RESUMO
OBJECTIVE: This study aimed to investigate the molecular mechanism of miR-150-5p regulating the malignant biological behavior of Human Epidermoid cancer cell (HEp-2) by targeting peptidyl-prolyl cis/trans isomerase NIMA-Interacting-1 (PIN1). METHODS: Firstly, qRT-PCR and Western blot were adopted to detect the expression levels of miR-150-5p and PIN1 in cancer tissue and paracancerous tissues of patients with LSCC, and those in human bronchial epithelial cells (16 HBE) and HEp-2. Next, the targeted relationship between miR-150-5p and PIN1 was assessed by bioinformatics website and dual-luciferase reporter assay, followed by their correlation analysis. Besides, after interfering with miR-150-5p or PIN1 expression in HEp-2 cells, CCK-8, cell colony formation assay, and transwell assay were utilized to detect the proliferation, viability, and invasion of cells, respectively. Subsequently, the protein levels of MMP-2, MMP-9, and EMT-related proteins in HEp-2 cells were checked by Western blot. RESULTS: Expression of miR-150-5p was down-regulated in LSCC tissues and HEp-2 cells. Moreover, miR-150-5p suppressed proliferation and invasion of HEp-2 cells, affected protein expression related to MMP and EMT, thereby inhibiting development of cancer. The expression of PIN1 was significantly increased in cancer tissues and HEp-2 cells, and there was a targeted relationship and negative correlation between miR-150-5p and PIN1 in cancer tissue. However, overexpression of PIN1 could reverse the effect of miR-150-5p on the proliferation and invasion of HEp-2 cells. CONCLUSION: In a nutshell, there is a targeted relationship between PIN1 and miR-150-5p. Besides, miR-150-5p can inhibit the proliferation and invasion of HEp-2 cells by regulating the expression of PIN1.
Assuntos
Neoplasias Laríngeas , Laringe , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/patologia , Laringe/patologia , Regulação Neoplásica da Expressão Gênica , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismoRESUMO
PIN1 is the only known enzyme capable of recognizing and isomerizing the phosphorylated Serine/ThreonineProline motif. Through this mechanism, PIN1 controls diverse cellular functions, including telomere maintenance. Both PIN1 overexpression and its involvement in oncogenic pathways are involved in several cancer types, including glioblastoma (GBM), a lethal disease with poor therapeutic resources. However, knowledge of the role of PIN1 in GBM is limited. Thus, the present work aimed to study the role of PIN1 as a telomere/telomerase regulator and its contribution to tumor biology. PIN1 knockout (KO) LN229 cell variant using CRISPR/Cas9 was developed and compared with PIN1 LN229 expressing cells. To study the effect of PIN1 absence, status of NFκB pathway was evaluated by luciferase reporter gene assay and quantitative PCR. Results revealed that PIN1 deletion in GBM cells diminished the active levels of NFκB and decrease the transcription of il8 and htert genes. Then, telomere/telomerase related processes were studied by RQTRAP assay and telomere length determination by qPCR, obtaining a reduction both in telomerase activity as in telomere length in PIN1 KO cells. In addition, measurement of SA ßgalactosidase and caspase3 activities revealed that loss of PIN1 triggers senescence and apoptosis. Finally, migration, cell cycle progression and tumorigenicity were studied by flow cytometry/western blot, Transwell assay and in vivo experiments, respectively. PIN1 deletion decreased migration as well as cell cycle progression by increasing doubling time and also resulted in the loss of LN229 cell ability to form tumors in mice. These results highlight the role of PIN1 in telomere homeostasis and GBM progression, which supports PIN1 as a potential molecular target for the development of novel therapeutic agents for GBM treatment.
Assuntos
Glioblastoma , Telomerase , Humanos , Animais , Camundongos , Glioblastoma/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Telomerase/metabolismo , Reação em Cadeia da Polimerase , Telômero/genética , Telômero/metabolismo , Linhagem Celular Tumoral , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismoRESUMO
PURPOSE: To investigate the role of peptidyl-prolyl cis/trans isomerase 1 (Pin1) on renal ischemia-reperfusion (I/R) injury and underlying mechanism. METHODS: By establishing the in vitro and in vivo models of renal I/R, the role of Pin1 was explored by using molecular assays. RESULTS: In renal I/R, endogenous Pin1 level was up-regulated in I/R-impaired kidney. Suppression of Pin1 with juglone afforded protection against I/R-mediated kidney dysfunction, and reduced I/R-induced endoplasmic reticulum (ER) stress in vivo. Consistent with the in vivo results, repression of Pin1 with juglone or gene knockdown with si-Pin1 conferred cytoprotection and restricted hypoxia/reoxygenation (H/R)-driven ER stress in HK-2 cells. Simultaneously, further study uncovered that Nrf-2/HO-1 signals was the association between Pin1 and ER stress in response to renal I/R. In addition, Nrf-2/HO-1 signal pathway was inactivated after kidney exposed to I/R, as indicated by the down-regulation of Nrf-2/HO-1 levels. Furthermore, inhibition of Pin1 remarkably rescued the inactivation ofNrf-2/HO-1. CONCLUSIONS: Pin1 modulated I/R-mediated kidney injury in ER stress manner dependent on Nrf2-HO-1 pathway in I/R injury.
Assuntos
Estresse do Retículo Endoplasmático , Nefropatias , Fator 2 Relacionado a NF-E2 , Peptidilprolil Isomerase de Interação com NIMA , Traumatismo por Reperfusão , Animais , Apoptose , Feminino , Heme Oxigenase (Desciclizante) , Rim/metabolismo , Rim/patologia , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Estresse Oxidativo , Ratos , Traumatismo por Reperfusão/metabolismoRESUMO
The parvulin PIN1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), is the only enzyme capable of isomerizing prolines of phospho-Serine/Threonine-Proline motifs. PIN1 binds to a subset of proteins and plays an essential role in regulating protein function post-phosphorylation control. Furthermore, the activity of PIN1 regulates the outcome of the signalling of proline-directed kinases (e.g. MAPK, CDK, or GSK3) and thus regulates cell proliferation and cell survival. For these reasons, PIN1 inhibitors are interesting since they may have therapeutic implications for cancer. Several authors have already reported that the non-structural point mutation Trp34Ala prevents PIN1 from interacting with its downstream effector proteins. In this work, we characterized PIN1 structurally, intending to explore new inhibition targets for the rational design of pharmacological activity compounds. Through a conformational diversity analysis of PIN1, we identified and characterized a highly specific druggable pocket around the residue Trp34. This pocket was used in a high-throughput docking screening of 450,000 drug-like compounds, and the top 10 were selected for re-docking studies on the previously used conformers. Finally, we evaluated the binding of each compound by thermal shift assay and found four molecules with a high affinity for PIN1 and potential inhibitory activity. Through this strategy, we achieved novel drug candidates with the ability to interfere with the phosphorylation-dependent actions of PIN1 and with potential applications in the treatment of cancer.Communicated by Ramaswamy H. Sarma.
Assuntos
Antineoplásicos , Inibidores Enzimáticos , Peptidilprolil Isomerase de Interação com NIMA , Neoplasias , Antineoplásicos/química , Antineoplásicos/farmacologia , Detecção Precoce de Câncer , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fosforilação , Prolina/metabolismoRESUMO
The prolyl isomerase Pin1 plays a key role in the modulation of proline-directed phosphorylation signaling by inducing local conformational changes in phosphorylated protein substrates. Extensive studies showed different roles for Pin1 in physiological processes and pathological conditions such as cancer and neurodegenerative diseases. However, there are still several unanswered questions regarding its biological role. Notably, despite evidences from cultured cells showing that Pin1 expression and activity may be regulated by different mechanisms, little is known on their relevance in vivo. Using Danio rerio (zebrafish) as a vertebrate model organism we showed that pin1 expression is regulated during embryogenesis to achieve specific mRNA and protein distribution patterns. Moreover, we found different subcellular distribution in particular stages and cell types and we extended the study of Pin1 expression to the adult zebrafish brain. The analysis of Pin1 overexpression showed alterations on zebrafish development and the presence of p53-dependent apoptosis. Collectively, our results suggest that specific mechanisms are operated in different cell types to regulate Pin1 function.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peixe-Zebra/embriologia , Animais , Peptidilprolil Isomerase de Interação com NIMA/genética , Especificidade por SubstratoRESUMO
BACKGROUND: Recent evidence suggests that the peptidyl-prolyl isomerase Pin1 is an oncoprotein that acts as a novel therapeutic target in a variety of tumors. In this study, we investigated the clinical significance of Pin1 and its function in prostate cancer (PCa) tumor progression. METHODS: Immunohistochemical and quantitative RT-PCR analyses were performed to detect the expression of Pin1 in 86 PCa tissue samples. The functional role of Pin1 was evaluated by small interfering RNA-mediated depletion in PCa cells followed by analyses of cell proliferation and migration. Furthermore, the association between expression of Pin1 and levels of ß-catenin and cyclin D1 was also evaluated. RESULTS: Our results showed that the high expression of Pin1 staining was 66 of 86 (76.74 %) PCa samples, and in 25 of 86 (29.07 %) BPH tissues, the difference was statistically significant (P < 0.001). Pin1 was significantly elevated in all PCa cell lines when compared to the normal RWPE-1 cells. We observed that proliferation and migration of LNCaP cells were inhibited by Pin1 knockdown. The levels of ß-catenin and cyclin D1 in clinical PCa specimens were positively associated with Pin1 expression. CONCLUSIONS: Our results suggest that Pin1 plays an important role in tumorigenesis of PCa, suggesting that targeting Pin1 pathway could represent a potential modality for treating PCa.