tissue, white adipose tissues (WAT) and brown adipose tissues (BAT). WAT is responsible for energy storage (Noriyoki et al., 2011) while BAT is responsible for burning fat and generation of energy (Ruiz-Ojeda et al., 2016). Both types of adipose tissue are formed from the differentiation of mesenchymal stem cells (MSC); this differentiation is regulated by complex gene regulation and interaction of different signaling pathways. The differentiation process consists of MSC differentiation into lipoblasts, then pre-adipocytes and finally mature adipocytes (Gregoire et al., 1998).
In order to treat obesity and its related disorders, the molecular mechanisms involved have to be elucidated. MicroRNA (miRNA) roles in obesity and obesity-related disorders have been of great interest in recent years (Ortega et al., 2013). miRNAs are small conserved non-coding RNA molecules that regulate mRNA by transcript degradation or inhibition of translation. Hence, they act as key regulators in diverse biological processes such as proliferation, apoptosis and cellular differentiation (Sousa-nunes and Somers, 2013). Furthermore, miRNAs regulate energy balance and homeostasis in adipose tissue by controlling several metabolic pathways (Hartig et al., 2015). MicroRNA miR-22-3p, located on 17p13.3 in human chromosome, was found to play a role in the differentiation of mesenchymal stem cells into neural progenitor cells (Huat et al., 2015). This study found that introduction of insulin growth factor (IGF1) enhanced the quality of bone marrow MSCs (BMSCs) derived from neural progenitor cells (NPCs). Microarray analysis has also revealed that introduction of IGF1 enhanced survivability and cell proliferation of NPC by down regulating key miRNAs. Among these, miR-22-3p was also downregulated (Huat et al., 2015). Furthermore, miR-22-3p has been studied as a tumor-suppressor in many types of human cancers (Luo et al., 2017) and in cellular senescence in breast cancer cells (Xiong et al., 2010).

O n l i n e F i r s t A r t i c l e
Signaling pathways are involved in cell differentiation processes and early development. MAPK and PI3-K signaling pathways play essential roles in adipocyte differentiation and obesity, especially early stem cell proliferation stages such as differentiation and adipogenesis (Xiong et al., 2010;Han et al., 2017). Our previous study showed that rosiglitazone enhances adipogenesis, particularly brown adipogenesis via the activation of PI3-K and MAPK pathways during the differentiation of telomerase-transformed mesenchymal stem cells into adipocytes (Fayyad et al., 2019).
A previous study has shown that human adipose derived mesenchymal stem cells (hADMSC) transfected with miR-22 inhibited lipid droplet formation and suppressed the expression of adipogenic transcription factors and specific genes (Huang et al., 2012). However, literature is lacking on the effect of miR-22-3p on telomerasetransformed mesenchymal stromal cells (iMSC3) during adipogenesis. Moreover, the effects of miR-22-3p during iMSC3 cell differentiation into adipocytes and regulation of the PI3-K and MAPK signaling pathways have not been elucidated yet. As mentioned earlier, our previous study reported that MAPK pathway activation is associated with enhanced adipogenesis (Fayyad et al., 2019). However, this pathway is not directly targeted by miR-22-3p but it is cross linked with the PI3-K pathway (Castellano and Downward, 2011;Aksamitiene et al., 2012). Hence, in this study, the inhibitory role of miR-22-3p in the in-vitro differentiation of iMSC3 was investigated. We also studied the expression of MAPK and AKT proteins during the differentiation process in order to check if these pathways exhibited inhibitory or stimulatory roles.

MATERIALS AND METHODS
All methods were performed as described previously (Fayyad et al., 2019).

Transfection and differentiation to adipocytes
For iMSC3 differentiation into adipocytes in the presence of miR-22-3p mimic, 250x10 3 cells were seeded onto a 6-well plate. After 24-48 h, cells were transfected at 60-80% confluency (Fig. 1). iMSC3 cells were treated in three groups, group 1 was transfected with hsa-miR-22-3p mimic (MC10203, Thermo-fisher scientific, USA) (miR-22-3p); group 2 was transfected with non-target miRNA sequence (4464058, Thermo-Fisher Scientific, USA) (negative control); group 3 was differentiated with no transfection as control. The cell cycle was synchronized on the day of transfection using media lacking Pen/Strep and FBS for cells (Zhenhui, 2012). The differentiation cycle started 24 h following transfection and serum starvation. Three differentiation cycles were carried out and each differentiation cycle lasted for four days. iMSC3 cells were differentiated into adipocytes in induction medium (DMI) [0.25μM/ mL Dexamethasone (Sigma-Aldrich, USA), 0.1μM/ mL Indomethacin (Sigma-Aldrich, USA), 0.5μM/m L3isobutyl-1-methylxanthine (Sigma-Aldrich, USA), and 1 μM/mL insulin] for 48 h. Subsequently, cells were washed with PBS and maintained in maintenance medium (DMII) of 1 μM/mL insulin for 48 h. The three differentiation cycles of induction and maintenance treatment lasted for 14 days. Cells were harvested for protein analysis at two time points: first cycle of differentiation (after 4 days) and at the third cycle (at the end of differentiation). The differentiation method utilized was similar to a previous study with some modifications (Zebisch et al., 2012).

Fixation of cells for staining
Cells were washed twice with PBS and then incubated with 2 ml cold 4% paraformaldehyde for 10 min. Paraformaldehyde was discarded and replaced with fresh 2 ml cold 4% paraformaldehyde, following which the cells were incubated for 1 h at room temperature.

Nile red staining
In Nile red staining, Nile red was used to stain fat droplets and DAPI to stain the nucleus of living cells. The staining method utilized was taken from a previous study with some modifications (Greenspan et al., 1985). Nile red stock solution was prepared in acetone (1 mg/ml), with the stain solution tightly sealed and protected from light at 4°C. Working Nile red solution was prepared by diluting stock solution to 100x using trizma-maleate (1 mM) (Sigma-Aldrich T3128, USA) and polyvinylpyrrolidone (3% w/v) (Sigma-Aldrich P2307, USA). Fixed cells were stained with 1:100 Nile red stain and DAPI stain (Life Technologies P36930, Eugene, OR, USA). Fat droplet morphology in cells was observed under fluorescent microscope using Cellsens standard software.

Oil red O staining
Oil red staining of fixed cells was performed to stain fat droplets in differentiated cells. The staining method was taken from a previous study with some modifications (Aguena et al., 2012). The derived adipocytes cells were washed twice with PBS, 2ml of cold 4% paraformaldehyde was added and the cells were incubated for 10 min at room temperature. Later, the solution was replaced with fresh 2 ml of 4% cold paraformaldehyde and incubated for 1 h at room temperature. Following incubation, paraformaldehyde was removed, and cells were washed twice with distilled water. 2 ml of 60% isopropanol was then added to cells for a further 10 min incubation period at room temperature. Cells were air-dried completely. Oil red O stain working solution (Sigma-Aldrich 1391, USA) was prepared by making 60% Oil red O stock solution and adding 2 ml of filtered stain to cells followed by incubation for 2 h on shaker at room temperature. Following incubation, cells were washed three times with distilled water and 2 ml of PBS was added for visualization under inverted light microscope using Optika vision lite software.

Lipid content quantification
Oil red O stain was eluted from cells using 100% isopropanol and was incubated on a shaker at room temperature for 15 min (Aguena et al., 2012). 100μl of sample absorbance was quantified at 500nm (O.D) to detect the amount of stain attached to differentiated cell fat droplets in control and miR22-3p transfected cells, respectively.

Fat droplet counting
Utilizing visualized oil red O stained pictures showing clear lipid droplets, pictures were divided using gridlines and lipid droplets were quantified under the different treatment groups. Picture counting was performed thrice in 3 different pictures and the average percentage was calculated.

Statistical analysis
All experiments were performed in triplicates and the results are expressed as the means ± standard deviation of the triplicate assays utilizing Microsoft Excel and the miR-22-30 suppresses adipocyte formation from iMSC O n l i n e

F i r s t A r t i c l e
Graphpad online calculator.

Transfection efficiency
The transfection efficiency of iMSC3 cells with nontarget miRNA labelled with CY3 was assessed before the transfection process. The percentage of transfection was detected by the shift in signal indicated (Figs. 2A, 2B). The obtained transfection efficiency was 77.8%, which indicated that miR-22-3p successfully transfected into iMSC3 cells under high efficiency.

miR-22-3p decreased adipocyte formation and lipid content in differentiated iMSC3
Morphological changes occur in iMSC3 cells following their differentiation into adipocytes. Lipid droplets start to form and can be visualized and compared between the control, negative control (transfected with non-target miRNA) and miR-22-3p transfected cell groups using fluorescent and inverted light microscopy. Oil red and Nile red stains were used to specifically stain fat or lipid droplets formed in adipocytes. DAPI was used to stain the cell nucleus. The lipid droplets from the control group are indicated by arrows in Figure 3A (1,2,3,4), forming in higher amounts and larger in size in comparison to miR-22-3p transfected cells shown in Figure 3A (5,6,7,8). Merged DAPI and Nile red stains clearly indicated that lipid droplets formed around the nucleus. The negative control group also demonstrated higher lipid formation in comparison to miR-22-3p transfected cells. Furthermore, lipid contents (Fig. 3B) were measured from eluted oil red stains using a spectrophotometer. We found significant decreases (p<0.05) in the amount of lipid in miR-22-3p transfected cells in comparison to control and negative control groups. Lipid counting was also performed on oil red stained fat droplets (Fig. 3C), where average fat droplet number (%) was significantly (p<0.01, p<0.001) decreased in miR-22-3p transfected cells in comparison to control and negative control groups.

iMSC3 cells transfected with miR-22-3p upregulates nontargeted MAPK signaling during its differentiation into adipocytes
In this study, the effect of miR-22-3p transfection of iMSC3 cells into adipocytes was studied at the protein level. Several signaling pathways are involved in the differentiation process. The role of miR-22-3p in the MAPK and PI3-K signaling pathways was investigated at two time points during differentiation. The MAPK pathway is activated by dual phosphorylation of its residues (Thr 202/ Tyr 204). This activation interacts with downstream effector proteins that are involved in downstream cellular processing. We studied MAPK regulation after the first (4 days) (Fig. 4A, B and Fig. 1) and third differentiation cycles (12 days), respectively. (Fig. 4C, D and Fig. 1). Interestingly, we found that p-MAPK/MAPK expression levels were significantly upregulated (p<0.05) at the above time intervals.

miR-22-3p did not regulate the PI3k/AKT signaling pathway during iMSC3 differentiation into adipocyte
The role of miR-22-3p in the PI3-K/Akt signaling pathway was investigated at two time points during differentiation. We studied PI3-K/Akt regulation after the first (4 days) (Fig. 5A, B and Fig. 1) and third differentiation cycles (12 days), respectively. (Fig. 5C, D and Fig. 1). We found that p-Akt(Thr)/Akt expression levels were neither down-nor upregulated at the discussed time intervals. The obtained results were not statistically significant.

DISCUSSION
Obesity is a disorder characterized by an increase in adipose tissue fat mass (Wang et al., 2014). The process of adipogenesis involves differentiation of MSCs into adipocytes. The MSCs can be differentiated into in vitro adipocytes as well as an in vitro model of obesity. In our study, we found that transfection of miR-22-3p significantly suppressed the differentiation of iMSC3 cells into adipocytes. The iMSC3 cells were transfected with non-target miRNA tagged with fluorescence (CY3) to assess transfection efficiency. The transfection rate was high, indicating cells could be easily transfected with microRNAs using RNAiMAX lipofectamine. A previous study reported the adipocyte inhibitory effects of miR-22 on adipose derived mesenchymal stem cells (hADMSC) (Huang et al., 2012). However, literature is lacking on the effect of miR-22-3p on the differentiation of iMSC3 into adipocytes. The transfection of miR-22-3p at the beginning of the process suppressed adipogenic differentiation as evident by the observed lower lipid content levels and low quantity of lipids in comparison to control and negative control groups. The lipid content assay confirmed that the transfection of miR-22-3p suppressed adipogenesis. Target prediction indicates that more than 500 genes are targeted by miR-22-3p (Target Scan, 2019), including AKT3, a component of the PI3-K pathway. The PI3-K and MAPK signaling pathways are involved in multiple physiological processes, including; cell proliferation, differentiation, metabolism, cytoskeleton reorganization, cell death and survival (Pearson et al., 2001;Vivanco and Sawyers, 2002). AKT and phospho-AKT (Thr 308) western blots revealed an insignificant p-value at the end of the first and third cycles of differentiation, respectively. Hence, despite being a target of miR-22-3p, the transfection process did not affect the regulation of the PI3-K/AKT. Similarly, the transfection of miR-22-3p into cancer cells also led to non-regulation of PI3-K (Lv et al., 2018). Our study also found the analogous effects of miR-22-3p on the PI3-K/AKT pathway. Western blots confirmed that there was neither consistent up-nor downregulation of the pathway during differentiation of iMSC3 into adipocytes following miR-22-3p transfection.
Although the MAPK/ERK pathway is not a known target of miR-22-3p, our previous study found that this pathway enhances adipogenesis of iMSC3 under rosiglitazone treatment (Fayyad et al., 2019). Furthermore, PI3-K has been shown to interact with the MAPK pathway (Aksamitiene et al., 2012). A previous study found that ERK1/2 is involved in early stages of adipocyte differentiation (Marquez et al., 2017). It was our initial hypothesis that MAPK/ERK would be downregulated following miR-22-3p transfection as the latter suppresses adipocytes. However, the present study indicated that MAPK was paradoxically upregulated after transfection. Any signaling pathway has diverse and often opposing effects in different cells (Lodish et al., 2000). Signaling pathways interact with effector proteins that have various effects depending on the context of the involved cells. As such, the regulation of more than 500 genes by miR-22-3p may also upregulate the MAPK/ERK pathway without having a direct effect on adipogenic differentiation.

CONCLUSIONS
Our study showed that miR-22-3p may have antiadipogenic effects on various cells. The transfection of miR-22-3p into iMSC3 cells suppressed adipogenesis without affecting the AKT3 protein, indicating no involvement of the PI-3/Akt pathway. At the same time, the MAPK pathway was observed to be indirectly upregulated following miR-22-3p transfection.

Statement of conflicts of interest
The authors have declared no conflict of interest.