scholarly journals Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)-Based  proteomic analysis of mRNA splicing relevant proteins in aging HSPCs

2019 ◽  
Author(s):  
Xiaolan Lian ◽  
Lina Zhang
Keyword(s):  
Proteomic Analysis ◽  
Aging Process ◽  
Mrna Splicing ◽  
Absolute Quantitation ◽  
Qrt Pcr ◽  
Isobaric Tags ◽  
Spliceostatin A

Abstract Background: HSPC aging was closely associated with the organism aging, senile diseases and hematopoietic related diseases. Therefore, study on HSPC aging born great significance to further elucidate the mechanisms of aging and to treat hematopoietic disease resulted from HSPC aging. Little attention had been paid to mRNA splicing as a mechanism underlying HSPC senescence. Results: We used our lab’s patented aging model of HSPCs in vitro to analyze mRNA splicing relevant proteins alterations with iTRAQ based proteomic analysis. We found that not only the notable mRNA splicing genes such as SR, hnRNP, WBP11, Sf3b1, Ptbp1 and U2AF1 but also the scarcely reported mRNA splicing relavant genes such as Rbmxl1, Dhx16, Pcbp2, Pabpc1 were significantly down-regulated. We further verified their genes expressions by qRT-PCR. In addition, we reported the effect of Spliceostatin A (SSA), which inhibits mRNA splicing in vivo and in vitro, on HSPC aging. Conclusions: It was concluded that mRNA splicing emerged as an important vulnerability of HSPC aging. This study improved our understanding of the role of mRNA splicing in the HSPC aging process.

scholarly journals MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma

Blood ◽  
2009 ◽  
Vol 113 (26) ◽  
pp. 6669-6680 ◽  
Author(s):  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
Brian Thompson ◽  
Xavier Leleu ◽  
Abdel Kareem Azab ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Expression Profiling ◽  
Map Kinases ◽  
Qrt Pcr ◽  
Ribosomal Protein S6 ◽  
Genomic Studies

Abstract Detailed genomic studies have shown that cytogenetic abnormalities contribute to multiple myeloma (MM) pathogenesis and disease progression. Nevertheless, little is known about the characteristics of MM at the epigenetic level and specifically how microRNAs regulate MM progression in the context of the bone marrow milieu. Therefore, we performed microRNA expression profiling of bone marrow derived CD138+ MM cells versus their normal cellular counterparts and validated data by qRT-PCR. We identified a MM-specific microRNA signature characterized by down-expression of microRNA-15a/-16 and overexpression of microRNA-222/-221/-382/-181a/-181b (P < .01). We investigated the functional role of microRNA-15a and -16 and showed that they regulate proliferation and growth of MM cells in vitro and in vivo by inhibiting AKT serine/threonine-protein-kinase (AKT3), ribosomal-protein-S6, MAP-kinases, and NF-κB-activator MAP3KIP3. Moreover, miRNA-15a and -16 exerted their anti-MM activity even in the context of the bone marrow milieu in vitro and in vivo. These data indicate that microRNAs play a pivotal role in the biology of MM and represent important targets for novel therapies in MM.

scholarly journals LncRNA LINC01303 Promotes the Progression of Oral Squamous Cell Carcinomas via the miR-429/ZEB1/EMT Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Bo Sun ◽  
Xianyu Zheng ◽  
Weilong Ye ◽  
Pengcheng Zhao ◽  
Guowu Ma
Keyword(s):  
Squamous Cell ◽  
Luciferase Reporter ◽  
Real Time Quantitative Pcr ◽  
Qrt Pcr ◽  
Cytoplasmic Rna ◽  
Transwell Invasion Assay ◽  
Dual Luciferase Reporter Assay

Objectives. The aim of this research was to uncover the biological role and mechanisms of LINC01303 in oral squamous cell carcinoma (OSCC). Materials and Methods. Real-time quantitative PCR (qRT-PCR) was used to determine LINC01303 expression in OSCC tissues. Subcellular distribution of LINC01303 was examined by nuclear/cytoplasmic RNA fractionation and FISH experiments. The role of LINC01303 in the growth of TSCCA and SCC-25 was examined by CCK-8 assay, colony formation, transwell invasion assay in vitro, and xenograft tumor experiment in vivo. Dual-luciferase reporter assay was used to verify the interaction between LINC01303 and miR-429. RNA pull‐down assay was used to discover miR-429‐interacted protein, which was further examined by qRT-PCR, western blot, and rescue experiments. Results. LINC01303 expression was higher in OSCC tissues compared with adjacent nontumor tissues. LINC01303 was found to be localized in the cytoplasm of OSCC cells. Knockdown of LINC01303 inhibited OSCC cell proliferation and invasion, whereas increasing the expression of LINC01303 showed the opposite effects. Furthermore, LINC01303 served as a miR-429 “sponge” and positively regulated ZEB1 expression. Moreover, LINC01303 promoted OSCC through miR-429/ZEB1 axis both in vivo and in vitro. Conclusions. LINC01303 plays an oncogenic role in OSCC and is a promising biomarker for OSCC patients.

scholarly journals METTL3-mediated N6-methyladenosine modification is critical for epithelial-mesenchymal transition and metastasis of gastric cancer

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Ben Yue ◽  
Chenlong Song ◽  
Linxi Yang ◽  
Ran Cui ◽  
Xingwang Cheng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Epithelial Mesenchymal Transition ◽  
Luciferase Reporter ◽  
Mesenchymal Transition ◽  
Qrt Pcr ◽  
Rna Immunoprecipitation ◽  
E Cadherin

Abstract Background As one of the most frequent chemical modifications in eukaryotic mRNAs, N6-methyladenosine (m6A) modification exerts important effects on mRNA stability, splicing, and translation. Recently, the regulatory role of m6A in tumorigenesis has been increasingly recognized. However, dysregulation of m6A and its functions in tumor epithelial-mesenchymal transition (EMT) and metastasis remain obscure. Methods qRT-PCR and immunohistochemistry were used to evaluate the expression of methyltransferase-like 3 (METTL3) in gastric cancer (GC). The effects of METTL3 on GC metastasis were investigated through in vitro and in vivo assays. The mechanism of METTL3 action was explored through transcriptome-sequencing, m6A-sequencing, m6A methylated RNA immunoprecipitation quantitative reverse transcription polymerase chain reaction (MeRIP qRT-PCR), confocal immunofluorescent assay, luciferase reporter assay, co-immunoprecipitation, RNA immunoprecipitation and chromatin immunoprecipitation assay. Results Here, we show that METTL3, a major RNA N6-adenosine methyltransferase, was upregulated in GC. Clinically, elevated METTL3 level was predictive of poor prognosis. Functionally, we found that METTL3 was required for the EMT process in vitro and for metastasis in vivo. Mechanistically, we unveiled the METTL3-mediated m6A modification profile in GC cells for the first time and identified zinc finger MYM-type containing 1 (ZMYM1) as a bona fide m6A target of METTL3. The m6A modification of ZMYM1 mRNA by METTL3 enhanced its stability relying on the “reader” protein HuR (also known as ELAVL1) dependent pathway. In addition, ZMYM1 bound to and mediated the repression of E-cadherin promoter by recruiting the CtBP/LSD1/CoREST complex, thus facilitating the EMT program and metastasis. Conclusions Collectively, our findings indicate the critical role of m6A modification in GC and uncover METTL3/ZMYM1/E-cadherin signaling as a potential therapeutic target in anti-metastatic strategy against GC.

scholarly journals LncRNA ASAP1-IT1 enhances cancer cell stemness via regulating miR-509-3p/YAP1 axis in NSCLC

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yantao Liu ◽  
Yuping Yang ◽  
Lingli Zhang ◽  
Jiaqiang Lin ◽  
Bin Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Growth ◽  
Cancer Stem Cells ◽  
Tumor Growth ◽  
Cancer Cell ◽  
Qrt Pcr

Abstract Background Non-small cell lung cancer (NSCLC) is a major cause of cancer-related death worldwide, and cancer stem cell is responsible for the poor clinical outcome of NSCLC. Previous reports indicated that long noncoding RNAs (lncRNAs) play important roles in maintaining cancer stemness, however, the underlying mechanisms remain unclear. This study investigates the role of ASAP1 Intronic Transcript 1 (ASAP1-IT1) in cancer cell stemness of NSCLC. Methods The expression of ASAP1-IT1, microRNA-509-3p (miR-509-3p) and apoptosis-/stemness-related genes was analyzed by qRT-PCR in NSCLC tissues, cancer cells and spheres of cancer stem cells. Knockdown of ASAP1-IT1 or overexpression of miR-509-3p in NSCLC cells by infection or transfection of respective plasmids. Sphere formation and colony formation were used to detect NSCLC stem cell-like properties and tumor growth in vitro. Luciferase reporter assays, RNA immunoprecitation (RIP) and qRT-PCR assays were used to analyze the interaction between lncRNA and miRNA. The expression of expression of regulated genes of ASAP1-IT1/miR-509-3p axis was evaluated by qRT-PCR and Western blot. The NSCLC xenograft mouse model was used to validate the role of ASAP1-IT1 in NSCLC stemness and tumor growth in vivo. Results ASAP1-IT1 was up-regulated in NSCLC tissues, cancer cells, and in spheres of A549-derived cancer stem cells. Downregulation of ASAP1-IT1 or overexpression of miR-509-3p significantly decreased cell colony formation and stem cell-like properties of A549-dereived stem cells with decreased expression of stem cell biomarkers SOX2, CD34, and CD133, and suppressing the expression of cell growth-related genes, Cyclin A1, Cyclin B1, and PCNA. Furthermore, knockdown of ASAP1-IT1 or overexpression of miR-509-3p repressed tumor growth in nude mice via reducing expression of tumorigenic genes. ASAP1-IT1 was found to interact with miR-509-3p. Moreover, overexpression of ASAP1-IT1 blocked the inhibition by miR-509-3p on stem cell-like properties and cell growth of A549-dereived stem cells both in vitro and in vivo. Finally, the level of YAP1 was regulated by ASAP1-IT1 and miR-509-3p. Conclusions YAP1-involved ASAP1-IT1/miR-509-3p axis promoted NSCLC progression by regulating cancer cell stemness, and targeting this signaling pathway could be is a promising therapeutic strategy to overcome NSCLC stemness.

scholarly journals The WW Domain-Containing Proteins Interact with the Early Spliceosome and Participate in Pre-mRNA Splicing In Vivo

2004 ◽  
Vol 24 (20) ◽  
pp. 9176-9185 ◽  
Author(s):  
Kai-Ti Lin ◽  
Ruei-Min Lu ◽  
Woan-Yuh Tarn
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Ww Domain ◽  
Ww Domains ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3′ part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3′ splice site recognition. Our results suggest an essential role of WW/FF domain-containing factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.

scholarly journals Rhabdovirus Infection Is Dependent on Serine/Threonine Kinase AP2-Associated Kinase 1

Life ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 170
Author(s):  
Jun Luo ◽  
Yue Zhang ◽  
Yang Wang ◽  
Qing Liu ◽  
Luman Chen ◽  
...  
Keyword(s):  
Absolute Quantitation ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
N2a Cells ◽  
Vesicular Stomatitis ◽  
Isobaric Tags ◽  
Potential Strategy ◽  
Prevention And Therapy

Rabies virus (RABV) causes a fatal neurological disease in both humans and animals. Understanding the mechanism of RABV infection is vital for prevention and therapy of virulent rabies infection. Our previous proteomics analysis based on isobaric tags for relative and absolute quantitation to identify factors revealed that RABV infection enhanced AP-2-associated protein kinase 1 (AAK1) in N2a cells. In this study, to further confirm the role of AAK1, we showed that RABV infection increased the transcription and expression of AAK1 in N2a cells. AAK1 knockdown significantly decreased RABV infection in both N2a and BHK-21 cells. AAK1 knockout inhibited RABV infection in N2a cells. Furthermore, inhibition of AAK1 kinase activity using sunitinib decreased RABV infection. However, AAK1 overexpression did not change RABV infection in vitro. Therapeutic administration of sunitinib did not significantly improve the survival rate of mice following lethal RABV challenge. In addition, AAK1 knockdown decreased infection in N2a cells by vesicular stomatitis virus, which is another rhabdovirus. These results indicate that rhabdovirus infection is dependent on AAK1 and inhibition of AAK1 is a potential strategy for the prevention and therapy of rabies.

scholarly journals METTL14 Suppresses Pyroptosis and Diabetic Cardiomyopathy by Down-Regulating TINCR lncRNA

2021 ◽  
Author(s):  
Liping Meng ◽  
Hui Lin ◽  
Xingxiao Huang ◽  
Jingfan Wen ◽  
Shengjie Wu
Keyword(s):  
Diabetic Cardiomyopathy ◽  
Epigenetic Regulation ◽  
Regulatory Mechanism ◽  
Rat Tissues ◽  
The Novel ◽  
Qrt Pcr ◽  
Rna Immunoprecipitation

Abstract Background: N6-methyladenosine (m6A) is one of the most important epigenetic regulation of RNAs, such as lncRNAs. However, the underlying regulatory mechanism of m6A in diabetic cardiomyopathy (DCM) is very limited. In this study, we sought to define the role of METTL14-mediated m6A modification in pyroptosis and DCM progression.Methods: DCM rat model was established and qRT-PCR, western blot and immunohistochemistry (IHC) were used to detect the expression of METTL14 and TINCR. Gain-and-loss functional experiments were performed to define the role of METTL14-TINCR-NLRP3 axis in pyroptosis and DCM. RNA pulldown and RNA immunoprecipitation (RIP) assays were carried out to verify the underlying interaction.Results: In vivo and in vitro studies showed that pyroptosis was tightly involved in DCM progression. METTL14 was downregulated in cardiomyocytes and hear tissues of DCM rat tissues. Functionally, METTL14 suppressed pyroptosis and DCM via downregulating lncRNA TINCR, which further decreased the expression of key pyroptosis-related protein, NLRP3. Mechanistically, METTL14 increased m6A methylation level of TINCR gene, resulting in its downregulation. Moreover, the m6A reader protein YTHDF2 was essential for m6A methylation and mediated the degradation of TINCR. Finally, TINCR positively regulated NLRP3 through increasing its mRNA stability.Conclusions: Our work revealed the novel role of METTL14-mediated m6A methylation and lncRNA regulation in pyroptosis and DCM, which could help extend our understanding the epigenetic regulation of pyroptosis in DCM progression.

Metabolomic Analysis of SCD During Goose Follicular Development: Implications for Lipid Metabolism

2020 ◽  
Author(s):  
Xin Yuan ◽  
Shenqiang Hu ◽  
Liang Li ◽  
Hehe Liu ◽  
Hua He ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Expression Patterns ◽  
Follicular Development ◽  
Pcr Analysis ◽  
Qrt Pcr ◽  
Preovulatory Follicles ◽  
Liquid Chromatography Tandem Mass

Abstract Background Stearoyl-CoA desaturase (SCD) is known to be an important rate-limiting enzyme in the production of MUFA. The role of this enzyme in goose follicular development is poorly understood. To investigate the metabolic mechanism of SCD during goose follicular development, we observed SCD expression patterns during follicular development in vivo and in vitro using quantitative reverse-transcription (qRT)-PCR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine a cellular model of SCD function in granulosa cells (GCs) via SCD overexpression and knockdown.Results qRT-PCR analysis showed that SCD was abundantly expressed in the GC layer, and was upregulated in preovulatory follicles. Peak expression was found in F1 and prehierarchal follicles with diameters of 4–6 mm and 8–10 mm, respectively. We further found the mRNA expression and corresponding enzyme activity to occur in a time-dependent oscillation in vitro, beginning on the first day of GC culture. By using LC-MS/MS, we identified numerous changes in metabolite activation and developed an overview of multiple metabolic pathways, ten of which were associated with lipid metabolism and enriched in both the overexpressed and the knockdown groups.ConclusionsWe confirmed cholesterol and pantothenol or pantothenate as potential metabolite biomarkers for studying SCD-related lipid metabolism in goose GCs.

scholarly journals Linc00460/miR-641 promoted promotes proliferation and autophagy of breast cancer

2020 ◽  
Author(s):  
Wei Zhang ◽  
Huimin Zhang ◽  
Bin Wang ◽  
Yu Ren ◽  
Jianjun He ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Luciferase Reporter ◽  
In Vitro Tests ◽  
Luciferase Reporter Gene ◽  
Qrt Pcr ◽  
In Vivo Tests ◽  
Worse Prognosis

Abstract This manuscript aimed to investigate the oncogenic role of linc00460 in breast cancer both in vivo and in vitro and then specify its downstream microRNAs to build a ceRNA network. As for in vivo tests, we used subgroup analysis and nomogram for survival analysis. As for in vitro tests, qRT-PCR, CCK-8 and Dual luciferase reporter gene were used. Linc00460 expressed highly in breast cancer. The nomogram indicated that linc00460 was associated with worse prognosis. Linc00460 might negatively regulate miR-641 to promote the proliferation and autophagy of breast cancer cell. In conclusion, linc00460 could be a risk factor for breast cancer by regulating miR-641; and it has the potential to be a novel biomarker.

Sign in / Sign up

Export Citation Format

Share Document