Weidong Li

Adipogenesis and osteoblastogenesis (adipo-osteoblastogenesis) are closely related processes involving with the phosphorylation of numerous cytoplasmic proteins and key transcription factors. Despite the recognition of the importance of protein phosphorylation in adipo-osteoblastocyte biology, relatively little is known about the specific kinases for adipo-osteoblastogenesis. Here, we constructed the comprehensive gene transcriptional landscapes of kinases at 3, 5, and 7 days during adipo-osteoblastogenesis from human bone marrow mesenchymal stem cells (hMSCs). We identified forty-four and eight significant DEGs (differentially expressed genes) separately for adipo-osteoblastogenesis. Five significant DEGs, namely CAMK2A, NEK10, PAK3, PRKG2, and PTK2B, were simultaneously shared by adipo-osteoblastogenic anecdotes. Using a lentivirus system, we confirmed that PTK2B (non-receptor protein tyrosine kinase 2 beta) simultaneously inhibited adipo-osteoblastogenesis through RNAi assays, and PRKG2 (protein kinase cGMP-dependent 2) facilitated adipogenesis and weakened osteoblastogenesis. The only certainty was that the identified candidate significant DEGs encoding kinases responsible for protein phosphorylation, especially PTK2B and PRKG2, were the potential molecular switches of cell fate determination for hMSCs. This study would provide novel study targets for hMSC differentiation and potential clues for the therapy of the adipo-osteoblastogenic balance-derived disorders.

MicroRNAs play an important role in the adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). How miR-100-3p influences such adipogenesis, however, remains uncertain. In this study, hMSC adipogenic differentiation was associated with miR-100-3p downregulation, and overexpressing this miRNA inhibited adipogenesis and the expression of adipogenic marker genes. Through bioinformatics approaches, miR-100-3p can bind the 3'-untranslated region (3'-UTR) of the mRNA encoding phosphoinositide 3-kinase regulatory subunit 1 (PIK3R1) such that miR-100-3p overexpression resulted in significant reductions in PIK3R1 expression. Importantly, overexpressing PIK3R1 was sufficient to reverse the anti-adipogenic effects of miR-100-3p overexpression. PIK3R1 is a critical component of the PI3K/AKT signaling pathway, and miR-100-3p overexpression resulted in reduced AKT phosphorylation in the context of adipogenesis. In addition, the adipogenic differentiation of hMSCs in which miR-100-3p was overexpressed was further enhanced upon treatment with the PI3K/AKT agonist 740Y-P relative to miR-100-3p overexpression alone. Taken together, these findings provide evidence that miR-100-3p inhibits the adipogenic differentiation of hMSCs by targeting PIK3R1 via the PI3K/AKT signaling pathway.

Transfer RNA‐derived small RNAs (tsRNAs), a novel type of non‐coding RNA derivative, are able to regulate a wide range of biological processes. What role these tsRNAs play in the regulation of human bone marrow mesenchymal stem cell (hMSCs) adipogenic differentiation remains uncertain. We induced the adipogenic differentiation of human bone marrow mesenchymal cells (hMSCs) and then performed small RNA transcriptomic sequencing, leading us to identify tsRNA‐06018 as a target of interest based upon resultant the tsRNA expression profiles. When tsRNA‐06018 was knocked down, this led to the inhibition of adipogenesis and a decrease in adipogenic marker expression. When STC2 was overexpressed, this impaired the adipogenic differentiation of these cells. We further used luciferase reporter assays to confirm that tsRNA‐06018 directly binds the 3′‐untranslated region (3′‐UTR) of STC2. In addition, we determined that both knocking down tsRNA‐06018 and overexpressing STC2 increased extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation within cells. We also assessed that the adipogenic differentiation of hMSCs in which tsRNA‐06018 was knocked down was further enhanced upon the addition of the ERK1/2 inhibitor U0126 as compared tsRNA‐06018 knockdown alone. Taken together, using small RNA sequencing we profiled tsRNAs in hMSCs during the process of adipogenesis, leading us to identify tsRNA‐06018 as a novel regulator of this differentiation process. This tsRNA was able to regulate adipogenic differentiation by targeting STC2 via the ERK1/2 signalling pathway.

Background: Transfer RNA-derived small RNAs (tsRNAs) are a recently discovered form of non-coding RNA capable of regulating myriad physiological processes. The role of tsRNAs in hMSC adipogenic differentiation, however, remains incompletely understood. The purpose of this study was to identify the novel tsRNA-16902 as a regulator of hMSC adipogenic differentiation.Methods: In this study, we conducted transcriptomic sequencing of hMSCs after inducing their adipogenic differentiation, and we were thereby able to clarify the molecular mechanism underlying the role of tsRNA-16902 in this context via a series of molecular biology methods.Results: When we knocked down tsRNA-16902 expression, this impaired hMSC adipogenic differentiation and associated marker gene expression. Bioinformatics analyses further revealed tsRNA-16902 to target retinoic acid receptor γ (RARγ). Luciferase reporter assays also confirmed the ability of tsRNA-16902 to bind to the RARγ 3′-untranslated region. Consistent with this, RARγ overexpression led to impaired hMSC adipogenesis. Further analyses revealed that Smad2/3 phosphorylation was increased in cells that either overexpressed RARγ or in which tsRNA-16902 had been knocked down. We also assessed the adipogenic differentiation of hMSCs in which tsRNA-16902 was knocked down and at the same time a Smad2/3 inhibitor was added to disrupt Smad2/3 phosphorylation. The adipogenic differentiation of hMSCs in which tsRNA-16902 was knocked down was further enhanced upon the addition of a Smad2/3 signaling inhibitor relative to tsRNA-16902 knockdown alone.Conclusions: Through a comprehensive profiling analysis of tsRNAs that were differentially expressed in the context of hMSC adipogenic differentiation, we were able to identify tsRNA-16902 as a previously uncharacterized regulator of adipogenesis. tsRNA-16902 is able to regulate hMSC adipogenic differentiation by targeting RARγ via the Smad2/3 signaling pathway. Together, our results may thus highlight novel strategies of value for treating obesity.

Background: The process of bone repair is heavily dependent on the ability of human bone marrow mesenchymal stem cells (hMSCs) to undergo osteogenic differentiation. MicroRNAs have been shown to regulate this osteogenic process. This study aimed to investigate the role of miR-765 in the osteogenic differentiation of hMSCs.Methods: We transfected hMSCs with lentiviral constructs to knock down or overexpress this miRNA, allowing us to assess its role in osteogenesis via assessing the expression of the relevant markers alkaline phosphatase (ALP), runt-related gene-2 (RUNX-2), and osteocalcin (OCN), with further functional measurements made via quantifying ALP activity and conducting Alizarin Red S staining. The targeting of the 3′-untranslated region (UTR) of BMP6 by miR-765 was examined via luciferase assay. We used hMSCs with altered miR-765 expression to assess p-Smad1/5/9 levels via Western blotting over the course of osteogenic differentiation. We also assessed the osteogenic differentiation of hMSCs in which miR-765 was knocked down and at the same time as a BMP/Smad signaling inhibitor was added to disrupt Smad1/5/9 phosphorylation.Results: We found miR-765 overexpression to inhibit osteogenesis-associated gene upregulation during osteogenic differentiation of hMSCs, whereas knockdown of this miRNA was associated with increased expression of these genes. Using luciferase reporter assays, we confirmed direct miR-765 binding to the 3′-untranslated region (UTR) of BMP6. We also found that miR-765 overexpression reduced Smad1/5/9 phosphorylation, and knockdown of this miRNA enhanced this phosphorylation on BMP6/Smad1/5/9 signaling. The osteogenic differentiation of hMSCs in which miR-765 had been knocked down was further weakened upon the addition of a BMP/Smad signaling inhibitor relative to miR-765 knockdown alone.Conclusions: Together, these results thus suggest that miR-765 is able to inhibit hMSC osteogenic differentiation by targeting BMP6 via regulation of the BMP6/Smad1/5/9 signaling pathway. Our findings may offer molecular insights of value for the development of novel therapeutic treatments for bone diseases including osteoporosis.

Metabolism homeostasis plays an important role in progenitor‐cell differentiation to adipocytes, but less is known about the whole transcriptional profiling of cellular metabolism during adipogenesis. We got the first insight into the whole transcriptional profiling of cellular metabolism during adipogenesis from human mesenchymal stem cells (hMSCs) by the RNA‐Seq technique. There were 1,998, 2,629, 3,112, and 3,054 differentially expressed genes (DEGs) at Days 7, 14, 21, and 28, respectively, during adipogenesis. The most enriched phosphatidylinositol 3′ kinase–serine/threonine kinase (PI3K‐Akt) signaling pathway stimulated and directly regulated cellular metabolism by priming glucose aerobic glycolysis, arginine and proline metabolism, glutathione metabolism, and arachidonic acid metabolism during adipogenesis, targeting the potential key genes, such as fatty acid synthase (FABP4), phosphoenolpyruvate carboxykinase 1 (PKC1), stearoyl‐CoA desaturase (SCD), and solute carrier family 2 member 1 of Gluts (SLC2A1). And it confirmed PCK1 as the key player for cellular metabolism by small interfering RNA. A comprehensive understanding of cellular metabolism and its regulatory axis of the signaling pathway during adipogenesis would reveal new study and therapy targets for fat metabolism disorders.

MicroRNAs (miRNAs), the potential regulator of adipogenesis, markedly characterized by lipid droplet (LD) formation, play an important role in progenitor‐cell differentiation into adipocytes. In recent years, it has excited interests in regulation of miRNAs in adipogenesis. However, no study is available, to our knowledge, regarding the expression of miRNAs on LD formation. Our study provides the first insight into the expression profiling of the miRNA targeting messenger RNAs (mRNAs) involving with LD formation during adipogenesis from human mesenchymal stem cells by RNA‐Seq transcriptome technique. It showed that 39, 105, 194, and 112 differentially expressed miRNA appeared at 7, 14, 21, and 28 days, respectively, for LD formation during adipogenesis. Nineteen miRNAs targeted 35 mRNA associated with LDs formation. Except for the known miRNA hsa‐miR‐1908 regulating adipogenesis, five miRNAs, including hsa‐miR‐146a‐3p, hsa‐miR‐4495, hsa‐miR‐4663, hsa‐miR‐6069, and hsa‐miR‐675‐3p are the latest potential biomarkers for LD formation, targeting ACSL1, APOB, METTL7A, PLIN1, and PLIN4. A comprehensive transcriptome profiling of miRNA reveals the regulatory relationship between miRNA and mRNA relating to LD formation during adipogenesis. Such candidates may represent biomarkers and therapeutic targets for metabolic syndromes like obesity, type‐2 diabetes, steatosis, atherosclerosis, and osteoporosis.

Adipogenesis, the developmental process of progenitor‐cell differentiating into adipocytes, leads to fat metabolic disorders. Alternative splicing (AS), a ubiquitous regulatory mechanism of gene expression, allows the generation of more than one unique messenger RNA (mRNA) species from a single gene. Till now, alternative splicing events during adipogenesis from human mesenchymal stem cells (hMSCs) are not yet fully elucidated. We performed RNA‐Seq coupled with bioinformatics analysis to identify the differentially expressed AS genes and events during adipogenesis from hMSCs. A global survey separately identified 1262, 1181, 1167, and 1227 ASE involved in the most common types of AS including cassette exon, alt3, and alt5, especially with cassette exon the most prevalent, at 7, 14, 21, and 28 days during adipogenesis. Interestingly, 122 differentially expressed ASE referred to 118 genes, and the three genes including ACTN1 (alt3 and cassette), LRP1 (alt3 and alt5), and LTBP4 (cassette, cassette_multi, and unknown), appeared in multiple AS types of ASE during adipogenesis. Except for all the identified ASE of LRP1 occurred in the extracellular topological domain, alt3 (84) in transmembrane domain significantly differentially expressed was the potential key event during adipogenesis. Overall, we have, for the first time, conducted the global transcriptional profiling during adipogenesis of hMSCs to identify differentially expressed ASE and ASE‐related genes. This finding would provide extensive ASE as the regulator of adipogenesis and the potential targets for future molecular research into adipogenesis‐related metabolic disorders.

Adipogenesis, a physiological process initiated with the committed preadipocytes expressing adipocyte‐specific genes and terminated in mature, differentiated and functional adipocytes, mainly involved with energy homeostasis. Abnormal distribution‐changes and dysfunctions in adipogenesis may lead to complex physiopathological disorders. However, it remains unclear for the key players working for the whole complex differentiating process of adipogenesis. Here, it investigated transcriptional profiling of adipogenesis from human mesenchymal stem cells (hMSCs) by RNA‐Seq transcriptome technique. Oil Red O staining assays were performed to assess adipogenic potential. Quantitative real‐time PCR (qRT‐PCR) and lentivirus transfection assays by small interference RNA (siRNA) were conducted to confirm the function of the candidate genes. A total of 1,078 differentially expressed genes shared at 7, 14, 21, and 28 days during adipogenesis from hMSCs, and 706 genes were significantly differentially expressed. It identified 20 potential key genes responsible for adipogenesis with four genes downregulating. The candidate gene, coagulation factor II thrombin receptor (F2R), encoding coagulation factor II thrombin receptor involving with a 7‐transmembrane receptor involved in the regulation of thrombotic response, also known as proteinase‐activated receptor‐1, contributed to adipogenesis, especially at Day 14, by Oil Red O staining, qRT‐PCR, and western blot after siRNA. A unique discovery shed new light to understand the key players of the whole processes of adipogenesis from hMSCs. The gene F2R might be used as an adipogenic marker to provide a potential target for understanding the metabolic syndromes like obesity, type‐2 diabetes, steatosis, atherosclerosis, and osteoporosis.