RNF38 inhibits osteosarcoma cell proliferation by binding to CRY1

2021 ◽  
pp. 1-7
Author(s):  
Jian Zhou ◽  
Zhen-yu Tang ◽  
Xiao-liang Sun
Keyword(s):  
Cell Proliferation ◽  
Western Blotting ◽  
Colony Formation ◽  
Akt Pathway ◽  
Osteosarcoma Cell ◽  
Mrna Levels ◽  
Osteosarcoma Cells ◽  
Protein Levels ◽  
Qrt Pcr

The PI3K/AKT pathway plays an important role in the development of osteosarcoma. RNF38 interferes with activation of the AKT pathway. Cryptochrome1 (CRY1) inhibits osteosarcoma proliferation through the AKT pathway. We aimed to clarify whether RNF38 affects the proliferation of osteosarcoma cells by regulating the PI3K/AKT pathway through its interaction with CRY1. The mRNA levels of RNF38 were determined using qRT-PCR. Protein levels of RNF38, p-p70S6, p70S6, +p-AKT, AKT, p-mTOR, mTOR, and CRY1 were detected by western blotting. The proliferation of osteosarcoma cells was detected using CCK-8 and colony formation assays. The interaction between CRY1 and RNF38 was detected by co-immunoprecipitation and GST pull-down assays. RNF38 expression was higher in Saos-2 and U20S cells than in hFOB cells. Overexpression of RNF38 promoted the proliferation of osteosarcoma cells, the number of colonies, and p-AKT and p-mTOR levels, suggesting that overexpression of RNF38 activated the PI3K/AKT pathway. In addition, RNF38 directly binds to the N-terminal of CRY1. The simultaneous knockdown of RNF38 and CRY1 restored the level of p-AKT, which was reduced by RNF38 knockdown alone. RNF38 affects the proliferation of osteosarcoma cells by regulating the PI3K/AKT pathway through its interaction with CRY1.

scholarly journals KIF21B Expression in Osteosarcoma and Its Regulatory Effect on Osteosarcoma Cell Proliferation and Apoptosis Through the PI3K/AKT Pathway

2021 ◽  
Vol 10 ◽  
Author(s):  
Songjia Ni ◽  
Jianjun Li ◽  
Sujun Qiu ◽  
Yingming Xie ◽  
Kaiqin Gong ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Western Blotting ◽  
Tumor Formation ◽  
Akt Pathway ◽  
Tunel Staining ◽  
Childhood And Adolescence ◽  
Osteosarcoma Cell ◽  
Regulatory Effect ◽  
Proliferation And Apoptosis

Osteosarcoma (OS) is the most common malignancy that occurs mainly during childhood and adolescence; however, no clear molecular or biological mechanism has been identified. In this study, we aimed to explore new biomarkers for the early diagnosis, targeted treatment, and prognostic determination of osteosarcoma. We first used bioinformatics analysis to show that KIF21B can be used as a biomarker for the diagnosis and prognosis of osteosarcoma. We then examined the expression of KIF21B in human osteosarcoma tissues and cell lines using immunohistochemistry, western blotting, and qRT-PCR. It was found that KIF21B expression was significantly upregulated in osteosarcoma tissues and cell lines. After knocking down the expression of KIF21B in the osteosarcoma cell lines 143B and U2-OS, we used cell fluorescence counting, CCK-8 assays, flow cytometry, and TUNEL staining to examine the effects of KIF21B on osteosarcoma cell proliferation and apoptosis. The results demonstrated that knocking down KIF21B in 143B and U2-OS cells could increase cell apoptosis, inhibit cell proliferation, and reduce tumor formation in nude mice. Subsequently, we used gene chips and bioinformatics to analyze the differential gene expression caused by knocking down KIF21B. The results showed that KIF21B may regulate OS cell proliferation and apoptosis by targeting the PI3K/AKT pathway. We then examined the expression of PI3K/AKT- and apoptosis-related proteins using western blotting. KIF21B knockdown inhibited the PI3K pathway, downregulated Bcl-2, and upregulated Bax. Moreover, the use of PI3K/AKT pathway agonists reversed the regulatory effect of KIF21B on the apoptosis and proliferation of 143B and U2-OS cells. In conclusion, our results indicated that KIF21B plays a key role in osteosarcoma. Low KIF21B expression might indirectly increase the apoptosis and inhibit the proliferation of osteosarcoma cells through the PI3K/AKT pathway.

BRCA1-Down-Regulation In Chronic Myeloid Leukemia (CML) Occurs Via The Deubiquitinase / Tumor Suppressor BRCA1-Associated Protein 1 (BAP1)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3813-3813
Author(s):  
Fatima Dkhissi ◽  
Djamel Aggoune ◽  
Julien Pontis ◽  
Marie Laure Bonnet ◽  
Marie Claude Meunier ◽  
...  
Keyword(s):  
Dna Repair ◽  
Tumor Suppressor ◽  
Western Blotting ◽  
Genetic Instability ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Down Regulation ◽  
Brca1 Protein ◽  
Protein Levels ◽  
Qrt Pcr

Abstract BCR-ABL has been shown to lead to a genetic instability in leukemic cells either directly by inducing oxidative stress or indirectly by compromising DNA repair mechanisms. BRCA1 is a major DNA repair gene as it promotes homologous recombination and plays thereby a critical role for preserving genomic integrity. We have previously reported that BCR-ABL down-regulates BRCA1 protein using a post-transcriptional mechanism (Deutsch et al, Blood 2003). The precise mechanism of this down regulation had not been established so far. BAP1 (BRCA1 associated protein-1) is a tumor suppressor gene that encodes a nuclear ubiquitin carboxy-terminal hydrolase, which interacts with BRCA1 protein and with many other cell cycle regulators. BAP1 is mutated in hereditary cancers and the overexpression of a deleted form of BAP1 has been shown to lead to a myelodysplastic syndrome in mice (Dey et al, Science 2012). In a gene profiling analysis of the human UT7 cells expressing BCR-ABL, we have discovered that the expression of BAP-1 is down-regulated as compared to parental UT7 cells. Using qRT-PCR and Western blotting analyses, we have confirmed the reduction of BAP1 transcript and protein levels in UT7 cells expressing BCR-ABL. This occurs in a tyrosine kinase dependent manner as exposure to Imatinib reverted BCR-ABL-associated BAP1 down-regulation. To determine the effects of BAP1 complementation in leukemic cells, we have transfected UT7-BCR-ABL cells with a BAP1 expression vector. The enforced expression of BAP1 in BCR-ABL expressing cells restored BRCA1 protein levels without affecting its mRNA level. As BAP1 is a deubiquitinase, we wondered whether there was an increased ubiquitination of BRCA1 in BCR-ABL expressing cells, due to a BAP1 deficiency. In the UT7-BCR-ABL model, we have performed immunoprecipitation of BRCA1 followed by Western blotting using anti-ubiquitin antibodies. These experiments revealed that BRCA1 was highly ubiquitinylated in BCR-ABL-expressing cells as compared to parental UT7 cells, explaining potentially its down-regulation in CML at the protein level via a proteasome-related mechanism. We next wished to validate these findings in primary human CML samples using qRT-PCR. In a cohort of newly diagnosed chronic phase CML patients before any therapy (n= 21) blood mRNA levels of BAP1 were significantly reduced ( p=0.0032, Mann Whitney Test ) as compared to normal controls (n= 8). Thus, our report reveals for the first time loss of BAP1 expression as a mechanism of the down-regulation of the DNA repair protein BRCA1 in CML. In addition to its contribution to genetic instability, BAP1 could be directly involved in the pathophysiology of CML due to its interactions with epigenetic factors such as polycomb proteins. The molecular mechanisms of BAP1 downregulation in BCR-ABL-expressing leukemic cells is under investigation. Disclosures: Guilhot: Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:BMS, Novartis: Honoraria, Research Funding.

scholarly journals LncRNA GAS5 Suppresses the Proliferation and Invasion of Osteosarcoma Cells via the miR-23a-3p/PTEN/PI3K/AKT Pathway

2020 ◽  
Vol 29 ◽  
pp. 096368972095309
Author(s):  
Jianmin Liu ◽  
Ming Chen ◽  
Longyang Ma ◽  
Xingbo Dang ◽  
Gongliang Du
Keyword(s):  
Cell Proliferation ◽  
Noncoding Rna ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
Luciferase Reporter Gene ◽  
Nude Mouse Model ◽  
Cell Functions ◽  
Proliferation And Invasion

Accumulating evidence has shown that long noncoding RNA GAS5 is a well-known tumor suppressor in the pathogenesis of a variety of human cancers. However, the detailed role of GAS5 in osteosarcoma is still largely unclear. In this study, we found that GAS5 was downregulated in human osteosarcoma tissues and cell lines compared with matched adjacent tissues and normal osteoblast cells. Overexpression of GAS5 could significantly suppress the growth and invasion of osteosarcoma cells, while downregulation of GAS5 promoted cell proliferation and invasion. We confirmed that GAS5 could directly bind with miR-23a-3p by using luciferase reporter gene and RNA immunoprecipitation and pull-down assay. Downregulation of miR-23a-3p repressed cell proliferation and invasion. Overexpression of miR-23a-3p counterbalanced the inhibition effect of GAS5 on cell proliferation and invasion. Further studies indicated that overexpression of GAS5 inhibited cell proliferation and metastasis by regulating phosphatase and tensin homolog (PTEN). PTEN was authenticated as a target of miR-23a-3p. Upregulation of GAS5 or silence of miR-23a-3p increased the level of PTEN, while downregulation of GAS5 or overexpression of miR-23a-3p suppressed the expression of PTEN. In addition, overexpression of GAS5 could neutralize the effect of downregulating PTEN on osteosarcoma cell functions. We proved that GAS5 regulated the viability and invasion of osteosarcoma cells through the PI3K/AKT pathway. Moreover, overexpression of GAS5 could inhibit tumor growth in a xenograft nude mouse model in vivo. In summary, GAS5 functions as a competing endogenous RNA, sponging miR-23a-3p, to promote PTEN expression and suppress cell growth and invasion in osteosarcoma by regulating the PI3K/AKT pathway.

Estrogen enhances growth hormone receptor expression and growth hormone action in rat osteosarcoma cells and human osteoblast-like cells

1997 ◽  
Vol 155 (1) ◽  
pp. 159-164 ◽  
Author(s):  
MC Slootweg ◽  
D Swolin ◽  
JC Netelenbos ◽  
OG Isaksson ◽  
C Ohlsson
Keyword(s):  
Growth Hormone ◽  
Cell Proliferation ◽  
Growth Hormone Receptor ◽  
Receptor Expression ◽  
Osteosarcoma Cell ◽  
Mrna Levels ◽  
Human Osteoblast ◽  
Osteosarcoma Cells ◽  
Gh Receptor ◽  
Hormone Receptor Expression

Postmenopausal bone loss is primarily due to estrogen deficiency. Recent clinical observation demonstrate that GH increases bone mass in GH deficient patients. The present study investigates whether estrogen regulates GH action and GH receptor expression in osteoblasts. 17 beta-estradiol or GH added to the culture medium as single substances did not influence rat osteosarcoma cell proliferation nor human osteoblast-like (hOB) cell proliferation. However, together they synergistically induced osteoblast proliferation (rat osteosarcoma cells 160.1 +/- 15.5% of control cells; human osteoblast-like cells 159.6 +/- 5.1% of control cells). 17 beta-estradiol stimulated 125I-GH binding and GH receptor (GHR) mRNA levels in rat osteosarcoma cells. The stimulatory effect of estradiol was time dependent, reaching a peak after 8 h of incubation with 17 beta-estradiol (binding 216.9 +/- 27.8% and mRNA 374.6 +/- 30.8% of control). The finding that estradiol stimulated 125I-GH binding was confirmed in human osteoblast-like cells. In these cells, 17 beta-estradiol (10(-12) M) increased 125I-GH binding to 203.8 +/- 3.6% of control levels. We conclude that estrogen stimulates GH activity as well as GH binding and GHR mRNA levels in osteoblasts. These findings indicate that estrogen potentiates the effect of GH at the receptor level.

scholarly journals MiR-489-3p inhibits cell proliferation, migration, and invasion, and induces apoptosis, by targeting the BDNF-mediated PI3K/AKT pathway in glioblastoma

2020 ◽  
Vol 15 (1) ◽  
pp. 274-283
Author(s):  
Bo Zheng ◽  
Tao Chen
Keyword(s):  
Cell Proliferation ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Migration And Invasion ◽  
Bdnf Mrna ◽  
Protein Levels ◽  
Rna Immunoprecipitation ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Protein Cell

AbstractAmong astrocyte tumors, glioblastoma (GBM) is the most malignant glioma, highly aggressive and invasive, with extremely poor prognosis. Previous research has reported that microRNAs (miRNAs) participate in the progression of many cancers. Thus, this study aimed to explore the role and the underlying mechanisms of microRNA (miR)-489-3p in GBM progression. The expression of miR-489-3p and brain-derived neurotrophic factor (BDNF) mRNA was measured by quantitative real-time polymerase chain reaction. Western blot analysis was used to detect BDNF protein and the PI3K/AKT pathway-related protein. Cell proliferation, apoptosis, migration, and invasion were analyzed using CKK-8 assay, flow cytometry, and transwell assay, respectively. The interaction between BDNF and miR-489-3p was explored by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. MiR-489-3p was down-regulated and BDNF was up-regulated in GBM tissues and cells. MiR-489-3p re-expression or BDNF knockdown inhibited GBM cell proliferation, migration, and invasion, and promoted apoptosis. BDNF was a target of miR-489-3p, and BDNF up-regulation reversed the effects of miR-489-3p on GBM cells. The protein levels of p-AKT and p-PI3K were notably reduced in GBM cells by overexpression of miR-489-3p, but were rescued following BDNF up-regulation. Therefore, miR-489-3p inhibited proliferation, migration, and invasion, and induced apoptosis, by targeting the BDNF-mediated PI3K/AKT pathway in GBM, providing new strategies for clinical treatment of GBM.

scholarly journals Assessment and Imaging of Intracellular Magnesium in SaOS-2 Osteosarcoma Cells and Its Role in Proliferation

Nutrients ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 1376
Author(s):  
Concettina Cappadone ◽  
Emil Malucelli ◽  
Maddalena Zini ◽  
Giovanna Farruggia ◽  
Giovanna Picone ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Magnesium Deficiency ◽  
Acid Synthesis ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
High Concentration ◽  
Proliferating Cells ◽  
Intracellular Magnesium ◽  
In Cells

Magnesium is an essential nutrient involved in many important processes in living organisms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2–3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth.

FOXD3 inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma through regulation of the PI3K–Akt pathway

2020 ◽  
Vol 98 (6) ◽  
pp. 653-660 ◽  
Author(s):  
Xiaoxing Xie ◽  
Gaoyun Xiong ◽  
Wenjun Chen ◽  
Hongdan Fu ◽  
Mingqian Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nasopharyngeal Carcinoma ◽  
Cell Apoptosis ◽  
Inhibitory Effect ◽  
Akt Pathway ◽  
Migration And Invasion ◽  
Inhibitory Effects ◽  
Protein Levels ◽  
Flow Cytometric ◽  
Phosphoinositide 3 Kinase

FOXD3 has been found previously to positively regulate miR-26b, a tumor inhibitor of nasopharyngeal carcinoma (NPC). However, FOXD3’s precise function and associated mechanism of action in NPC have not yet been investigated. In this study, the expression of FOXD3 mRNA and protein was evaluated using RT-qPCR, western blotting, and immunohistochemistry. Protein levels involved in the phosphoinositide 3-kinase – protein kinase B (PI3K–Akt) pathway were assessed by western blot, and cell proliferation was determined by MTT and colony forming assays. Additionally, cell apoptosis was assessed by flow cytometric assay. Finally, the migration and invasion capabilities of the NPC cells were determined using wound healing and Transwell assays. We found that FOXD3 levels were relatively low in NPC tissue and cells, while an increase caused the inhibition of the PI3K–Akt pathway. Functional experiments found that overexpression of FOXD3 suppressed cell proliferation, migration, and invasion and enhanced cell apoptosis in NPC C6661 cells. IGF-1, an activator of the PI3K–Akt pathway, reversed the inhibitory effect of FOXD3. Furthermore, we found upregulation of the PI3K–Akt pathway and upregulation of the inhibitory effects of FOXD3 on C6661 cellular activities. In conclusion, FOXD3 negatively affected the PI3K–Akt pathway to restrain the processes involved in C6661 cell pathology. These findings further exposed the function and downstream axis of FOXD3 in NPC and displayed a promising new target for NPC therapy.

A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event

1994 ◽  
Vol 14 (5) ◽  
pp. 3085-3093
Author(s):  
L A Chandler ◽  
C P Ehretsmann ◽  
S Bourgeois
Keyword(s):  
Posttranscriptional Regulation ◽  
Tail Length ◽  
Common Mechanism ◽  
Osteosarcoma Cell ◽  
Mrna Levels ◽  
Ras Oncogene ◽  
Down Regulation ◽  
Regulate Gene Expression ◽  
Protein Levels ◽  
Human Osteosarcoma Cell

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

Abstract 3653: Akt3 Offers Stronger Protection than Akt1 Against Brain Injury by Differentially Promoting Mtor Protein Levels In Both In Vitro and In Vivo Stroke Models

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Rong Xie ◽  
Michelle Cheng ◽  
Mei Li ◽  
Robert Sapolsky ◽  
Heng Zhao
Keyword(s):  
Gene Transfer ◽  
Western Blotting ◽  
Ischemic Injury ◽  
Akt Pathway ◽  
Over Expression ◽  
Protein Levels ◽  
Akt Isoforms ◽  
The Brain

Background and Objective: Akt is a serine-threonine kinase that plays critical role in promoting cell survival. Akt consists of three isoforms (Akt1, 2, 3), with Akt3 predominantly expressed in the brain. Although Akt pathway has been shown to mediate neuronal survival in cerebral ischemic injury, it is unclear how these Akt isoforms contribute to neuronal protection, and whether exogenous Akt can protect the brain against ischemic injury or not. In this study, we over-expressed Akt isoforms and its downstream signaling proteins such as FKHR and PRAS40 to investigate the role of the Akt pathway along with its potential relationship with the mTOR pathway in stroke. Methods: Sprauge Dawley rats (250∼280g) were used for all studies. A lentiviral vector consists of a CMV promoter driving IRES-eGFP was used to clone an active Akt 1 and 3 (cAKt 1 and 3), dominant-negative Akt (AktDN), active FKHR (AAA FKHR), and PRAS40. Lentivirus expressing these genes were added to primary mixed cortical cultures for two days prior to oxygen glucose deprivation (OGD) (MOI=1:5). Neuronal survival was measured by LDH release. Lentivirus were stereotaxically injected into the cortex, and rats were subjected to focal ischemia induced by distal MCA occlusion combined with bilateral CCA occlusion. Western blotting and immunofluorescent confocal microscopy were used to detect the expression of Akt isoforms and other proteins in both the Akt and mTOR pathways. Results: Western blotting analysis showed that both endogenous Akt1 and 3 proteins degraded as early as 1 h after stroke, while Akt2 protein remained unchanged until 24 h after stroke. In vitro studies showed that over-expression of both constitutively active cAkt1 and cAkt3 decreased LDH release after OGD, while AktDN worsened neuronal death ( P <0.05). In vivo over-expression of cAkt1, cAkt3 and PRAS40 reduced infarct size after stroke ( P <0.01). Gene transfer of cAkt1 and 3 also promoted protein levels of pAkt (phosphorylated Akt), pPRAS40, pFKHR, pPTEN, pmTOR, but not pGSK3β. Both in vitro and in vivo studies showed that over-expression of cAkt3 resulted in a stronger protection than cAkt1 ( P <0.05). Interestingly, cAkt3 gene transfer preserved both endogenous protein levels of Akt1 and 3, whereas cAkt1 gene transfer only preserved endogenous Akt1. Furthermore, cAkt3 promoted higher pmTOR levels than cAkt1. Treatment of rapamycin, an mTOR inhibitor, blocked the protective effects of both cAkt1 and cAkt3 both in vitro and in vivo. Conclusion: Lentiviral-mediated overexpression of cAkt3 confers stronger protection than that of cAkt1, by maintaining both endogenous Akt1 and Akt3, as well as promoting higher mTOR activities after stroke.

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