Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 52
Filtrar
1.
iScience ; 24(12): 103382, 2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34841227

RESUMO

GLP-1 analogs are a class of glucose-lowering agents with multiple benefits in diabetes, but its role in adipose tissues remains to be elucidated. The aim of this study was to determine the action of recombinant human GLP-1 (rhGLP-1) Beinaglutide (BN) in the insulin sensitivity and lipid metabolism of adipose tissues. We have shown that, after BN injection, obese mice displayed lower body weight, fat mass, and plasma lipid levels. In addition, BN promoted the insulin sensitivity in the white adipose tissues. Furthermore, we have found that the BN treatment caused significant changes in content and composition of different lipid classes, including glycerolipids, glycerophospholipids, and sphingolipids, as well as expression of genes in lipid metabolic pathways in the adipose tissues. Taken together, our data demonstrate that BN could resist HFD-induced obesity by targeting the composition of major lipid classes and the expression of genes in lipid metabolism of adipose tissues.

2.
Chem Sci ; 12(35): 11762-11768, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34659713

RESUMO

The addition of sulfonyl radicals to alkenes and alkynes is a valuable method for constructing useful highly functionalized sulfonyl compounds. The underexplored alkoxy- and fluorosulfonyl radicals are easily accessed by CF3 radical addition to readily available allylsulfonic acid derivatives and then ß-fragmentation. These substituted sulfonyl radicals add to aryl alkyl alkynes to give vinyl radicals that are trapped by trifluoromethyl transfer to provide tetra-substituted alkenes bearing the privileged alkoxy- or fluorosulfonyl group on one carbon and a trifluoromethyl group on the other. This process exhibits broad functional group compatibility and allows for the late-stage functionalization of drug molecules, demonstrating its potential in drug discovery and chemical biology.

3.
Genes Dis ; 8(6): 814-826, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34522710

RESUMO

Intestinal cancers are developed from intestinal epithelial stem cells (ISCs) in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes. ISCs play a key role in maintaining the homeostasis of gut epithelium. In 2009, Sato et al established a three-dimensional culture system, which mimicked the niche microenvironment by employing the niche factors, and successfully grew crypt ISCs into organoids or Mini-guts in vitro. Since then, the intestinal organoid technology has been used to delineate cellular signaling in ISC biology. However, the cultured organoids consist of heterogeneous cell populations, and it was technically challenging to introduce genomic changes into three-dimensional organoids. Thus, there was a technical necessity to develop a two-dimensional ISC culture system for effective genomic manipulations. In this study, we established a conditionally immortalized mouse intestinal crypt (ciMIC) cell line by using a piggyBac transposon-based SV40 T antigen expression system. We showed that the ciMICs maintained long-term proliferative activity under two-dimensional niche factor-containing culture condition, retained the biological characteristics of intestinal epithelial stem cells, and could form intestinal organoids in three-dimensional culture. While in vivo cell implantation tests indicated that the ciMICs were non-tumorigenic, the ciMICs overexpressing oncogenic ß-catenin and/or KRAS exhibited high proliferative activity and developed intestinal adenoma-like pathological features in vivo. Collectively, these findings strongly suggested that the engineered ciMICs should be used as a valuable tool cell line to dissect the genetic and/or epigenetic underpinnings of intestinal tumorigenesis.

4.
Genes Dis ; 8(6): 931-938, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34522719

RESUMO

Methyltetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism, and its single nucleotide polymorphism (SNP) site C677T may be associated with gastrointestinal cancer. However, the relationship between MTHFR C677T polymorphism and gastrointestinal tumor markers carcinoma embryonic antigen (CEA), carbohydrate antigen 199 (CA199) and carbohydrate antigen 724 (CA724) in Helicobacter pylori (H. pylori) infection is not specified. This study aims to identify the association between MTHFR C677T polymorphism and gastrointestinal tumor markers (CEA, CA199 and CA724) in H. pylori infection. The relationship between MTHFR C677T polymorphism and gastrointestinal tumor markers in 58 patients with H. pylori infection and 94 non-infected patients was studied. We found that TT genotype was a susceptibility factor of H. pylori infection, which was also associated with increased CEA and CA724 levels. Moreover, there was a negative additive interaction between MTHFR gene C677T polymorphism and CEA levels in H.pylori infection. Meanwhile, there were significant differences in CEA levels between MTHFR C677T polymorphism and H.pylori infection. The presence of T allele led to a decrease in CEA levels when 13C urea breath test (13C-UBT) was positive, while the presence of T allele led to an increase in CEA levels when 13C-UBT was negative. Therefore, we suggest that healthy people should take MTHFR C677T polymorphism screening, combined with 13C-UBT and gastrointestinal tumor markers detection, which can screen out the susceptible population of H. pylori, and help to detect gastrointestinal cancer in the early stage.

5.
Genes Dis ; 8(3): 298-306, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33997177

RESUMO

Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5-10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

6.
Genes Dis ; 8(1): 8-24, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33569510

RESUMO

Notch is a cell-cell signaling pathway that is involved in a host of activities including development, oncogenesis, skeletal homeostasis, and much more. More specifically, recent research has demonstrated the importance of Notch signaling in osteogenic differentiation, bone healing, and in the development of the skeleton. The craniofacial skeleton is complex and understanding its development has remained an important focus in biology. In this review we briefly summarize what recent research has revealed about Notch signaling and the current understanding of how the skeleton, skull, and face develop. We then discuss the crucial role that Notch plays in both craniofacial development and the skeletal system, and what importance it may play in the future.

7.
Bioorg Chem ; 119: 105575, 2021 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-34995979

RESUMO

Since androgen receptor (AR) can bind to BRD4 protein and this binding can be blocked by BRD4 inhibitors, targeting BRD4 has emerged as a promising approach for the treatment of prostate cancer (PC). Herein, we designed and synthesized a series of 5-(1-benzyl-1H-indazol-6-yl)-4-ethoxy-1-methylpyridin-2(1H)-one derivatives as novel BRD4 inhibitors for prostate cancer. Among them, compound 13 displayed the most robust BRD4 inhibitory activity with an IC50 value of 18 nM. Furthermore, 13 showed potent anti-proliferative activity against enzalutamide-resistant 22RV1 cells. The mechanism of action studies demonstrated that 13 induced cell apoptosis by regulating Bcl-2/Bax proteins and activating caspase-3 signaling pathway. In addition, the c-Myc level was significantly reduced in 22RV1 cells on the western blot assay. These findings collectively suggested that compound 13 might find potential use for the treatment of prostate cancer.

8.
Mol Ther Nucleic Acids ; 22: 885-899, 2020 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-33230483

RESUMO

RNA interference (RNAi) is mediated by an ∼21-nt double-stranded small interfering RNA (siRNA) and shows great promise in delineating gene functions and in developing therapeutics for human diseases. However, effective gene silencing usually requires the delivery of multiple siRNAs for a given gene, which is often technically challenging and time-consuming. In this study, by exploiting the type IIS restriction endonuclease-based synthetic biology methodology, we developed the fast assembly of multiplex siRNAs (FAMSi) system. In our proof-of-concept experiments, we demonstrated that multiple fragments containing three, four, or five siRNA sites targeting common Smad4 and/or BMPR-specific Smad1, Smad5, and Smad8 required for BMP9 signaling could be assembled efficiently. The constructed multiplex siRNAs effectively knocked down the expression of Smad4 and/or Smad1, Smad5, and Smad8 in mesenchymal stem cells (MSCs), and they inhibited all aspects of BMP9-induced osteogenic differentiation in bone marrow MSCs (BMSCs), including decreased expression of osteogenic regulators/markers, reduced osteogenic marker alkaline phosphatase (ALP) activity, and diminished in vitro matrix mineralization and in vivo ectopic bone formation. Collectively, we demonstrate that the engineered FAMSi system provides a fast-track platform for assembling multiplexed siRNAs in a single vector, and thus it may be a valuable tool to study gene functions or to develop novel siRNA-based therapeutics.

9.
J Exp Clin Cancer Res ; 39(1): 115, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32552756

RESUMO

An amendment to this paper has been published and can be accessed via the original article.

10.
Front Pharmacol ; 11: 492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32372963

RESUMO

Traditional Chinese medicine is an accepted and integral part of clinical cancer management alongside Western medicine in China. However, historically TCM physicians were unaware of the chemical constituents of their formulations, and the specific biological targets in the body. Through HPLC, flow cytometry, and other processes, researchers now have a much clearer picture of how herbal medicine works in conjunction with the immune system in cancer therapy. Among them, the regulation of tumor-related T cells plays the most important role in modulating tumor immunity by traditional Chinese medicine. Encouraging results have been well-documented, including an increase in T cell production along with their associated cytokines, enhanced regulation of Tregs and important T cell ratios, the formation and function of Tregs in tumor microenvironments, and the promotion of the number and function of normal T Cells to reduce conventional cancer therapy side effects. Chinese herbal medicine represents a rich field of research from which to draw further inspiration for future studies. While promising agents have already been identified, the vast majority of Chinese herbal mechanisms remain undiscovered. In this review, we summarize the effects and mechanisms of specific Chinese herbs and herbal decoctions on tumor related T cells.

11.
J Adv Res ; 24: 239-250, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32373357

RESUMO

RNA sequencing (RNA-seq)-based whole transcriptome analysis (WTA) using ever-evolving next-generation sequencing technologies has become a primary tool for coding and/or noncoding transcriptome profiling. As WTA requires RNA-seq data for both coding and noncoding RNAs, one key step for obtaining high-quality RNA-seq data is to remove ribosomal RNAs, which can be accomplished by using various commercial kits. Nonetheless, an ideal rRNA removal method should be efficient, user-friendly and cost-effective so it can be adapted for homemade RNA-seq library construction. Here, we developed a novel reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) method. We demonstrated that RTR2D was simple and efficient, and depleted human or mouse rRNAs with high specificity without affecting coding and noncoding transcripts. RNA-seq data analysis indicated that RTR2D yielded highly correlative transcriptome landscape with that of NEBNext rRNA Depletion Kit at both mRNA and lncRNA levels. In a proof-of-principle study, we found that RNA-seq dataset from RTR2D-depleted rRNA samples identified more differentially expressed mRNAs and lncRNAs regulated by Nutlin3A in human osteosarcoma cells than that from NEBNext rRNA Depletion samples, suggesting that RTR2D may have lower off-target depletion of non-rRNA transcripts. Collectively, our results have demonstrated that the RTR2D methodology should be a valuable tool for rRNA depletion.

12.
BMC Cardiovasc Disord ; 20(1): 222, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404177

RESUMO

BACKGROUND: Uncertainty still remains on the correlation of methylenetetrahydrofolate reductase (MTHFR) variant C677T with risk of carotid atherosclerosis (CAS), and there is a lack of reports on C677T/MTHFR in the Asian population. The association of C677T/MTHFR polymorphisms with CAS in the Chinese Han population in Chongqing was investigated in the present study. METHODS: Subjects (n = 730, 214 females and 516 males, Han ethnicity) who provided an informed consent were randomly selected from the general population of Chongqing, China. Polymerase chain reaction-restriction fragment length polymorphism and Sanger sequencing genotyping assays were used to determine the MTHFR genotypes. The atherosclerosis index of the intima-media thickness (IMT) was measured by high-resolution ultrasound to evaluate the CAS. Less than 1.0 mm was considered as normal for IMT, 1.0-1.5 mm was considered as thickening, and ≥ 1.5 mm and a local bulge thickened in the lumen was considered as CAS. According to the carotid ultrasonography results, these subjects were divided into two groups: CAS-group (IMT ≥ 1.0 mm) and control group (IMT < 1.0 mm). RESULTS: The frequency of C/T heterozygotes, T/T homozygotes genotype was significantly higher in the subjects with CAS (62% vs. 36.9%; 16.2% vs. 9.5%; 47.2% vs. 27.9%, P < 0.05), while the frequency of C/C homozygotes and C allele was significantly lower (21.8% vs. 53.7%; 52.8% vs. 72.1%, P < 0.05), when compared to the control group. The risk of CAS was higher for subjects with C/T heterozygotes and T/T homozygotes (OR = 4.06, 95% CI: 2.76-5.98, P < 0.001 and OR = 3.14, 95% CI: 1.73-5.69, P < 0.001, respectively), when compared to the subjects with the C/C genotype. In the model 1 (CT + TT versus CC), C677T/MTHFR was significantly associated with the prevalence of CAS, and the all adjusted OR values for CAS were 3.87 (95% CI, 2.67 to 5.62) in all, 17.18 (95% CI, 7.27 to 40.49) in women and 2.57 (95% CI, 1.65 to 3.99) in men after adjusting for potential confounding factors. CONCLUSIONS: The present study suggests that a mutation in the methylenetetrahydrofolate reductase gene is a risk factor of CAS in the Chinese Han population.


Assuntos
Doenças das Artérias Carótidas/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo Genético , Adulto , Doenças das Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/etnologia , Espessura Intima-Media Carotídea , China/epidemiologia , Estudos Transversais , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Medição de Risco , Fatores de Risco , Ultrassonografia Doppler em Cores
13.
J Exp Clin Cancer Res ; 39(1): 79, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32381034

RESUMO

BACKGROUND: The poor prognosis of patients with ovarian cancer is mainly due to cancer progression. γ-Synuclein (SNCG) has reported as a critical player in cancer metastasis. However, its biological roles and mechanism are yet incompletely understood in ovarian cancer, especially in high-grade serous ovarian cancer (HGSOC). METHODS: This is a retrospective study of 312 patients with ovarian cancer at a single center between 2006 and 2016. Ovarian cancer tissues were stained by immunohistochemistry to analyze the relationship between SNCG expression and clinicopathologic factors. The clinical outcomes versus SNCG expression level were evaluated by Kaplan-Meier method and multiple Cox regression analysis. Next, systematical functional experiments were given to examine the proliferation and metastatic abilities of SNCG both in vitro and in vivo using loss- and gain- of function approaches. Furthermore, the mechanisms of SNCG overexpression were examined by human phospho-kinase array kit and western blot analysis. RESULTS: Clinically, the expression of SNCG was significantly upregulated in ovarian cancer compared with the borderline and benign tumor, normal ovary, and fallopian tube. Notably, the high level of SNCG correlated with high-risk clinicopathologic features and showed poor survival for patients with HGSOC, indicating an independent prognostic factor for these patients. Functionally, we observed that overexpression of SNCG promoted cell proliferation, tumor formation, migration, and invasion both in vitro and in vivo. Mechanistically, we identified that SNCG promoted cancer cell metastasis through activating the PI3K/AKT signaling pathway. CONCLUSIONS: Our results reveal SNCG up-regulation contributes to the poor clinical outcome of patients with HGSOC and highlight the metastasis-promoting function of SNCG via activating the PI3K/Akt signaling pathway in HGSOC.

14.
Future Med Chem ; 12(9): 813-833, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32208930

RESUMO

Cyclin-dependent kinase 7 (CDK7) plays crucial roles in the regulation of cell cycle and transcription that are tightly associated with cancer development and metastasis. The recent identification of the first covalent inhibitor which possesses high specificity against CDK7 prompts intense studies on designing highly selective CDK7 inhibitors and exploring their applications in cancer treatments. This review summarizes the latest biological studies on CDK7 and reviews the development of CDK7 inhibitors in preclinical and clinical evaluations, along with the prospects and potential challenges in this research area. CDK7 is an attractive anticancer target, and the discovery and development of CDK7 inhibitors has received much attention.


Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/química , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Humanos , Modelos Moleculares , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/química
15.
Genes Dis ; 7(2): 235-244, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32215293

RESUMO

Bone morphogenetic protein 9 (BMP9) (or GDF2) was originally identified from fetal mouse liver cDNA libraries. Emerging evidence indicates BMP9 exerts diverse and pleiotropic functions during postnatal development and in maintaining tissue homeostasis. However, the expression landscape of BMP9 signaling during development and/or in adult tissues remains to be analyzed. Here, we conducted a comprehensive analysis of the expression landscape of BMP9 and its signaling mediators in postnatal mice. By analyzing mouse ENCODE transcriptome datasets we found Bmp9 was highly expressed in the liver and detectable in embryonic brain, adult lungs and adult placenta. We next conducted a comprehensive qPCR analysis of RNAs isolated from major mouse tissues/organs at various ages. We found that Bmp9 was highly expressed in the liver and lung tissues of young adult mice, but decreased in older mice. Interestingly, Bmp9 was only expressed at low to modest levels in developing bones. BMP9-associated TGFß/BMPR type I receptor Alk1 was highly expressed in the adult lungs. Furthermore, the feedback inhibitor Smads Smad6 and Smad7 were widely expressed in mouse postnatal tissues. However, the BMP signaling antagonist noggin was highly expressed in fat and heart in the older age groups, as well as in kidney, liver and lungs in a biphasic fashion. Thus, our findings indicate that the circulating BMP9 produced in liver and lungs may account for its pleiotropic effects on postnatal tissues/organs although possible roles of BMP9 signaling in liver and lungs remain to be fully understood.

16.
Stem Cells Dev ; 29(8): 498-510, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32041483

RESUMO

Mesenchymal stem cells (MSCs) are multipotent progenitors that have the ability to differentiate into multiple lineages, including bone, cartilage, and fat. We previously demonstrated that the least known bone morphogenetic protein (BMP)9 (also known as growth differentiation factor 2) is one of the potent osteogenic factors that can induce both osteogenic and adipogenic differentiation of MSCs. Nonetheless, the molecular mechanism underlying BMP9 action remains to be fully understood. Leptin is an adipocyte-derived hormone in direct proportion to the amount of body fat, and exerts pleiotropic functions, such as regulating energy metabolism, bone mass, and mineral density. In this study, we investigate the potential effect of leptin signaling on BMP9-induced osteogenic differentiation of MSCs. We found that exogenous leptin potentiated BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo, while inhibiting BMP9-induced adipogenic differentiation. BMP9 was shown to induce the expression of leptin and leptin receptor in MSCs, while exogenous leptin upregulated BMP9 expression in less differentiated MSCs. Mechanistically, we demonstrated that a blockade of JAK signaling effectively blunted leptin-potentiated osteogenic differentiation induced by BMP9. Taken together, our results strongly suggest that leptin may potentiate BMP9-induced osteogenesis by cross-regulating BMP9 signaling through the JAK/STAT signaling pathway in MSCs. Thus, it is conceivable that a combined use of BMP9 and leptin may be explored as a novel approach to enhancing efficacious bone regeneration and fracture healing.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento/metabolismo , Janus Quinases/metabolismo , Leptina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
17.
Cancer Gene Ther ; 27(6): 424-437, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31222181

RESUMO

MicroRNAs (miRNAs) are ~22 nucleotide noncoding RNAs that are involved in virtually all aspects of cellular process as their deregulations are associated with many pathological conditions. Mature miRNAs (mMIRs) are generated through a series of tightly-regulated nuclear and cytoplasmic processing events of the transcribed primary, precursor and mMIRs. Effective manipulations of miRNA expression enable us to gain insights into miRNA functions and to explore potential therapeutic applications. Currently, overexpression of miRNAs is achieved by using chemically-synthesized miRNA mimics, or shRNA-like stem-loop vectors to express primary or precursor miRNAs, which are limited by low transfection efficacy or rate-limiting miRNA processing. To overcome rate-limiting miRNA processing, we developed a novel strategy to express mMIRs which are driven by converging U6/H1 dual promoters. As a proof-of-concept study, we constructed mMIR expression vectors for hsa-miR-223 and hsa-Let-7a-1, and demonstrated that the expressed mMIRs effectively silenced target gene expression, specifically suppressed miRNA reporter activity, and significantly affected cell proliferation, similar to respective primary and precursor miRNAs. Furthermore, these mMIR expression vectors can be easily converted into retroviral and adenoviral vectors. Collectively, our simplified mMIR expression system should be a valuable tool to study miRNA functions and/or to deliver miRNA-based therapeutics.


Assuntos
MicroRNAs/administração & dosagem , Animais , Proliferação de Células , Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Mamíferos , MicroRNAs/biossíntese , MicroRNAs/genética , Transfecção
18.
Aging (Albany NY) ; 11(24): 12476-12496, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31825894

RESUMO

Understanding the bone and musculoskeletal system is essential to maintain the health and quality of life of our aging society. Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiate into multiple tissue types including bone. We demonstrated that BMP9 is the most potent osteogenic factors although molecular mechanism underlying BMP9 action is not fully understood. Long noncoding RNAs (lncRNAs) play important regulatory roles in many physiological and/or pathologic processes. Here, we investigated the role of lncRNA Rmst in BMP9-induced osteogenic differentiation of MSCs. We found that Rmst was induced by BMP9 through Smad signaling in MSCs. Rmst knockdown diminished BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro, and attenuated BMP9-induced ectopic bone formation. Silencing Rmst decreased the expression of Notch receptors and ligands. Bioinformatic analysis predicted Rmst could directly bind to eight Notch-targeting miRNAs, six of which were downregulated by BMP9. Silencing Rmst restored the expression of four microRNAs (miRNAs). Furthermore, an activating Notch mutant NICD1 effectively rescued the decreased ALP activity caused by Rmst silencing. Collectively, our results strongly suggest that the Rmst-miRNA-Notch regulatory axis may play an important role in mediating BMP9-induced osteogenic differentiation of MSCs.


Assuntos
Fator 2 de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , RNA Longo não Codificante , Receptores Notch/metabolismo , Diferenciação Celular , Fator 2 de Diferenciação de Crescimento/genética , Células HEK293 , Humanos , Receptores Notch/genética , Transfecção
19.
ACS Synth Biol ; 8(9): 2092-2105, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31465214

RESUMO

As an important post-transcriptional regulatory machinery mediated by ∼21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing ß-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced ß-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of ß-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Clonagem Molecular , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Fator 2 de Diferenciação de Crescimento/antagonistas & inibidores , Fator 2 de Diferenciação de Crescimento/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteogênese , RNA Interferente Pequeno/genética , Proteína Smad4/antagonistas & inibidores , Proteína Smad4/genética , Proteína Smad4/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/antagonistas & inibidores , beta Catenina/genética , beta Catenina/metabolismo
20.
Environ Sci Pollut Res Int ; 26(18): 18181-18190, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31037529

RESUMO

Chromium is used in daily life and has a wide range of functions. It plays an important role in protein synthesis and carbohydrate and lipid metabolism. Chromium is found in trivalent Cr(III) and hexavalent Cr(VI) form; Cr(III) is relatively stable and intimately participates with many phenomena of metabolisms. Whereas, Cr(VI) is toxic, which results in growth inhibition and leading to changes in components of antioxidant systems as well as secondary metabolites. However, the molecular mechanism that is involved in Cr (VI)-induced hepatotoxicity is still unclear. For this purpose, 40 chickens were randomly assigned into two groups: the normal group (feeding the basic diet and clear water), the chromium group (16%LD50, 74.24 mg/kg/day K2Cr2O7 ). The samples were subjected to pathological examination and UHPLC-QE-MS non-target metabolomics method for metabolomics analysis of broiler liver using principal component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA). The central venous cells of the broiler liver in the chromium poisoning group showed turbidity and flaky necrosis, nuclear condensation, nuclear rupture, and even nuclear dissolution. The differential metabolite analysis between the chromium poisoning and the control group showed that 32 differential metabolites were upregulated and 15 were downregulated in positive ion mode. Whereas,17 differential metabolites were downregulated, and 35 were downregulated in negative ion mode (P ≤ 0.05). The potential marker substances are oleic acidamide, farnesylacetone, betaine, taurine, choline, and galactinol. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that the lipid metabolism, carbohydrate metabolism, nucleotide metabolism, amino acid metabolism, energy metabolism, membrane transport, digestive system, and nervous system were the most important metabolic pathways in the liver. This study provides a theoretical basis for the future understanding of the pathogenesis of chromium poisoning and a new insight of the subsequent molecular mechanism of chromium hepatotoxicity.


Assuntos
Carcinógenos Ambientais/toxicidade , Galinhas , Cromo/toxicidade , Fígado/efeitos dos fármacos , Metabolômica , Animais , Biomarcadores/metabolismo , Metabolismo dos Carboidratos , Galinhas/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Redes e Vias Metabólicas , Distribuição Aleatória
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...