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1.
Int J Mol Sci ; 24(2)2023 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-36674747

RESUMO

Radioresistance compromises the efficacy of radiotherapy for glioblastoma multiforme (GBM), the most devastating and common brain tumor. The present study investigated the relationship between radiation tolerance and formation of polyploid/multinucleated giant (PGCC/MGCC) and quiescent/senescent slow-cycling cancer cells in human U-87, LN-229, and U-251 cell lines differing in TP53/PTEN status and radioresistance. We found significant enrichment in MGCC populations of U-87 and LN-229 cell lines, and generation of numerous small mononuclear (called Raju cells, or RJ cells) U-87-derived cells that eventually form cell colonies, in a process termed neosis, in response to X-ray irradiation (IR) at single acute therapeutic doses of 2-6 Gy. For the first time, single-cell high-content imaging and analysis of Ki-67- and EdU-coupled fluorescence demonstrated that the IR exposure dose-dependently augments two distinct GBM cell populations. Bifurcation of Ki-67 staining suggests fast-cycling and slow-cycling populations with a normal-sized nuclear area, and with an enlarged nuclear area, including one resembling the size of PGCC/MGCCs, that likely underlie the highest radioresistance and propensity for repopulation of U-87 cells. Proliferative activity and anchorage-independent survival of GBM cell lines seem to be related to neosis, low level of apoptosis, fraction of prematurely stress-induced senescent MGCCs, and the expression of p63 and p73, members of p53 family transcription factors, but not to the mutant p53. Collectively, our data support the importance of the TP53wt/PTENmut genotype for the maintenance of cycling radioresistant U-87 cells to produce a significant amount of senescent MGCCs as an IR stress-induced adaptation response to therapeutic irradiation doses.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/radioterapia , Glioblastoma/metabolismo , Raios X , Proteína Supressora de Tumor p53/genética , Antígeno Ki-67/metabolismo , Linhagem Celular Tumoral , Tolerância a Radiação/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
2.
J Phys Chem B ; 127(3): 634-647, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36626331

RESUMO

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tightly regulated dual-specificity phosphatase and key regulator of the PI3K/AKT/mTOR signaling pathway. PTEN phosphorylation at its carboxy-terminal tail (CTT) serine/threonine cluster negatively regulates its tumor suppressor function by inducing a stable, closed, and inactive conformation. Germline PTEN mutations predispose individuals to PTEN hamartoma tumor syndrome (PHTS), a rare inherited cancer syndrome and, intriguingly, one of the most common causes of autism spectrum disorder (ASD). However, the mechanistic details that govern phosphorylated CTT catalytic conformational dynamics in the context of PHTS-associated mutations are unknown. Here, we utilized a comparative protein structure network (PSN)-based approach to investigate PTEN CTT phosphorylation-induced conformational dynamics specific to PTEN-ASD compared to PTEN-cancer phenotypes. Results from our study show differences in structural flexibility, inter-residue contacts, and allosteric communication patterns mediated by CTT phosphorylation, differentiating PTEN-ASD and PTEN-cancer phenotypes. Further, we identified perturbations among global metapaths and community network connections within the active site and inter-domain regions, indicating the significance of these regions in transmitting information across the PSN. Together, our studies provide a mechanistic underpinning of allosteric regulation through the coupled interplay of CTT phosphorylation conformational dynamics in PTEN-ASD and PTEN-cancer mutations. Importantly, the detailed atomistic interactions and structural consequences of PTEN variants reveal potential allosteric druggable target sites as a viable and currently unexplored treatment approach for individuals with different PHTS-associated mutations.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Neoplasias , Humanos , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Mutação , Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/metabolismo
3.
BMC Neurosci ; 24(1): 8, 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36707796

RESUMO

BACKGROUND: Circular RNAs (circRNAs) can act as microRNA (miRNA) sponges, thus regulating gene expression. The role of circRNAs in the process of oxygen-glucose deprivation/reoxygenation (OGD/R) is unclear. Here, we explored the mechanism underlying Circ VRK1 in human brain microvascular endothelial cells (HBMVECs) injury induced by OGD/R. METHODS: The OGD/R cell model was established in HBMVECs. The microarray was applied to detect differentially expressed circRNAs, followed by subcellular fractionation assay. Colony formation assay, flow cytometry, ELISA, tube formation, Transwell and western blot assays were performed for loss-of-function assay. HE staining, TTC staining, immunohistochemistry and western blot were performed in an established mouse model. The relationships between Circ VRK1 and miR-17, and between miR-17 and PTEN were detected by bioinformatics and dual-luciferase assays. Rescue experiments were conducted in vitro and in vivo, and PI3K/AKT activity was detected by Western Blot. RESULTS: Circ VRK1, predominantly present in the cytoplasm of cells, was upregulated in the HBMVECs exposed to OGD/R. Circ VRK1 downregulation decreased proliferation, migration, tube formation, inflammatory factors and oxidative stress, while increased apoptosis in HBMVECs. Moreover, Circ VRK1 silencing reduced neurological damage, cerebral infarct size, CD34-positive cell counts and VEGF expression in mice. Circ VRK1 mediated PTEN expression and the PI3K/AKT pathway by targeting miR-17. Deletion of miR-17 inhibited the effects of Circ VRK1 siRNA, and silencing of PTEN suppressed the effects of miR-17 inhibitor. CONCLUSION: Circ VRK1 was upregulated during OGD/R. Circ VRK1 downregulation regulates PTEN expression by targeting miR-17, thereby promoting PI3K/AKT pathway activity to alleviate OGD/R injury.


Assuntos
MicroRNAs , Oxigênio , Humanos , Camundongos , Animais , Oxigênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Endoteliais/metabolismo , Glucose/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Fosfatidilinositol 3-Quinases , MicroRNAs/genética , Encéfalo/metabolismo , Apoptose/fisiologia , Reperfusão , Proliferação de Células , PTEN Fosfo-Hidrolase/metabolismo
4.
J Orthop Surg Res ; 18(1): 34, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635778

RESUMO

OBJECTIVE: This study aims to investigate the correlation of long non-coding RNA HOX transcript antisense RNA (lncRNA HOTAIR) with the PTEN/PI3K/AKT pathway and clinical-related indicators in osteoarthritis (OA) and determine the effect of baicalin intervention. METHODS: The levels of clinical lipid metabolism indexes and immune-inflammatory indexes in OA patients and normal controls was detected. OA chondrocytes (OA-CHs) were induced with peripheral blood mononuclear cells (PBMCs), followed by baicalin treatment (50 ug/mL). RT-qPCR was performed to measure lncRNA HOTAIR expression. The levels of inflammatory cytokines and adiponectin were detected using ELISA kits. CCK-8 assay was used to assess the viability of CHs. The related protein expression was measured using Western blot analysis. RESULTS: LncRNA HOTAIR might act as a biomarker of OA in vivo. LncRNA HOTAIR was positively correlated with TC, hs-CRP, IgA, TNF-α, and VAS score. Overexpression of lncRNA HOTAIR in vitro inhibited cell proliferation, reduced IL-10 and PTEN expression, but augmented TNF-α, p-PI3K, and p-AKT proteins in OA-CHs stimulated by OA-PBMCs. The changes of above indexes were also observed in OA-CHs stimulated by OA-PBMCs treated with si-lncRNA HOTAIR or baicalin, implying the synergistic effects of baicalin and lncRNA HOTAIR silencing on OA. CONCLUSIONS: Conclusively, lncRNA HOTAIR was highly expressed in OA-CHs, which facilitated OA inflammatory responses by orchestrating inflammatory cytokines and the PTEN/PI3K/AKT pathway. Baicalin exerted therapeutic effects by inhibiting the expression of lncRNA HOTAIR, decreasing the protein levels of p-PI3K and p-AKT, and increasing the protein levels of PTEN, APN, and ADIPOR1.


Assuntos
Osteoartrite , RNA Longo não Codificante , Humanos , Apoptose/genética , Condrócitos/metabolismo , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Osteoartrite/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
5.
Eur J Pharmacol ; 940: 175465, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36566915

RESUMO

Liver cancer is a kind of malignant tumor with poor sensitivity to chemotherapy. It is urgent to investigate approaches to improve the outcome of chemotherapy. KDM5A has been reported to be an oncogene in various cancers and is associated with drug resistance. However, the functions of KDM5A in chemotherapeutic sensitivity of liver cancer not been well illustrated. In this study, we found that KDM5A was upregulated in liver cancer tissue and cell lines. KDM5A knockdown using a gene interference strategy suppressed the growth of liver cancer in vitro and in vivo. CPI-455, a pharmacological inactivation of KDM5A enhanced the cytotoxicity of cisplatin (CDDP) in liver cells. CPI-455 and CDDP cotreatment resulted in apoptosis and mitochondrial dysfunction. We also found that knockdown or inactivation of KDM5A resulted in the downregulation of ROCK1, an oncogene regulating the activation of the PTEN/AKT signaling pathway. In particular, overexpression of ROCK1 or SF1670, a pharmacological inhibitor of PTEN, alleviated the cytotoxicity of CPI-455 and CDDP cotreatment. In HCCLM3 xenografts, CPI-455 and CDDP cotreatment dramatically inhibited the growth of xenograft tumor compared to CPI-455 or CDDP treatment alone. In conclusion, this study suggested that targeting the inactivation of KDM5A is an efficient strategy to enhance the chemosensitivity of liver cancer cells to CDDP by modulating the ROCK1/PTEN/AKT signaling pathway.


Assuntos
Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Transdução de Sinais , Apoptose , Neoplasias Hepáticas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Quinases Associadas a rho/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
6.
Int J Exp Pathol ; 104(1): 33-42, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36576072

RESUMO

Ras homologue family member C (RhoC) is an oncogene in diverse types of human cancers, whereas its regulatory mechanisms involving macrophage polarization is rarely investigated. This study is designed to explore the regulatory role of RhoC in colon cancer and the underlying molecular mechanisms involving macrophage polarization. We detected RhoC expression by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, and analysed the biological function of RhoC knockdown in CC cells by the MTT, wound healing and transwell assay. Macrophage polarization-associated markers, genes associated with migration, phosphatase and tensin homologue (PTEN) and forkhead box O (FOXO) were determined by qRT-PCR and western blot. The xenograft tumour mouse model was used to assess the role of RhoC in vivo. RhoC is highly expressed in CC cells. The cell viability, invasion and migration abilities of CC cells were reduced by knockdown of RhoC. RhoC knockdown promoted M1 polarization, inhibited M2 polarization and decreased levels of genes associated with migration (matrix metalloproteinase-2 and matrix metalloproteinase-9). Silencing of RhoC inhibited tumour growth and expression of genes associated with migration in the xenografted model. In addition, silencing of RhoC promoted PTEN/FOXO1 expression, and PTEN inhibitor (SF1670) reversed the inhibitory effects of RhoC silencing. We demonstrated that silencing of RhoC reduced CC cells invasion and migration, and tumour growth by suppressing M2 macrophage polarization via regulating the PTEN/FOXO1 pathway.


Assuntos
Neoplasias do Colo , Proteína Forkhead Box O1 , Macrófagos , PTEN Fosfo-Hidrolase , Proteína de Ligação a GTP rhoC , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/farmacologia , Regulação Neoplásica da Expressão Gênica , Macrófagos/patologia , Metaloproteinase 2 da Matriz/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteína de Ligação a GTP rhoC/genética , Proteína de Ligação a GTP rhoC/metabolismo
7.
Pharm Biol ; 61(1): 23-29, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36524761

RESUMO

CONTEXT: Salvianolic acid B (SAB) can alleviate renal fibrosis and improve the renal function. OBJECTIVE: To investigate the effect of SAB on renal tubulointerstitial fibrosis and explore its underlying mechanisms. MATERIALS AND METHODS: Male C57 mice were subjected to unilateral ureteric obstruction (UUO) and aristolochic acid nephropathy (AAN) for renal fibrosis indication. Vehicle or SAB (10 mg/kg/d, i.p.) were given consecutively for 2 weeks in UUO mice while 4 weeks in AAN mice. The serum creatinine (Scr) and blood urine nitrogen (BUN) were measured. Masson's trichrome staining and the fibrotic markers (FN and α-SMA) were used to evaluate renal fibrosis. NRK-49F cells exposed to 2.5 ng/mL TGF-ß were treated with SAB in the presence or absence of 20 µM 3-DZNep, an inhibitor of EZH2. The protein expression of EZH2, H3k27me3 and PTEN/Akt signaling pathway in renal tissue and NRK-49F cells were measured by Western blots. RESULTS: SAB significantly improved the levels of Scr by 24.3% and BUN by 35.7% in AAN mice. SAB reduced renal interstitial collagen deposition by 34.7% in UUO mice and 72.8% in AAN mice. Both in vivo and in vitro studies demonstrated that SAB suppressed the expression of FN and α-SMA, increased PTEN and decreased the phosphorylation of Akt, which were correlated with the down-regulation of EZH2 and H3k27me3. The inhibition of EZH2 attenuated the anti-fibrotic effects of SAB in NRK-49Fs. CONCLUSION: SAB might have therapeutic potential on renal fibrosis of CKD through inhibiting EZH2, which encourages further clinical trials.


Assuntos
Nefropatias , Animais , Masculino , Camundongos , Fibrose/tratamento farmacológico , Fibrose/patologia , Histonas/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/tratamento farmacológico , Nefropatias/prevenção & controle , Nefropatias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Obstrução Ureteral/complicações , Obstrução Ureteral/tratamento farmacológico , Benzofuranos/farmacologia , Benzofuranos/uso terapêutico , Depsídeos/farmacologia , Depsídeos/uso terapêutico , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo
8.
J Clin Lab Anal ; 37(1): e24802, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36478207

RESUMO

BACKGROUND: LncRNA LINC00534 has been found to be differentially expressed in placental tissue samples of preeclampsia (PE), but the exact mechanism is still unclear. METHODS: In vitro assays were carried out in HTR-8/SVneo cells using various methods, including cell counting kit-8 (CCK-8), transwells, flow cytometry, and Western blotting (WB) and quantitative polymerase chain reaction. RNA pull-down and bioinformatics analysis were applied to examine other potential underlying mechanisms involved. RESULTS: We found that there was a high expression of LINC00534 in the placental tissues of patients with PE. LINC00534 overexpression (OE) significantly inhibited cell proliferation and migration as well as accelerated cell apoptosis in HTR8/SVneo cells. The knockdown of LINC00534 produced an opposite trend. Mechanistically, LINC00534 promoted the expressions of PTEN (Phosphatase and tensin homolog) through decreasing miR-494-3p. Further rescue studies showed that LINC00534 played a role by targeting mir-494-3p, which controlled the growth and migration of HTR-8/SVneo trophoblast cells via regulating PTEN/PI3K/AKT (Phosphatidylinositol3-kinase/protein kinase B). Moreover, lncRNA pull-down assay identified 198 potential bound proteins for LINC00534. Those proteins were mostly involved in RNA processing and modification, posttranslational modification, protein turnover, and chaperones. CONCLUSION: Overall, by suppressing HTR8/SVneo cell growth and migration via the miR-494-3p/PTEN axis and other mechanisms, LINC00534 offers new insight into PE pathogenesis.


Assuntos
MicroRNAs , Pré-Eclâmpsia , RNA Longo não Codificante , Humanos , Gravidez , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , Placenta/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Trofoblastos/metabolismo , Proliferação de Células/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
9.
Front Biosci (Landmark Ed) ; 27(11): 299, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36472099

RESUMO

BACKGROUND: To investigate the effect and potential molecular mechanisms of Dipsacoside B (DB), an herb monomer extracted from Dipsacusasper or Lonicera macranthoides, on the migration and proliferation of vascular smooth muscle cells (VSMCs) and balloon-induced neointimal formation. METHODS: In vivo, rat abdominal aorta balloon injury model was utilized to investigate the effect of DB on the neointimal formation. In vitro, cultured VSMCs were used to investigate the effect of DB on Angiotensin-II (Ang-II)-induced migration and proliferation of VSMCs. Western blot and immunofluorescence were used to measure PTEN expression. RESULTS: As compared to vehicle control balloon-injury group, DB treatment significantly inhibited the neointimal formation together up-regulated the expression of phosphatase and tension homolog deleted on chromosome 10 (PTEN). Cell proliferations (MTT and Edu incorporation) assays and wound migration measurement further revealed that treatment with DB significantly blunted Ang-II-induced proliferation and migration potential of VSMCs. Western blot analysis exhibited that DB upregulated the expression of PTEN in vivo and in vitro. CONCLUSIONS: DB treatment suppresses the proliferation and migration of VSMCs and reduces neointimal formation by the mechanisms involving regulating the phenotype switch of VSMCs via upregulating PTEN expression.


Assuntos
Músculo Liso Vascular , Miócitos de Músculo Liso , Ratos , Animais , Movimento Celular , Neointima/metabolismo , Proliferação de Células , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Células Cultivadas , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
10.
Biochemistry (Mosc) ; 87(11): 1310-1326, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36509719

RESUMO

Tumor-suppressive effects of PTEN are well-known, but modern evidence suggest that they are not limited to its ability to inhibit pro-oncogenic PI3K/AKT signaling pathway. Features of PTEN structure facilitate its interaction with substrates of different nature and display its activity in various ways both in the cytoplasm and in cell nuclei, which makes it possible to take a broader look at its ability to suppress tumor growth. The possible mechanisms of the loss of PTEN effects are also diverse - PTEN can be regulated at many levels, leading to change in the protein activity or its amount in the cell, while their significance for the development of malignant tumors has yet to be studied. Here we summarize the current data on the PTEN structure, its functions and changes in its regulatory mechanisms during malignant transformation of the cells, focusing on one of the most sensitive to the loss of PTEN types of malignant tumors - endometrial cancer.


Assuntos
Neoplasias do Endométrio , Fosfatidilinositol 3-Quinases , Feminino , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transformação Celular Neoplásica/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Genes Supressores de Tumor , Transdução de Sinais , Proteínas Proto-Oncogênicas c-akt/metabolismo
11.
Cell Death Dis ; 13(12): 1075, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36575176

RESUMO

Nutrient-limiting conditions are common during cancer development. The coordination of cellular glucose levels and cell survival is a fundamental question in cell biology and has not been completely understood. 4EBP1 is known as a translational repressor to regulate cell proliferation and survival by controlling translation initiation, however, whether 4EBP1 could participate in tumor survival by other mechanism except for translational repression function, especially under glucose starvation conditions remains unknown. Here, we found that protein levels of 4EBP1 was up-regulated in the central region of the tumor which always suffered nutrient deprivation compared with the peripheral region. We further discovered that 4EBP1 was dephosphorylated by PTPMT1 under glucose starvation conditions, which prevented 4EBP1 from being targeted for ubiquitin-mediated proteasomal degradation by HERC5. After that, 4EBP1 translocated to cytoplasm and interacted with STAT3 by competing with JAK and ERK, leading to the inactivation of STAT3 in the cytoplasm, resulting in apoptosis under glucose withdrawal conditions. Moreover, 4EBP1 knockdown increased the tumor volume and weight in xenograft models by inhibiting apoptosis in the central region of tumor. These findings highlight a novel mechanism for 4EBP1 as a new cellular glucose sensor in regulating cancer cell death under glucose deprivation conditions, which was different from its classical function as a translational repressor.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Glucose , Neoplasias Pulmonares , Humanos , Morte Celular , Proliferação de Células , Glucose/metabolismo , Neoplasias Pulmonares/genética , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo
12.
Aging (Albany NY) ; 14(24): 10081-10092, 2022 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-36575044

RESUMO

This study aims to explore the specific mechanisms of SALL4 on the migration, invasion and proliferation of HCC. HepG2 and SMMC-7721 cells were transfected with SALL4 NC, mimics and inhibitors. The proliferation capability and cell cycle progression of HCC cells were detected through CCK8 assay and flow cytometry, and their migration and invasion capabilities were detected by wound healing assay and Transwell assay. In SALL4 inhibitor NC group and SALL4 inhibitor group, the PTEN inhibitor SF1670 was added, and the expression levels of PI3K/AKT, migration, invasion and proliferation-related proteins were detected by Western blotting. Results showed that after up-regulation of SALL4, the migration distance of HCC cells increased, the numbers of migrated cells and the number of colonies formed significantly rosed, and there were fewer cells in G1 phase but significantly more cells in S phase, thereby down-regulation of SALL4, the opposite results. The results of Western blotting revealed that after SF1670, the specific PTEN inhibitor was added in SALL4 inhibitor group and SALL4 inhibitor NC group, the protein expression of PTEN in HCC cells significantly declined, while the protein expressions of p-PI3K, p-AKT, MMP2, MMP9, CyclinD, CyclinA1, PCNA and P62 significantly rose. In conclusion, SALL4 activates the PI3K/AKT signaling pathway through targeting PTEN, thereby facilitating the migration, invasion and proliferation of HCC cells.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Humanos , Carcinoma Hepatocelular/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , MicroRNAs/metabolismo , Neoplasias Hepáticas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Transdução de Sinais/fisiologia , Movimento Celular , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fatores de Transcrição/genética
13.
Folia Histochem Cytobiol ; 60(4): 323-334, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36504133

RESUMO

INTRODUCTION: As one of the basic components of Astragalus, Astragaloside IV (AS-IV) has a protective effect on endothelial injury caused by diabetes. AS-IV stimulated endothelial progenitor cells (EPCs) to secrete exosomes loaded with miR-21. This study aimed to investigate the effects of AS-IV-mediated EPCs exosomal miR-21 (EPC-exos-miR-21) on high glucose (HG) damaged endothelial cells. MATERIALS AND METHODS: After the isolation of EPCs derived from fetal umbilical cord blood, exosomes of EPCs were obtained by differential centrifugation. The morphology of exosomes was observed by electron microscopy. The particle size distribution of exosomes was detected by Nanoparticle Tracking Analysis. Human umbilical vein endothelial cells (HUVECs) were treated with 33 mM glucose to establish an HG injury model. Flow cytometry and TUNEL assay were used to characterize the surface markers of primary EPCs and the apoptosis of HUVECs. The gene and protein expression were detected by qPCR, immunofluorescence, and Western blotting. A dual luciferase assay was used to verify the targeting relationship of miR-21 with PTEN. RESULTS: HG environment led to time- and dose-dependent inhibition and enhancement of autophagy and apoptosis in HUVECs. AS-IV stimulated EPCs to secrete exosomes loaded with miR-21. Exosomes secreted by EPCs pretreated with AS-IV [EPC-exo(ASIV)] promoted autophagy and inhibited apoptosis in HG-impaired HUVECs. PTEN is a target of miR-21. MiR-21 carried by EPC-exo(ASIV) repressed PTEN expression in HG-impaired HUVECs. In contrast, p-AKT, p-mTOR, p-PI3K, cleaved PARP and PARP levels were upregulated. Compared to the HG group, the expression of autophagy regulatory genes (ATG5, beclin1 and LC3) was enhanced in the EPC-exo(ASIV) group and EPC-exo(ASIV)-miR-21 mimic group. In contrast, apoptosis-positive regulatory genes (Bax, caspase-3 and caspase-9) were attenuated. Further overexpression of PTEN reversed the expression of these genes. CONCLUSIONS: AS-IV-mediated EPC-exos-miR-21 could enhance autophagy and depress apoptosis in HG-damaged endothelial cells via the miR-21/PTEN axis.


Assuntos
Células Progenitoras Endoteliais , Exossomos , MicroRNAs , Humanos , Células Progenitoras Endoteliais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Exossomos/genética , Exossomos/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Apoptose , Autofagia , Glucose/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
14.
Discov Med ; 34(171): 45-58, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36494326

RESUMO

miR-21 is involved in the mechanisms of inflammatory bowel disease (IBD). It negatively regulates PTEN, which is an upstream regulatory gene of the PI3K/Akt/mTOR signaling pathway. But the relationship between miR-21 and immune tolerance and intestinal epithelial injury, and the mechanism by which miR-21 participates in Crohn's disease (CD) have not been studied. We aimed to address these two questions. The results of the present study showed that the levels of miR-21 and PTEN respectively decreased and increased significantly in the intestinal mucosa from active CD compared with control ones. Transfection with miR-21-5p mimics significantly downregulated the expression of PTEN and upregulated PI3K-Akt-mTOR signaling and the downstream pathway in PBMCs, while transfection with a miR-21-5p inhibitor antagomiR-21had the opposite effect. Moreover, the ratio of Treg/Th1 cells differentiated from peripheral blood mononuclear cells (PBMCs) decreased after being transfected with mimics, and increased with the inhibitor. AntagomiR-21 significantly relieved the lesions in colons of mice with TNBS-induced colitis, accompanied by the upregulation of PTEN and downregulation of mTOR. Inhibition of miR-21 also effectively suppressed the process of epithelial-mesenchymal transition (EMT) in vivo. In conclusion, the level of miR-21 decreased in CD, resulting in an upregulated PI3K-Akt-mTOR signaling pathway, compromised immune tolerance, and elevated inflammation.


Assuntos
Doença de Crohn , MicroRNAs , Camundongos , Animais , Transição Epitelial-Mesenquimal/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Doença de Crohn/genética , Leucócitos Mononucleares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Tolerância Imunológica
15.
BMC Urol ; 22(1): 200, 2022 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-36496361

RESUMO

OBJECTIVE: The aim of the present study was to explore the effect of cytoplasmic transduction peptide (CTP)-phosphatase and tensin homolog (PTEN) on the proliferation, cell cycle, apoptosis, migration and invasion of bladder cancer cells and the underlying molecular mechanism. METHODS: A eukaryotic expression vector, pTT5-CTP-PTEN, was constructed. The constructed vector was transfected into HEK 293-6E cells to express a fusion protein, CTP-PTEN. The fusion protein was purified. 5637 bladder cancer cells were cocultured with purified CTP-PTEN fusion protein. Target gene expression, protein expression, cell proliferation, cell cycle, apoptosis, cell invasion and cell migration were examined by reverse transcription polymerase chain reaction (RT-PCR), western blot, MTT assay, flow cytometry, Transwell assay, and cell scratch assay, respectively. RESULTS: Both PTEN and CTP-PTEN fusion protein inhibited the proliferation, cell cycle, invasion and migration of bladder cancer cells and promoted the apoptosis of bladder cancer cells. The effect of CTP-PTEN was more significant. CONCLUSIONS: The fused expression of CTP and PTEN significantly increased the penetrability of the tumor suppressor gene PTEN into cancer cells. The CTP-PTEN fusion protein exhibited a significant carcinostatic effect on 5637 bladder cancer cells.


Assuntos
MicroRNAs , Neoplasias da Bexiga Urinária , Humanos , Células HEK293 , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proliferação de Células , Movimento Celular , Apoptose , Neoplasias da Bexiga Urinária/genética , Linhagem Celular Tumoral , Peptídeos/genética , Peptídeos/metabolismo , MicroRNAs/genética , Regulação Neoplásica da Expressão Gênica
16.
Exp Mol Med ; 54(11): 1814-1821, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36385557

RESUMO

PTEN is among the most commonly lost or mutated tumor suppressor genes in human cancer. PTEN, a bona fide lipid phosphatase that antagonizes the highly oncogenic PI3K-AKT-mTOR pathway, is considered a major dose-dependent tumor suppressor. Although PTEN function can be compromised by genetic mutations in inherited syndromes and cancers, posttranslational modifications of PTEN may also play key roles in the dynamic regulation of its function. Notably, deregulated ubiquitination and deubiquitination lead to detrimental impacts on PTEN levels and subcellular partitioning, promoting tumorigenesis. While PTEN can be targeted by HECT-type E3 ubiquitin ligases for nuclear import and proteasomal degradation, studies have shown that several deubiquitinating enzymes, including HAUSP/USP7, USP10, USP11, USP13, OTUD3 and Ataxin-3, can remove ubiquitin from ubiquitinated PTEN in cancer-specific contexts and thus reverse ubiquitination-mediated PTEN regulation. Researchers continue to reveal the precise molecular mechanisms by which cancer-specific deubiquitinases of PTEN regulate its roles in the pathobiology of cancer, and new methods of pharmacologically for modulating PTEN deubiquitinases are critical areas of investigation for cancer treatment and prevention. Here, we assess the mechanisms and functions of deubiquitination as a recently appreciated mode of PTEN regulation and review the link between deubiquitinases and PTEN reactivation and its implications for therapeutic strategies.


Assuntos
Neoplasias , Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Carcinogênese/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Enzimas Desubiquitinantes , Tioléster Hidrolases/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
17.
Zhonghua Zhong Liu Za Zhi ; 44(11): 1168-1174, 2022 Nov 23.
Artigo em Chinês | MEDLINE | ID: mdl-36380665

RESUMO

Objective: To explore the effect of growth arrest-specific5 (GAS5) inhibition on the proliferation, colony formation, invasion, migration andepithelial-mesenchymal transition(EMT), cancer cell stem of HCT-116 and its mechanism. Methods: The colorectal carcinoma (CRC) cell HCT116 was divided into blank control, negative control (NC), si-GAS5 and si-GAS5+ miR-21 inhibitor groups. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expressions of miR-21 and GAS5 at 48 h after transfection. The binding site of GAS5 and miR-21 was determined by luciferase reporter array. Cell proliferation ability was detected by CCK-8 assay. Cell colony ability was detected by colony formation assay. Cell invasion and migration abilities were detected by Transwell assay. Cell cycle and apoptosis were examined by flow cytometer (FCM). The protein levels of EMT associated factors including Snail, N-cadherin, vimentin, E-cadherin, stem cell related factors including CD44, SOX2, Oct2, and PTEN/Akt signal pathway associated factors were examined by western blotting. Results: The expression levels of miR-21 in blank, NC, si-GAS5 group were 1.00±0.10, 1.00±0.10, 1.80±0.20, the absorbance values were 0.51±0.02, 0.50±0.01 and 0.65±0.01, the cell clones were 90±4, 91±5, 200±8, the invaded cells were 118±3, 119±3, 150±4, the migrated cells were 110±2, 108±2, 127±2, the cell ratios in G(1) phase were (49.3±2.1)%, (50.1±2.0)% and (42.2±1.1)%, the cell ratios in S phase were (19.2±1.2)%, (20.2±1.1)% and (28.3±2.2)%, the cell apoptotic ratios were (14.4±2.2)%, (14.5±2.1)% and (7.2±1.3)%. These results indicated that inhibition of GAS5 up regulated the expression level of miR-21, promoted cell proliferation, invasion and migration, decreased G(1)-phase cells and increased S-phase cells, and suppressed cell apoptosis (P<0.05). Moreover, inhibition of GAS5 up regulated the expressions of Snail, N-cadherin, vimentin, Sox2, CD44, Oct2 and p-Akt in HCT-116 cells (P<0.05), while down regulated the expressions of E-cadherin and PTEN (P<0.05). Inhibition of miR-21 reversed the impact of GAS5 knockdown on PTEN/Akt signaling pathway (P<0.05). Conclusion: GAS5 can act as a competing endogenous RNA for miR-21, and down regulation of GAS5 can promote the development of CRC by activating the miR-21/PTEN/Akt signaling pathway and promoting the acquisition of EMT and tumor cell stemness.


Assuntos
Neoplasias Colorretais , MicroRNAs , Humanos , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Vimentina/metabolismo
18.
J Neurosci ; 42(41): 7848-7860, 2022 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-36414008

RESUMO

Mutations in PTEN-induced kinase 1 (PINK1) contribute to autosomal recessive Parkinson's disease with cognitive and neuropsychiatric comorbidities. Disturbances in dendritic and spine architecture are hallmarks of neurodegenerative and neuropsychiatric conditions, but little is known of the impact of PINK1 on these structures. We used Pink1 -/- mice to study the role of endogenous PINK1 in regulating dendritic architecture, spine density, and spine maturation. Pink1 -/- cortical neurons of unknown sex showed decreased dendritic arborization, affecting both apical and basal arbors. Dendritic simplification in Pink1 -/- neurons was primarily driven by diminished branching with smaller effects on branch lengths. Pink1 -/- neurons showed reduced spine density with a shift in morphology to favor filopodia at the expense of mushroom spines. Electrophysiology revealed significant reductions in miniature EPSC (mEPSC) frequency in Pink1 -/- neurons, consistent with the observation of decreased spine numbers. Transfecting with human PINK1 rescued changes in dendritic architecture, in thin, stubby, and mushroom spine densities, and in mEPSC frequency. Diminished spine density was also observed in Golgi-Cox stained adult male Pink1 -/- brains. Western blot study of Pink1 -/- brains of either sex revealed reduced phosphorylation of NSFL1 cofactor p47, an indirect target of PINK1. Transfection of Pink1 -/- neurons with a phosphomimetic p47 plasmid rescued dendritic branching and thin/stubby spine density with a partial rescue of mushroom spines, implicating a role for PINK1-regulated p47 phosphorylation in dendrite and spine development. These findings suggest that PINK1-dependent synaptodendritic alterations may contribute to the risk of cognitive and/or neuropsychiatric pathologies observed in PINK1-mutated families.SIGNIFICANCE STATEMENT Loss of PINK1 function has been implicated in both familial and sporadic neurodegenerative diseases. Yet surprisingly little is known of the impact of PINK1 loss on the fine structure of neurons. Neurons receive excitatory synaptic signals along a complex network of projections that form the dendritic tree, largely at tiny protrusions called dendritic spines. We studied cortical neurons and brain tissues from mice lacking PINK1. We discovered that PINK1 deficiency causes striking simplification of dendritic architecture associated with reduced synaptic input and decreased spine density and maturation. These changes are reversed by reintroducing human PINK1 or one of its downstream mediators into PINK1-deficient mouse neurons, indicating a conserved function, whose loss may contribute to neurodegenerative processes.


Assuntos
Espinhas Dendríticas , Doença de Parkinson , Humanos , Animais , Camundongos , Espinhas Dendríticas/metabolismo , Neurônios/fisiologia , Doença de Parkinson/metabolismo , Fosforilação , Proteínas Quinases/genética , PTEN Fosfo-Hidrolase/metabolismo
19.
BMC Cancer ; 22(1): 1145, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36344947

RESUMO

BACKGROUND: Cervical cancer (CC) is a common gynecological malignancy worldwide. Some patients perform serious resistance after chemotherapy, and long-stranded non-coding RNA MEG3 is reported to be involved in the regulation of chemoresistance in many solid tumors. However, its involvement in cervical adenocarcinoma has not been reported. METHODS: Hela cell lines, cisplatin-resistant cell lines (Hela-CR) and nude mice were used in this study. After MEG3 was overexpressed or knocked down in cells by the lentivirus vector, cell growth was detected by the CCK-8 assay, and cell migration was evaluated using Transwell assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of MEG3, miR-21 and PTEN mRNA. Apoptosis was detected by flow cytometry. The targeting relationship between mRNAs was predicted and verified using dual-luciferase reporter gene experiments. Western blot was executed to examine Bax, cleaved-caspase 3, Bcl-2, PTEN and GAPDH expression. Cells were injected into the mice to form xenograft tumors to compare tumorigenesis capacity. RESULTS: We demonstrated that MEG3 was down-regulated in cervical cancer by analyzing the TCGA database. Moreover, knockdown of MEG3 promoted CC cell proliferation, migration and inhibited the apoptosis. These changes of CC cells were more pronounced under cisplatin treatment. Further studies showed that the MEG3/miR-21/PTEN axis affected cisplatin sensitivity in cervical cancer cells, and these results of recue assay were used to confirm this conclusion. CONCLUSIONS: MEG3 performing as ceRNA promotes cisplatin sensitivity in CC cells through the miR-21/PTEN axis.


Assuntos
MicroRNAs , RNA Longo não Codificante , Neoplasias do Colo do Útero , Feminino , Humanos , Camundongos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Camundongos Nus , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Células HeLa , MicroRNAs/genética , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , RNA Mensageiro , Regulação Neoplásica da Expressão Gênica , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
20.
Cell Rep ; 41(5): 111574, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36323257

RESUMO

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of AKT/mTOR signaling pathway. Mutations in PTEN are found in patients with autism, epilepsy, or macrocephaly. In mouse models, Pten loss results in neuronal hypertrophy, hyperexcitability, seizures, and ASD-like behaviors. The underlying molecular mechanisms of these phenotypes are not well delineated. We determined which of the Pten loss-driven aberrations in neuronal form and function are orchestrated by downstream mTOR complex 1 (mTORC1). Rapamycin-mediated inhibition of mTORC1 prevented increase in soma size, migration, spine density, and dendritic overgrowth in Pten knockout dentate gyrus granule neurons. Genetic knockout of Raptor to disrupt mTORC1 complex formation blocked Pten loss-mediated neuronal hypertrophy. Electrophysiological recordings revealed that genetic disruption of mTORC1 rescued Pten loss-mediated increase in excitatory synaptic transmission. We have identified an essential role for mTORC1 in orchestrating Pten loss-driven neuronal hypertrophy and synapse formation.


Assuntos
Neurônios , Sinapses , Animais , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Sinapses/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Hipertrofia/metabolismo
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