The medium was removed, and the cells washed twice with PBS. both miRNAs are novel regulators of the SVCT2 transporter and play an important role in the osteogenic differentiation of BMSCs. strong class=”kwd-title” Keywords: Vitamin C transporter, SVCT2, miRNA-141, miRNA-200a, BMSCs, Osteogenic differentiation Introduction Bone marrow stromal cells (BMSCs) are progenitor cells that differentiate into osteoblasts, osteocytes, adipocytes, and chondrocytes (Prockop, 1997; Pittenger et al., 1999). Differentiation of BMSCs is an important aspect of musculoskeletal development and maintenance during a lifetime. Lineage progression of BMSCs is controlled by various factors including DNA methylation, microRNA, transcription, growth factors and nutrition. Vitamin C is an essential nutrient that is required for normal bone marrow stromal cell differentiation, collagen synthesis, and bone formation (Bellows et al., 1986, Lee et al 1992, Choi et al 2008, Maehata et al 2007, Urban et al 2012, Wei et al 2012). It is readily available in the diet and its deficiency is generally rare in developed countries (Richardson et al 2002), except in certain groups such as the elderly, alcoholics and smokers (Alcantara-Martos et al 2007, Oeffinger et al 1998, Schectman et al 1989, Schleicher et al 2009, We et al 2001, Lykkesfeldt et al 1989). Vitamin C is highly water-soluble; it cannot simply diffuse across the hydrophobic lipid bilayer of the plasma membrane to gain access into cells; specific transport systems exist in the plasma membrane to mediate the entry process. We recently reported that vitamin C is transported into BMSCs through a sodium-dependent Vitamin C Transporter 2 (SVCT2) (Fulzele et al 2013). We also reported that this transporter isoform is expressed in bone and intervertebral disc cells (Fulzele et al 2013, Chothe et al 2013). Importantly, we demonstrated that knockdown of the SVCT2 transporter inhibits differentiation of BMSCs into the osteogenic lineage (Fulzele et al 2013). Given the importance of the SVCT2 transporter in osteogenic differentiation, understanding the molecular mechanisms underlying the regulation of its expression is necessary. Previous studies have shown the regulation of SVCT2 expression at a transcriptional level in various cell types (Savini et al 2007, Portugal et al 2012, Qiao et al 2011, 2012). So far, regulation of SVCT2 expression at a post-transcriptional level has not yet been explored. Post-transcriptional regulation of gene expression can be controlled by 3-Methylglutaric acid various mechanisms such as RNA interference (Smialowska et al 2014, Cernilogar et al 2013), ribozymes (Klauser et al 2014) and microRNAs (miRs) (Zeng et al 2003). In the last decade, miRNAs have emerged as important regulators of gene expression. MicroRNAs are small noncoding RNAs that down-regulate expression of their target genes by sequence-specific binding to the 3-untranslated regions (3-UTRs) of target 3-Methylglutaric acid mRNAs through inducing mRNA degradation or inhibiting translation (Zeng et al 2003). Although the role of most miRNAs remains elusive, several studies indicate that miRNAs act as key regulators in cell differentiation (Chen et al 2014, Kane et al 2014,), cell proliferation (Selcuklu et al 2012, Li et al 2013), and bone formation (Hwang et al 2014, Xie et al 2014). In the present study, we investigated the post-transcriptional regulation of the SVCT2 transporter in mouse BMSCs. Materials and Methods Isolation of mouse Bone Marrow Stromal Cells (BMSCs) the mouse BMSCs were isolated according to our published method (Fulzele et al 2013). Briefly, mouse BMSCs were isolated from the long bones of 6 month-old C57BL/6 mice (n=6). The mice were euthanized and the femurs and humeri removed. The marrow was flushed with phosphate-buffered saline (PBS) and the cellular material harvested. The cellular material was centrifuged, the supernatant discarded and the pellet washed with PBS. The cells were plated in 100-cm2 culture plates with DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin/streptomycin, and 2 mML-glutamine. After 24 h, the supernatant was removed and the adherent stromal cells trypsinized for negative selection. A negative selection process was used to deplete hematopoietic cell lineages (T- and B-lymphocytic, myeloid and erythroid cells) using a commercially available kit (BD biosciences), thus retaining the progenitor (stem) cell population. The positive fractions were collected using the following parameters: negative for CD3e (CD3 chain), CD11b (integrin M chain), CD45R/B220, Ly-6G and Ly-6C (Gr-1), and TER-119/Erythroid Cells (Ly-76). Positive 3-Methylglutaric acid selections were then performed.Although the role of most miRNAs remains elusive, several studies indicate that miRNAs act as key regulators in cell differentiation (Chen et al 2014, Kane et al 2014,), cell proliferation (Selcuklu et al 2012, Li et al 2013), and bone formation (Hwang et al 2014, Xie et al 2014). that miR-141 and miR-200a decreased osteogenic differentiation. Furthermore, miRNA inhibitors increased SVCT2 and osteogenic gene expression in BMSCs. Taken together, these results indicate that both miRNAs are novel regulators of the SVCT2 transporter and play an important role in the osteogenic differentiation of BMSCs. strong class=”kwd-title” Keywords: Vitamin C transporter, SVCT2, miRNA-141, miRNA-200a, BMSCs, Osteogenic differentiation Introduction Bone marrow stromal cells (BMSCs) are progenitor cells that differentiate into osteoblasts, osteocytes, adipocytes, and chondrocytes (Prockop, 1997; Pittenger et al., 1999). Differentiation of BMSCs is an important SPRY1 aspect of musculoskeletal development and maintenance during a lifetime. Lineage progression of BMSCs is definitely controlled by 3-Methylglutaric acid various factors including DNA methylation, microRNA, transcription, growth factors and nourishment. Vitamin C is an essential nutrient that is required for normal bone marrow stromal cell differentiation, collagen synthesis, and bone formation (Bellows et al., 1986, Lee et al 1992, Choi et al 2008, Maehata et al 2007, Urban et al 2012, Wei et al 2012). It is readily available in the diet and its deficiency is generally rare in developed countries (Richardson et al 2002), except in 3-Methylglutaric acid certain groups such as the seniors, alcoholics and smokers (Alcantara-Martos et al 2007, Oeffinger et al 1998, Schectman et al 1989, Schleicher et al 2009, We et al 2001, Lykkesfeldt et al 1989). Vitamin C is highly water-soluble; it cannot just diffuse across the hydrophobic lipid bilayer of the plasma membrane to gain access into cells; specific transport systems exist in the plasma membrane to mediate the access process. We recently reported that vitamin C is transferred into BMSCs through a sodium-dependent Vitamin C Transporter 2 (SVCT2) (Fulzele et al 2013). We also reported that this transporter isoform is definitely expressed in bone and intervertebral disc cells (Fulzele et al 2013, Chothe et al 2013). Importantly, we shown that knockdown of the SVCT2 transporter inhibits differentiation of BMSCs into the osteogenic lineage (Fulzele et al 2013). Given the importance of the SVCT2 transporter in osteogenic differentiation, understanding the molecular mechanisms underlying the rules of its manifestation is necessary. Earlier studies have shown the rules of SVCT2 manifestation at a transcriptional level in various cell types (Savini et al 2007, Portugal et al 2012, Qiao et al 2011, 2012). So far, rules of SVCT2 manifestation at a post-transcriptional level has not yet been explored. Post-transcriptional rules of gene manifestation can be controlled by various mechanisms such as RNA interference (Smialowska et al 2014, Cernilogar et al 2013), ribozymes (Klauser et al 2014) and microRNAs (miRs) (Zeng et al 2003). In the last decade, miRNAs have emerged as important regulators of gene manifestation. MicroRNAs are small noncoding RNAs that down-regulate manifestation of their target genes by sequence-specific binding to the 3-untranslated areas (3-UTRs) of target mRNAs through inducing mRNA degradation or inhibiting translation (Zeng et al 2003). Even though role of most miRNAs remains elusive, several studies indicate that miRNAs act as key regulators in cell differentiation (Chen et al 2014, Kane et al 2014,), cell proliferation (Selcuklu et al 2012, Li et al 2013), and bone formation (Hwang et al 2014, Xie et al 2014). In the present study, we investigated the post-transcriptional rules of the SVCT2 transporter in mouse BMSCs. Materials and Methods Isolation of mouse Bone Marrow Stromal Cells (BMSCs) the mouse BMSCs were isolated according to our published method (Fulzele et al 2013). Briefly, mouse BMSCs were isolated from your long bones of 6 month-old C57BL/6 mice (n=6). The mice were euthanized and the femurs and humeri eliminated. The marrow was flushed with phosphate-buffered saline (PBS) and the cellular material harvested. The cellular material was centrifuged, the supernatant discarded and the pellet washed with PBS. The cells were plated in 100-cm2 tradition plates with DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin/streptomycin, and 2 mML-glutamine. After 24 h, the supernatant was eliminated and the adherent stromal cells trypsinized for bad selection. A negative selection process was used to deplete hematopoietic cell lineages (T- and B-lymphocytic, myeloid and erythroid cells) using a.
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