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1.
Braz. j. biol ; 84: e250151, 2024. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1350306

ABSTRACT

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.


Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.


Subject(s)
Humans , Vitreous Body , Cell Proliferation , Cell Dedifferentiation , Tretinoin , Tumor Cells, Cultured , Cell Differentiation , Cell Division , Cell Line
2.
Int. j. morphol ; 41(5): 1348-1356, oct. 2023.
Article in English | LILACS | ID: biblio-1521029

ABSTRACT

SUMMARY: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in various types of cancers including breast cancer. However, the role of AhR with its endogenous ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the progression of breast cancer remains poorly understood. We aimed to investigate cell proliferation and migration states in breast cancer after activating AhR with the endogenous ligand ITE. Breast cancer tissue was evaluated by cell lines, immunohistochemistry, reverse transcription-polymerase chain reaction, cell proliferation, flow cytometry, migration assays and western blot techniques. We found that AhR was widely expressed in breast cancer tissues and metastasis lymph node tissues, but not in normal tissues. The expression AhR was independent between the age, grades and TNM classifications for breast cancer tissues. ITE treatment significantly induced the activation of AhR in a time-dependent manner in both MCF-7 and T47D breast cancer cell lines. Meanwhile, ITE did not affect the cell migration but significantly suppressed the cell proliferation in estrogen receptor positive (ER+) MCF-7 andT47D cells, which probably attribute to the induction of cell cycle arrest in G1 phase and shortened S phase. Further mechanism study showed that ERK1/2 and AKT signaling were required for the activation of AhR in MCF-7 cells. These data suggest that AhR is a potential new target for treating patients with breast cancer. ITE may be more potentially used for therapeutic intervention for breast cancer with the kind of ER(+).


El receptor de hidrocarburo de arilo (AhR) es un factor de transcripción activado por ligando que se expresa en gran medida en varios tipos de cáncer, incluido el cáncer de mama. Sin embargo, el papel de AhR con su ligando endógeno 2- (1'H-indol-3'-carbonil)-tiazol-4-ácido carboxílico metil éster (ITE) en la progresión del cáncer de mama sigue siendo poco conocido. Nuestro objetivo fue investigar la proliferación celular y los estados de migración en el cáncer de mama después de activar AhR con el ligando endógeno ITE. El tejido de cáncer de mama se evaluó mediante líneas celulares, inmunohistoquímica, reacción en cadena de la polimerasa con transcriptasa inversa, proliferación celular, citometría de flujo, ensayos de migración y técnicas de transferencia Western. Descubrimos que AhR se expresó ampliamente en tejidos de cáncer de mama y en linfonodos con metástasis, pero no en tejidos normales. La expresión AhR fue independiente entre la edad, grados y clasificaciones TNM para tejidos de cáncer de mama. El tratamiento con ITE indujo significativamente la activación de AhR de manera dependiente del tiempo en las líneas celulares de cancer de mama MCF-7 y T47D. Mientras tanto, ITE no afectó la migración celular, pero suprimió significativamente la proliferación celular en células MCF-7 y T47D con receptor de estrógeno positivo (ER+), lo que probablemente se atribuye a la inducción de la detención del ciclo celular en la fase G1 y la fase S acortada. Un estudio adicional del mecanismo mostró que las señales de ERK1/2 y AKT eran necesarias para la activación de AhR en las células MCF-7. Estos datos sugieren que AhR es un nuevo objetivo potencial para el tratamiento de pacientes con cáncer de mama. ITE puede ser utilizado más potencialmente en la intervención terapéutica para el cáncer de mama con el tipo de ER (+).


Subject(s)
Humans , Female , Thiazoles/administration & dosage , Breast Neoplasms/pathology , Receptors, Aryl Hydrocarbon/drug effects , Indoles/administration & dosage , Thiazoles/pharmacology , Immunohistochemistry , Receptors, Estrogen , Blotting, Western , Cytochrome P-450 CYP1A1/genetics , Cell Line, Tumor/drug effects , Cell Proliferation/drug effects , Cell Migration Assays , Cytochrome P-450 CYP1B1/genetics , Flow Cytometry , Indoles/pharmacology
3.
Rev. Odontol. Araçatuba (Impr.) ; 44(1): 17-21, jan.-abr. 2023.
Article in English | BBO, LILACS | ID: biblio-1427816

ABSTRACT

Os hemangiomas são neoplasias benignas dos tecidos moles. São lesões caracterizadas pela proliferação de células endoteliais. Objetivos: O objetivo deste trabalho é demonstrar por meio de uma revisão da literatura os principais hemangiomas de interesse odontológico e suas características clínicas e abordagens terapêuticas. Revisão de literatura: O estudo mostrou prevalência de até 6% na população geral, sendo o tumor mais comum na infância. Essas lesões apresentam rápido crescimento pós-natal, que pode levar meses a dois anos em média, mas geralmente após esse período ocorre sua involução. O diagnóstico é comumente baseado nas características clínicas e na história do paciente. É importante entender que o estudo histopatológico pode ser necessário nos casos em que o diagnóstico é incerto, para diferenciá-lo de outras neoplasias graves. Destacam-se as principais complicações relacionadas a lesões, ulcerações e hemorragias, além de infecções secundárias que podem causar alto índice de morbidade. Assim, é fundamental que o dentista reconheça essas patologias e tenha capacidade para tratá-las. Considerações finais: Foi possível observar que os hemangiomas são manifestações vasculares incomuns para o cirurgião-dentista, porém o profissional deve saber diagnosticá-los e tratá-los. Dentre as áreas acometidas, essas lesões são frequentes na cavidade oral e o tratamento consiste em acompanhamento com intervenções conservadoras(AU)


Hemangiomas are benign soft tissue neoplasms. These are lesions characterized by the proliferation of endothelial cells. Objectives: The objective of this work is to demonstrate through a literature review about the main hemangiomas of dental interest and clinical characteristics and therapeutic approaches. Literature review: The study showed a prevalence of up to 6% in the general population, being the most common tumor in childhood. These lesions presents a rapid postnatal growth, which may take months until two years on average, but usually after this period their involution occurs. The diagnosis is commonly based on clinical characteristics and patient's history. It is important to understand that the histopathological study may be necessary in cases that the diagnostic is uncertainty, in order to differentiate it from other serious neoplasms. Main complications related to injuries, ulceration and hemorrhage can be highlighted, in addition to secondary infections that can cause a high rate of morbidity. Thus, it is essential that dentists recognize such pathologies and be able to treat them. Final considerations: Was possible do observe that hemangiomas are uncommon vascular manifestations for the dental surgeon, however, the professional must know to diagnose and treat them. Among the affected areas, these lesions are frequently seen in the oral cavity and the treatment consists of following up with conservative interventions(AU)


Subject(s)
Soft Tissue Neoplasms , Mouth Neoplasms , Hemangioma , Hemangioma/diagnosis , Hemangioma/therapy , Cell Proliferation , Mouth/injuries , Neoplasms
4.
J. oral res. (Impresa) ; 12(1): 86-99, abr. 4, 2023. ilus
Article in English | LILACS | ID: biblio-1512278

ABSTRACT

Objective: The objective of this study was to investigate the morphology, proliferation, and differentiation of gingival mesenchymal stem cells (GMSCs) irradiated with a 970 nm Diode Laser (LLLT). It is essential to validate the efficacy of treatment, optimize irradiation conditions and guarantee the safety and quality of stem cells for future use in dental applications. Materials and Methods: GMSCs were cultured in standard conditions and irradiated with a Diode laser (970 nm, 0.5W) with an energy density of 9J/cm2. Cell proliferation was assessed with the WST-1 proliferation kit. GMSCs were differentiated into chondrogenic and osteogenic lineages. Cell morphology was performed with Hematoxylin/eosin staining, and quantitative nuclear analysis was done. Cell viability was monitored with trypan blue testing. Results: GMSCs subjected to irradiation demonstrated a significant increase in proliferation at 72 hours compared to the non-irradiated controls (p=0.027). This indicates that the 970 nm diode laser has a stimulatory effect on the proliferation of GMSCs. LLLT-stimulated GMSCs exhibited the ability to differentiate into chondrogenic and osteogenic lineages. A substantial decrease in cell viability was observed 24 hours after irradiation (p=0.024). However, after 48 hours, the cell viability recovered without any significant differences. This indicates that there might be a temporary negative impact on cell viability immediately following irradiation, but the cells were able to recover and regain their viability over time. Conclusions: This study support that irradiation with a 970 nm diode laser could stimulate the proliferation of GMSCs, maintain their ability to differentiate into chondrogenic and osteogenic lineages, and has minimal impact on the mor- phological characteristics of the cells. These results support the potential use of NIR Lasers in combination with GMSCs as a promising strategy for dental treatments.


Objetivo: El objetivo de este estudio fue investigar la morfología, proliferación y diferenciación de las células madre mesenquimatosas (GMSC) irradiadas con un láser de diodo de 970 nm (LLLT). Es fundamental validar la eficacia del tratamiento, optimizar las condiciones de irradiación y garantizar la seguridad y calidad de las células madre para su uso futuro en aplicaciones dentales.Materiales y Métodos: Las GMSC se cultivaron en condiciones estándar y se irradiaron con un láser de diodo (970 nm, 0,5 W) con una densidad de energía de 9 J/cm2. La proliferación celular se evaluó con el kit de proliferación WST-1. Las GMSC se diferenciaron en linajes condrogénicos y osteogénicos. La morfología celular se realizó con tinción de hematoxilina/eosina y se realizó un análisis nuclear cuantitativo. La viabilidad celular se controló con prueba de azul de tripano. Resultados: Las GMSC sometidas a irradiación demostraron un aumento significativo en la proliferación a las 72 horas en comparación con los controles no irradiados (p=0,027). Esto indica que el láser de diodo de 970 nm tiene un efecto estimulante sobre la proliferación de GMSC. Las GMSC estimuladas con LLLT exhibieron la capacidad de diferenciarse en linajes condrogénicos y osteogénicos. Se observó una disminución sustancial de la viabilidad celular 24 horas después de la irradiación (p=0,024). Sin embargo, después de 48 horas, la viabilidad celular se recuperó sin diferencias significativas. Esto indica que podría haber un impacto negativo temporal en la viabilidad de las células inmediatamente después de la irradiación, pero las células pudieron recuperarse y recuperar su viabilidad con el tiempo. Conclusión: En conclusión, este estudio respalda que la irradiación con un láser de diodo de 970 nm podría estimular la proliferación de GMSC, mantener su capacidad para diferenciarse en linajes condrogénicos y osteogénicos y tiene un impacto mínimo en las características morfológicas de las células. Estos resultados respaldan el uso potencial de láseres NIR en combinación con GMSC como una estrategia prometedora para tratamientos dentales.


Subject(s)
Humans , Low-Level Light Therapy , Cell Proliferation/radiation effects , Lasers, Semiconductor , Mesenchymal Stem Cells/radiation effects , In Vitro Techniques , Gingiva/radiation effects
5.
Int. j. morphol ; 41(1): 308-318, feb. 2023. ilus, tab, graf
Article in English | LILACS | ID: biblio-1430503

ABSTRACT

SUMMARY: Gastrin plays a vital role in the development and progression of gastric cancer (GC). Its expression is up-regulated in GC tissues and several GC cell lines. Yet, the underlying mechanism remains to be investigated. Here, we aim to investigate the role and mechanism of gastrin in GC proliferation. Gastrin-overexpressing GC cell model was constructed using SGC7901 cells. Then the differentially expressed proteins were identified by iTRAQ analysis. Next, we use flow cytometry and immunofluorescence to study the effect of gastrin on the mitochondrial potential and mitochondria-derived ROS production. Finally, we studied the underlying mechanism of gastrin regulating mitochondrial function using Co-IP, mass spectrometry and immunofluorescence. Overexpression of gastrin promoted GC cell proliferation in vitro and in vivo. A total of 173 proteins were expressed differently between the controls and gastrin- overexpression cells and most of these proteins were involved in tumorigenesis and cell proliferation. Among them, Cox17, Cox5B and ATP5J that were all localized to the mitochondrial respiratory chain were down-regulated in gastrin-overexpression cells. Furthermore, gastrin overexpression led to mitochondrial potential decrease and mitochondria-derived ROS increase. Additionally, gastrin-induced ROS generation resulted in the inhibition of cell apoptosis via activating NF-kB, inhibiting Bax expression and promoting Bcl-2 expression. Finally, we found gastrin interacted with mitochondrial membrane protein Annexin A2 using Co-IP and mass spectrometry. Overexpr ession of gastrin inhibits GC cell apoptosis by inducing mitochondrial dysfunction through interacting with mitochondrial protein Annexin A2, then up-regulating ROS production to activate NF-kB and further leading to Bax/Bcl-2 ratio decrease.


La gastrina juega un papel vital en el desarrollo y progresión del cáncer gástrico (CG). Su expresión está regulada al alza en tejidos de CG y en varias líneas celulares de CG. Sin embargo, el mecanismo subyacente aun no se ha investigado. El objetivo de este estudio fue investigar el papel y el mecanismo de la gastrina en la proliferación de CG. El modelo de células CG que sobre expresan gastrina se construyó usando células SGC7901. Luego, las proteínas expresadas diferencialmente se identificaron mediante análisis iTRAQ. A continuación, utilizamos la citometría de flujo y la inmunofluorescencia para estudiar el efecto de la gastrina en el potencial mitocondrial y la producción de ROS derivada de las mitocondrias. Finalmente, estudiamos el mecanismo subyacente de la gastrina que regula la función mitocondrial utilizando Co-IP, espectrometría de masas e inmunofluorescencia. La sobreexpresión de gastrina promovió la proliferación de células CG in vitro e in vivo. Un total de 173 proteínas se expresaron de manera diferente entre los controles y las células con sobreexpresión de gastrina y la mayoría de estas proteínas estaban implicadas en la tumorigenesis y la proliferación celular. Entre estas, Cox17, Cox5B y ATP5J, todas localizadas en la cadena respiratoria mitocondrial, estaban reguladas a la baja en las células con sobreexpresión de gastrina. Además, la sobreexpresión de gastrina provocó una disminución del potencial mitocondrial y un aumento de las ROS derivadas de las mitocondrias. Por otra parte, la generación de ROS inducida por gastrina resultó en la inhibición de la apoptosis celular mediante la activación de NF-kB, inhibiendo la expresión de Bax y promoviendo la expresión de Bcl-2. Finalmente, encontramos que la gastrina interactuaba con la proteína de membrana mitocondrial Anexina A2 usando Co-IP y espectrometría de masas. La sobreexpresión de gastrina inhibe la apoptosis de las células CG al inducir la disfunción mitocondrial a través de la interacción con la proteína mitocondrial Anexina A2, luego regula el aumento de la producción de ROS para activar NF-kB y conduce aún más a la disminución de la relación Bax/Bcl-2.


Subject(s)
Animals , Mice , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Gastrins/metabolism , Annexin A2/metabolism , Mitochondria/pathology , Mass Spectrometry , NF-kappa B , Fluorescent Antibody Technique , Reactive Oxygen Species , Apoptosis , Cell Line, Tumor , Immunoprecipitation , Cell Proliferation , Carcinogenesis , Flow Cytometry
6.
Int. j. morphol ; 41(1): 286-296, feb. 2023. ilus, tab, graf
Article in English | LILACS | ID: biblio-1430539

ABSTRACT

SUMMARY: Cancer is the second leading cause of death in the world and colorectal cancer is the only cancer that has shown a sustained increase in mortality in the last decade. In the search for new chemotherapeutic agents against cancer, extremophilic microorganisms have shown to be a potential source to obtain molecules of natural origin and with selective cytotoxic action towards cancer cells. In this work we analyzed the ability of a collection of Antarctic soil bacteria, isolated on Collins Glacier from the rhizosphere of Deschampsia antarctica Desv plant, to secrete molecules capable of inhibiting cell proliferation of a colorectal cancer tumor line. Our results demonstrated that culture supernatants from the Antarctic bacteria K2I17 and MI12 decreased the viability of LoVo cells, a colorectal adenocarcinoma cell line. Phenotypic and genotypic characterization of the Antarctic bacteria showed that they were taxonomically related and nucleotide identity analysis based on the 16S rRNA gene sequence identified the bacterium K2I17 as a species belonging to the genus Bacillus.


El cáncer es la segunda causa de muerte en el mundo y el cáncer colorrectal es el único que presenta un aumento sostenido de la mortalidad en la última década. En la búsqueda de nuevos agentes quimioterapeúticos contra el cáncer, se ha propuesto a los microorganismos extremófilos como una fuente potencial para obtener moléculas de origen natural y con acción citotóxica selectiva hacia las células cancerígenas. En este trabajo analizamos la capacidad de una colección de bacterias de suelo antártico, aisladas en el glaciar Collins desde rizosfera de la planta de Deschampsia antarctica Desv, de secretar moléculas capaces de inhibir la proliferación celular de una línea tumoral de cáncer colorrectal. Nuestros resultados demostraron que los sobrenadantes de cultivo de las bacterias antárticas K2I17 y MI12 disminuyeron la viabilidad de la línea celular de adenocarcinoma colorrectal LoVo, en un ensayo de reducción metabólica de MTT. La caracterización fenotípica y genotípica de las bacterias antárticas, demostró que estaban relacionadas taxonómicamente y el análisis de la identidad nucleotídica en base a la secuencia del gen ARNr 16S identificó a la bacteria K2I17 como una especie perteneciente al género Bacillus.


Subject(s)
Humans , Soil Microbiology , Bacillus/physiology , Colorectal Neoplasms/drug therapy , Cell Proliferation/drug effects , Phenotype , Bacillus/isolation & purification , Bacillus/genetics , In Vitro Techniques , RNA, Ribosomal, 16S , Adenocarcinoma/drug therapy , Cell Survival/drug effects , Polymerase Chain Reaction , Cell Line, Tumor/drug effects , Genotype , Antarctic Regions
7.
Article in Chinese | WPRIM | ID: wpr-987025

ABSTRACT

OBJECTIVE@#To investigate the regulatory effects of miR-30e-5p on biological behaviors of colorectal cancer cells and the role of PTEN/CXCL12 axis in mediating these effects.@*METHODS@#Bioinformatic analysis was performed to explore the differential expression of miR-30e-5p between colorectal cancer tissues and normal tissues. RT-qPCR was used to detect the differential expression of miR-30e-5p in intestinal epithelial cells and colorectal cancer cells. Bioinformatics and dual luciferase assay were used to predict and validate the targeting relationship between miR-30e-5p and PTEN. Human and murine colorectal cancer cell lines were transfected with miR-30e-5p mimics, miR-30e-5p inhibitor, miR-30e-5p mimics+LV-PTEN, or miR-30e-5p inhibitor + si-PTEN. The changes in biological behaviors of the cells were detected using plate clone formation assay, CCK-8 assay, flow cytometry, scratch healing and Transwell assays. PTEN and CXCL12 expressions in the cancer cells were detected by Western blotting. The effects of miR-30e-5p inhibitor on colorectal carcinogenesis and development were observed in nude mice.@*RESULTS@#Bioinformatic analysis showed that miR-30e-5p expression was significantly elevated in colorectal cancer tissues compared with the adjacent tissue (P < 0.01). Higher miR-30e-5p expression was detected in colorectal cancer cell lines than in intestinal epithelial cells (P < 0.01). Dual luciferase assay confirmed the targeting relationship between miR-30e-5p and PTEN (P < 0.05). Transfection with miR-30e-5p mimics significantly enhanced proliferation and metastasis and inhibited apoptosis of the colorectal cancer cells (P < 0.05), and co-transfection with LV-PTEN obviously reversed these changes (P < 0.05). MiR-30e-5p mimics significantly inhibited PTEN expression and enhanced CXCL12 expression in the cancer cells (P < 0.01), and miR-30e-5p inhibitor produced the opposite effect. Transfection with miR-30e-5p inhibitor caused cell cycle arrest in the cancer cells, which was reversed by co-transfection with si-PTEN (P < 0.05). In the in vivo experiments, the colorectal cancer cells transfected with miR-30e-5p inhibitor showed significantly lowered tumorigenesis.@*CONCLUSION@#Overexpression of miR-30e-5p promotes the malignant behaviors of colorectal cancer cells by downregulating PTEN to activate the CXCL12 axis.


Subject(s)
Humans , Animals , Mice , MicroRNAs/metabolism , Cell Line, Tumor , Cell Proliferation/physiology , Mice, Nude , Cell Movement/physiology , Colorectal Neoplasms/pathology , Luciferases/metabolism , Gene Expression Regulation, Neoplastic , PTEN Phosphohydrolase/metabolism , Chemokine CXCL12/metabolism
8.
Article in Chinese | WPRIM | ID: wpr-987024

ABSTRACT

OBJECTIVE@#To investigate the effect of lactic acid-induced upregulation of PLEKHA4 expression on biological behaviors of glioma cells and the possible molecular mechanism.@*METHODS@#GEO database and GEPIA2 website were used to analyze the relationship between PLEKHA4 expression level and the pathological grade of glioma. A specific PLEKHA4 siRNA was transfected in glioma U251 and T98G cells, and the changes in cell proliferation ability were assessed by real-time cell analysis technology and Edu experiment. The colony-forming ability of the cells was evaluated using plate cloning assay, and cell cycle changes and cell apoptosis were analyzed with flow cytometry. The mRNA expression of PLEKHA4 was detected by PCR in glioma samples and controls and in glioma cells treated with lactic acid and glucose. Xenograft mice in vivo was used to detect tumor formation in nude mice; Western blotting was used to detect the expressions of cyclinD1, CDK2, Bcl2, β-catenin and phosphorylation of the key proteins in the MAPK signaling pathway.@*RESULTS@#The results of GEO database and online website analysis showed that PLEKHA4 was highly expressed in glioma tissues and was associated with poor prognosis; PLEKHA4 knockdown obviously inhibited the proliferation and attenuated the clone-forming ability of the glioma cells (P < 0.05). Flow cytometry showed that PLEKHA4 knockdown caused cell cycle arrest in G1 phase and promoted apoptosis of the cells (P < 0.01). PLEKHA4 gene mRNA expression was increased in glioma samples and glioma cells after lactate and glucose treatment (P < 0.01). PLEKHA4 knockdown, tumor formation ability of nude mice decreased; PLEKHA4 knockdown obviously lowered the expression of cyclinD1, CDK2, Bcl2 and other functional proteins, inhibited the phosphorylation of ERK and p38 and reduced the expression of β-catenin protein (P < 0.01).@*CONCLUSION@#PLEKHA4 knockdown inhibited the proliferation of glioma cells and promoted apoptosis by inhibiting the activation of the MAPK signaling pathway and expression of β-catenin. Lactic acid produced by glycolysis upregulates the expression of PLEKHA4 in glioma cells.


Subject(s)
Humans , Animals , Mice , Up-Regulation , beta Catenin/metabolism , Mice, Nude , Brain Neoplasms/pathology , Lactic Acid , Cell Line, Tumor , Glioma/pathology , Cell Proliferation , Apoptosis , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , Gene Expression Regulation, Neoplastic
9.
Article in Chinese | WPRIM | ID: wpr-987011

ABSTRACT

OBJECTIVE@#To investigate the expression of four-jointed box kinase 1 (FJX1) in gastric cancer (GC), its correlation with survival outcomes of the patients, and its role in GC progression.@*METHODS@#The expression level of FJX1 in GC tissues and normal gastric mucosal tissues and its correlation with the survival outcomes of GC patients were analyzed using TCGA and GEO database GC cohort. Immunohistochemistry was used to detect FJX1 expression level in clinical specimens of GC tissue, and its correlations with the patients' clinicopathological parameters and prognosis were analyzed. Bioinformatic analysis was performed to identify the potential pathways of FJX1 in GC. The effects of FJX1 overexpression or FJX1 silencing on GC cell proliferation and expressions of proliferation-related proteins, PI3K, AKT, p-PI3K, and p-AKT were evaluated using CCK-8 assay and Western blotting. The effect of FJX1 overexpression on GC cell tumorigenicity was evaluated in nude mice.@*RESULTS@#GC tissues showed significantly higher expressions of FJX1 mRNA and protein compared with normal gastric mucosa tissues (P < 0.05). The high expression of FJX1 was associated with poor prognosis of GC patients (P < 0.05) and served as an independent risk factor for poor survival outcomes in GC (P < 0.05). FJX1 was expressed mainly in the cytoplasm of GC cells in positive correlation with Ki67 expression (R=0.34, P < 0.05), and was correlated with CA199 levels, depth of tumor infiltration and lymph node metastasis of GC (P < 0.05). In the cell experiment, FJX1 level was shown to regulate the expressions of Ki67 and PCNA and GC cell proliferation (P < 0.05). Gene set enrichment analysis indicated that the PI3K/AKT pathway potentially mediated the effect of FJX1, which regulated the expressions of PI3K and AKT and their phosphorylated proteins. In nude mice, FJX1 overexpression in GC cells significantly promoted the growth of the transplanted tumors (P < 0.05).@*CONCLUSION@#FJX1 is highly expressed in GC tissues and is correlated with poor prognosis of GC patients. FJX1 overexpression promotes GC cell proliferation through the PI3K/AKT signaling pathway, and may serve as a potential prognostic biomarker and therapeutic target for GC.


Subject(s)
Animals , Mice , Humans , Cell Proliferation , Ki-67 Antigen , Mice, Nude , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , Stomach Neoplasms/pathology , Intercellular Signaling Peptides and Proteins/genetics
10.
Article in Chinese | WPRIM | ID: wpr-987007

ABSTRACT

OBJECTIVE@#To investigate the mechanism by which conditioned medium of colorectal cancer cells promotes the formation of cancer-associated fibroblasts (CAFs).@*METHODS@#Normal human colorectal fibroblasts (CCD-18Co cells) in logarithmic growth phase were treated with the conditioned media of colorectal cancer HCT116 cells (HCT116-CM) or Caco-2 cells (Caco-2-CM) alone or in combination with 300 nmol/L ERK inhibitor SCH772984. The expression levels of CAFs-related molecular markers were detected in the treated cells with real-time quantitative PCR (RT- qPCR) and immunofluorescence assay, and the changes in cell proliferation, colony formation and migration were assessed with RTCA, colony formation and wound healing assays; Western blotting was performed to detect the activated signaling pathways in the fibroblasts and the changes in CAFs formation after blocking of the signaling pathway.@*RESULTS@#HCT116-CM and Caco-2-CM significantly upregulated mRNA expression levels of CAFs markers (including α-SMA, FAP, FN and TGF-β) in CCD-18Co cells, and strongly promoted fibroblast transformation into CAFs (P < 0.05). The two conditioned media also promoted the proliferation, colony formation and migration of CCD-18Co cells (P < 0.05) and significantly increased the levels of α-SMA protein and ERK phosphorylation in the cells (P < 0.05). The ERK inhibitor SCH772984 obviously inhibited the expression of α-SMA and the transformation of CCD-18Co cells into CAFs induced by the conditioned medium of colorectal cancer cells (P < 0.05).@*CONCLUSION@#Colorectal cancer cells may induce the formation of colorectal CAFs by activating the ERK pathway in the fibroblasts.


Subject(s)
Humans , Cancer-Associated Fibroblasts/metabolism , Culture Media, Conditioned/pharmacology , MAP Kinase Signaling System , Caco-2 Cells , Fibroblasts , Signal Transduction , Cell Proliferation , Cell Line, Tumor , Colorectal Neoplasms/genetics , Cell Movement
11.
Article in Chinese | WPRIM | ID: wpr-986983

ABSTRACT

OBJECTIVE@#To investigate the effects of expression levels of S100 calcium-binding protein A10 (S100A10) in lung adenocarcinoma (LUAD) on patient prognosis and the regulatory role of S100A10 in lung cancer cell proliferation and metastasis.@*METHODS@#Immunohistochemistry was used to detect the expression levels of S100A10 in LUAD and adjacent tissues, and the relationship between S100A10 expression and clinicopathological parameters and prognosis of the patients was statistically analyzed. The lung adenocarcinoma expression dataset in TCGA database was analyzed using gene enrichment analysis (GSEA) to predict the possible regulatory pathways of S100A10 in the development of lung adenocarcinoma. Lactate production and glucose consumption of lung cancer cells with S100A10 knockdown or overexpression were analyzed to assess the level of glycolysis. Western blotting, CCK-8 assay, EdU-594 assay, and Transwell assays were performed to determine the expression level of S100A10 protein, proliferation and invasion ability of lung cancer cells. A549 cells with S100A10 knockdown and H1299 cells with S100A10 overexpression were injected subcutaneously in nude mice, and tumor growth was observed.@*RESULTS@#The expression level of S100A10 was significantly upregulated in LUAD tissues as compared with the adjacent tissues, and an elevated S100A10 expression level was associated with lymph node metastasis, advanced tumor stage and distant organ metastasis (P < 0.05), but not with tumor differentiation or the patients' age or gender (P > 0.05). Survival analysis showed that elevated S100A10 expressions in the tumor tissue was associated with a poor outcome of the patients (P < 0.001). In the lung cancer cells, S100A10 overexpression significantly promoted cell proliferation and invasion in vitro (P < 0.001). GSEA showed that the gene sets of glucose metabolism, glycolysis and mTOR signaling pathway were significantly enriched in high expressions of S100A10. In the tumor-bearing nude mice, S100A10 overexpression significantly promoted tumor growth, while S100A10 knockdown obviously suppressed tumor cell proliferation (P < 0.001).@*CONCLUSION@#S100A10 overexpression promotes glycolysis by activating the Akt-mTOR signaling pathway to promote proliferation and invasion of lung adenocarcinoma cells.


Subject(s)
Animals , Mice , Humans , Adenocarcinoma of Lung/pathology , Cell Proliferation , Lung Neoplasms/pathology , Mice, Nude , Proto-Oncogene Proteins c-akt , Signal Transduction , TOR Serine-Threonine Kinases , S100 Proteins/genetics
12.
Article in Chinese | WPRIM | ID: wpr-986979

ABSTRACT

OBJECTIVE@#To investigate the molecular mechanism underlying the inhibitory effect of aloin on the proliferation and migration of gastric cancer cells.@*METHODS@#Human gastric cancer MGC-803 cells treated with 100, 200 and 300 μg/mL aloin were examined for changes in cell viability, proliferation and migration abilities using CCK-8, EdU and Transwell assays. HMGB1 mRNA level in the cells was detected with RT-qPCR, and the protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 were determined using Western blotting. JASPAR database was used to predict the binding of STAT3 to HMGB1 promoter. In a BALB/c-Nu mouse model bearing subcutaneous MGC-803 cell xenograft, the effect of intraperitoneal injection of aloin (50 mg/kg) on tumor growth was observed. The protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 in the tumor tissue was examined using Western blotting, and tumor metastasis in the liver and lung tissues was detected using HE staining.@*RESULTS@#Treatment with aloin concentration-dependently inhibited the viability of MGC-803 cells (P < 0.05), significantly reduced the number of EdU-positive cells (P < 0.01), and attenuated the migration ability of the cells (P < 0.01). Aloin treatment dose-dependently down-regulated HMGB1 mRNA expression (P < 0.01), lowered the protein expressions of HMGB1, cyclin B1, cyclin E1, MMP-2, MMP-9 and p-STAT3, and up-regulated E-cadherin expression in MGC-803 cells. Prediction based on JASPAR database suggested that STAT3 could bind to the promoter region of HMGB1. In the tumor-bearing mice, aloin treatment significantly reduced the tumor size and weight (P < 0.01), lowered the protein expressions of cyclin B1, cyclin E1, MMP-2, MMP-9, HMGB1 and p-STAT3 and increased the expression of E-cadherin in the tumor tissue (P < 0.01).@*CONCLUSION@#Aloin attenuates the proliferation and migration of gastric cancer cells by inhibiting the STAT3/HMGB1 signaling pathway.


Subject(s)
Humans , Animals , Mice , Stomach Neoplasms , Cyclin B1 , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , HMGB1 Protein , Signal Transduction , Cell Proliferation , STAT3 Transcription Factor
13.
Article in Chinese | WPRIM | ID: wpr-986965

ABSTRACT

OBJECTIVE@#Bo investigate the regulatory relationship between NKD1 and YWHAE and the mechanism of NKD1 for promoting tumor cell proliferation.@*METHODS@#HCT116 cells transfected with pcDNA3.0-NKD1 plasmid, SW620 cells transfected with NKD1 siRNA, HCT116 cells with stable NKD1 overexpression (HCT116-NKD1 cells), SW620 cells with nkd1knockout (SW620-nkd1-/- cells), and SW620-nkd1-/- cells transfected with pcDNA3.0-YWHAE plasmid were examined for changes in mRNA and protein expression levels of YWHAE using qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NKD1 to the promoter region of YWHAE gene. The regulatory effect of NKD1 on YWHAE gene promoter activity was analyzed by dual-luciferase reporter gene assay, and the interaction between NKD1 and YWHAE was analyzed with immunofluorescence assay. The regulatory effect of NKD1 on glucose uptake was examined in the tumor cells.@*RESULTS@#In HCT116 cells, overexpression of NKD1 significantly enhanced the expression of YWHAE at both the mRNA and protein levels, while NKD1 knockout decreased its expression in SW620 cells (P < 0.001). ChIP assay showed that NKD1 protein was capable of binding to the YWHAE promoter sequence; dual luciferase reporter gene assay showed that NKD1 overexpression (or knockdown) in the colon cancer cells significantly enhanced (or reduced) the transcriptional activity of YWHAE promoter (P < 0.05). Immunofluorescence assay demonstrated the binding of NKD1 and YWHAE proteins in colon cancer cells. NKD1 knockout significantly reduced glucose uptake in colon cancer cells (P < 0.01), while YWHAE overexpression restored the glucose uptake in NKD1-knockout cells (P < 0.05).@*CONCLUSION@#NKD1 protein activates the transcriptional activity of YWHAE gene to promote glucose uptake in colon cancer cells.


Subject(s)
Humans , Colonic Neoplasms , HCT116 Cells , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , RNA, Messenger , Glucose , Calcium-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , 14-3-3 Proteins/metabolism
14.
Article in Chinese | WPRIM | ID: wpr-986962

ABSTRACT

OBJECTIVE@#To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells.@*METHODS@#Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated.@*RESULTS@#Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing.@*CONCLUSION@#RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.


Subject(s)
Humans , rab GTP-Binding Proteins/metabolism , Triple Negative Breast Neoplasms , Cell Line, Tumor , rab27 GTP-Binding Proteins/metabolism , RNA, Small Interfering/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic
15.
Article in Chinese | WPRIM | ID: wpr-986961

ABSTRACT

OBJECTIVE@#To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes (FLSs) from patients with in rheumatoid arthritis (RA) and explore the mechanism.@*METHODS@#The inhibitory effect of 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method. Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine (30 μmol/L) on apoptosis of 25 ng/mL TNF-α- induced RA-FLSs, and Western blotting was performed to detect the changes in the expression levels of autophagy- and apoptosis-related proteins. The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B. RA-FLSs were treated with the reactive oxygen species (ROS) mimic H2O2 or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed.@*RESULTS@#The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time- and concentration-dependent manner. Flow cytometry and JC-1 staining showed that berberine (30 μmol/L) significantly increased apoptosis rate (P < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs (P < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax (P < 0.05) and LC3B-II/I (P < 0.01) and increased the expression of p62 protein in the cells (P < 0.05). Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs. Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR (P < 0.01); this effect was regulated by ROS level, and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs (P < 0.01).@*CONCLUSION@#Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.


Subject(s)
Humans , Synoviocytes , Berberine/metabolism , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/metabolism , Hydrogen Peroxide/metabolism , Sincalide/metabolism , Cell Proliferation , Arthritis, Rheumatoid/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Apoptosis , Fibroblasts , Autophagy , Cells, Cultured
16.
Article in Chinese | WPRIM | ID: wpr-986960

ABSTRACT

OBJECTIVE@#To analyze the expression of hydroxysteroid dehydrogenase like 2 (HSDL2) in rectal cancer tissues and the effect of changes in HSDL2 expression level on proliferation of rectal cancer cells.@*METHODS@#Clinical data and tissue samples of 90 patients with rectal cancer admitted to our hospital from January 2020 to June 2022 were collected from the prospective clinical database and biological specimen database. The expression level of HSDL2 in rectal cancer and adjacent tissues was detected by immunohistochemistry, and based on the median level of HSDL2 expression, the patients were divided into high expression group (n=45) and low expression group (n=45) for analysis the correlation between HSDL2 expression level and the clinicopathological parameters. GO and KEGG enrichment analyses were performed to explore the role of HSDL2 in rectal cancer progression. The effects of changes in HSDL2 expression levels on rectal cancer cell proliferation, cell cycle and protein expressions were investigated in SW480 cells with lentivirus-mediated HSDL2 silencing or HSDL2 overexpression using CCK-8 assay, flow cytometry and Western blotting.@*RESULTS@#The expressions of HSDL2 and Ki67 were significantly higher in rectal cancer tissues than in the adjacent tissues (P < 0.05). Spearman correlation analysis showed that the expression of HSDL2 protein was positively correlated with Ki67, CEA and CA19-9 expressions (P < 0.01). The rectal cancer patients with high HSDL2 expressions had significantly higher likelihood of having CEA ≥5 μg/L, CA19-9 ≥37 kU/L, T3-4 stage, and N2-3 stage than those with a low HSDL2 expression (P < 0.05). GO and KEGG analysis showed that HSDL2 was mainly enriched in DNA replication and cell cycle. In SW480 cells, HSDL2 overexpression significantly promoted cell proliferation, increased cell percentage in S phase, and enhanced the expression levels of CDK6 and cyclinD1 (P < 0.05), and HSDL2 silencing produced the opposite effects (P < 0.05).@*CONCLUSION@#The high expression of HSDL2 in rectal cancer participates in malignant progression of the tumor by promoting the proliferation and cell cycle progress of the cancer cells.


Subject(s)
Humans , CA-19-9 Antigen , Ki-67 Antigen/metabolism , Prospective Studies , Cell Line, Tumor , Cell Proliferation/genetics , Rectal Neoplasms/genetics , Cell Cycle , Gene Expression Regulation, Neoplastic , Hydroxysteroid Dehydrogenases/metabolism
17.
Article in Chinese | WPRIM | ID: wpr-986959

ABSTRACT

OBJECTIVE@#To investigate the expression of microRNA miR-431-5p in gastric cancer (GC) tissues and its effects on apoptosis and mitochondrial function in GC cells.@*METHODS@#The expression level of miR-431-5p in 50 clinical samples of GC tissues and paired adjacent tissues was detected using real-time fluorescence quantitative PCR, and its correlation with the clinicopathological features of the patients was analyzed. A cultured human GC cell line (MKN-45 cells) were transfected with a miR-431-5p mimic or a negative control sequence, and the cell proliferation, apoptosis, mitochondrial number, mitochondrial potential, mitochondrial permeability transition pore (mPTP), reactive oxygen species (ROS) production and adenosine triphosphate (ATP) content were detected using CCK-8 assay, flow cytometry, fluorescent probe label, or ATP detection kit. The changes in the expression levels of the apoptotic proteins in the cells were detected with Western blotting.@*RESULTS@#The expression level of miR-431-5p was significantly lower in GC tissues than in the adjacent tissues (P < 0.001) and was significantly correlated with tumor differentiation (P=0.0227), T stage (P=0.0184), N stage (P=0.0005), TNM stage (P=0.0414) and vascular invasion (P=0.0107). In MKN-45 cells, overexpression of miR-431-5p obviously inhibited cell proliferation and induced cell apoptosis, causing also mitochondrial function impairment as shown by reduced mitochondrial number, lowered mitochondrial potential, increased mPTP opening, increased ROS production and reduced ATP content. Overexpression of miR-431-5p significantly downregulated the expression of Bcl-2 and increased the expressions of pro-apoptotic proteins p53, Bcl-2 and cleaved caspase-3 protein.@*CONCLUSION@#The expression of miR-431-5p is down-regulated in GC, which results in mitochondrial function impairment and promotes cell apoptosis by activating the Bax/Bcl-2/caspase3 signaling pathway, suggesting the potential role of miR-431-5p in targeted therapy for GC.


Subject(s)
Humans , Apoptosis/genetics , bcl-2-Associated X Protein , Caspase 3 , Cell Line, Tumor , Cell Proliferation/genetics , MicroRNAs/metabolism , Mitochondria/metabolism , Mitochondrial Permeability Transition Pore , Reactive Oxygen Species , Stomach Neoplasms/pathology
18.
Article in Chinese | WPRIM | ID: wpr-986958

ABSTRACT

OBJECTIVE@#To investigate the role of myosin heavy chain 9 (MYH9) in regulation of cell proliferation, apoptosis, and cisplatin sensitivity of non-small cell lung cancer (NSCLC).@*METHODS@#Six NSCLC cell lines (A549, H1299, H1975, SPCA1, H322, and H460) and a normal bronchial epithelial cell line (16HBE) were examined for MYH9 expression using Western blotting. Immunohistochemical staining was used to detect MYH9 expression in a tissue microarray containing 49 NSCLC and 43 adjacent tissue specimens. MYH9 knockout cell models were established in H1299 and H1975 cells using CRISPR/Cas9 technology, and the changes in cell proliferation cell were assessed using cell counting kit-8 (CCK8) and clone formation assays; Western blotting and flow cytometry were used to detect apoptosis of the cell models, and cisplatin sensitivity of the cells was evaluated using IC50 assay. The growth of tumor xenografts derived from NSCLC with or without MYH9 knockout was observed in nude mice.@*RESULTS@#MYH9 expression was significantly upregulated in NSCLC (P < 0.001), and the patients with high MYH9 expression had a significantly shorter survival time (P=0.023). In cultured NSCLC cells, MYH9 knockout obviously inhibited cell proliferation (P < 0.001), promoted cell apoptosis (P < 0.05), and increased their chemosensitivity of cisplatin. In the tumor-bearing mouse models, the NSCLC cells with MYH9 knockout showed a significantly lower growth rate (P < 0.05). Western blotting showed that MYH9 knockout inactivated the AKT/c- Myc axis (P < 0.05) to inhibit the expression of BCL2- like protein 1 (P < 0.05), promoted the expression of BH3- interacting domain death agonist and the apoptosis regulator BAX (P < 0.05), and activated apoptosis-related proteins caspase-3 and caspase-9 (P < 0.05).@*CONCLUSION@#High expression of MYH9 contributes to NSCLC progression by inhibiting cell apoptosis via activating the AKT/c-Myc axis.


Subject(s)
Animals , Humans , Mice , Apoptosis , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Proliferation , Cisplatin/pharmacology , Cytoskeletal Proteins/metabolism , Lung Neoplasms/metabolism , Mice, Nude , Myosin Heavy Chains/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction
19.
Article in Chinese | WPRIM | ID: wpr-986842

ABSTRACT

OBJECTIVE@#To identify and characterize read-through RNAs and read-through circular RNAs (rt-circ-HS) derived from transcriptional read-through hypoxia inducible factor 1α (HIF1α) and small nuclear RNA activating complex polypeptide 1 (SNAPC1) the two adjacent genes located on chromosome 14q23, in renal carcinoma cells and renal carcinoma tissues, and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α.@*METHODS@#Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells. Tissue microarrays of 437 different types of renal cell carcinoma (RCC) were constructed, and chromogenic in situ hybridization (ISH) was used to investigate expression of rt-circ-HS in different RCC types. Small interference RNA (siRNA) and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation, migration and invasiveness by cell counting kit 8 (CCK8), EdU incorporation and Transwell cell migration and invasion assays. RT-PCR and Western blot were used to exa-mine expression of HIF1α and SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA, respectively. The binding of rt-circ-HS with microRNA 539 (miR-539), and miR-539 with HIF1α 3' untranslated region (3' UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays.@*RESULTS@#We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 2-6 and SNAPC1 exon 2-4. Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells. ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5% (295/437), and the expression was different in different types of RCCs. Mechanistically, rt-circ-HS promoted renal carcinoma cell proliferation, migration and invasiveness by functioning as a competitive endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α.@*CONCLUSION@#The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.


Subject(s)
Humans , Carcinoma, Renal Cell/pathology , Cell Proliferation , Hypoxia , Kidney Neoplasms , MicroRNAs/genetics , Neoplasm Invasiveness/genetics , RNA, Circular/metabolism , RNA, Small Interfering , Hypoxia-Inducible Factor 1, alpha Subunit/genetics
20.
Chinese Journal of Hepatology ; (12): 408-414, 2023.
Article in Chinese | WPRIM | ID: wpr-986144

ABSTRACT

Objective: To investigate the effect of 1-acyl-sn-glycerol-3-phosphate acyltransferaseδ (APGAT4) on the growth and lenvatinib resistance of hepatocellular carcinoma (HCC), and provide novel targets for HCC treatment. Methods: Using the bioinformatics methods to screen out upregulated genes in lenvatinib resistant cell lines from GEO dataset and survival related genes from TCGA dataset. Immumohistochemical staining was used to detect the expression AGPAT4 in HCC tissues, and its correlation with patients' survival. CCK8, EdU, cell cycle, and cell apoptosis assays were used to investigate the impact of role AGPAT4 on the proliferation and lenvatinib reistance of HCC cells. AGPAT4 stable knockdown cell line and subcutaneous nude mouse model were established to test the therapeutic effects of Lenvatinib. Analysis of variance was used to compare the differences between data sets. Results: APGAT4 was the common factor that predicted poor survival and Lenvatinib resistance. The mRNA and protein levels of APGAT4 were significantly upregulated in HCC tissues compared to the para-tumor tissues (P < 0.05). Using siRNA could significantly knocked down the mRNA and protein expression of APGAT4 in HCC cell lines Hep3B and HCCLM3. Compared with the control group, the proliferation ability of HCC cell lines (Hep3B and HCCLM3) in APGAT4 knockdown group was significantly inhibited, and the cell cycle was arrested in G2/M phase (P < 0.05). In addition, compared to the control group, HCC cell lines (Hep3B and HCCLM3) in APGAT4 knockdown group showed significant decrease in the Lenvatinib half maximal inhibitory concentration, and were more sensitive to lenvatinib-induced apoptosis (P < 0.05). In HCC subcutaneous nude mouse model, compared to the control group, the growth of tumor in APGAT4 knockdown group was significantly suppressed, and more apoptosis cells were induced (P < 0.05). Conclusion: APGAT4 promotes the growth and Lenvatinib resistance of HCC, which is a potential target for HCC treatment. Targeting APGAT4 treatment is conducive to inhibit the growth and Lenvatinib resistance of HCC.


Subject(s)
Animals , Mice , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Mice, Nude , Cell Line, Tumor , Cell Proliferation , RNA, Messenger , Gene Expression Regulation, Neoplastic
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