miR-26b-5p/TCF-4 Controls the Adipogenic Differentiation of Human Adipose-derived Mesenchymal Stem Cells

In this study, we assessed the ability of miR-26b-5p to regulate T cell factor 4 (TCF-4) expression and thereby control human adipose-derived mesenchymal stem cell (hADMSC) adipogenic differentiation. Adipogenic medium was used to induce hADMSC differentiation over a 6-d period. The ability of miR-26b-5p to interact with the TCF-4 mRNA was confirmed through both predictive bioinformatics analyses and luciferase reporter assays. Immunofluorescent staining was used to visualize the impact of miR-26b-5p inhibition or overexpression on TCF-4 and β-catenin levels in hADMSCs. Further functional analyses were conducted by transfecting these cells with siRNAs specific for TCF-4 and β-catenin. Adipogenic marker and Wnt/β-catenin pathway gene expression levels were assessed via real-time polymerase chain reaction and western blotting. β-catenin localization was assessed via immunofluorescent staining. As expected, our adipogenic media induced the adipocytic differentiation of hADMSCs. In addition, we confirmed that TCF-4 is an miR-26b-5p target gene in these cells, and that protein levels of both TCF-4 and β-catenin were reduced when these cells were transfected with miR-26b-5p mimics. Overexpression of this microRNA also enhanced hADMSC adipogenesis, whereas TCF-4 and β-catenin overexpression inhibited this process. The enhanced hADMSC adipogenic differentiation that was observed following TCF-4 or β-catenin knockdown was partially reversed when miR-26b-5p expression was inhibited. We found that miR-26b-5p serves as a direct negative regulator of TCF-4 expression within hADMSCs, leading to inactivation of the Wnt/β-catenin pathway and thereby promoting the adipogenic differentiation of these cells in vitro.

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

10.21203/rs.2.23419/v1 ◽

2020 ◽

Author(s):

Yadong Luo ◽

Huan Ji ◽

Yan Cao ◽

Xu Ding ◽

Meng Li ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Negative Regulator ◽

Protein Levels ◽

Interfering Rna ◽

The Relationship ◽

Low Expression

Abstract 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.

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Long noncoding RNA SNHG14 promotes hepatocellular carcinoma progression by regulating miR-876-5p/SSR2 axis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01838-5 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Zhibin Liao ◽

Hongwei Zhang ◽

Chen Su ◽

Furong Liu ◽

Yachong Liu ◽

...

Keyword(s):

Noncoding Rna ◽

Animal Experiments ◽

Luciferase Reporter ◽

Western Blot Assay ◽

Downstream Target ◽

Protein Levels ◽

Elevated Expression ◽

Rna Immunoprecipitation

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

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Cell-derived Extracellular Matrix as maintaining Biomaterial for adipogenic differentiation

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2020-3106 ◽

2020 ◽

Vol 6 (3) ◽

pp. 410-413

Author(s):

Petra J. Kluger ◽

Svenja Nellinger ◽

Simon Heine ◽

Ann-Cathrin Volz

Keyword(s):

Tissue Engineering ◽

Extracellular Matrix ◽

Adipogenic Differentiation ◽

Cellular Activity ◽

Adipose Tissue Engineering ◽

Physical Support ◽

Supporting Effect ◽

Brdu Assay ◽

The Impact

AbstractThe extracellular matrix (ECM) naturally surrounds cells in humans, and therefore represents the ideal biomaterial for tissue engineering. ECM from different tissues exhibit different composition and physical characteristics. Thus, ECM provides not only physical support but also contains crucial biochemical signals that influence cell adhesion, morphology, proliferation and differentiation. Next to native ECM from mature tissue, ECM can also be obtained from the in vitro culture of cells. In this study, we aimed to highlight the supporting effect of cell-derived- ECM (cdECM) on adipogenic differentiation. ASCs were seeded on top of cdECM from ASCs (scdECM) or pre-adipocytes (acdECM). The impact of ECM on cellular activity was determined by LDH assay, WST I assay and BrdU assay. A supporting effect of cdECM substrates on adipogenic differentiation was determined by oil red O staining and subsequent quantification. Results revealed no effect of cdECM substrates on cellular activity. Regarding adipogenic differentiation a supporting effect of cdECM substrates was obtained compared to control. With these results, we confirm cdECM as a promising biomaterial for adipose tissue engineering.

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Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽

10.3233/tub-200072 ◽

2021 ◽

Vol 43 (1) ◽

pp. 11-26

Author(s):

Maike Busch ◽

Natalia Miroschnikov ◽

Jaroslaw Thomas Dankert ◽

Marc Wiesehöfer ◽

Klaus Metz ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Cancer Progression ◽

Treated Patient ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Gene Assay ◽

The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

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Impact of Curcumin on Microsomal Enzyme Activities: Drug Interaction and Chemopreventive Studies

Current Medicinal Chemistry ◽

10.2174/0929867328666210329123449 ◽

2021 ◽

Vol 28 ◽

Author(s):

Habibeh Mashayekhi-Sardoo ◽

Adeleh Mashayekhi‐Sardoo ◽

Basil D. Roufogalis ◽

Tannaz Jamialahmadi ◽

Amirhossein Sahebkar

Keyword(s):

Curcuma Longa ◽

Significant Inhibition ◽

Microsomal Enzyme ◽

Scientific Databases ◽

Protein Levels ◽

Curcumin Analogs ◽

The Us ◽

Natural Polyphenol ◽

The Impact

: Curcumin, a yellow pigment in Asian spice, is a natural polyphenol component of Curcuma longa rhizome. Curcuminoid components include curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Previous studies established curcumin as a safe agent based on preclinical and clinical evaluations and curcuminoids have been approved by the US Food and Drug Administration (FDA) as “Generally Recognized as Safe” (GRAS). The present review collects and summarizes clinical and preclinical studies of curcumin interactions, with an emphasis on the effect of curcumin and curcumin analogs on the mRNA and protein levels of microsomal CYP450 enzymes (phase I metabolism) and their interactions with toxicants, drugs and drug probes. The literature search was conducted using keywords in various scientific databases, including Web of Science, Scopus, PubMed, and Google Scholar. Studies concerning the impact of curcumin and curcumin analogs on microsomal enzyme activity are reviewed and include oral, topical, and systemic treatment in humans and experimental animals, as well as studies from in vitro research. When taken together the data identified some inconsistent results between various studies. The findings showed significant inhibition of CYP450 enzymes by curcumin and its analogs. However such effects often differed when curcumin and curcumin analogs were coadministered with toxicant and other drugs and drug probes. We conclude from this review that herb-drug interactions should be considered when curcumin and curcumin analogs are consumed.

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Effect of Substrate Elasticity on In Vitro Aging of Human Mesenchymal Stem Cells

MRS Proceedings ◽

10.1557/opl.2012.1678 ◽

2012 ◽

Vol 1498 ◽

pp. 39-45

Author(s):

Courtney E. LeBlon ◽

Caitlin R. Fodor ◽

Tony Zhang ◽

Xiaohui Zhang ◽

Sabrina S. Jedlicka

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Elastic Modulus ◽

Myogenic Differentiation ◽

Human Mesenchymal Stem Cells ◽

Immunofluorescent Staining ◽

Differentiation Potential ◽

Gene And Protein Expression ◽

The Impact

ABSTRACTHuman mesenchymal stem cells (hMSCs) were routinely cultured on tissue-culture polystyrene (TCPS) to investigate the in vitro aging and cell stiffening. hMSCs were also cultured on thermoplastic polyurethane (TPU), which is a biocompatible polymer with an elastic modulus of approximately 12.9MPa, to investigate the impact of substrate elastic modulus on cell stiffening and differentiation potential. Cells were passaged over several generations on each material. At each passage, cells were subjected to osteogenic and myogenic differentiation. Local cell elastic modulus was measured at every passage using atomic force microscopy (AFM) indentation. Gene and protein expression was examined using qRT-PCR and immunofluorescent staining, respectively, for osteogenic and myogenic markers. Results show that the success of myogenic differentiation is highly reliant on the elastic modulus of the undifferentiated cells. The success of osteogenic differentiations is most likely somewhat dependent on the cell elastic modulus, as differentiations were more successful in earlier passages, when cells were softer.

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miR-125b Disrupts Mitochondrial Dynamics via Targeting Mitofusin 1 in Cisplatin-Induced Acute Kidney Injury

Kidney Diseases ◽

10.1159/000520140 ◽

2021 ◽

pp. 1-11

Author(s):

Yue Zhao ◽

Yue Lang ◽

Mingchao Zhang ◽

Shaoshan Liang ◽

Xiaodong Zhu ◽

...

Keyword(s):

Acute Kidney Injury ◽

Molecular Mechanisms ◽

Mitochondrial Dynamics ◽

Kidney Injury ◽

Immunofluorescent Staining ◽

Luciferase Reporter ◽

Mitochondrial Fragmentation ◽

Tubular Cells ◽

Renal Tubular Cells

Background: Mitochondria are dynamic organelles whose structure are maintained by continuous fusion and fission. During acute kidney injury (AKI) progression, mitochondrial fission in renal tubular cells was elevated, characterized by mitochondrial fragmentation. It is tightly associated with mitochondrial dysfunction, which has been proven as a critical mechanism responsible for AKI. However, the initiating factor for the disruption of mitochondrial dynamics in AKI was not well understood. Objectives: To explore the molecular mechanisms of mitochondrial disorders and kidney damage. Methods: We established cisplatin-induced AKI model in C57BL/6 mice and proximal tubular cells, and detected the expression of miR-125b by qPCR. Then we delivered miR-125b antagomir after cisplatin treatment in mice via hydrodynamic-based gene transfer technique. Subsequently, we performed luciferase reporter and immunoblotting assays to prove miR-125b could directly modulate mitofusin1 (MFN1) expression. We also tested the role of miR-125b in mitochondrial and renal injury through immunofluorescent staining, qPCR, and immunoblotting assays. Results: miR-125b levels were induced in cisplatin-challenged mice and cultured tubular cells. Anti-miR-125b could effectively alleviate cisplatin-induced mitochondrial fragmentation and kidney injury both in vitro and in vivo. Furthermore, miR-125b could directly regulate MFN1, which is a key regulator of mitochondrial fusion. Our study indicated that miR-125b is upregulated during cisplatin-induced AKI. Inhibition of miR-125b may suppress mitochondrial and renal damage through upregulating MFN1. This study suggests that miR-125b could be a potential therapeutic target in AKI.

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An Integrated Analysis of miRNA and Gene Expression Changes in Response to an Obesogenic Diet to Explore the Impact of Transgenerational Supplementation with Omega 3 Fatty Acids

Nutrients ◽

10.3390/nu12123864 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3864

Author(s):

Karla Fabiola Corral-Jara ◽

Laura Cantini ◽

Nathalie Poupin ◽

Tao Ye ◽

Jean Paul Rigaudière ◽

...

Keyword(s):

Gene Expression ◽

Fatty Acids ◽

Negative Regulator ◽

Integrated Analysis ◽

Omega 3 ◽

Biological Variables ◽

Biological Insight ◽

Obesogenic Diet ◽

The Impact

Insulin resistance decreases the ability of insulin to inhibit hepatic gluconeogenesis, a key step in the development of metabolic syndrome. Metabolic alterations, fat accumulation, and fibrosis in the liver are closely related and contribute to the progression of comorbidities, such as hypertension, type 2 diabetes, or cancer. Omega 3 (n-3) polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), were identified as potent positive regulators of insulin sensitivity in vitro and in animal models. In the current study, we explored the effects of a transgenerational supplementation with EPA in mice exposed to an obesogenic diet on the regulation of microRNAs (miRNAs) and gene expression in the liver using high-throughput techniques. We implemented a comprehensive molecular systems biology approach, combining statistical tools, such as MicroRNA Master Regulator Analysis pipeline and Boolean modeling to integrate these biochemical processes. We demonstrated that EPA mediated molecular adaptations, leading to the inhibition of miR-34a-5p, a negative regulator of Irs2 as a master regulatory event leading to the inhibition of gluconeogenesis by insulin during the fasting–feeding transition. Omics data integration provided greater biological insight and a better understanding of the relationships between biological variables. Such an approach may be useful for deriving innovative data-driven hypotheses and for the discovery of molecular–biochemical mechanistic links.

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Decreased phosphatase PTEN amplifies PI3K signaling and enhances proinflammatory cytokine release in COPD

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00382.2016 ◽

2017 ◽

Vol 313 (2) ◽

pp. L230-L239 ◽

Cited By ~ 23

Author(s):

Satoru Yanagisawa ◽

Jonathan R. Baker ◽

Chaitanya Vuppusetty ◽

Peter Fenwick ◽

Louise E. Donnelly ◽

...

Keyword(s):

Epithelial Cells ◽

Pi3k Pathway ◽

Negative Regulator ◽

Pi3k Signaling ◽

Pten Protein ◽

Protein Levels ◽

Enhanced Production ◽

Phosphorylated Akt ◽

Peripheral Lung ◽

The Impact

The phosphatidylinositol 3-kinase (PI3K) pathway is activated in chronic obstructive pulmonary disease (COPD), but the regulatory mechanisms for this pathway are yet to be elucidated. The aim of this study was to determine the expression and role of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the PI3K pathway, in COPD. PTEN protein expression was measured in the peripheral lung of COPD patients compared with smoking and nonsmoking controls. The direct influence of cigarette smoke extract (CSE) on PTEN expression was assessed using primary lung epithelial cells and a cell line (BEAS-2B) in the presence or absence of l-buthionine-sulfoximine (BSO) to deplete intracellular glutathione. The impact of PTEN knockdown by RNA interference on cytokine production was also examined. In peripheral lung, PTEN protein was significantly decreased in patients with COPD compared with the subjects without COPD ( P < 0.001) and positively correlated with the severity of airflow obstruction (forced expiratory volume in 1-s percent predicted; r = 0.50; P = 0.0012). Conversely, phosphorylated Akt, as a marker of PI3K activation, showed a negative correlation with PTEN protein levels ( r = −0.41; P = 0.0042). In both primary bronchial epithelial cells and BEAS-2B cells, CSE decreased PTEN protein, which was reversed by N-acetyl cysteine treatment. PTEN knockdown potentiated Akt phosphorylation and enhanced production of proinflammatory cytokines, such as IL-6, CXCL8, CCL2, and CCL5. In conclusion, oxidative stress reduces PTEN protein levels, which may result in increased PI3K signaling and amplification of inflammation in COPD.

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miR-19a protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis via PTEN/PI3K/p-Akt pathway

Bioscience Reports ◽

10.1042/bsr20170899 ◽

2017 ◽

Vol 37 (6) ◽

Cited By ~ 19

Author(s):

Guochao Sun ◽

Ying Lu ◽

Yingxia Li ◽

Jun Mao ◽

Jun Zhang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Ischemic Injury ◽

Cardiomyocyte Apoptosis ◽

Luciferase Reporter ◽

H9c2 Cells ◽

Rat Cardiomyocytes ◽

Protein Levels ◽

Myocardial Ischemic Injury ◽

Hypoxia Reoxygenation

miRNAs have been implicated in processing of cardiac hypoxia/reoxygenation (H/R)-induced injury. Recent studies demonstrated that miR-19a might provide a potential cardioprotective effect on myocardial disease. However, the effect of miR-19a in regulating myocardial ischemic injury has not been previously addressed. The present study was to investigate the effect of miR-19a on myocardial ischemic injury and identified the potential molecular mechanisms involved. Using the H/R model of rat cardiomyocytes H9C2 in vitro, we found that miR-19a was in low expression in H9C2 cells after H/R treatment and H/R dramatically decreased cardiomyocyte viability, and increased lactate dehydrogenase (LDH) release and cardiomyocyte apoptosis, which were attenuated by co-transfection with miR-19a mimic. Dual-luciferase reporter assay and Western blotting assay revealed that PTEN was a direct target gene of miR-19a, and miR-19a suppressed the expression of PTEN via binding to its 3′-UTR. We further identified that overexpression of miR-19a inhibited the expression of PTEN at the mRNA and protein levels. Moreover, PTEN was highly expressed in H/R H9C2 cells and the apoptosis induced by H/R was associated with the increase in PTEN expression. Importantly, miR-19a mimic significantly increased p-Akt levels under H/R. In conclusion, our findings indicate that miR-19a could protect against H/R-induced cardiomyocyte apoptosis by inhibiting PTEN /PI3K/p-Akt signaling pathway.

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