lncRNA MALAT1 Regulates Mouse Granulosa Cell Apoptosis and 17β-Estradiol Synthesis via Regulating miR-205/CREB1 Axis

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a known long noncoding RNA, was reported to play a crucial role in follicular growth and ovarian disease. However, the physiological function of MALAT1 in mouse granulosa cells (mGCs) remains largely unclear. The aims of this study were to determine the biological function and molecular mechanism of MALAT1 in mGCs. We knocked down MALAT1 in mGCs by using siRNA against MALAT1. We found that knockdown of MALAT1 promoted apoptosis and caspase-3/9 activities in mGCs. Enzyme-linked immunosorbent assay demonstrated that knockdown of MALAT1 significantly decreased the production of estradiol (E2) and progesterone (P4) in mGCs. Mechanistically, MALAT1 serves as a competing endogenous RNA (ceRNA) to sponge microRNA-205 (miR-205), thereby facilitating its downstream target of cyclic AMP response element- (CRE-) binding protein 1 (CREB1). Furthermore, CREB1 overexpression or miR-205 downregulation partially recovered the effect of MALAT1 depletion in mGCs. In summary, these findings suggested that MALAT1 regulated apoptosis and estradiol synthesis of mGCs through the miR-205/CREB1 axis.

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LncRNA-H19 Drives Cardiomyocyte Senescence by Targeting miR-19a/socs1/p53 Axis

Frontiers in Pharmacology ◽

10.3389/fphar.2021.631835 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuting Zhuang ◽

Tingting Li ◽

Hongwen Xiao ◽

Jiaxu Wu ◽

Shuang Su ◽

...

Keyword(s):

Molecular Mechanism ◽

Noncoding Rna ◽

Physiological Function ◽

Luciferase Reporter ◽

Aged Mouse ◽

Progressive Decline ◽

Downstream Target ◽

Novel Target ◽

Related Proteins

Purpose: Cardiomyocyte senescence is associated with a progressive decline in cardiac physiological function and the risk of cardiovascular events. lncRNA H19 (H19), a well-known long noncoding RNA (lncRNA), is involved in the pathophysiological process of multiple cardiovascular disease such as heart failure, cardiac ischemia and fibrosis. However, the role of H19 in cardiomyocyte senescence remains to be further explored.Methods: Senescence-associated β-galactosidases (SA-β-gal) staining was used to detect cardiomyocyte senescence. Western blot, qRT-PCR and luciferase reporter assay were employed to evaluate the role of H19 in cardiomyocyte senescence and its underling molecular mechanism.Results: H19 level was significantly increased in high glucose-induced senescence cardiomyocytes and aged mouse hearts. Overexpression of H19 enhanced the number of SA-β-gal-positive cells, and the expression of senescence-related proteins p53 and p21, whereas H19 knockdown exerted the opposite effects. Mechanistically, H19 was demonstrated as a competing endogenous RNA (ceRNA) for microRNA-19a (miR-19a): H19 overexpression downregulated miR-19a level, while H19 knockdown upregulated miR-19a. The expression of SOSC1 was dramatically increased in senescence cardiomyocytes and aged mouse hearts. Further experiments identified SOCS1 as a downstream target of miR-19a. H19 upregulated SOCS1 expression and activated the p53/p21 pathway by targeting miR-19a, thus promoting the cardiomyocytes senescence.Conclusion: Our results show that H19 is a pro-senescence lncRNA in cardiomyocytes acting as a ceRNA to target the miR-19a/SOCS1/p53/p21 pathway. Our research reveals a molecular mechanism of cardiomyocyte senescence regulation and provides a novel target of the therapy for senescence-associated cardiac diseases.

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lncRNA MALAT1 regulated ATAD2 to facilitate retinoblastoma progression via miR-655-3p

Open Medicine ◽

10.1515/med-2021-0290 ◽

2021 ◽

Vol 16 (1) ◽

pp. 931-943

Author(s):

Yuxin Zhao ◽

Zhaoxia Wang ◽

Meili Gao ◽

Xuehong Wang ◽

Hui Feng ◽

...

Keyword(s):

Noncoding Rna ◽

Epithelial Mesenchymal Transition ◽

Xenograft Tumor ◽

Competing Endogenous Rna ◽

Mesenchymal Transition ◽

Downstream Target ◽

Lncrna Malat1 ◽

Xenograft Tumor Growth

Abstract Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was reported as an oncogene in many tumors including retinoblastoma (RB). This research mainly focused on the functions and mechanism of MALAT1 in RB. MALAT1 was upregulated in RB tissues and cells, and it served as a competing endogenous RNA (ceRNA) and inhibited miRNA-655-3p (miR-655-3p) expression, which eventually regulated the expression of miR-655-3p downstream target ATPase Family AAA Domain Containing 2 (ATAD2). The level of ATAD2 significantly increased, while that of miR-655-3p remarkably decreased in RB tissues and cells. MALAT1 depletion inhibited cell proliferation, metastasis, and epithelial–mesenchymal transition (EMT), but promoted apoptosis in vitro and blocked xenograft tumor growth in vivo. MALAT1 exerted its oncogenic functions in RB by regulating miR-655-3p/ATAD2 axis.

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MicroRNA-205 affects mouse granulosa cell apoptosis and estradiol synthesis by targeting CREB1

10.1101/301523 ◽

2018 ◽

Author(s):

Pengju Zhang ◽

Jun Wang ◽

Hongyan Lang ◽

Weixia Wang ◽

Xiaohui Liu ◽

...

Keyword(s):

Caspase 3 ◽

Follicular Development ◽

Luciferase Reporter ◽

Follicular Atresia ◽

Qrt Pcr ◽

Reporter Assays ◽

Cyclic Amp Response Element ◽

Cytochrome P450 Family ◽

Estradiol Synthesis

ABSTRACTMicroRNAs-205 (miR-205), were reportedly to be involved in various physiological and pathological processes, but its biological function in follicular atresia remain unknown. In this study, we investigated the expression of miR-205 in mouse granulosa cells (mGCs), and explored its functions in primary mGCs using a serial of in vitro experiments. The result of qRT-PCR demonstrated that miR-205 expression was significantly increased in early atretic follicles (EAF), and progressively atretic follicles (PAF) compared to healthy follicles (HF). Our results also revealed that overexpression of miR-205 in mGCs significantly promoted apoptosis, caspas-3/9 activities, and inhibited estrogen E2 release, and cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1, a key gene in E2 production) expression. Bioinformatics and luciferase reporter assays revealed that the gene of cyclic AMP response element (CRE)-binding protein 1 (CREB1) was a potential target of miR-205. qRT-PCR and western blot assays revealed that overexpression of miR-205 inhibited the expression of CREB1 in mGCs. Importantly, CREB1 upregulation partially rescued the effects of miR-205 on apoptosis, caspase-3/9 activities, E2 production and CYP19A1 expression in mGCs. Our results indicate that miR-205 may play an important role in ovarian follicular development and provide new insights into follicular atresia.

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Downregulation of long noncoding RNA DLEU1 attenuates hypersensitivity in chronic constriction injury-induced neuropathic pain in rats by targeting miR-133a-3p/SRPK1 axis

Molecular Medicine ◽

10.1186/s10020-020-00235-6 ◽

2020 ◽

Vol 26 (1) ◽

Author(s):

Zhen Li ◽

Aiyuan Li ◽

Liping Yan ◽

Tian Yang ◽

Wei Xu ◽

...

Keyword(s):

Spinal Cord ◽

Neuropathic Pain ◽

Noncoding Rna ◽

Target Genes ◽

Luciferase Reporter ◽

Enzyme Linked Immunosorbent Assay ◽

Withdrawal Threshold ◽

Downstream Target ◽

Tnf Α ◽

Close Relationship

Abstract Background Neuropathic pain belongs to chronic pain and is caused by the primary dysfunction of the somatosensory nervous system. Long noncoding RNAs (lncRNAs) have been reported to regulate neuronal functions and play significant roles in neuropathic pain. DLEU1 has been indicated to have close relationship with neuropathic pain. Therefore, our study focused on the significant role of DLEU1 in neuropathic pain rat models. Methods We first constructed a chronic constrictive injury (CCI) rat model. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were employed to evaluate hypersensitivity in neuropathic pain. RT-qPCR was performed to analyze the expression of target genes. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the concentrations of interleukin‐6 (IL-6), tumor necrosis factor‐α (TNF-α) and IL-1β. The underlying mechanisms of DLEU1 were investigated using western blot and luciferase reporter assays. Results Our findings showed that DLEU1 was upregulated in CCI rats. DLEU1 knockdown reduced the concentrations of IL‐6, IL‐1β and TNF‐α in CCI rats, suggesting that neuroinflammation was inhibited by DLEU1 knockdown. Besides, knockdown of DLEU1 inhibited neuropathic pain behaviors. Moreover, it was confirmed that DLEU1 bound with miR-133a-3p and negatively regulated its expression. SRPK1 was the downstream target of miR-133a-3p. DLEU1 competitively bound with miR-133a-3p to upregulate SRPK1. Finally, rescue assays revealed that SRPK1 overexpression rescued the suppressive effects of silenced DLEU1 on hypersensitivity in neuropathic pain and inflammation of spinal cord in CCI rats. Conclusion DLEU1 regulated inflammation of the spinal cord and mediated hypersensitivity in neuropathic pain in CCI rats by binding with miR-133a-3p to upregulate SRPK1 expression.

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Treatment with Melittin Induces Apoptosis and Autophagy of Fibroblast-like Synoviocytes in Patients with Rheumatoid Arthritis

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666191210110826 ◽

2020 ◽

Vol 21 (8) ◽

pp. 734-740 ◽

Cited By ~ 1

Author(s):

Shou-di He ◽

Ning Tan ◽

Chen-xia Sun ◽

Kang-han Liao ◽

Hui-jun Zhu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Caspase 3 ◽

Joint Destruction ◽

Joint Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Caspase 9 ◽

Inflammatory Processes ◽

Related Proteins ◽

Local Stimulation ◽

Fibroblast Like Synoviocytes

Background: Melittin, the major medicinal component of honeybee venom, exerts antiinflammatory, analgesic, and anti-arthritic effects in patients with Rheumatoid Arthritis (RA). RA is an inflammatory autoimmune joint disease that leads to irreversible joint destruction and functional loss. Fibroblast-Like Synoviocytes (FLS) are dominant, special mesenchymal cells characterized by the structure of the synovial intima, playing a crucial role in both the initiation and progression of RA. Objective: In this study, we evaluated the effects of melittin on the viability and apoptosis of FLS isolated from patients with RA. Methods: Cell viability was determined using CCK-8 assays; apoptosis was evaluated by flow cytometry, and the expression levels of apoptosis-related proteins (caspase-3, caspase-9, BAX, and Bcl-2) were also determined. To explore whether melittin alters inflammatory processes in RA-FLS, IL-1β levels were determined using an enzyme-linked immunosorbent assay (ELISA). Furthermore, we performed GFP-LC3 punctate fluorescence dot assays and western blotting (for LC3, ATG5, p62, and Beclin 1) to assess autophagy in RA-FLS. Results: Our results show that melittin can significantly impair viability, promote apoptosis and autophagy, and inhibit IL-1β secretion in RA-FLS. Conclusion: Melittin may be useful in preventing damage to the joints during accidental local stimulation.

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The Effects of D-aspartate on Neurosteroids, Neurosteroid Receptors, and Inflammatory Mediators in Experimental Autoimmune Encephalomyelitis

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530318666181005093459 ◽

2019 ◽

Vol 19 (3) ◽

pp. 316-325

Author(s):

Mahdi Goudarzvand ◽

Yaser Panahi ◽

Reza Yazdani ◽

Hosein Miladi ◽

Saeed Tahmasebi ◽

...

Keyword(s):

Experimental Autoimmune Encephalomyelitis ◽

Inflammatory Mediators ◽

Mouse Serum ◽

Enzyme Linked Immunosorbent Assay ◽

Oral Gavage ◽

Autoimmune Encephalomyelitis ◽

Beneficial Effects ◽

17Β Estradiol ◽

The Brain ◽

Experimental Autoimmune

Objective: Experimental autoimmune encephalomyelitis (EAE) is a widely used model for multiple sclerosis. The present study has been designed to compare the efficiencies of oral and intraperitoneal (IP) administration of D-aspartate (D-Asp) on the onset and severity of EAE, the production of neurosteroids, and the expression of neurosteroid receptors and inflammatory mediators in the brain of EAE mice. Methods: In this study, EAE was induced in C57BL/6 mice treated with D-Asp orally (D-Asp-Oral) or by IP injection (D-Asp-IP). On the 20th day, brains (cerebrums) and cerebellums of mice were evaluated by histological analyses. The brains of mice were analyzed for: 1) Neurosteroid (Progesterone, Testosterone, 17β-estradiol) concentrations; 2) gene expressions of cytokines and neurosteroid receptors by reverse transcription polymerase chain reaction, and 3) quantitative determination of D-Asp using liquid chromatography-tandem mass spectrometry. Further, some inflammatory cytokines and matrix metalloproteinase-2 (MMP-2) were identified in the mouse serum using enzyme-linked immunosorbent assay kits. Results: Our findings demonstrated that after D-Asp was administered, it was taken up and accumulated within the brain. Further, IP injection of D-Asp had more beneficial effects on EAE severity than oral gavage. The concentration of the testosterone and 17β-estradiol in D-Asp-IP group was significantly higher than that of the control group. There were no significant differences in the gene expression of cytokine and neurosteroid receptors between control, D-Asp-IP, and D-Asp-Oral groups. However, IP treatment with D-Asp significantly reduced C-C motif chemokine ligand 2 and MMP-2 serum levels compared to control mice. Conclusion: IP injection of D-Asp had more beneficial effects on EAE severity, neurosteroid induction and reduction of inflammatory mediators than oral gavage.

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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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Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding

Antibodies ◽

10.3390/antib10030031 ◽

2021 ◽

Vol 10 (3) ◽

pp. 31

Author(s):

Ann Christina Bergmann ◽

Cecilie Kyllesbech ◽

Rimantas Slibinskas ◽

Evaldas Ciplys ◽

Peter Højrup ◽

...

Keyword(s):

Amino Acids ◽

Monoclonal Antibodies ◽

Epitope Mapping ◽

Biological Function ◽

Specific Binding ◽

Enzyme Linked Immunosorbent Assay ◽

Potential Therapeutic Target ◽

Charged Amino Acids ◽

Chaperone Protein ◽

Terminal Amino

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

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NEAT1 aggravates sepsis-induced acute kidney injury by sponging miR-22-3p

Open Medicine ◽

10.1515/med-2020-0401 ◽

2020 ◽

Vol 15 (1) ◽

pp. 333-342

Author(s):

Yawei Feng ◽

Jun Liu ◽

Ranliang Wu ◽

Peng Yang ◽

Zhiqiang Ye ◽

...

Keyword(s):

Acute Kidney Injury ◽

Western Blot ◽

Cell Apoptosis ◽

Noncoding Rna ◽

Cell Injury ◽

Kidney Injury ◽

Inflammatory Factors ◽

Luciferase Reporter ◽

Enzyme Linked Immunosorbent Assay ◽

Western Blot Assay

AbstractBackground and aimAcute kidney injury (AKI) is a common complication of sepsis. Long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) plays a vital role in various diseases, including AKI. This study aimed to investigate the function and mechanism of NEAT1 in sepsis-induced AKI.Materials and methodsA septic AKI model was established by treating HK-2 cells with lipopolysaccharide (LPS). The levels of NEAT1 and miR-22-3p were measured by quantitative real-time PCR. Cell apoptosis was assessed by flow cytometry. The levels of apoptosis-related protein and autophagy-related factors were examined by the western blot assay. An enzyme-linked immunosorbent assay was used to calculate the contents of inflammatory factors. The interaction between NEAT1 and miR-22-3p was validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA pull-down assay. The levels of nuclear factor (NF)-κB pathway-related proteins were evaluated by the western blot assay.ResultsNEAT1 was upregulated, while miR-22-3p was downregulated in patients with sepsis and in LPS-stimulated HK-2 cells. LPS treatment triggered cell apoptosis, autophagy, and inflammatory response in HK-2 cells. NEAT1 knockdown attenuated LPS-induced cell injury. NEAT1 modulated LPS-triggered cell injury by targeting miR-22-3p. Furthermore, NEAT1 regulated the NF-κB pathway by modulating miR-22-3p.ConclusionDepletion of NEAT1 alleviated sepsis-induced AKI via regulating the miR-22-3p/NF-κB pathway.

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LncRNA HAND2-AS1 exerts anti-oncogenic effects on bladder cancer via restoration of RARB as a sponge of microRNA-146

Cancer Cell International ◽

10.1186/s12935-021-02063-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Liping Shan ◽

Wei Liu ◽

Yunhong Zhan

Keyword(s):

Cell Proliferation ◽

Bladder Cancer ◽

Noncoding Rna ◽

Caspase 3 ◽

Retinoic Acid Receptor ◽

Human Bladder Cancer ◽

Human Bladder ◽

Link Type ◽

Retinoic Acid Receptor Beta ◽

Receptor Beta

Abstract Background Growing evidence has shown that long noncoding RNA: microRNA: mRNA is implicated in tumor initiation, development, and progression. Long noncoding RNA HAND2-AS1 exhibits anti-cancer effects in diverse cancers. However, the knowledge of HAND-AS1 in bladder cancer development remains unknown. Methods LncRNA and miRNA microarray was conducted to explore different expressed RNA in primary bladder cancer specimens. RNA-RNA interaction prediction tools miRcode (http://www.mircode.org/), DIANA-lncBase v2 (https://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-experimental), DIANA-TarBase v.8 (https://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=tarbasev8%2Findex) and miRDB (http://www.mirdb.org/) were employed to predict the interactions between RNA. Bladder cancer cell lines were used to perform cell proliferation and apoptosis assays. Western blot and quantitative Real-time Polymerase Chain Reaction were used to determine the expression of protein and RNA separately. Dual-luciferase assay was conducted to determine the activity of three prime untranslated region of retinoic acid receptor beta (RARB). Furthermore, 5637 human bladder cancer mouse models were established to investigate the interactions of lncRNA: miRNA: mRNA in vivo. Results Based on the RT2 lncRNA PCR Arrays analysis, we validated HAND2-AS1 declined in bladder cancer and negatively correlated with the depth of invasion and grades. The overexpression of HAND2-AS1 in human bladder cancer cells 5637 and RT4 hampered cell proliferation by provoking Caspase 3-triggered cell apoptosis. Besides, one of the HAND2-AS1 sponges, miR-146, elevated in bladder cancer and targeted the tumor suppressor, retinoic acid receptor beta (RARB). We further demonstrated that the HAND2-AS1: miR-146: RARB complex promoted Caspase 3-mediated apoptosis by suppressing COX-2 expression. Finally, the results gained in mouse xenografts suggested that HAND2-AS1 diminished miR-146 expression, thereby reversing the suppression of miR-146 on RARB-mediated apoptosis and contributing to bladder cancer regression. Conclusion The present study sheds light on the fact that lncRNA HAND2-AS1 exerted as a tumor suppressor by releasing RARB from miR-146, leading to tumor proliferation and invasion inhibition. The findings expanded HAND2-AS-mediated regulatory networks' knowledge and provided novel insights to improve the RARB-targeted regimens against bladder cancer.

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