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BACKGROUND: Patients with colorectal cancer (CRC) with liver metastasis or drug resistance have a poor prognosis. Previous research has demonstrated that PPP2R1B inactivation results in the development of CRC. However, the role of PPP2R1B in CRC metastasis and drug resistance is unclear. METHODS: Venny 2.1 was used to determine the intersection between survival-related differentially expressed genes (DEGs) and liver metastasis-related DEGs according to RNA-seq data from The Cancer Genome Atlas (TCGA) and the GEO database (GSE179979). LCâMS/MS and coimmunoprecipitation were performed to predict and verify the substrate protein of PPP2R1B. Gene Set Variation Analysis (GSVA) was subsequently utilized to assess pathway enrichment levels. The predictive performance of PPP2R1B was assessed by regression analysis, Kaplan-Meier (KM) survival analysis and drug sensitivity analysis. Immunohistochemistry (IHC), qRT-PCR and western blotting were performed to measure the expression levels of related mRNAs or proteins. Biological features were evaluated by wound healing, cell migration and invasion assays and CCK-8 assays. A mouse spleen infection liver metastasis model was generated to confirm the role of PPP2R1B in the progression of liver metastasis in vivo. RESULTS: According to bioinformatics analysis, PPP2R1B is significantly associated with liver metastasis and survival in CRC patients, and these findings were further verified via immunohistochemistry (IHC), qPCR and Western blotting. Pathway enrichment and LCâMS/MS analysis revealed that PPP2R1B is negatively associated with the MAPK/ERK signalling pathway. Additionally, PD98059, a MAPK/ERK pathway inhibitor, inhibited EMT in vitro by reversing the changes in key proteins involved in EMT signalling (ZEB1, E-cadherin and Snail) and ERK/MAPK signalling (p-ERK) mediated by PPP2R1B. Oxaliplatin sensitivity prediction and validation revealed that PPP2R1B silencing decreased Oxaliplatin chemosensitivity, and these effects were reversed by PD98059 treatment. Moreover, PPP2R1B was coimmunoprecipitated with p-ERK in vitro. A negative correlation between PPP2R1B and p-ERK expression was also observed in clinical CRC samples, and the low PPP2R1B/high p-ERK coexpression pattern indicated a poor prognosis in CRC patients. In vivo, PPP2R1B silencing significantly promoted liver metastasis. CONCLUSIONS: This study revealed that PPP2R1B induces dephosphorylation of the p-ERK protein, inhibits liver metastasis and increases Oxaliplatin sensitivity in CRC patients and could be a potential candidate for therapeutic application in CRC.
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Protein phosphatase 2A (PP2A) represses many oncogenic signaling pathways and is an important tumor suppressor. PP2A comprises three distinct subunits and forms through a highly regulated biogenesis process, with the scaffolding A subunit existing as two highly related isoforms, Aα and Aß. PP2A's tumor-suppressive functions have been intensely studied, and PP2A inactivation has been shown to be a prerequisite for tumor formation. Interestingly, although partial loss of the Aα isoform is growth promoting, complete Aα loss has no transformative properties. Additionally, in cancer patients, Aα is found to be inactivated in a haploinsufficient manner. Using both cellular and in vivo systems, colorectal and endometrial cancer cell lines, and biochemical and cellular assays, here we examined why the complete loss of Aα does not promote tumorigenesis. CRISPR/Cas9-mediated homozygous Aα deletion resulted in decreased colony formation and tumor growth across multiple cell lines. Protein expression analysis of PP2A family members revealed that the Aα deletion markedly up-regulates Aß protein expression by increasing Aß protein stability. Aß knockdown in control and Aα knockout cell lines indicated that Aß is necessary for cell survival in the Aα knockout cells. In the setting of Aα deficiency, co-immunoprecipitation analysis revealed increased binding of specific PP2A regulatory subunits to Aß, and knockdown of these regulatory subunits restored colony-forming ability. Taken together, our results uncover a mechanism by which PP2A Aα regulates Aß protein stability and activity and suggests why homozygous loss of Aα is rarely seen in cancer patients.
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Péptidos beta-Amiloides/biosíntesis , Regulación de la Expresión Génica , Proteína Fosfatasa 2/metabolismo , Péptidos beta-Amiloides/genética , Animales , Sistemas CRISPR-Cas , Femenino , Células HCT116 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Unión Proteica , Proteína Fosfatasa 2/genética , Estabilidad ProteicaRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are a class of small, well-conserved, non-coding RNAs that regulate the translation of RNAs. They have a role in biological and pathological process including cell differentiation, apoptosis, proliferation and metabolism. Since their discovery, they have been shown to have a potential role in cancer pathogenesis through their function as oncogenes or tumor suppressors. A substantial number of miRNAs show differential expression in esophageal cancer tissues, and so have been investigated for possible use in diagnosis. Furthermore, there is increasing interest in their use as prognostic markers and determining treatment response, as well as identifying their downstream targets and understanding their mode of action. METHODS: We analyzed the most recent studies on miRNAs in esophageal cancer and/or Barrett's esophagus (BE). The publications were identified by searching in PuBMed for the following terms: Barrett's esophagus and microRNA; esophageal cancer and microRNA. RESULTS: Four miRNAs (mi-R-25, -99a, -133a and -133b) showed good potential as diagnostic markers and interestingly five (mi-R-21, -27b, -126, - 143 and -145) appeared to be useful both as diagnostic and prognostic/predictive markers. CONCLUSION: The data so far on miRNAs in esophageal carcinogenesis is promising but further work is required to determine whether miRNAs can be used as biomarkers, not only in the clinical setting or added to individualized treatment regimes but also in non-invasive test by making use of miRNAs identified in blood.
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Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , MicroARNs/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/terapia , Animales , Esófago de Barrett/genética , Esófago de Barrett/metabolismo , Esófago de Barrett/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Neoplasias Esofágicas/terapia , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Pronóstico , Resultado del TratamientoRESUMEN
Osteogeinc differentiation from mesenchymal stem cells (MSCs) into osteoblasts is a key step for bone tissue engineering in regenerative medicine. The insight into regulatory mechanism of osteogenesis of MSCs facilitates achieving better recovery effect. Long non-coding RNAs are regarded as a family of important moderators in osteogenesis. In this study, we found a novel lncRNA, lnc-PPP2R1B was up-regulated during osteogenesis of MSCs by Illumina HiSeq transcritome sequencing. We demonstrated lnc-PPP2R1B overexpression promoted osteogenesis and knockdown of lnc-PPP2R1B inhibited osteogenesis of MSCs. Mechanically, it physically interacted with and up-regulated heterogeneous nuclear ribonucleoprotein L Like (HNRNPLL), which is a master regulator of activation-induced alternative splicing in T cells. We found lnc-PPP2R1B knockdown or HNRNPLL knockdown decreased transcript-201 of Protein Phosphatase 2A, Regulatory Subunit A, Beta Isoform (PPP2R1B) while increased transcript-203 of PPP2R1B, and did not affect transcript-202/204/206. PPP2R1B is a constant regulatory subunit of protein phosphatase 2 (PP2A), which activates Wnt/ß-catenin pathway by removing phosphorylation and stabilization of ß-catenin and translocation into nucleus. The transcript-201 retained exon 2 and 3, compared to transcript-203. And it was reported the exon 2 and 3 of PPP2R1B were one part of B subunit binding domain on A subunit in PP2A trimer, and therefore retaining exon 2 and 3 promised formation and enzyme function of PP2A. Finally, lnc-PPP2R1B promoted ectopic osteogenesis in vivo. Conclusively, lnc-PPP2R1B mediated alternative splicing of PPP2R1B through retaining exon 2 and 3 by interacting with HNRNPLL and then promoted osteogenesis, which may facilitate an in-depth understanding of function and mechanism of lncRNAs in osteogenesis. Lnc-PPP2R1B interacted with HNRNPLL, and regulated alternative splicing of PPP2R1B through retaining exon 2 and 3, which preserved enzyme function of PP2A and enhanced dephosphorylation and nuclear translocation of ß-catenin, thereby promoting Runx2 and OSX expression and then osteogenesis. And it provided experimental data and potential target for promoting bone formation and bone regeneration.
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Ribonucleoproteína Heterogénea-Nuclear Grupo L , Células Madre Mesenquimatosas , Empalme Alternativo/genética , beta Catenina/genética , beta Catenina/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo L/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo L/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo L/farmacología , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/farmacología , HumanosRESUMEN
Thyroid cancer is a prevalent human endocrine tumour. Surgical resection is a primary approach for well-differentiated thyroid cancers. Currently, the combination of chemotherapy with subsequent irradiation is a therapeutic strategy for the late stage or metastatic thyroid cancer. Yet, drug resistance and side-effects greatly limit widely clinical applications of chemotherapeutic drugs. The long non-coding RNA IQCH antisense RNA 1 (IQCH-AS1) is correlated with survival and diagnosis of cancer patients. Currently, the precise roles of IQCH-AS1 in thyroid cancer and the chemosensitivity of doxorubicin remain unclear. Here, we report IQCH-AS1 was significantly down-regulated in thyroid cancer tissues and cell lines. Overexpression of IQCH-AS1 effectively sensitized thyroid cancer cells to doxorubicin. From the established doxorubicin-resistant thyroid cancer cell line, 8505 C Doxo R, we detected that IQCH-AS1 was remarkedly suppressed in doxorubicin-resistant cells. Bioinformatics analysis, RNA pull-down and luciferase assays illustrated that IQCH-AS1 functioned as a ceRNA of miR-196a-5p which showed an oncogenic role in thyroid cancer. Overexpression of miR-196a-5p, which was upregulated in 8505 C Doxo R cells, significantly de-sensitized thyroid cancer cells to doxorubicin. Furthermore, PPP2R1B, which encode the protein phosphatase 2 A regulatory subunit A, was directly targeted by miR-196a-5p in thyroid cancer cells. Rescue experiments validated that recovery of miR-196a-5p in IQCH-AS1-overexpressing 8508 C doxorubicin resistant cells successfully reversed the IQCH-AS1-promoted doxorubicin sensitization via targeting PPP2R1B. Summarily, our study revealed new functions and molecular targets of the lncRNA-IQCH-AS1-mediated chemosensitivity of thyroid cancer, contributing to the development of anti-chemoresistant strategies against thyroid cancer.
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MicroARNs , ARN Largo no Codificante , Neoplasias de la Tiroides , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Doxorrubicina/farmacología , Línea Celular TumoralRESUMEN
Vemurafenib, a BRAF V600E inhibitor, provides therapeutic benefits for patients with melanoma, but the frequent emergence of drug resistance remains a challenge. An understanding of the mechanisms underlying vemurafenib resistance may generate novel therapeutic strategies for patients with melanoma. Here, we showed that eIF3a, a translational regulatory protein, was an important mediator involved in vemurafenib resistance. eIF3a was expressed at significantly lower levels in vemurafenib-resistant A375 melanoma cells (A375R) than in parental A375 cells. Overexpression of eIF3a enhanced the sensitivity to BRAF inhibitors by reducing p-ERK levels. Furthermore, eIF3a controlled ERK activity by regulating the expression of the phosphatase PPP2R1B via a translation mechanism, thus determining the sensitivity of melanoma cells to vemurafenib. In addition, a positive correlation between eIF3a and PPP2R1B expression was also observed in tumor samples from the Human Protein Atlas and TCGA databases. In conclusion, our studies reveal a previously unknown molecular mechanism of BRAF inhibitor resistance, which may provide a new strategy for predicting vemurafenib responses in clinical treatment.
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BACKGROUND: Chemoresistance is the main challenge for treating tongue squamous cell carcinoma (TSCC). MiR-200c is an important regulator of chemoresistance. Exosomes are a promising molecule-delivery system for cancer treatment. Thus, this study aimed to investigate the role of miR-200c in chemoresistance of TSCC and whether exosomes could effectively deliver miR-200c to chemo-resistant cells and regulate cellular activities. RESULTS: The results showed that the downregulation of miR-200c increased resistance to DTX, migration, and invasion and decreased apoptosis, which was reversed by the overexpression of miR-200c. The NTECs-derived exosomes transported miR-200c to HSC-3DR, increasing the sensitivity to DTX in vitro and in vivo. Also, epithelial-to-mesenchymal transition (EMT) and DNA damage responses were involved in DTX resistance. Furthermore, miR-200c regulated DTX resistance by targeting TUBB3 and PPP2R1B. CONCLUSION: Exosome-mediated miR-200c delivery may be an effective and promising strategy to treat chemoresistance in TSCC. METHODS: Docetaxel (DTX) resistant HSC-3 cells (HSC-3DR) were transfected with miR-200c lentivirus and cocultured with exosomes derived from normal tongue epithelial cells (NTECs) that were overexpressed with miR-200c. The roles of miR-200c and exosomal miR-200c in vitro and in vivo were determined by RNA-Seq, qRT-PCR, western blots, transmission electron microscopy, and flow cytometry, fluorescence, CCK8, Transwell, and wound healing assays.
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Docetaxel/uso terapéutico , Exosomas/fisiología , MicroARNs/fisiología , Proteína Fosfatasa 2/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de la Lengua/tratamiento farmacológico , Tubulina (Proteína)/genética , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , RNA-Seq , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/patologíaRESUMEN
microRNAs (miRNAs) are a class of small non-coding RNAs that post-transcriptionally regulate gene expression. Increasing evidence has shown that the deregulation of miRNAs is linked to cancer. The overexpression of miR-224 has been reported in several human cancers. The aim of the present study was to investigate the function of miR-224 in the pathogenetic process of hepatocellular carcinoma (HCC), and the precise mechanism underlying its function. Both gain-of-function and loss-of function assays were conducted through transfection with miR-224 mimics and miR-224 inhibitors in the HepG2 liver carcinoma cell line. The data revealed that miR-224 exerts a significant role in promoting cell proliferation, migration and invasion. Western blot analysis showed that the phosphorylation levels of AKT positively correlated with endogenous levels of miR-224. In addition, results from a dual luciferase reporter assay showed that the expression of the serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A ß isoform (PPP2R1B) is inhibited by miR-224; thus, it appears that PPP2R1B is a candidate target of miR-224 in HCC. These data suggest that miR-224 plays a significant role in HCC, possibly through the activation of the AKT signaling pathway by targeting PPP2R1B.