MicroRNA-455-3p promotes TGF-β signaling and inhibits osteoarthritis development by directly targeting PAK2

Abstract MicroRNAs (miRNAs, miR) play a key role in the pathogenesis of osteoarthritis (OA). Few studies have examined the regulatory role of P21-activated kinases (PAKs), a family of serine/threonine kinases, in OA. The aim of this study was to determine whether miR-455-3p can regulate cartilage degeneration in OA by targeting PAK2. MiR-455-3p knockout mice showed significant degeneration of the knee cartilage. MiR-455-3p expression increased and PAK2 expression decreased in the late stage of human adipose-derived stem cell (hADSC) chondrogenesis and in chondrocytes affected by OA. Furthermore, in both miR-455-3p-overexpressing chondrocytes and PAK2-suppressing chondrocytes, cartilage-specific genes were upregulated, and hypertrophy-related genes were downregulated. A luciferase reporter assay confirmed that miR-455-3p regulates PAK2 expression by directly targeting the 3′-untranslated regions (3′UTRs) of PAK2 mRNA. IPA-3, a PAK inhibitor, inhibited cartilage degeneration due to OA. Moreover, suppressing PAK2 promoted R-Smad activation in the TGF/Smad signaling pathway in chondrocytes. Altogether, our results suggest that miR-455-3p promotes TGF-β/Smad signaling in chondrocytes and inhibits cartilage degeneration by directly suppressing PAK2. These results thus indicate that miR-455-3p and PAK2 are novel potential therapeutic agents and targets, respectively, for the treatment of OA.

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Bax Targeted by miR-29a Regulates Chondrocyte Apoptosis in Osteoarthritis

BioMed Research International ◽

10.1155/2019/1434538 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Guiqiang Miao ◽

Xuehui Zang ◽

Huige Hou ◽

Hui Sun ◽

Lihui Wang ◽

...

Keyword(s):

Cartilage Degeneration ◽

Degenerative Joint Disease ◽

Joint Disease ◽

Luciferase Reporter ◽

Chondrocyte Apoptosis ◽

Reporter Assay ◽

Proapoptotic Protein ◽

Regulation Mechanisms ◽

Direct Target

Osteoarthritis (OA) is a chronic degenerative joint disease, where chondrocyte apoptosis is responsible for cartilage degeneration. Bax is a well-known proapoptotic protein of the Bcl-2 family, involved in a large number of physiological and pathological processes. However, the regulation mechanisms of Bax underlying chondrocyte apoptosis in OA remain unknown. In the present study, we determined the role of Bax in human OA and chondrocyte apoptosis. The results showed that Bax was upregulated in chondrocytes from the articular cartilage of OA patients and in cultured chondrocyte-like ATDC5 cells treated by IL-1β. Bax was identified to be the direct target of miR-29a by luciferase reporter assay and by western blotting. Inhibition of miR-29a by the mimics protested and overexpression by miR-29a inhibitors aggravated ATDC5 apoptosis induced by IL-1β. These data reveal that miR-29a/Bax axis plays an important role in regulating chondrocyte apoptosis and suggest that targeting the proapoptotic protein Bax and increasing expression levels of miR-29a emerge as potential approach for protection against the development of OA.

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MicroRNA-384 Inhibits the Growth and Invasion of Renal Cell Carcinoma Cells by Targeting Astrocyte Elevated Gene 1

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504017x15035025554553 ◽

2018 ◽

Vol 26 (3) ◽

pp. 457-466 ◽

Cited By ~ 6

Author(s):

Haitao Song ◽

Yanwei Rao ◽

Gang Zhang ◽

Xiangbo Kong

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

Bioinformatic Analysis ◽

Carcinoma Cells ◽

Luciferase Reporter ◽

Reporter Assay ◽

Antitumor Effects ◽

Potential Therapeutic Target

MicroRNAs (miRNAs) are emerging as pivotal regulators in the development and progression of various cancers, including renal cell carcinoma (RCC). MicroRNA-384 (miR-384) has been found to be an important cancer-related miRNA in several types of cancers. However, the role of miR-384 in RCC remains unclear. In this study, we aimed to investigate the potential function of miR-384 in regulating tumorigenesis in RCC. Here we found that miR-384 was significantly downregulated in RCC tissues and cell lines. Overexpression of miR-384 significantly inhibited the growth and invasion of RCC cells, whereas inhibition of miR-384 had the opposite effects. Bioinformatic analysis and luciferase reporter assay showed that miR-384 directly targeted the 3′-untranslated region of astrocyte elevated gene 1 (AEG-1). Further data showed that miR-384 could negatively regulate the expression of AEG-1 in RCC cells. Importantly, miR-384 expression was inversely correlated with AEG-1 expression in clinical RCC specimens. Moreover, miR-384 regulates the activation of Wnt signaling. Overexpression of AEG-1 significantly reversed the antitumor effects of miR-384. Overall, these findings suggest that miR-384 suppresses the growth and invasion of RCC cells via downregulation of AEG-1, providing a potential therapeutic target for the treatment of RCC.

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LncRNA NEAT1 promotes proliferation, migration, invasion and epithelial-mesenchymal transition process in TGF-β2-stimulated lens epithelial cells through regulating the miR-486-5p/SMAD4 axis

Cancer Cell International ◽

10.1186/s12935-020-01619-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Huajun Wang ◽

Guangying Zheng

Keyword(s):

Epithelial Cells ◽

Signaling Pathway ◽

Luciferase Reporter ◽

Posterior Capsular Opacification ◽

Lens Epithelial Cells ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Smad Signaling ◽

Smad Signaling Pathway ◽

Mesenchymal Transformation

Abstract Background Abnormal proliferation, metastasis and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs) are direct factors of posterior capsular opacification (PCO). Nuclear enriched abundant transcript 1 (NEAT1) has been shown to promote cell proliferation, metastasis and EMT, but whether it affects the progression of PCO is unclear. Methods The expression of NEAT1, microRNA-486-5p (miR-486-5p) and Drosophila mothers against decapentaplegic 4 (SMAD4) was determined using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation of cells was measured via 3-(4, 5-dimethyl-2 thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Transwell assay was employed to detect the migration and invasion of cells. The levels of EMT marker proteins, SMAD4 protein and transforming growth factor-β (TGF-β)/SMAD signaling pathway-related proteins were assessed by western blot (WB) analysis. Further, the relationship between miR-486-5p and NEAT1 or SMAD4 was confirmed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and biotin-labeled RNA pull-down assay. Results NEAT1 is upregulated and miR-486-5p is downregulated in the posterior capsular tissues of PCO patients and TGF-β2-induced LECs. Interference of NEAT1 reverses the promoting effect of TGF-β2 on the proliferation, migration, invasion and EMT of LECs. MiR-486-5p can be sponged by NEAT1, and its inhibitor reverses the suppression effect of NEAT1 silencing on the progression of TGF-β2-induced LECs. SMAD4 functions as a target of miR-486-5p, and its overexpression recovers the inhibition effect of miR-486-5p overexpression on the progression of TGF-β2-induced LECs. The activity of the TGF-β/SMAD signaling pathway is regulated by the NEAT1/miR-486-5p/SMAD4 axis. Conclusion Our study shows that NEAT1 has a positive effect on the progression of PCO and is expected to become a new target for PCO treatment.

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miR-6869-5p Inhibits Glioma Cell Proliferation and Invasion via Targeting PGK1

Mediators of Inflammation ◽

10.1155/2020/9752372 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Fakai Wang ◽

Huanjun Zhang ◽

Bing Liu ◽

Wei Liu ◽

Zengchao Zhang

Keyword(s):

Cell Proliferation ◽

Glioma Cell ◽

Glioma Cells ◽

Negative Association ◽

Luciferase Reporter ◽

Reporter Assay ◽

Glioma Cell Proliferation ◽

Precise Role ◽

Proliferation And Invasion

Accumulating studies have suggested the dysregulated microRNAs (miRNAs) play important roles in brain tumors, including glioma. miR-6869-5p has been documented to be aberrantly expressed in diverse cancers. However, the precise role of miR-6869-5p in glioma remains poorly understood. This study is aimed at evaluating its modifying effects on glioma. Significantly decreased expression of miR-6869-5p was found in glioma tissues and cells. Negative association was documented between miR-6869-5p and PGK1 in glioma cells, and PGK1 was demonstrated to be a targeted gene of this miRNA by luciferase reporter assay. miR-6869-5p regulated glioma cell proliferation and invasion via targeting PGK1. In addition, the survival analysis had suggested that low miR-6869-5p expression predicted poor prognosis of glioma patients. This study has suggested that miR-6869-5p is a useful tumor suppressor and prognostic marker in glioma.

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ER stress triggers MCP-1 expression through SET7/9-induced histone methylation in the kidneys of db/db mice

AJP Renal Physiology ◽

10.1152/ajprenal.00697.2012 ◽

2014 ◽

Vol 306 (8) ◽

pp. F916-F925 ◽

Cited By ~ 42

Author(s):

Jigang Chen ◽

Yanhong Guo ◽

Wei Zeng ◽

Li Huang ◽

Qi Pang ◽

...

Keyword(s):

Gene Silencing ◽

Er Stress ◽

Regulatory Mechanism ◽

Luciferase Reporter ◽

Dependent Manner ◽

Reporter Assay ◽

Monocyte Chemoattractant Protein 1 ◽

Monocyte Chemoattractant ◽

Glucose Regulated Protein

Epigenetics plays a key role in the pathogenesis of diabetic nephropathy (DN), although the precise regulatory mechanism is still unclear. Here, we examined the role of endoplasmic reticulum (ER) stress in histone H3 lysine 4 (H3K4) methyltransferase SET7/9-induced monocyte chemoattractant protein-1 (MCP-1) expression in the kidneys of db/db mice. Our results indicate that the expression of MCP-1 significantly increased in the kidneys of db/db mice in a time-dependent manner. An increased chromatin mark associated with an active gene (H3K4me1) at MCP-1 promoters accompanied this change in expression. The expression of SET7/9 and the recruitment to these promoters were also elevated. SET7/9 gene silencing with small interfering (si) RNAs significantly attenuated the expression of H3K4me1 and MCP-1. Furthermore, expression of signaling regulator glucose-regulated protein 78 (GRP78), a monitor of ER stress, significantly increased in the kidneys of db/db mice. The expression of spliced X-box binding protein 1 (XBP1s), an ER stress-inducible transcription factor, and recruitment to the SET7/9 promoters were also increased. XBP1s gene silencing with siRNAs significantly attenuated the expression of SET7/9, H3K4me1, and MCP-1. The chaperone betaine not only effectively downregulated the GRP78 and XBP1s expression levels but also markedly decreased the SET7/9, H3K4me1, and MCP-1 levels. Luciferase reporter assay demonstrated that XBP1s participated in ER stress-induced SET7/9 transcription, Taken together, these results reveal that ER stress can trigger the expression of MCP-1, in part through the XBP1s-mediated induction of SET7/9.

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MicroRNA-381 Negatively Regulates TLR4 Signaling in A549 Cells in Response to LPS Stimulation

BioMed Research International ◽

10.1155/2015/849475 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 7

Author(s):

Zhihao Xu ◽

Dapeng Dong ◽

Xiaofei Chen ◽

Huaqiong Huang ◽

Shenglan Wen

Keyword(s):

Target Gene ◽

Target Genes ◽

Bioinformatics Analysis ◽

Respiratory Infections ◽

A549 Cells ◽

Luciferase Reporter ◽

Reporter Assay ◽

Elisa Kit ◽

Cox 2

It is widely reported that miR-381 is dysregulated in various tumors. However, the specific role of miR-381 in respiratory infections has not been reported. To probe this role, A549 cells were pretreated with 1 μg/mL LPS for 24 h. The level of miR-381 was detected using RT-qPCR. The expression of proinflammatory cytokines was determined using an ELISA kit and western blotting. Bioinformatics analysis was used to predict the target genes of miR-381, and a luciferase reporter assay was used to validate the expression of the target genes. miR-381 expression was increased in A549 cells treated with LPS, which is a ligand of TLRs. Further study revealed that the overexpression of miR-381 increased the activity of NF-κB signaling, thereby increasing the expression of IL-6, TNFα, and COX-2. Further study revealed that IκBα was a target gene of miR-381. The upregulation of miR-381 under LPS stimulation contributes to respiratory infections mainly by targeting IκBα.

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Role of Tenascin-X in regulating TGF-β/Smad signaling pathway in pathogenesis of slow transit constipation

World Journal of Gastroenterology ◽

10.3748/wjg.v26.i7.717 ◽

2020 ◽

Vol 26 (7) ◽

pp. 717-724

Author(s):

Yi-Chao Zhang ◽

Bao-Xiang Chen ◽

Xiao-Yu Xie ◽

Yan Zhou ◽

Qun Qian ◽

...

Keyword(s):

Signaling Pathway ◽

Slow Transit Constipation ◽

Smad Signaling ◽

Smad Signaling Pathway ◽

Slow Transit ◽

Transit Constipation

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Erratum

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504021x16137463165433 ◽

2021 ◽

Vol 28 (6) ◽

pp. 683-684

Author(s):

Yang Sun ◽

Jun-Gong Jin ◽

Wei-Yang Mi ◽

Hao-Wu ◽

Shi-Rong Zhang ◽

...

Keyword(s):

Cell Proliferation ◽

Glioma Cell ◽

Bioinformatics Analysis ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter Assay ◽

Glioma Tissue ◽

Glioma Cell Proliferation ◽

Complementary Binding

Glioma is the most common and lethal malignant intracranial tumor. Long noncoding RNAs (lncRNAs) have been identified as pivotal regulators in the tumorigenesis of glioma. However, the role of lncRNA urothelial carcinoma-associated 1 (UCA1) in glioma genesis is still unknown. The purpose of this study was to investigate the underlying function of UCA1 on glioma genesis. The results demonstrated that UCA1 was upregulated in glioma tissue and indicated a poor prognosis. UCA1 knockdown induced by si-UCA1 significantly suppressed the proliferative, migrative, and invasive activities of glioma cell lines (U87 and U251). Bioinformatics analysis and luciferase reporter assay verified the complementary binding within UCA1 and miR-122 at the 3-UTR. Functional experiments revealed that UCA1 acted as an miR-122 sponge to modulate glioma cell proliferation, migration, and invasion via downregulation of miR-122. Overall, the present study demonstrated that lncRNA UCA1 acts as an endogenous sponge of miR-122 to promote glioma cell proliferation, migration, and invasion, which provides a novel insight and therapeutic target in the tumorigenesis of glioma.

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Knockdown of LincRNA PADNA Promotes Bupivacaine-Induced Neurotoxicity by miR-194/FBXW7 Axis

10.21203/rs.3.rs-30999/v1 ◽

2020 ◽

Author(s):

Fan Yuning ◽

Chen Liang ◽

Wang Tenghuan ◽

Nan Zhenhua ◽

Shengkai Gong

Keyword(s):

Functional Analysis ◽

Western Blot ◽

Cell Viability ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Drg Neurons ◽

Dual Luciferase Reporter Assay

Abstract The aim of the study was to explore the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Mouse DRG neurons were cultured in vitro and treated with bupivacaine to establish the neurotoxicity model. Caspase3 activity, cell viability, tunel assay were analyzed to assess the role of lincRNA PADNA. Dual-luciferase reporter assay was used to determine the binding target of lincRNA PANDA. The expression of lincRNA PADNA was significantly increased with the increasing concentration of bupivacaine. Functional analysis revealed that knockdown of lincRNA PADNA accelerated the caspase3 activity and inhibited the cell viability. Western blot showed that knockdown of lincRNA PADNA promoted the occurrence of cleaved-caspase3. We also proved that lincRNA PADNA may bind with miR-194. Overexpression of miR-194 could rescued the function of lincRNA PADNA, suggesting that lincRNA PADNA may sponge miR-194. In addition, we provided new evidences that lincRNA PADNA/miR-194/FBXW7 axis play an important role in the neurotoxicity process. We performed comprehensive experiments to verify the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Our study provided new evidences and clues for prevention of neurotoxicity.

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Circular RNA hsa_circ_0004872 inhibits gastric cancer progression via the miR-224/Smad4/ADAR1 successive regulatory circuit

Molecular Cancer ◽

10.1186/s12943-020-01268-5 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Cunying Ma ◽

Xiaoying Wang ◽

Fenghua Yang ◽

Yichen Zang ◽

Jiansong Liu ◽

...

Keyword(s):

Gastric Cancer ◽

Nude Mice ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Invasion And Migration ◽

Potential Biomarker ◽

And Migration

Abstract Background Emerging evidence has shown that circular RNAs (circRNAs) play a crucial regulatory role in the occurrence and development of cancer. Exploring the roles and mechanisms of circRNAs in tumorigenesis and progression may help to identify new diagnostic markers and therapeutic targets. In the present study, we investigated the role and regulatory mechanism of hsa_circ_0004872 in gastric cancer (GC). Methods qRT-PCR was used to determine the expression of hsa_circ_0004872 in GC tissues and cells. EdU, CCK-8, transwell and scratch wound healing assays were used to assess the role of hsa_circ_0004872 in GC cell proliferation, invasion and migration, respectively. Subcutaneous and tail vein tumor injections in nude mice were used to assess the role of hsa_circ_0004872 in vivo. RIP assay, biotin-coupled probe pull-down assay, FISH and luciferase reporter assay were performed to confirm the relationship between hsa_circ_0004872 and the identified miRNA. ChIP assay, luciferase reporter assay and western blot were used to determine the direct binding of Smad4 to the promoter of the ADAR1 gene. Results In this study, we found that hsa_circ_0004872 was dramatically downregulated in GC tissues compared with adjacent noncancerous tissues. The expression level of hsa_circ_0004872 was associated with tumor size and local lymph node metastasis. Enforced expression of hsa_circ_0004872 inhibited the proliferation, invasion and migration of GC cells, whereas knockdown of hsa_circ_0004872 had the opposite effects. Nude mice experiments showed that ectopic expression of hsa_circ_0004872 dramatically inhibited tumor growth and metastasis in vivo. Moreover, we demonstrated that hsa_circ_0004872 acted as a “molecular sponge” for miR-224 to upregulate the expression of the miR-224 downstream targets p21 and Smad4. Importantly, we found that the RNA-editing enzyme ADAR1 inhibited hsa_circ_0004872 expression and further led to the upregulation of miR-224. Smad4, the downstream target of miR-224, could further affect hsa_circ_0004872 levels by directly binding to the promoter region of ADAR1 to inhibit ADAR1 expression. Conclusions Our findings showed that hsa_circ_0004872 acted as a tumor suppressor in GC by forming a negative regulatory loop consisting of hsa_circ_0004872/miR-224/Smad4/ADAR1. Thus, hsa_circ_0004872 may serve as a potential biomarker and therapeutic target for GC.

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