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Diabetic retinopathy is one of the microvascular complications in diabetic patients and the leading cause of blindness worldwide. The levels of METTL3, lncRNA SNHG7, KHSRP, MKL1, endothelial and mesenchymal markers were determined by RT-qPCR or western blot assays in vitro and in vivo. H&E staining was used to observe the retinal structure in a mouse model of DR. The expression levels of METTL3 and SNHG7 were significantly downregulated in DR patients, DR mice and high glucose-induced HRMECs cells. Notably, METTL3 installed the m6A modification and enhanced the stability of SNHG7. Besides, METTL3 inhibited HRMECs EndoMT by promoting the expression of SNHG7. Additionally, SNHG7 was found to weaken MKL1 mRNA stability by binding to the RNA-binding protein KHSRP. Furthermore, we verified that METTL3 regulated EndoMT in DR through the SNHG7/MKL1 axis. We conclude that METTL3 regulates endothelial-mesenchymal transition in DR via the SNHG7/KHSRP/MKL1 axis, providing a new target for DR treatment.
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Diabetes Mellitus , Retinopatia Diabética , RNA Longo não Codificante , Camundongos , Animais , Transferases , Retinopatia Diabética/genética , RNA Longo não Codificante/genética , Metiltransferases/genéticaRESUMO
Manganese neurotoxicity has been reported to cause a neurodegenerative disease known as parkinsonism. Previous reports have shown that the expression of the KH-type splicing regulatory protein (KHSRP), a nucleic acid-binding protein, and NLRP3 is increased upon Mn exposure. However, the relation between these two during Mn toxicity has not been fully deduced. The mouse neuroblastoma (N2a) and SD rats are treated with LPS and MnCl2 to evaluate the expression of KHSRP and NLRP3. Further, the effect of the NLRP3 inhibitor MCC950 is checked on the expression of NLRP3, KHSRP and pro-inflammatory markers (TNFα, IL-18 and IL-1ß) as well as the caspase-1 enzyme. Our results demonstrated an increment in NLRP3 and KHSRP expression post-MnCl2 exposure in N2a cells and rat brain, while on the other hand with LPS exposure only NLRP3 expression levels were elevated and KHSRP was found to be unaffected. An increased expression of KHSRP, NLRP3, pro-inflammatory markers and the caspase-1 enzyme was observed to be inhibited with MCC950 treatment in MnCl2-exposed cells and rats. Manganese exposure induces NLRP3 and KHSRP expression to induce neuroinflammation, suggesting a correlation between both which functions in toxicity-related pathways. Furthermore, MCC950 treatment reversed the role of KHSRP from anti-inflammatory to pro-inflammatory.
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Manganês , Proteína 3 que Contém Domínio de Pirina da Família NLR , Doenças Neuroinflamatórias , Animais , Camundongos , Ratos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Inflamassomos/metabolismo , Lipopolissacarídeos/toxicidade , Manganês/toxicidade , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/etiologia , Doenças Neuroinflamatórias/induzido quimicamente , Doenças Neuroinflamatórias/etiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos Sprague-DawleyRESUMO
K-homology (KH)-type splicing regulatory protein (KHSRP) is an RNA binding protein that participates in RNA variable splicing and stability, and facilitates the biogenesis of miRNAs that target mRNA. However, to date, the role of KHSRP in colorectal cancer (CRC) progression has not been reported. In this study, the function of KHSRP in CRC proliferation and 5-fluoruracil (5-FU) resistance was investigated. The upregulation of KHSRP expression was confirmed in CRC patient tissues and two CRC cell lines. Manipulating KHSRP expression altered cell proliferation and 5-FU resistance in CRC cells. ERRFI1, a downstream effector of KHSRP in CRC cells, reduced CRC cell proliferation. Sensitivity to 5-FU mediated by KHSRP knockdown was reversed by ERRFI1 knockdown. We found that KHSRP decreased ERRFI1 mRNA expression indirectly. By screening KHSRP-regulated miRNAs, we further found that miR-501-5p directly combines with KHSRP in CRC cells. Mechanistically, the results of a luciferase assay suggested that miR-501-5p directly binds to the ERRFI1 3'-untranslated region. Taken together, our data indicated that modification of ERRFI1 by KHSRP occurs through miR-501-5p, an essential mechanism driving CRC proliferation and 5-FU resistance. Insight into this mechanism may provide novel targets for overcoming drug resistance in CRC.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Transativadores/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3'UTRs of cyclin-dependent kinase inhibitor 1B (CDKN1B/p27Kip1) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell's response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that p27Kip1 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3'UTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation.
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Regiões 3' não Traduzidas , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Sequências de Repetição em Tandem , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Processamento Pós-Transcricional do RNA , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/química , Proteínas de Ligação a RNA/genéticaRESUMO
MicroRNAs (miRNAs) are short (21-23nt long) RNAs that post-transcriptionally regulate gene expression in plants and animals. They are key regulators in all biological processes. In mammalian cells miRNAs are loaded into one of the four members of the Argonaute (Ago) protein family to form the RNA-induced silencing complex (RISC). RISCs inhibit the translation of mRNAs that share sequence complementarity with their loaded miRNAs. miRNA processing and miRNA-mediated gene regulation are highly regulated processes and involve many RNA-binding proteins as auxiliary factors. Here we show that the two RNA-binding proteins, p72 and KHSRP, both with known roles in promoting miRNA biogenesis, regulate the protein level of human Ago2 in transformed human cells. We determined that p72 and KHSRP influence Ago2 stability by regulating miRNA levels in the cell and that loss of p72/KHSRP results in a decrease of unloaded Ago2.
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Proteínas Argonautas/genética , RNA Helicases DEAD-box/genética , MicroRNAs/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Proteínas Argonautas/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Transdução de Sinais , Transativadores/metabolismo , TransfecçãoRESUMO
BACKGROUND: MicroRNAs (miRNAs) are important regulators involved in diverse physiological and pathological processes including cancer. SUMO (small ubiquitin-like modifier) is a reversible protein modifier. We recently found that SUMOylation of TARBP2 and DGCR8 is involved in the regulation of the miRNA pathway. KHSRP is a single stranded nucleic acid binding protein with roles in transcription and mRNA decay, and it is also a component of the Drosha-DGCR8 complex promoting the miRNA biogenesis. METHODS: The in vivo SUMOylation assay using the Ni2+-NTA affinity pulldown or immunoprecipitation (IP) and the in vitro E.coli-based SUMOylation assay were used to analyze SUMOylation of KHSRP. Nuclear/Cytosol fractionation assay and immunofluorescent staining were used to observe the localization of KHSRP. High-throughput miRNA sequencing, quantantive RT-PCR and RNA immunoprecipitation assay (RIP) were employed to determine the effects of KHSRP SUMO1 modification on the miRNA biogenesis. The soft-agar colony formation, migration, vasculogenic mimicry (VM) and three-dimensional (3D) cell culture assays were performed to detect the phenotypes of tumor cells in vitro, and the xenograft tumor model in mice was conducted to verify that SUMO1 modification of KHSRP regulated tumorigenesis in vivo. RESULTS: KHSRP is modified by SUMO1 at the major site K87, and this modification can be increased upon the microenvironmental hypoxia while reduced by the treatment with growth factors. SUMO1 modification of KHSRP inhibits its interaction with the pri-miRNA/Drosha-DGCR8 complex and probably increases its translocation from the nucleus to the cytoplasm. Consequently, SUMO1 modification of KHSRP impairs the processing step of pre-miRNAs from pri-miRNAs which especially harbor short G-rich stretches in their terminal loops (TL), resulting in the downregulation of a subset of TL-G-Rich miRNAs such as let-7 family and consequential tumorigenesis. CONCLUSIONS: Our data demonstrate how the miRNA biogenesis pathway is connected to tumorigenesis and cancer progression through the reversible SUMO1 modification of KHSRP.
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Carcinogênese/genética , Carcinogênese/metabolismo , MicroRNAs/genética , Proteínas de Ligação a RNA/metabolismo , Proteína SUMO-1/metabolismo , Transativadores/metabolismo , Composição de Bases , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Peróxido de Hidrogênio/metabolismo , Hipóxia/metabolismo , Lisina/metabolismo , MicroRNAs/química , Modelos Biológicos , Conformação de Ácido Nucleico , Ligação Proteica , Transporte Proteico , Sumoilação , Sequências Repetidas TerminaisRESUMO
Ischemic events, common in many diseases, result from decreased blood flow and impaired delivery of oxygen and glucose to tissues of the body. While much is known about the cellular transcriptional response to ischemia, much less is known about the posttranscriptional response to oxygen and glucose deprivation. The goal of this project was to investigate one such posttranscriptional response, the regulation of mRNA stability. To that end, we have identified several novel ischemia-related mRNAs that are synergistically stabilized by oxygen and glucose deprivation including VEGF, MYC, MDM2, and CYR61. This increase in mRNA half-life requires the synergistic effects of both low oxygen (1%) as well as low glucose (≤ 1 g/L) conditions. Oxygen or glucose deprivation alone fails to initiate the response, as exposure to either high glucose (4 g/L) or normoxic conditions inhibits the response. Furthermore, in response to hypoxia/hypoglycemia, the identified mRNAs are released from the RNA binding protein KHSRP which likely contributes to their stabilization.
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Hipoglicemia/genética , Estabilidade de RNA/genética , Hipóxia Celular/genética , Inativação Gênica , Glucose/deficiência , Glucose/farmacologia , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Humanos , Isquemia/genética , Concentração Osmolar , Oxigênio , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein that has a role in tumorigenesis of small cell lung cancer. The KHSRP protein level was shown to be significantly increased in small cell lung cancer (SCLC) tumor tissues compared with normal lung tissues by immunohistochemical staining. Moreover, KHSRP protein levels were strongly associated with T stage in patients with SCLC. Using in vitro assays, we found that knockdown of the KHSRP gene inhibited cell proliferation and increased cell apoptosis but had no effect on cell migration and invasion. We also showed that down-regulation of the KHSRP gene suppressed tumor growth in vivo. Further analyses indicated that KHSRP was involved in miR-26a maturation and inhibited the expression of PTEN in SCLC cells. Taken together, these findings suggested that KHSRP plays an important role in SCLC tumorigenesis and could be a potential novel therapeutic target for SCLC treatment.
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Transformação Celular Neoplásica/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Transativadores/metabolismo , Adulto , Idoso , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Transativadores/genéticaRESUMO
Androgen-regulated DNA damage response (DDR) is one of the essential mechanisms in prostate cancer (PCa), a hormone-sensitive disease. The heterogeneous nuclear ribonucleoprotein K (hnRNPK)-homology splicing regulatory protein known as far upstream element-binding protein 2 (KHSRP) is an RNA-binding protein that can attach to AU-rich elements in the 3' untranslated region (3'-UTR) of messenger RNAs (mRNAs) to mediate mRNA decay and emerges as a critical regulator in the DDR to preserve genome integrity. Nevertheless, how KHSRP responds to androgen-regulated DDR in PCa development remains unclear. This study found that androgen can significantly induce acetylation of KHSRP, which intrinsically drives tumor growth in xenografted mice. Moreover, enhanced KHSRP acetylation upon androgen stimuli impedes KHSRP-regulated DDR gene expression, as seen by analyzing RNA sequencing (RNA-seq) and Gene Set Enrichment Analysis (GSEA) datasets. Additionally, NAD-dependent protein deacetylase sirtuin-7 (SIRT7) is a promising deacetylase of KHSRP, and androgen stimuli impairs its interaction with KHSRP to sustain the increased KHSRP acetylation level in PCa. We first report the acetylation of KHSRP induced by androgen, which interrupts the KHSRP-regulated mRNA decay of the DDR-related genes to promote the tumorigenesis of PCa. This study provides insight into KHSRP biology and potential therapeutic strategies for PCa treatment, particularly that of castration-resistant PCa.
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Breast cancer has become the most common type of cancers worldwide. Its high prevalence and malignant features are associated with various environmental factors and molecules. The KH-type splicing regulatory protein (KHSRP) participates in the development of breast cancer, while the underlying mechanisms are largely unknown. In this study, we silenced KHSRP expression in MDA-MB-231 cells by small interfering RNA (siKHSRP), and then assessed its effects on cellular features. Finally, we performed whole transcriptome sequencing (RNA-seq) experiments to explore the downstream targets of KHSRP, and validated their changed pattern using quantitative polymerase chain reaction. We found KHSRP showed higher expression level and was associated with worse prognosis in breast cancer patients. In siKHSRP samples, the proliferation, invasion, and migration abilities were significantly repressed compared with negative control (NC) samples, while the apoptosis level was increased. By investigating the RNA-seq data, we found KHSRP globally regulates the expression and alternative splicing profiles of MDA-MB-231 cells by identifying 1632 differentially expressed genes (DEGs) and 1630 HKSRP-regulated AS events (RASEs). Functional enriched analysis of DEGs demonstrated that cilium assembly and movement and extracellular matrix organization pathways were specifically enriched in up DEGs, consistent with the repressed migration and invasion abilities in siKHSRP cells. Interestingly, the cell cycle and DNA damage and repair associated pathways were enriched in both down DEGs and RASE genes, suggesting that KHSRP may modulate cell proliferation by regulating genes in these pathways. Finally, we validated the changed expression and AS patterns of genes in cell cycle and DNA damage/repair pathways. Expression levels of BIRC5, CCNA2, CDK1, FEN1, FOXM1, PTTG1, and UHRF1 were downregulated in siKHSRP samples. The AS patterns of PARK7, ERCC1, CENPX, and UBE2A were also dysregulated in siKHSRP samples and confirmed PCR experiments. In summary, our study comprehensively explored the downstream targets and their functions of KHSRP in breast cancer cells, highlighting the molecular mechanisms of KHSRP on the oncogenic features of breast cancer. The identified molecular targets could be served as potential therapeutic targets for breast cancer in future.
Assuntos
Processamento Alternativo , Neoplasias da Mama , Proliferação de Células , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Reparo do DNA/genética , Linhagem Celular Tumoral , Feminino , Proliferação de Células/genética , Movimento Celular/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Apoptose/genética , Carcinogênese/genética , Células MDA-MB-231RESUMO
OBJECTIVE: To investigate the role of JAK1/STAT3/KHSRP axis in mediating the regulatory effect of LINC00626 on progression of esophagogastric junction adenocarcinoma. METHODS: We collected surgical tumor and adjacent tissue specimens from 64 patients with esophagogastric junction adenocarcinoma and examined the expression levels of LINC00626 and KHSRP. qRT-PCR was used to detect the expressions of LINC00626 and KHSRP in 6 esophageal adenocarcinoma cell lines (OE-19, TE-7, Bic-1, Flo-1, SK-GT-4, and BE-3) and a normal esophageal epithelial cell line (HET-1A). OE-19 and TE-7 cell lines with stable LINC00626 knockdown and FLO-1 and SK-GT-4 cells stably overexpressing LINC00626 were constructed by lentiviral transfection, and the changes in proliferation, migration and invasion of the cells were evaluated using Cell Counting Kit-8 (CCK-8) assay and Transwell migration/invasion assay. The expressions of KHSRP and JAK/STAT pathway proteins in the transfected cells were detected with Western blotting. The effects of LINC006266 knockdown and overexpression on subcutaneous tumor formation and lung metastasis of OE-19 and FLO-1 cell xenografts were tested in nude mice. RESULTS: The expression levels of LINC00626 and KHSRP were significantly increased in esophagogastric junction adenocarcinoma tissues and in esophageal adenocarcinoma cells. LINC00626 knockdown obviously inhibited the proliferation, migration and invasion of esophageal adenocarcinoma cells in vitro and decreased their tumor formation and lung metastasis abilities in nude mice, while overexpression of LINC00626 produced the opposite effects. In esophageal adenocarcinoma cells, LINC0626 knockdown significantly decreased and LINC00626 overexpression strongly enhanced the phosphorylation of JAK1 and STAT3. CONCLUSION: High LINC00626 expression promotes esophageal-gastric junction adenocarcinoma metastasis by activating the JAK1/STAT3/KHSRP signal axis.
Assuntos
Adenocarcinoma , Neoplasias Esofágicas , Janus Quinase 1 , Neoplasias Pulmonares , Proteínas de Ligação a RNA , Animais , Humanos , Camundongos , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Junção Esofagogástrica/metabolismo , Junção Esofagogástrica/patologia , Regulação Neoplásica da Expressão Gênica , Janus Quinases/metabolismo , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Transdução de Sinais , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT3/metabolismo , Transativadores , RNA Longo não Codificante/genéticaRESUMO
RNA-binding proteins (RBPs) are widely involved in the transcriptional and posttranscriptional regulation of multiple biological processes. The transcriptional regulatory ability of RBPs was indicated by the identification of chromatin-enriched RBPs (Che-RBPs). One of these proteins, KH-type splicing regulatory protein (KHSRP), is a multifunctional RBP that has been implicated in mRNA decay, alternative splicing, and miRNA biogenesis and plays an essential role in myeloid differentiation by facilitating the maturation of miR-129. In this study, we revealed that KHSRP regulates monocytic differentiation by regulating gene transcription and RNA splicing. KHSRP-occupied specific genomic sites in promoter and enhancer regions to regulate the expression of several hematopoietic genes through transcriptional activation and bound to pre-mRNA intronic regions to modulate alternative splicing during monocytic differentiation. Of note, KHSRP had co-regulatory effects at both the transcriptional and posttranscriptional levels on MOGOH and ADARB1. Taken together, our analyses revealed the dual DNA- and RNA-binding activities of KHSRP and have provided a paradigm to guide the analysis of other functional Che-RBPs in different biological systems.
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Wilms tumor (WT) is the most common renal malignancy in children, and the survival rate of high-risk WT patients was still low despite multimodality therapy. KHSRP, an RNA-binding protein, has been proved to be relative to tumor progression in different kinds of malignancies, but the function of KHSRP in WT remained unclear. Here, our study aimed to explore and clarify the function of KHSRP in WT cells and its molecular mechanism. Thus, our results showed that KHSRP was highly expressed in WT tumor tissues compared to normal kidney tissues and correlated with poor prognosis in WT patients. Downregulation of KHSRP using siRNAs in WT cell line SK-NEP-1 and Wit49 resulted in inhibition of cell proliferation and cell cycle arrest via stabilizing and upregulating p27 protein. Furthermore, mechanistic analyses revealed that KHSRP bound to 3'UTR of PPP2CA mRNA and modulating its mRNA stability, resulting in regulation of the phosphorylation level and protein stability of p27 in WT cell lines. In conclusion, our results demonstrated that KHSRP played an important role in WT and modulated cell proliferation and cell cycle via regulating the expression of PPP2CA and p27.
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Cervical cancer (CC) is one of the deadliest female malignancies worldwide. Long non-coding RNAs (lncRNAs) are essential regulators for cancer progression. This study aimed to elucidate the role of lncRNA LINC01305 in the progression of CC. We found where LINC01305 was expressed in CC tissues and its correlation with the survival rate of CC patients. Functional experiments were performed to elucidate the effect of LINC01305 on CC. The results showed that LINC01305 was increased in CC tumor tissues and was correlated with a lower survival rate. The overexpression and knockdown of LINC01305 enhanced and inhibited the progression of CC, respectively. Additionally, the upregulation of LINC01305 promoted tumor growth in xenograft mice. Moreover, the effect of LINC01305 on CC was mediated through interacting with the RNA-binding protein, KHSRP. Furthermore, LINC01305 was mainly distributed in exosomes and was transferred to recipient cells to enhance CC progression. Lastly, LINC01305 may participate in the regulation of the stemness of CC. Taken together, the results suggest that LINC01305 promotes the progression of CC through KHSRP and that LINC01305 is released through exosomes and is involved in the stemness of CC. This study sheds light on the molecular mechanism underlying the progression of CC.
Assuntos
Exossomos , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Neoplasias do Colo do Útero/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Long intergenic noncoding RNAs (lincRNAs) are critical regulators involved in diverse biological processes. However, the roles and related mechanisms of lincRNAs in axon development are largely unknown. Here we report an axon-enriched lincRNA regulating axon elongation, referred to as ALAE. Profiling of highly expressed lincRNAs detected abundant and enriched ALAE in the axons of dorsal root ganglion (DRG) neurons, where it locally promoted axon elongation. Mechanically, ALAE directly interacted with the KH3-4 domains of KH-type splicing regulatory protein (KHSRP) and impeded its association with growth-associated protein 43 (Gap43) mRNA. Knockdown of ALAE reduced the protein but not the mRNA level of GAP43 in the axons of DRG neurons. Furthermore, local disruption of the interaction between ALAE and KHSRP in the axon significantly inhibited Gap43 mRNA translation, impairing axon elongation. This study implies crucial roles of axon-enriched lincRNAs in spatiotemporal regulation of local translation during axon development.
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Axônios/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , HumanosRESUMO
BACKGROUND: De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction, and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents; however, in most cases, the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors. METHODS: We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents' peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences and calcium (Ca2+) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines. RESULTS: NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca2+ reactivity to glutamate. Moreover, we could observe increased cellular proliferation and alterations in mitochondrial quantity and morphology. CONCLUSIONS: The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family. LIMITATIONS: Our study investigated only one family; therefore, the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage.
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Transtorno Autístico , Células-Tronco Pluripotentes Induzidas , Esquizofrenia , Humanos , Leucócitos Mononucleares , Masculino , Mutação , Fenótipo , Proteínas de Ligação a RNA , Esquizofrenia/genética , Sialoglicoproteínas , TransativadoresRESUMO
BACKGROUND: KH-type splicing regulatory protein (KHSRP) plays an important role in cancer invasion, but the relevant mechanism is not well known. In the present study, we investigated the function and potential molecular mechanism of KHSRP in non-small cell lung cancer (NSCLC) metastasis and elucidated its clinical significance. METHODS: Isobaric tags for relative and absolute quantitation and the SWATH™ approach were combined with nanoliquid chromatography-tandem mass spectrometry analysis to identify metastasis-associated nucleoproteins in NSCLC. Real-time PCR and Western blot were used to screen for metastasis-associated candidate molecules. Gene knockdown and overexpression were used to investigate their functions and molecular mechanisms in lung cancer cells. Coimmunoprecipitation (Co-IP) experiments were performed to identify the interactions between candidate molecules and their interacting proteins. Gene expression and its association with multiple clinicopathologic characteristics were analyzed by immunohistochemistry (IHC) and Western blot in human lung cancer specimens. RESULTS: KHSRP was identified as a metastasis-associated candidate molecule. In NSCLC cell lines, knockdown of KHSRP significantly reduced lung cancer cell proliferation, migration, and invasion in vitro and in vivo, whereas overexpression of KHSRP did the opposite. Mechanistically, the protein heterogeneous nuclear ribonucleoprotein C (C1/C2) (HNRNPC) was identified to interact with KHSRP using Co-IP experiments. In NSCLC cell lines, overexpression of HNRNPC significantly promoted lung cancer cell proliferation, migration, and invasion in vitro and in vivo. KHSRP and HNRNPC may induce human lung cancer cell invasion and metastasis by activating the IFN-α-JAK-STAT1 signaling pathway. Drastically higher expression levels of KHSRP and HNRNPC were observed in lung cancer tissues compared to those in adjacent noncancerous tissues. Increased KHSRP and HNRNPC expression was significantly associated with advanced tumor stages and metastasis (both lymph node and distant). Kaplan-Meier survival analysis showed that patients with high KHSRP and HNRNPC expression levels were predicted to have the shortest survival times and to have a poor prognosis. CONCLUSIONS: KHSRP plays an important role in NSCLC metastasis and may serve as a potential prognostic marker and novel therapeutic target for lung cancer metastasis treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , Proteínas de Ligação a RNA/genética , Transativadores/genética , TransfecçãoRESUMO
KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC.
RESUMO
K-homology splicing regulator protein (KHSRP), a multifunctional RNA-binding protein, was originally thought to primarily control mRNA decay. KHSRP was shown to be involved in p38MAPK, NF-κB and the JAK2-STAT-1a pathways. Besides, KHSRP regulated neuronal localization of beta-actin and microtubule-associated protein 2 (MAP2) mRNAs, respectively. However, the expression and roles of KHSRP in peripheral system lesions and repair are still unknown. In our study, we found that KHSRP levels were relatively higher in the crushed sciatic nerves, significantly reached a highest level at day 5. Spatially, we observed that KHSRP had a major colocalization with Schwann cells (SCs) and neurons. KHSRP was connected with promyelinating SCs marker. KHSRP promoted the decay of beta-catenin (ß-catenin) mRNA which was inactivated by PI3K-AKT signaling. We doubted that KHSRP might participate in Schwann cells differentiation by regulation of ß-catenin mRNA decay. In vitro, in cyclic adenosine monophosphate (cAMP)-induced Schwann cells differentiation system, we detected the increased KHSRP in cytoplasm and decreased ß-catenin at protein and mRNA level. In differentiation model of rat pheochromocytoma cells (PC12) induced by nerve growth factor (NGF) and primary dorsal root ganglion (DRG) cell culture, KHSRP also acted on neuronal differentiation. Specially, KHSRP-specific siRNA-transfected cells did not show morphological change, which was similar to ß-catenin overexpressed SCs. During SC/neuron co-cultures, KHSRP was transported to cytoplasm and involved in SCs myelination. In conclusion, we speculated that KHSRP was involved in SCs and neuronal differentiation by inducing ß-catenin mRNA decay.
Assuntos
Neurônios/metabolismo , Proteínas de Ligação a RNA/biossíntese , Células de Schwann/metabolismo , Nervo Isquiático/lesões , Transativadores/biossíntese , Animais , Diferenciação Celular/fisiologia , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Neurônios/patologia , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Células de Schwann/patologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Transativadores/genéticaRESUMO
MicroRNAs (miRNAs), a family of small nonprotein-coding RNAs, play a critical role in posttranscriptional gene regulation by acting as adaptors for the miRNA-induced silencing complex to inhibit gene expression by targeting mRNAs for translational repression and/or cleavage. miR-155-5p and miR-155-3p are processed from the B-cell Integration Cluster (BIC) gene (now designated, MIR155 host gene or MIR155HG). MiR-155-5p is highly expressed in both activated B- and T-cells and in monocytes/macrophages. MiR-155-5p is one of the best characterized miRNAs and recent data indicate that miR-155-5p plays a critical role in various physiological and pathological processes such as hematopoietic lineage differentiation, immunity, inflammation, viral infections, cancer, cardiovascular disease, and Down syndrome. In this review we summarize the mechanisms by which MIR155HG expression can be regulated. Given that the pathologies mediated by miR-155-5p result from the over-expression of this miRNA it may be possible to therapeutically attenuate miR-155-5p levels in the treatment of several pathological processes.