scholarly journals Cimifugin Suppresses NF-κB Signaling to Prevent Osteoclastogenesis and Periprosthetic Osteolysis

2021 ◽  
Author(s):  
Juan Duan ◽  
Xuantao Hu ◽  
Tao Li ◽  
Pengcheng Dou
Keyword(s):  
Signaling Pathway ◽  
Western Blotting ◽  
Bone Erosion ◽  
Osteoclast Differentiation ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Periprosthetic Osteolysis ◽  
Gene Assay

Abstract Background: Aseptic loosening of prosthesis (ALP) is one of the most common long-term complication of knee and hip arthroplasty. Wear paticle-induced osteoclastogenesis and subsequent periprosthetic osteolysis accounts for the morbidity of ALP. Here, we investigate cimifugin (CIM), a natural extract from Cimicifuga Racemosa and Saposhnikovia Divaricata, as a bone protective drug in treatment of ALP.Method: First, we deployed cell viability and osteoclast formation assays to detect the effect of noncytotoxic CIM on osteoclast differentiation in vitro. Bone slice resorption evaluation and F-actin ring immunofluorescence assays were adopted to measure bone-resorbing function affected by CIM. Then, we introduce quantitative real-time polymerase chain reaction (qRT-PCR) analysis to further identify the repressed osteoclastogenesis by CIM in gene expression level. To reveal underlying mechanism of findings above, we used Western blotting and luciferase reporter gene assay to determine the regulation manner of CIM in NF-κB and MAPKs signaling pathways. Moreover, Ti particle-induced murine calvarial osteolysis model and following histomorphometric analysis via micro-CT and immunohistochemical staining were used to demonstrate the effect of CIM on periprosthetic osteolysis in vivo. Result: CIM administration dose-dependently inhibited both bone marrow-derived macrophages (BMMs) and RAW264.7 cells derived osteoclastogenesis and bone resorption pit formation in vitro, which further supported by reduced expression of F-actin and osteoclast specific genes. According to the Western blotting analysis, inhibition of IkBa phosphorylation in NF-κB signaling pathway, not the phosphorylation of MAPKs, was detected as the suppressive effect of CIM on osteoclastogenesis. In vivo animal experiment demonstrated that CIM alleviated Ti particle-induced bone erosion and osteoclast accumulation in murine calvaria. Conclusion: The current study for the first time proved that CIM could dose-dependently inhibit RANKL-induced osetoclastogenesis via suppressing NF-κB signaling pathway in vitro and prevent periprosthetic osteolysis in vivo. These findings suggest the potential therapeutic use of CIM in ALP.

scholarly journals Cimifugin Suppresses NF-κB Signaling to Prevent Osteoclastogenesis and Periprosthetic Osteolysis

2021 ◽  
Vol 12 ◽  
Author(s):  
Juan Duan ◽  
Xuantao Hu ◽  
Tao Li ◽  
Gen Wu ◽  
Pengcheng Dou ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Western Blot ◽  
Signaling Pathway ◽  
Osteoclast Differentiation ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Periprosthetic Osteolysis

Background: Aseptic loosening of prosthesis (ALP) is one of the most common long-term complications of knee and hip arthroplasty. Wear particle-induced osteoclastogenesis and subsequent periprosthetic osteolysis account for the morbidity of ALP. Here, we investigate the potential of cimifugin (CIM), a natural extract from Cimicifuga racemosa and Saposhnikovia divaricata, as a bone-protective drug in the treatment of ALP.Method: First, we performed cell viability and osteoclast formation assays to assess the effect of noncytotoxic CIM on osteoclast differentiation in vitro. Bone slice resorption and F-actin ring immunofluorescence assays were adopted to assess the effects of CIM on bone-resorption function. Then, quantitative real-time polymerase chain reaction (qRT–PCR) analysis was performed to further assess the repressive effects of CIM on osteoclastogenesis at the gene expression level. To elucidate the mechanisms underlying the above findings, Western blot and luciferase reporter gene assays were used to assess the regulatory effects of CIM on the NF-κB and MAPK signaling pathways. Moreover, a Ti particle-induced murine calvarial osteolysis model and subsequent histomorphometric analysis via micro-CT and immunohistochemical staining were used to elucidate the effect of CIM on periprosthetic osteolysis in vivo.Result: CIM dose-dependently inhibited both bone marrow-derived macrophage (BMM)- and RAW264.7 cell-derived osteoclastogenesis and bone resorption pit formation in vitro, which was further supported by the reduced expression of F-actin and osteoclast-specific genes. According to the Western blot analysis, inhibition of IκBα phosphorylation in the NF-κB signaling pathway, not the phosphorylation of MAPKs, was responsible for the suppressive effect of CIM on osteoclastogenesis. Animal experiments demonstrated that CIM alleviated Ti particle-induced bone erosion and osteoclast accumulation in murine calvaria.Conclusion: The current study suggested for the first time that CIM can inhibit RANKL-induced osetoclastogenesis by suppressing the NF-κB signaling pathway in vitro and prevent periprosthetic osteolysis in vivo. These findings suggest the potential of CIM as a therapeutic in ALP.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals Oncogene APOL1 promotes proliferation and inhibits apoptosis via activating NOTCH1 signaling pathway in pancreatic cancer

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Jiewei Lin ◽  
Zhiwei Xu ◽  
Junjie Xie ◽  
Xiaxing Deng ◽  
Lingxi Jiang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Signaling Pathway ◽  
Density Lipoprotein ◽  
Human Pancreatic Cancer ◽  
Luciferase Reporter ◽  
Notch1 Signaling ◽  
In Vivo Experiments ◽  
Notch1 Signaling Pathway

AbstractAPOL1 encodes a secreted high-density lipoprotein, which has been considered as an aberrantly expressed gene in multiple cancers. Nevertheless, the role of APOL1 in the regulatory mechanisms of pancreatic cancer remains unknown and should be explored. We identified APOL1 was abnormally elevated in human pancreatic cancer tissues compared with that in adjacent tissues and was associated with poor prognosis. The effects of APOL1 in PC cell proliferation, cell cycle, and apoptosis was verified via functional in vitro and in vivo experiments. The results showed that knockdown of APOL1 significantly inhibited the proliferation and promoted apoptosis of pancreatic cancer. In addition, we identified APOL1 could be a regulator of NOTCH1 signaling pathway using bioinformatics tools, qRT-PCR, dual-luciferase reporter assay, and western blotting. In summary, APOL1 could function as an oncogene to promote proliferation and inhibit apoptosis through activating NOTCH1 signaling pathway expression in pancreatic cancer; therefore, it may act as a novel therapeutic target for pancreatic cancer.

scholarly journals Has-miR-875-5p Inhibits Tumorigenesis by Targeting USF2 via TGF-β Signaling Pathway in Gastric Cancer

2021 ◽  
Author(s):  
Shenshuo Gao ◽  
Zhikai Zhang ◽  
Xubin Wang ◽  
Yan Ma ◽  
Chensheng Li ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Signaling Pathway ◽  
Research Direction ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Gastric Cancer Cells ◽  
New Research

Abstract Background: Gastric cancer (GC) is one of the most common malignancies, and more and more evdiences show that the pathogenesis is regulated by various miRNAs.In this study, we investigated the role of miR-875 in GC. Methods:The expression of miR-875-5p was detected in human GC specimens and cell lines by miRNA RT-PCR. The effect of miR-875-5p on GC proliferation was determined by CCK-8 proliferation assay and EDU assay. Migration and invasion were examined by transwell migration and invasion assay and wound healing assay. The interaction between miR-875-5p and its target gene USF2 was verified by a dual luciferase reporter assay. The effects of miR-875-5p in vivo were studied in xenograft nude mice models.Related proteins were detected by Western blot.Results:The results showed that miR-875-5p inhibited the proliferation, migration and invasion of gastric cancer cells in vitro, and inhibited tumorigenesis in vivo. USF2 proved to be a direct target of miR-875-5p. Knockdown of USF2 partially counteracts the effects of miR-875-5p inhibitors.Overexpression of miR-875-5p can inhibit proliferation, migration, and invasion through the TGF-β signaling pathway by down-regulation of USF2 in GC, providing a new research direction for the diagnosis and targeted therapy of GC.Conclusions: MiR-875-5pcan inhibited the progression of GC by directly targeting USF2 and negatively regulating TGF-β signaling pathway.In the future, miR-875-5p is expected to be used as a potential therapeutic target for GC therapy.

scholarly journals Fucoidan Prevents RANKL-Stimulated Osteoclastogenesis and LPS-Induced Inflammatory Bone Loss via Regulation of Akt/GSK3β/PTEN/NFATc1 Signaling Pathway and Calcineurin Activity

Marine Drugs ◽  
2019 ◽  
Vol 17 (6) ◽  
pp. 345 ◽  
Author(s):  
Sheng-Hua Lu ◽  
Yi-Jan Hsia ◽  
Kuang-Chung Shih ◽  
Tz-Chong Chou
Keyword(s):  
Bone Loss ◽  
Molecular Mechanisms ◽  
Bone Erosion ◽  
Osteoclast Differentiation ◽  
Bone Diseases ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Calcineurin Activity

Excessive osteoclast differentiation and/or function plays a pivotal role in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis. Here, we examined whether fucoidan, a sulfated polysaccharide present in brown algae, attenuates receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and lipopolysaccharide (LPS)-induced bone resorption in vivo, and investigated the molecular mechanisms involved. Our results indicated that fucoidan significantly inhibited osteoclast differentiation in RANKL-stimulated macrophages and the bone resorbing activity of osteoclasts. The effects of fucoidan may be mediated by regulation of Akt/GSK3β/PTEN signaling and suppression of the increase in intracellular Ca2+ level and calcineurin activity, thereby inhibiting the translocation of nuclear factor-activated T cells c1 (NFATc1) into the nucleus. However, fucoidan-mediated NFATc1 inactivation was greatly reversed by kenpaullone, a GSK3β inhibitor. In addition, using microcomputer tomography (micro-CT) scanning and bone histomorphometry, we found that fucoidan treatment markedly prevented LPS-induced bone erosion in mice. Collectively, we demonstrated that fucoidan was capable of inhibiting osteoclast differentiation and inflammatory bone loss, which may be modulated by regulation of Akt/GSK3β/PTEN/NFATc1 and Ca2+/calcineurin signaling cascades. These findings suggest that fucoidan may be a potential agent for the treatment of osteoclast-related bone diseases.

scholarly journals MiR-203 Determines Poor Outcome and Suppresses Tumor Growth by Targeting TBK1 in Osteosarcoma

2015 ◽  
Vol 37 (5) ◽  
pp. 1956-1966 ◽  
Author(s):  
Shiping Liu ◽  
Peng Feng
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Cancer Progression ◽  
Human Cancer ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Poor Overall Survival ◽  
Gene Assay

Background/Aims: Increasing evidence has shown that miR-203 plays important role in human cancer progression. However, little is known about the function of miR-203 in osteosarcoma (OS). Methods: The expression of miR-203 in OS tissues and cell lines were examined by qRT-PCR. The biological role of miR-20 in OS cell proliferation was examined in vitro and in vivo. The targets of miR-203 were identified by a luciferase reporter gene assay. Results: miR-203 was down regulated in OS tissues and cell lines; decreased miR-203 was associated with a poor overall survival in OS patients. Restoration of miR-203 expression reduced cell growth in vitro and suppressed tumorigenicity in vivo. In contrast, inhibition of miR-203 stimulated OS cell growth both in vitro and in vivo. In addition, TANK binding kinase 1 (TBK1) was identified as a direct target of miR-203; overexpression of TBK1 partly reversed the suppressive effects of miR-203. Furthermore, TBK1 was found up-regulated and inversely correlated with miR-203 in OS tissues. Conclusion: Taken together, these findings suggest that miR-203 acts as a tumor suppressor via regulation of TBK1 expression in OS progression, and miR-203 may be a promising therapeutic target for OS.

scholarly journals PRPF6 Induces the Alternative Splicing of lncRNA SNHG16 To Promote Progression and Paclitaxel Resistance of Ovarian Cancer via Regulating GATA3 Transcription

2021 ◽  
Author(s):  
Han Wang ◽  
Yingying Zhou ◽  
Siyang Zhang ◽  
Ya Qi ◽  
Min Wang
Keyword(s):  
Ovarian Cancer ◽  
Alternative Splicing ◽  
Epithelial To Mesenchymal Transition ◽  
Binding Protein ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mesenchymal Transition ◽  
Gene Assay

Abstract Background Small nucleolar RNA host gene 16 (SNHG16) and pre-mRNA processing factor 6(PRPF6) play vital roles in regulatory mechanisms of multiple cancers, but the mechanisms in ovarian cancer (OC) remains poorly understood. Methods The expression of SNHG16 transcripts-SNHG16-L/S in OC tissues were analyzed by real-time PCR (RT-PCR). The expression of PRPF6 in OC tissues were detected by Immunohistochemistry (IHC). Tumorigenesis, epithelial-to-mesenchymal transition (EMT) and PTX-resistance were detected by western blot, transwell, CCK-8 assays, colony formation assays and flow cytometry analyses. Molecular interactions were examined by dual-luciferase reporter gene assay, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP). Results The results indicated the expression of SNHG16-L/S was opposite in chemo-resistance and chemo-sensitivity tissues of OC. And SNHG16-L/S had different effects on the progression and PTX-resistance of OC cells. SNHG16-L inhibited GATA binding protein 3 (GATA3) transcription through CCAAT/enhancer-binding protein b (CEBPB) to further promote tumorigenesis, EMT and PTX-resistance of OC. Moreover, PRPF6 was upregulated in chemo-resistance tissues of OC. PRPF6 promoted tumorigenesis and PTX-resistance in vitro and in vivo. Mechanistically, PRPF6 induced the alternative splicing of SNHG16 to downregulate SNHG16-L, which further mediated progression and PTX-resistance through upregulating GATA3 in OC. Conclusions Totally, the results demonstrated that PRPF6 promoted progression and PTX-resistance in OC through SNHG16-L/CEBPB/GATA3 axis. Thus, PRPF6 may become a valuable target for OC therapy.

scholarly journals miR-495-3p Depresses Cell Proliferation and Migration By Down-Regulating HMGB1 in Colorectal Cancer

2021 ◽  
Author(s):  
Zhang Jieling ◽  
Li Kai ◽  
Zheng Huifen ◽  
Zhu Yiping
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
And Migration ◽  
Cancer Tissues

Abstract Background: MicroRNAs play an important role in the genesis and progression of tumors, including colorectal cancer (CRC), which has a high morbidity and mortality rate. In this research, the role of miR-495-3p and HMGB1 in CRC was investigated.Methods: We performed qRT-PCR to detect the expression of miR-495-3p in colorectal cancer tissues and cell lines. Functional experiments such as CCK-8 assay, EDU assay, Transwell assay and apoptosis assay were conducted to explore the effects of miR-495-3p on the proliferation, migration and apoptosis of CRC cells in vitro. Then, the use of database prediction, dual-luciferase reporter gene assay and functional experiments verified the role of miR-495-3p target gene HMGB1 in CRC. Finally, rescue experiments was performed to investigate whether overexpression of HMGB1 could reverse the inhibitory effect of miR-495-3p on CRC cell proliferation in vivo and in vitro.Results: miR-495-3p was down-regulated in colorectal cancer tissues and cell lines, and could inhibit the proliferation and migration of colorectal cancer cells, and promote cell apoptosis. The database prediction and dual-luciferase reporter gene assay showed that HMGB1 was the downstream target gene of miR-495-3p. We finally demonstrated that miR-495-3p inhibited CRC cell proliferation by targeting HMGB1 in vitro and in vivo.Conclusion: Our research shows that miR-495-3p inhibits the progression of colorectal cancer by down-regulating the expression of HMGB1, which indicates that miR-495-3p may become a potential therapeutic target for colorectal cancer.

scholarly journals EPDR1, Which Is Negatively Regulated by miR-429, Suppresses Epithelial Ovarian Cancer Progression via PI3K/AKT Signaling Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhendan Zhao ◽  
Zhiling Wang ◽  
Pengling Wang ◽  
Shujie Liu ◽  
Yingwei Li ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Epithelial Ovarian Cancer ◽  
Signaling Pathway ◽  
Cancer Progression ◽  
Figo Stage ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Gynecology And Obstetrics

Epithelial ovarian cancer (EOC) is the main pathological type of ovarian cancer. In this study, we found that ependymin-related 1 (EPDR1) was remarkably downregulated in EOC tissues, and low EPDR1 expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage, metastasis, and poor prognosis. We confirmed that EPDR1 overexpression dramatically suppressed EOC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, EPDR1 inhibited EOC tumorigenesis and progression, at least in part, through the repression of the PI3K (Phosphoinositide 3-kinase)/AKT (AKT Serine/Threonine Kinase 1) signaling pathway. Furthermore, the expression and function of EPDR1 were regulated by miR-429, as demonstrated by luciferase reporter assays and rescue experiments. In conclusion, our study validated that EPDR1, negatively regulated by miR-429, played an important role as a tumor-suppressor gene in EOC development via inhibition of the PI3K/AKT pathway. The miR-429/EPDR1 axis might provide novel therapeutic targets for individualized treatment of EOC patients in the future.

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