Adipogenic differentiation of human adipose-derived mesenchymal stem cells regulated by microRNA-26b-5p/TCF-4 CURRENT STATUS:

Background: Our study was designed to investigate the role of miR-26b-5p on TCF-4, affecting the adipogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs). METHODS: The adipogenic differentiation of hADMSCs was induced by adipogenic medium for 6 days (d). Bioinformatic and dual-luciferase analyses were used to confirm the relationship between TCF-4 and miR-26b-5p. Immunofluorescence was used to detect the effect of miR-26b-5p on TCF-4 and β-catenin in hADMSCs transfected with miR-26b-5p mimic and inhibitor. Mimic, inhibitor, and small interfering RNA (siRNA) transfected in hADMSCs to against LEF1 and β-catenin. Quantitative real-time PCR and western blotting were used to examine the adipogenic markers and Wnt/β-catenin pathway at the mRNA and protein levels, respectively. Immunofluorescence was performed to locate β-catenin. RESULTS: hADMSCs could differentiate toward adipocytes by the adipogenic medium. The results of bioinformatic and dual-luciferase analyses show that TCF-4 is a potential target of miR-26b-5p. The immunofluorescence intensity of TCF4 and β-catenin were inhibited by miR-26b-5p in hADMSCs. Overexpression of miR-26b-5p promotes the adipogenic differentiation of hADMSCs. Overexpression of TCF-4 and β-catenin inhibits the adipogenic differentiation of hADMSCs. The adipogenic differentiation of hADMSCs that promoted by knocking down TCF4 could be weakened by low-expression of miR-26b-5p. The stimulative effect of β-catenin low-expression in adipogenic differentiation was inhibited by miR-26b-5p inhibitor. Conclusions: miR-26b-5p is a negative regulator to inhibit TCF-4 directly, and then inactivated Wnt/β-catenin pathway, which promotes the adipogenic differentiation of hADMSCs in vitro.


Introduction
Trauma and tumors occurred in the oral and maxillofacial region may cause facial soft tissue defects, which is a common problem faced by oral and maxillofacial and plastic surgeons. Flap transplantation, artificial material, and adipose tissue transplantation are often used to reconstruct defects in soft tissue defects of the oral and maxillofacial region [1,2]. But these methods have some application-level problems. Flap transplantation caused tissue damage and even to defects in the donor site [3]; artificial implant materials usually caused rejection reaction [4]; adipose tissue transplantation faced early absorption, sometimes the patients need a surgical treatment many times [5].
The development of tissue engineering technology provides a new method for repairing soft tissue defects [6], and one of key points is the research of seed cells [7]. Zuk et al. first verified that adipose stem cells have the multi-directional differentiation potential for differentiation into adipocytes, osteoblasts, chondrocytes and myocytes, and become an important seed cell source for tissue regeneration [8]. Adipose-derived mesenchymal stem cells (ADMSCs) have sufficient sources, simple acquisition, high yield, rapid proliferation, multi-directional differentiation potential, can secrete various cytokines, promote angiogenesis, exert immunomodulatory effects, and recruit other cells [9]. Research on ADMSCs as seed cells has become one of the focuses of soft tissue repair and reconstruction.
How to promote adipogenic differentiation of ADMSCs and avoid fat absorption has become a key area of orthopedic research. Many studies have shown that Wnt/β-catenin plays a critical regulatory role in the adipogenic differentiation of adipose-derived mesenchymal stem cells. Wnt3a blocks the differentiation of mouse adipose precursor cell line 3T3-L1 into adipocytes by inhibiting peroxisome proliferator-activated receptor γ (PPARγ) expression [10]. LPR-6-deficient mouse fibroblasts spontaneously differentiate into adipocytes [10]. The above results indicate that Wnt/β-catenin plays a negative regulatory role in adipogenic differentiation. Current research confirms that microRNAs are involved in the regulation of a variety of biological processes, which can affect the adipogenic differentiation process of adiposederived mesenchymal stem cells by regulating signaling pathways. Some microRNAs involved in adipogenic differentiation, such as hsa-miR-15a-5p, hsa-miR-27a-3p, hsa-miR-106b-5p, miR-17-5p, miR-17, etc., can be promoted adipogenic differentiation of ADMSCs by different signaling pathways [11,12,13]. Although many studies have revealed that microRNAs regulate the adipogenic differentiation of adipose stem cells through the Wnt/ β-catenin signaling pathway, the diversity of microRNAs and the diversity of their target genes. Research needs to go further.
hsa-miR-26b-5p was upregulated in hADMSCs during adipogenic differentiation in our preexperiment. The bioinformatic analysis indicated that the target of hsa-miR-26b-5p is most likely TCF4 (T cell factor 4, Gene TCF7L2) in the Wnt/β-catenin pathway. We hypothesize that down-regulating has-miR-26b-5p activates the Wnt/β-catenin pathway, thereby inhibiting adipogenic differentiation of hADMSCs. This study intends to validate this hypothesis by cell biology and molecular biology methods (Fig. 1.).

Materials And Methods
The Obtain method and characterization of hADMSCs The obtain method and characterization of hADMSCs were illustrated in our previous study [14].
The adipogenic differentiation of hADMSCs hADMSCs were seeded into 6-well plates at a density of 9000/cm 2 .After 48 h, the adipogenic medium was added into each well in 6-well plates, and the medium changed every 2 d. The control groups were just cultured in Alpha-modified Eagle's medium(α-MEM; Gibco BRL, USA) with 10% fetal bovine serum (FBS; Gibco BRL, USA). Both the adipogenic group and the control group were cultured at 37 °C and 5% CO 2 incubator for 4 d. On the fourth day, hADMSCs were collected for Microarray for miRNA, immunofluorescence, Realtime Quantitative PCR (qPCR) and western blot analyses.

Statistical analysis
The differences between multiple groups were analyzed by one-way analysis of variance (ANOVA). The differences between the two groups were analyzed by two-way ANOVA. SPSS 20.0 software (IBM SPSS Statistics, Armonk, NY: IBM-Corp.) was used to analyze the differences mentioned above, p < 0.05 was set to the statistical significance.

Results
The adipogenic differentiation of hADMSCs After cultured in the adipogenic medium at 37 °C and 5% CO2 for 4 d, hADMSCs were harvested to detect the adipogenic differentiation and Wnt/β-catenin pathway by the qPCR and western blot analysis. The group of hADMSCs set as control, which was cultured in α-MEM with 10% FBS at 37 °C and 5% CO2.
Comparing the control group, overexpression of miR-26b-5p could not change the luciferase activity of reporter gene for TCF7L2 mRNA 3'-UTR mutated. Comparing the control group, low expression of miR-26b-5p significantly increased the luciferase activity to 1.36 ± 0.83-fold (p = 0.036). Comparing the control group, low expression of miR-26b-5p could not change the luciferase activity of reporter gene for TCF7L2 mRNA 3'-UTR mutated. The results were shown in Fig. 4. C and D.
Based upon the results above, the stimulative effect of β-catenin low-expression in adipogenic differentiation was inhibited by of miR-26b-5p inhibitor.

Discussion
Adipose tissue is abundant and extensive in the body, and they have a sufficient tissue supply to repair soft tissue defects [9]. Coleman introduced a procedure based on a methodology and the use of specific material for adipose filling [15,16]. However, autologous adipose tissue may lose their volume of up to 60% after transplantation because of necrosis [17,18]. So, it is hard to achieve the goal of soft tissue augmentation.
How to reduce the absorption of fat tissue is the crucial point of fat tissue transplantation.
The transplantation of ADSMCs mixed with fat particles can effectively reduce the absorption of transplanted fat [19,20]. The softer texture of transplanted adipose tissue with mixed ADSMCs; a fat structure remains intact; fat lobules and leaflet intervals are kept well. ADSMCs secrete a variety of pro-angiogenic cytokines, such as VEGF, bFGF, HGF, IGF-1, etc, which could promote blood supply and early graft survival [21,22]. The addition of ADMSCs to fat granules for transplantation has a higher survival rate than single fat granule transplantation [23,24].
Although ADSMCs could retain the volume of transplanted adipose tissue, there were some potential risks for postoperative soft tissue restoration in patients with malignant tumors.
At present, there is no sufficient evidence that ASCs have a potential carcinogenic effect.
But in many studies proved that ADMSCs could secrete VEGF,c-Kit, PDGF could promote endothelial proliferation and neoangiogenesis, and supporting tumor growth and metastasis in many types of malignant tumors [25,26]. Wnt signaling pathway plays an inhibition role in adipogenic differentiation of MSCs [27].
3T3-L1 preadipocytes were maintained in an undifferentiated state by increased expression of Wnt10b, which inhibited the expression of PPARγ and C/EBP-α [28,29]. The activation of Wnt/β-catenin pathway via the ectopic expression of Wnt1 and Wnt3a will prevent the adipogenic differentiation of MSCs by suppression of PPARγ [27,30,31].

Wnt10a and Wnt6 as endogenous regulators of adipogenesis and overexpression of Wnt6
and Wnt10a blocked adipogenesis [32]. The overexpression fo PPARγ may inhibit the activity of Wnt/β-catenin pathway, but overexpression of PPARγ and/or C/EBPα could not rescue Wnt/β-catenin-mediated inhibition of adipogenesis [33,34]. adipogenesis in cardiac stem cells [36]. Embelin has the potential to prevent body weight gain; after Embelin treated, both the proliferation and adipogenic differentiation were inhibited in ST2 and C3H10T1/2 cells, nuclear protein levels of β-catenin and TCF-4 were increased [37]. Myostatin enhanced nuclear translocation of β-catenin and formation of the Smad3-β-catenin-TCF4 complex, together with the altered expression of a number of Wnt/β-catenin pathway genes in hMSCs. The inhibitory effects of myostatin on adipogenesis were blocked by RNAi silencing of β-catenin and diminished by overexpression of dominant-negative TCF4 [38]. Overexpression of dominant-negative TCF4 inactivated Wnt signaling pathway in preadipocytes; on the contrary, these cells differentiate into adipocytes [27].
Existing research indicates that miR-26b-3p is involved in the regulation of a variety of biological processes. miR-26b-3p was significantly upregulated during serial in vitro passage of human umbilical cord-derived mesenchymal stem cells and was correlated with cellular senescence and cell cycle genes; overexpression of miR-26b-3p inhibited the proliferation of these cells in vitro [44]. Lin reported that miR-26b-3p suppresses osteoblast differentiation of MC3T3-E1 cells via directly targeting estrogen receptor α [45]. miR-26b-3p was significantly upregulated in whole blood in patients with rheumatoid arthritis and their asymptomatic first-degree relatives [46]. Ginsenoside Rh2 inhibited the expression of miR-26b-3p in liver cancer cells [47]. miR-26b-3p were upregulated in patients with Alzheimer's disease than normal people [48].

Conclusions
Suppressive expression of miR-26b-5p and low-expression of TCF-4 promoting the adipogenic differentiation of hADMSCs in vitro. miR-26b-5p is a negative regulator to inhibit TCF-4 directly, and then inactivated Wnt/β-catenin pathway, which promotes the adipogenic differentiation of hADMSCs in vitro.

Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due but are available from the corresponding author upon reasonable request.  Figure 1 Experimental procedure in this study.      Comparing to hADMSCs cotransfected with miR-26b-5p mimic and EX-Ctrl group, the following proteins in hADMSCs cotransfected with miR-26b-5p mimic and EX-TCF7L2 were increased significantly: C/EBP α 1.28 ± 0.08-fold (p = 0.048), PPARγ