MiR-1246 Involves in the Proliferation, Apoptosis and Inflammation of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Targeting Axis Inhibition Protein 2 and Glycogen Synthetase Kinase-3β via Wnt/β-Catenin Pathway in Rheumatoid Arthritis

2021 ◽  
Vol 11 (11) ◽  
pp. 2246-2253
Author(s):  
Siyin Guo ◽  
Shichang Hu ◽  
Guoyuan Zhao ◽  
Weijian Hu ◽  
Yiling Liao ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Bioinformatic Analysis ◽  
Vital Role ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Regulatory Effects ◽  
Apoptosis And Inflammation

Background and Objective: Accumulating evidence supports that fibroblast-like synovial cells (FLS) plays a vital role in the pathogenesis of rheumatoid arthritis (RA). miR-1246 has been reported to be up-regulated in the sera of RA patients. The purpose of the present research was to determine the potential role of miR-1246 in RA and the underlying mechanisms. Methods: miR-1246, GSK3β and AXIN2 levels were determined by RT-qPCR. By exploiting miR-1246 inhibitor, shRNA-GSK3β and shRNA-AXIN2, we detected the effects of miR-1246, GSK3β and AXIN2 on cell proliferation, apoptosis and inflammation in RA-FLSs. Bioinformatics analysis predicted the binding sites of miR-1246 to AXIN2, miR-1246 to GSK3β. Moreover, luciferase reporter assay verified the binding relationship. Results: In comparison with normal human FLSs, higher levels of miR-1246 existed in RA-FLSs. Downregulation of miR-1246 inhibited cell proliferation, inflammation and β-catenin expression, and promoted cell apoptosis. Furthermore, bioinformatic analysis and dual-luciferase reporter assay identified AXIN2 or GSK3β as a target gene of miR-1246. Downregulation of miR-1246 enhanced AXIN2 and GSK3β expression in RA-FLSs. Besides, co-transfection with shRNA-GSK3β or shRNA-AXIN2 partly reversed the regulatory effects of miR-1246 inhibitor in RA-FLSs. Conclusions: Collectively, our in vitro experiments proved that downregulation of miR-1246 might alleviate RA pathogenesis by targeting AXIN2 and GSK3β via Wnt/β-Catenin axis.

MicroRNA-16-5p Promoted Fibroblast-Like Synoviocyte Proliferation and Suppressed Apoptosis via Targeting Suppressor of Cytokine Signaling 6 (SOCS6) in Human Rheumatoid Arthritis

2021 ◽  
Vol 11 (9) ◽  
pp. 1744-1751
Author(s):  
Deqian Meng ◽  
Wenyou Pan ◽  
Ju Li
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Cytokine Signaling ◽  
Human Rheumatoid Arthritis ◽  
Reporter Assay ◽  
Functional Roles ◽  
Proliferation And Apoptosis ◽  
Fibroblast Like Synoviocytes

Accumulating evidence have indicated that MicroRNAs (miRNAs) are key regulators in human rheumatoid arthritis (RA). The aim of this study was to explore the functional roles of miR-16-5p in proliferation, inflammation, and apoptosis of fibroblast-like synoviocytes (FLS). The expression of miR-16-5p and SOCS6 in FLA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter assay was used to verify the direct target of miR-16-5p. Western blot analysis was performed to analysis the levels of SOCS6, Bcl-2, Bax and cleaved caspase 3. miR-16-5p expression was significantly upregulated while SOCS6 level was decreased in RA-FLS compared with normal FLS. In addition, luciferase reporter assay confirmed that SOCS6 was the target of miR-16-5p. Silencing of miR-16-5p inhibited cell proliferation, releases of TNF-α, IL-1β, IL-6 and IL-8, and induced the apoptosis. The effects of miR-16-5p silencing on RA-FLS were reversed by downregulation of SOCS6. In summary, knockdown of miR-16-5p could suppress cell proliferation and accelerate the apoptosis of RA-FLS through targeting SOCS6, which may provide a potential therapeutic target for patients with RA.

scholarly journals Knockdown of Long Noncoding RNA HOXA-AS2 Suppresses Chemoresistance of Acute Myeloid Leukemia via the miR-520c-3p/S100A4 Axis

2018 ◽  
Vol 51 (2) ◽  
pp. 886-896 ◽  
Author(s):  
Xiaoya Dong ◽  
Zhigang Fang ◽  
Mingxue Yu ◽  
Ling Zhang ◽  
Ruozhi Xiao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Online Programs ◽  
Acute Myeloid

Background/Aims: Among different molecular candidates, there is growing data to support that long noncoding RNAs (lncRNAs) play a significant role in acute myeloid leukemia (AML). HOXA-AS2 is significantly overexpressed in a variety of tumors and associated with anti-cancer drug resistance, however, little is known regarding the expression and function of HOXA-AS2 in the chemoresistance of AML. In this study, we aimed to determine the role and molecular mechanism of HOXA-AS2 in adriamycin-based chemotherapy resistance in AML cells. Methods: Quantitative real-time PCR was used to detect HOXA-AS2 expression in the BM samples and ADR cell lines, U/A and T/A cells. Furthermore, the effects of HOXA-AS2 silencing on cell proliferation and apoptosis were assessed in vitro by CCK8 and flow cytometry, and on tumor growth in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in AML. Results: In this study, we showed that HOXA-AS2 is significantly upregulated in BM samples from AML patients after treatment with adriamycin-based chemotherapy and in U/A and T/A cells. Knockdown of HOXA-AS2 inhibited ADR cell proliferation in vitro and in vivo and promoted apoptosis. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3’-UTR with complementary binding sites, which was validated using luciferase reporter assay and anti-Ago2 RIP assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in ADR cells. S100A4 was predicted as a downstream target of miR-520c-3p, which was confirmed by luciferase reporter assay. Conclusion: Our results suggest that HOXA-AS2 plays an important role in the resistance of AML cells to adriamycin. Thus, HOXA-AS2 may represent a therapeutic target for overcoming resistance to adriamycin-based chemotherapy in AML.

scholarly journals FENDRR Sponges miR-424-5p to Inhibit Cell Proliferation, Migration and Invasion in Colorectal Cancer

2020 ◽  
Vol 19 ◽  
pp. 153303382098010
Author(s):  
Chuan Cheng ◽  
Huixia Li ◽  
Jiujian Zheng ◽  
Jie Xu ◽  
Peng Gao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Differential Analysis ◽  
Inhibit Cell Proliferation ◽  
Dual Luciferase Reporter Assay

Objective: LncRNAs are non-coding RNAs exerting vital roles in the occurrence and development of various cancer types. This study tended to describe the expression pattern of FENDRR in colorectal cancer (CRC), and further investigate the role of FENDRR in CRC cell biological behaviors. Methods: Gene expression profile of colon cancer was accessed from the TCGA database, and then processed for differential analysis for identification of differentially expressed lncRNAs and miRNAs. Some in vitro experiments like qRT-PCR, MTT, colony formation assay, wound healing assay and Transwell assay were performed to assess the effect of FENDRR on cell biological behaviors. Dual-luciferase reporter assay was conducted to further validate the targeting relationship between FENDRR and miR-424-5p, and rescue experiments were carried out for determining the mechanism of FENDRR/miR-424-5p underlying the proliferation, migration and invasion of CRC cells. Results: Bioinformatics analysis suggested that FENDRR was significantly down-regulated in CRC tissue, and low FENDRR was intimately correlated to poor prognosis. FENDRR overexpression could greatly inhibit cell proliferation, migration and invasion. Besides, there was a negative correlation between FENDRR and miR-424-5p. Dual-luciferase reporter assay indicated that miR-424-5p was a direct target of FENDRR. Rescue experiments discovered that FENDRR exerted its role in cell proliferation, migration and invasion in CRC via targeting miR-424-5p. Conclusion: FENDRR is poorly expressed in CRC tissue and cells, and low FENDRR is responsible for the inhibition of cell proliferation, migration and invasion of CRC by means of targeting miR-424-5p.

scholarly journals miR-136 Inhibits Malignant Progression of Hepatocellular Carcinoma Cells by Targeting Cyclooxygenase 2

2018 ◽  
Vol 26 (6) ◽  
pp. 967-976 ◽  
Author(s):  
Haiyan Jia ◽  
Hong Wang ◽  
Yanfen Yao ◽  
Chunlei Wang ◽  
Pibao Li
Keyword(s):  
Hepatocellular Carcinoma ◽  
Vital Role ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Vitro Assay ◽  
Proliferation And Metastasis ◽  
Relationship Of

MicroRNAs (miRNAs) play a vital role in regulating tumor progression. Dysregulated miR-136 expression was linked to the development of various human cancers. In the present study, we investigated the expression and relationship of miR-136 and COX2 in hepatocellular carcinoma (HCC) using relevant experiments, involving CCK-8, Transwell assay, and luciferase reporter assay. We demonstrated that miR-136 expression is obviously decreased in HCC tissues and cells, and negatively correlated with the expression of COX2 mRNA. In vitro assay revealed that overexpression of miR-136 significantly changed the expression of proliferation- and metastasis-related proteins and inhibited the proliferation, migration, and invasion of HepG2 and Hep3B cells. Dual-luciferase reporter assay validated that the 3′-untranslated region (3′-UTR) of COX2 is a direct target of miR-136. Furthermore, COX2 siRNA partially enhanced the miR-136 overexpression-induced inhibitory effects. In conclusion, miR-136 was vital in the regulation of HCC cell proliferation and metastasis by targeting COX2. Thus, our findings provided novel evidence that miR-136 might be recommended as a potential target for the diagnosis and treatment of HCC patients.

scholarly journals HOXA-AS2 Promotes Proliferation and Induces Epithelial-Mesenchymal Transition via the miR-520c-3p/GPC3 Axis in Hepatocellular Carcinoma

2018 ◽  
Vol 50 (6) ◽  
pp. 2124-2138 ◽  
Author(s):  
Ying Zhang ◽  
Jianliang Xu ◽  
Shaoquan Zhang ◽  
Jun An ◽  
Jin Zhang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Online Programs ◽  
Hcc Cells

Background/Aims: Previous studies have demonstrated that long non-coding RNAs (lncRNAs) may play critical roles in cancer biology, including Hepatocellular carcinoma (HCC). The HOXA cluster antisense RNA2 (HOXA-AS2) lncRNA plays an important role in carcinogenesis, however, the underlying role of HOXA-AS2 in HCC remains unknown. The present study examined the effects of HOXA-AS2 on the progression of HCC, and explored the underlying molecular mechanisms. Methods: Quantitative real-time PCR was used to detect HOXA-AS2 expression in HCC tissues and cell lines. Furthermore, the effects of HOXA-AS2 silencing and overexpression on cell proliferation, cell cycle, apoptosis, migration, and invasion were assessed in HCC in vitro and in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in HCC cells. Results: We observed that HOXA-AS2 was up-regulated in HCC tissues and cell lines. In vitro experiments revealed that HOXA-AS2 knockdown significantly inhibited HCC cells proliferation by causing G1 arrest and promoting apoptosis, whereas HOXA-AS2 overexpression promoted cell growth. Further functional assays indicated that HOXA-AS2 significantly promoted HCC cell migration and invasion by promoting EMT. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3’-UTR with complementary binding sites, which was validated using luciferase reporter assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in HCC cells. MiR-520c-3p was down-regulated and inversely correlated with HOXA-AS2 expression in HCC tissues. miR-520c-3p suppressed cell proliferation, invasion and migration in HCC cells, and enforced expression of miR-520c-3p attenuated the oncogenic effects of HOXA-AS2 in HCC cells. By bioinformatic analysis and dual-luciferase reporter assay, we found that miR-223-3p directly targeted the 3’-untranslated region (UTR) of Glypican-3 (GPC3), one of the key players in HCC. GPC3 was up-regulated in HCC tissues, and was negatively correlated with miR-520c-3p expression and positively correlated with HOXA-AS2 expression. Conclusion: In summary, our results suggested that the HOXA-AS2/miR-520c-3p/GPC3 axis may play an important role in the regulation of PTC progression, which could serve as a biomarker and therapeutic target for HCC.

scholarly journals miR-520c-3p regulates IL-1β-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GAS2

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Le Peng ◽  
Ming Deng ◽  
Yonggang Ma ◽  
Wei Hu ◽  
Fan Liang
Keyword(s):  
Real Time ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Reporter Assay ◽  
Oa Chondrocytes ◽  
Apoptosis And Inflammation ◽  
Dual Luciferase Reporter Assay

Abstract Background MicroRNAs (miRNAs) have been shown to be associated with osteoarthritis (OA) progression. This study aimed to explore the role of miR-520c-3p in OA progression. Methods Expression levels of miR-520c-3p and Growth arrest-specific 2 (GAS2) were detected using quantitative real-time PCR. The proliferation and apoptosis of cells were measured using cell counting kit 8 (CCK8) assay and flow cytometry. Furthermore, the protein levels of apoptosis-related markers, extracellular degradation markers, inflammatory response markers, and GAS2 were tested using quantitative real-time polymerase chain reaction (RT-PCR) and western blot (WB) analysis. In addition, the interaction between miR-520c-3p and GAS2 was examined using dual luciferase reporter assay. Results GAS2 was highly expressed, and miR-520c-3p was lowly expressed in OA cartilage tissues. miR-520c-3p could promote the proliferation and inhibit the apoptosis and inflammation of OA chondrocytes. miR-520c-3p could be sponged by GAS2, and its inhibitor could reverse the regulation of GAS2 on the biological functions of OA chondrocytes. GAS2 was a target of miR-520c-3p, which was identified by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of GAS2 could inhibit the proliferation and promoted the apoptosis and inflammation of OA chondrocytes. Conclusion Our data showed that miR-520c-3p might regulate the GAS2 to inhibit the progression of OA.

scholarly journals Sp1-Induced Upregulation of LBX2-AS1 Aggravates The Progression of Glioblastoma By Targeting The miR-491-5p/LIF Axis

2021 ◽  
Author(s):  
Wentao Li ◽  
Ismatullah Soufiany ◽  
Xiao Lyu ◽  
Lin Zhao ◽  
Chenfei Lu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Epithelial To Mesenchymal Transition ◽  
Luciferase Reporter ◽  
Mesenchymal Transition ◽  
Stat3 Signaling ◽  
Regulatory Effects

Abstract Background: Mounting evidences have shown the importance of lncRNAs in tumorigenesis and cancer progression. LBX2-AS1 is an oncogenic lncRNA that has been found abnormally expressed in gastric cancer and lung cancer samples. Nevertheless, the biological function of LBX2-AS1 in glioblastoma (GBM) and potential molecular mechanism are largely unclear. Methods: Relative levels of LBX2-AS1 in GBM samples and cell lines were detected by qRT-PCR and FISH. In vivo and in vitro regulatory effects of LBX2-AS1 on cell proliferation, epithelial-to-mesenchymal transition (EMT) and angiogenesis in GBM were examined through xenograft models and functional experiments, respectively. The interaction between Sp1 and LBX2-AS1 was assessed by ChIP. Through bioinformatic analyses, dual-luciferase reporter assay, RIP and Western blot, the regulation of LBX2-AS1 and miR-491-5p on the target gene leukemia Inhibitory factor (LIF) was identified. Results: LBX2-AS1 was upregulated in GBM samples and cell lines, and its transcription was promoted by binding to the transcription factor Sp1. As a lncRNA mainly distributed in the cytoplasm, LBX2-AS1 upregulated LIF, and activated the LIF/STAT3 signaling by exerting the miRNA sponge effect on miR-491-5p, thus promoting cell proliferation, EMT and angiogenesis in GBM. Besides, LBX2-AS1 was unfavorable to the progression of glioma and the survival. Conclusion: Upregulated by Sp1, LBX2-AS1 promotes the progression of GBM by targeting the miR-491-5p/LIF axis. It is suggested that LBX2-AS1 may be a novel diagnostic biomarker and therapeutic target of GBM.

scholarly journals The Immunomodulatory Effects of AlbuminIn VitroandIn Vivo

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Derek S. Wheeler ◽  
John S. Giuliano ◽  
Patrick M. Lahni ◽  
Alvin Denenberg ◽  
Hector R. Wong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lethal Dose ◽  
Intraperitoneal Administration ◽  
Peritoneal Macrophages ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Dose Dependent

Albumin appears to have proinflammatory effectsin vitro. We hypothesized that albumin would induce a state of tolerance to subsequent administration of lipopolysaccharide (LPS)in vitroandin vivo. RAW264.7 and primary peritoneal macrophages were treated with increasing doses of bovine serum albumin (BSA) and harvested for NF-κB luciferase reporter assay or TNF-αELISA. In separate experiments, RAW264.7 cells were preconditioned with 1 mg/mL BSA for 18 h prior to LPS (10 μg/mL) treatment and harvested for NF-κB luciferase reporter assay or TNF-αELISA. Finally, C57Bl/6 mice were preconditioned with albumin via intraperitoneal administration 18 h prior to a lethal dose of LPS (60 mg/kg body wt). Blood was collected at 6 h after LPS administration for TNF-αELISA. Albumin produced a dose-dependent and TLR-4-dependent increase in NF-κB activation and TNF-αgene expressionin vitro. Albumin preconditioning abrogated the LPS-mediated increase in NF-κB activation and TNF-αgene expressionin vitroandin vivo. The clinical significance of these findings remains to be elucidated.

scholarly journals MiR-214 Attenuates Osteogenic Differentiation of Mesenchymal Stem Cells via Targeting FGFR1

2016 ◽  
Vol 38 (2) ◽  
pp. 809-820 ◽  
Author(s):  
Lei Yang ◽  
Dawei Ge ◽  
Xiaojian Cao ◽  
Yingbin Ge ◽  
Hongtao Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Postmenopausal Osteoporosis ◽  
Osteoblast Differentiation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Matrix Mineralization ◽  
Direct Target

Background/Aims: Postmenopausal osteoporosis is closely associated with reduction in the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Previous studies have demonstrated that miR-214 plays an important role in the genesis and development of postmenopausal osteoporosis. Here, we performed this study to investigate the potential mechanism by which miR-214 regulates osteoblast differentiation of MSCs. Methods: First, we explored the expression of miR-214 in MSCs of osteoporotic mice. Next, we examined the change of miR-214 during osteoblast differentiation of MSCs. Then, MSCs were infected with lentiviral vectors expressing miR-214 or miR-214 sponge to investigate the effect of miR-214 on osteoblast differentiation of MSCs. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-214. Results: MiR-214 was up-regulated in MSCs of osteoporotic mice and down-regulated during osteoblast differentiation of MSCs. Furthermore, overexpression of miR-214 inhibited osteoblast differentiation of MSCs in vitro, whereas inhibition of miR-214 function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that FGFR1 is a direct target of miR-214. Conclusions: MiR-214 attenuates osteogenesis by inhibiting the FGFR1/FGF signaling pathway. Our findings suggest that targeting miR-214 promises to be a potential therapy in treatment of postmenopausal osteoporosis.

scholarly journals MiR-3064 in Epicardial Adipose-Derived Exosomes Targets Neuronatin to Regulate Adipogenic Differentiation of Epicardial Adipose Stem Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Wenkai Yang ◽  
Hanjian Tu ◽  
Kai Tang ◽  
Haozhong Huang ◽  
Shi Ou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Target Gene ◽  
Adipogenic Differentiation ◽  
Adipose Stem Cells ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Sequencing Analysis ◽  
Reporter Assay ◽  
Significant Difference

Backgroud: The metabolism of epicardial adipose tissue (EAT) is closely related to coronary atherosclerotic heart disease (CAHD), but the specific mechanism is not fully understood. In this study, we investigated the effects of EAT microenvironment on adipose metabolism from the viewpoint of EAT-derived exosomes and epicardial adipose stem cells (EASCs).Methods: EAT samples from CAHD patients and non-CAHD patients were collected to obtain exosomes via tissue culture. MiRNA sequencing was performed to analyze differences in miRNA expression in exosomes between groups. Luciferase reporter assay was then performed to verify the miRNA target gene. EAT was digested by collagenase to obtain EASCs, which were induced to mature adipocytes in vitro. Immunochemical staining and western blotting were performed to detect protein expression levels.Results: The results showed that CAHD patients had higher levels of EASCs in EAT, and no significant difference in the adipogenic differentiation ability of EASCs was observed between CAHD and non-CAHD patients in vitro. This indicates that the EAT microenvironment is a key factor affecting the adipogenic differentiation of EASCs. The EAT-derived exosomes from CAHD patients inhibited adipogenic differentiation of EASCs in vitro. Sequencing analysis showed that miR-3064-5p was highly expressed in EAT-derived exosomes in CAHD patients, and its inhibitor could improve the adipogenic differentiation of EASCs. Luciferase reporter assay results showed that the target gene of miR-3064-5p is neuronatin (Nnat). Nnat remained silent in EASCs and was less expressed in EAT of CAHD patients.Conclusion: Abovementioned results suggest that Nnat is the key to regulating the adipogenic differentiation of EASCs, and miR-3064-5p in EAT-derived exosomes can inhibit the expression of Nnat by targeting its mRNA, thereby affecting the adipogenic differentiation of EASCs.

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