Overexpression SPRY2 Suppresses Esophageal Squamous Cell Carcinoma Progression Via Inactivation of ERK/AKT Signaling / La Sobreexpresión SPRY2 Suprime la Evolución del Carcinoma de Células Escamosas de Esófago Mediante la Inactivación de la Señalización ERK/AKT

SUMMARY: Esophageal cancer is one of the most aggressive gastrointestinal cancers. Invasion and metastasis are the main causes of poor prognosis of esophageal cancer. SPRY2 has been reported to exert promoting effects in human cancers, which controls signal pathways including PI3K/AKT and MAPKs. However, the expression of SPRY2 in esophageal squamous cell carcinoma (ESCC) and its underlying mechanism remain unclear. In the present study, we aimed to investigate the detailed role of SPRY2 in the regulation of cell proliferation, invasion and ERK/AKT signaling pathway in ESCC. It was identified that the expression level of SPRY2 in ESCC was remarkably decreased compared with normal tissues, and it was related to clinicopathologic features and prognosis ESCC patients. The upregulation of SPRY2 expression notably inhibited the proliferation, migration and invasion of Eca-109 cells. In addition, the activity of ERK /AKT signaling was also suppressed by the SPRY2 upregulation in Eca-109 cells. Our study suggests that overexpression of SPRY2 suppress cancer cell proliferation and invasion of by through suppression of the ERK/AKT signaling pathways in ESCC. Therefore, SPRY2 may be a promising prognostic marker and therapeutic target for ESCC.

El cáncer de esófago es uno de los cánceres gastrointestinales más agresivos. La invasión y la metástasis son las principales causas de mal pronóstico del cáncer de esófago. Se ha informado que SPRY2 ejerce efectos promotores en los cánceres humanos, que controla las vías de señales, incluidas PI3K/AKT y MAPK. Sin embargo, la expresión de SPRY2 en el carcinoma de células escamosas de esófago (ESCC) y su mecanismo subyacente aún no están claros. En el presente estudio, nuestro objetivo fue investigar el papel detallado de SPRY2 en la regulación de la proliferación celular, la invasión y la vía de señalización ERK/AKT en ESCC. Se identificó que el nivel de expresión de SPRY2 en ESCC estaba notablemente disminuido en comparación con los tejidos normales, y estaba relacionado con las características clínico-patológicas y el pronóstico de los pacientes con ESCC. La regulación positiva de la expresión de SPRY2 inhibió notablemente la proliferación, migración e invasión de células Eca-109. Además, la actividad de la señalización de ERK/AKT también fue suprimida por la regulación positiva de SPRY2 en las células Eca-109. Nuestro estudio sugiere que la sobreexpresión de SPRY2 suprime la proliferación y la invasión de células cancerosas mediante la supresión de las vías de señalización ERK/AKT en ESCC. Por lo tanto, SPRY2 puede ser un marcador de pronóstico prometedor y un objetivo terapéutico para la ESCC.

Cell softness reveals tumorigenic potential via ITGB8/AKT/glycolysis signaling in a mice model of orthotopic bladder cancer / 中华医学杂志(英文版)

BACKGROUND@#Bladder cancer, characterized by a high potential of tumor recurrence, has high lifelong monitoring and treatment costs. To date, tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types. Nonetheless, the existence of soft tumor cells in bladder tumors remains elusive. Thus, our study aimed to develop a micro-barrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells.@*METHODS@#The stiffness of bladder cancer cells was determined by atomic force microscopy (AFM). The modified microfluidic chip was utilized to separate soft cells, and the 3D Matrigel culture system was to maintain the softness of tumor cells. Expression patterns of integrin β8 (ITGB8), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were determined by Western blotting. Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59 (TRIM59). The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models.@*RESULTS@#Using our newly designed microfluidic approach, we identified a small fraction of soft tumor cells in bladder cancer cells. More importantly, the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens, in which the number of soft tumor cells was associated with tumor relapse. Furthermore, we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells. Simultaneously, we detected a remarkable up-regulation in ITGB8, TRIM59, and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts.@*CONCLUSIONS@#The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness. Meanwhile, the soft tumor cells become more sensitive to chemotherapy after stiffening, that offers new insights for hampering tumor progression and recurrence.

MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1 / 国际口腔科学杂志·英文版

Mesenchymal stem cell (MSC)-based therapy has emerged as a promising treatment for spinal cord injury (SCI), but improving the neurogenic potential of MSCs remains a challenge. Mixed lineage leukemia 1 (MLL1), an H3K4me3 methyltransferases, plays a critical role in regulating lineage-specific gene expression and influences neurogenesis. In this study, we investigated the role and mechanism of MLL1 in the neurogenesis of stem cells from apical papilla (SCAPs). We examined the expression of neural markers, and the nerve repair and regeneration ability of SCAPs using dynamic changes in neuron-like cells, immunofluorescence staining, and a SCI model. We employed a coimmunoprecipitation (Co-IP) assay, real-time RT-PCR, microarray analysis, and chromatin immunoprecipitation (ChIP) assay to investigate the molecular mechanism. The results showed that MLL1 knock-down increased the expression of neural markers, including neurogenic differentiation factor (NeuroD), neural cell adhesion molecule (NCAM), tyrosine hydroxylase (TH), βIII-tubulin and Nestin, and promoted neuron-like cell formation in SCAPs. In vivo, a transplantation experiment showed that depletion of MLL 1 in SCAPs can restore motor function in a rat SCI model. MLL1 can combine with WD repeat domain 5 (WDR5) and WDR5 inhibit the expression of neural markers in SCAPs. MLL1 regulates Hairy and enhancer of split 1 (HES1) expression by directly binds to HES1 promoters via regulating H3K4me3 methylation by interacting with WDR5. Additionally, HES1 enhances the expression of neural markers in SCAPs. Our findings demonstrate that MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1. These results provide a potential therapeutic target for promoting the recovery of motor function in SCI patients.

Genetic diagnosis of Branchio-Oto syndrome pedigree due to a de novo heterozygous deletion of EYA1 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a Chinese pedigree affected with Branchio-Oto syndrome (BOS).@*METHODS@#A pedigree with BOS which had presented at the Genetics and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University in May 2021 was selected as the study subject. Clinical data of the pedigree was collected. Peripheral blood samples of the proband and her parents were collected. Whole exome sequencing (WES) was carried out for the proband. Multiplex ligation-dependent probe amplification (MLPA) was used to verify the result of WES, short tandem repeat (STR) analysis was used to verify the relationship between the proband and her parents, and the pathogenicity of the candidate variant was analyzed.@*RESULTS@#The proband, a 6-year-old girl, had manifested severe congenital deafness, along with inner ear malformation and bilateral branchial fistulae. WES revealed that she has harbored a heterozygous deletion of 2 466 kb at chromosome 8q13.3, which encompassed the EYA1 gene. MLPA confirmed that all of the 18 exons of the EYA1 gene were lost, and neither of her parents has carried the same deletion variant. STR analysis supported that both of her parents are biological parents. Based on the guidelines from the American College of Medical Genetics and Genomics, the deletion was classified as pathogenic (PVS1+PS2+PM2_Supporting+PP4).@*CONCLUSION@#The heterozygous deletion of EYA1 gene probably underlay the pathogenicity of BOS in the proband, which has provided a basis for the clinical diagnosis.

Analysis of Significant Genes and Pathways in Esophageal Cancer Based on Gene Expression Omnibus Database / 中国医学科学杂志(英文版)

Objective To screen antigen targets for immunotherapy by analyzing over-expressed genes, and to identify significant pathways and molecular mechanisms in esophageal cancer by using bioinformatic methods such as enrichment analysis, protein-protein interaction (PPI) network, and survival analysis based on the Gene Expression Omnibus (GEO) database.Methods By screening with highly expressed genes, we mainly analyzed proteins MUC13 and EPCAM with transmembrane domain and antigen epitope from TMHMM and IEDB websites. Significant genes and pathways associated with the pathogenesis of esophageal cancer were identified using enrichment analysis, PPI network, and survival analysis. Several software and platforms including Prism 8, R language, Cytoscape, DAVID, STRING, and GEPIA platform were used in the search and/or figure creation.Results Genes MUC13 and EPCAM were over-expressed with several antigen epitopes in esophageal squamous cell carcinoma (ESCC) tissue. Enrichment analysis revealed that the process of keratinization was focused and a series of genes were related with the development of esophageal cancer. Four genes including ALDH3A1, C2, SLC6A1,and ZBTB7C were screened with significant P value of survival curve.Conclusions Genes MUC13 and EPCAM may be promising antigen targets or biomarkers for esophageal cancer. Keratinization may greatly impact the pathogenesis of esophageal cancer. Genes ALDH3A1, C2, SLC6A1,and ZBTB7C may play important roles in the development of esophageal cancer.

Genetic research progress in branchio-oto syndrome/ branchio-oto-renal syndrome / 中南大学学报(医学版)

Branchio-oto syndrome (BOS)/branchio-oto-renal syndrome (BORS) is a kind of autosomal dominant heterogeneous disorder. These diseases are mainly characterized by hearing impairment and abnormal phenotype of ears, accompanied by renal malformation and branchial cleft anomalies including cyst or fistula, with an incidence of 1/40 000 in human population. Otic anormalies are one of the most obvious clinical manifestations of BOS/BORS, including deformities of external, middle, inner ears and hearing loss with conductive, sensorineural or mix, ranging from mild to profound loss. Temporal bone imaging could assist in the diagnosis of middle ear and inner ear malformations for clinicians. Multiple methods including direct sequencing combined with next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), or array-based comparative genomic hybridization (aCGH) can effectively screen and identify pathogenic genes and/or variation types of BOS/BORS. About 40% of patients with BOS/BORS carry aberrations of EYA1 gene which is the most important cause of BOS/BORS. A total of 240 kinds of pathogenic variations of EYA1 have been reported in different populations so far, including frameshift, nonsense, missense, aberrant splicing, deletion and complex rearrangements. Human Endogenous Retroviral sequences (HERVs) may play an important role in mediating EYA1 chromosomal fragment deletion mutations caused by non-allelic homologous recombination. EYA1 encodes a phosphatase-transactivator cooperated with transcription factors of SIX1, participates in cranial sensory neurogenesis and development of branchial arch-derived organs, then regulates the morphological and functional differentiation of the outer ear, middle ear and inner ear toward normal tissues. In addition, pathogenic mutations of SIX1 and SIX5 genes can also cause BOS/BORS. Variations of these genes mentioned above may cause disease by destroying the bindings between SIX1-EYA1, SIX5-EYA1 or SIX1-DNA. However, the role of SIX5 gene in the pathogenesis of BORS needs further verification.

PRKCDBP Methylation is a Potential and Promising Candidate Biomarker for Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#The occurrence and development of lung cancer are closely linked to epigenetic modification. Abnormal DNA methylation in the CpG island region of genes has been found in many cancers. Protein kinase C delta binding protein (PRKCDBP) is a potential tumor suppressor and its epigenetic changes are found in many human malignancies. This study investigated the possibility of PRKCDBP methylation as a potential biomarker for non-small cell lung cancer (NSCLC).@*METHODS@#We measured the methylation levels of PRKCDBP in the three groups of NSCLC tissues. Promoter activity was measured by the dual luciferase assay, with 5'-aza-deoxycytidine to examine the effect of demethylation on the expression level of PRKCDBP.@*RESULTS@#The methylation levels of PRKCDBP in tumor tissues and 3 cm para-tumor were higher than those of distant (>10 cm) non-tumor tissues. Receiver operating characteristic (ROC) curve analysis between tumor tissues and distant non-tumor tissues showed that the area under the line (AUC) was 0.717. Dual luciferase experiment confirmed that the promoter region was able to promote gene expression. Meanwhile, in vitro methylation of the fragment (PRKCDBP_Me) could significantly reduce the promoter activity of the fragment. Demethylation of 5'-aza-deoxycytidine in lung cancer cell lines A549 and H1299 showed a significant up-regulation of PRKCDBP mRNA levels.@*CONCLUSIONS@#PRKCDBP methylation is a potential and promising candidate biomarker for non-small cell lung cancer.

Effects of Monoammonium Glycyrrhizinate on Stem Cell-like Characteristics, Oxidative Stress and Mitochondrial Function of Acute Promyelocytic Leukemia NB4 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of monoammonium glycyrrhizinate on the stem cell-like characteristics, oxidative stress and mitochondrial function of acute promyelocytic leukemia cells NB4.@*METHODS@#CCK-8 method was used to detect the viability of acute promyelocytic leukemia cells NB4, and the appropriate dose was screened; Cloning method was used to detect the proliferation rate of NB4 cell; Western blot was used to detect the expression of cell cycle-related protein; flow cytometry was used to detect cell apoptosis and sort NB4 stem cells positive (CD133+); Stem cell markers (Oct4, ABCG2, Dclk1) were detected by RT-PCR; ROS was detected by fluorescence; The kit was used to detect the level of oxidative stress markers (MDA); The flow cytometry was used to detect the change of mitochondrial membrane potential; Western blot was used to detect the expression of mitochondrial damage index-related proteins (Bax/BCL-2).@*RESULTS@#Compared with the control group, if the concentration of MAG was less than 5 μmol/L, the cell NB4 viability showed no significant difference; if the concentration was higher than 5 μmol/L, the inhibitory effect on the growth of cell NB4 increased and showed significant difference (P<0.05), according to the results of CCK-8 experiment, four groups were set based on the concentration of MAG 0 μmol/L, MAG 5 μmol/L, MAG 10 μmol/L, and MAG 20 μmol/L; compared with the control group (MAG 0 μmol/L), the cells in MAG 5 μmol/L group showed no significant difference, while the proliferation rate, cyclin expression, mitochondrial membrane potential, stem cell CD133+ ratio, and marker mRNA level ( Oct4, ABCG2, Dclk1) of NB4 cell were significantly reduced (P<0.05); the apoptosis rate, reactive oxygen species, MDA content and Bax/BCL-2 expression of NB4 cell significantly increased (P<0.05).@*CONCLUSION@#Monoammonium glycyrrhizinate has a significant inhibitory effect on acute promyelocytic leukemia cells NB4, which may be related to the regulation of stem cell-like characteristics, oxidative stress and mitochondrial function.

Genetic analysis of a Chinese pedigree affected with branchiootic syndrome due to a nonsense variant of EYA1 gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical phenotype and genetic basis for a Chinese pedigree suspected for branchiootic syndrome (BOS).@*METHODS@#The proband was subjected to target-capture high-throughput sequencing to detect potential variant of deafness-associated genes. Candidate variants were verified by Sanger sequencing of the family members.@*RESULTS@#The proband was found to harbor a c.1627C>T (p.Gln543Ter) nonsense variant of the EYA1 gene. Sanger sequencing confirmed that all of the 4 patients with the BOS phenotype from the pedigree have harbored the same heterozygous variant. Based on the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic (PVS1+PS+PP3+PP4).@*CONCLUSION@#The c.1627C>T (p.Gln543Ter) variant of the EYA1 gene probably underlay the BOS phenotype in this pedigree. Above finding has provided a basis for its clinical diagnosis.

Narcolepsy: the impact of aging, hypocretin deficiency, and years of formal education in olfactory function and abdominal obesity / Narcolepsia: o impacto da idade, deficiência de hipocretina e anos de estudo na função olfatória e na obesidade central

ABSTRACT Background: Narcolepsy is a disease resulting from the loss of hypocretin-producing cells or other dysfunctions of the hypocretinergic system. In addition to sleep disorders, affected patients may experience increased weight gain, olfactory changes, and poorer quality of life. Methods: This study aimed to investigate the relationship between narcolepsy and weight gain, years of study, sleep parameters, and olfactory dysfunction in patients with narcolepsy type 1 and narcolepsy type 2. Anthropometric, olfactory, socioeducational, and excessive daytime sleepiness evaluations were performed in 77 patients. Results: Greater weight gain and abdominal obesity were observed in patients with type 1 narcolepsy. Patients with higher education level had lower scores of daytime sleepiness, higher scores on the olfactory function test, and lower rates of abdominal obesity. Discussion: Patients with narcolepsy type 1 showed an increased body weight and abdominal obesity when compared to narcolepsy type 2. The patients with a higher schooling level showed a reduction of the daytime sleepiness scores, lower rates of abdominal obesity, and better scores on the olfactory function test. Conclusion: Among all the patients with narcolepsy, the data indicated that aging and hypocretin deficiency are associated with abdominal obesity, while years of study is the variable that mostly influences olfaction function.

RESUMO Antecedentes: A narcolepsia é resultante da perda de células produtoras de hipocretina ou da disfunção do sistema hipocretinérgico. Além dos distúrbios do sono característicos da doença, os pacientes afetados podem apresentar também aumento de peso, alterações olfatórias e pior qualidade de vida. Métodos: O objetivo do estudo é investigar a relação entre a narcolepsia e o ganho de peso, anos de estudo, parâmetros do sono e a disfunção olfatória em pacientes com narcolepsia tipo 1 e narcolepsia tipo 2. Foram realizadas avaliações antropométricas, do olfato, sociais, educacionais e da sonolência excessiva diurna nos 77 indivíduos participantes da pesquisa. Resultados: Foram observados, nos pacientes com narcolepsia tipo 1, maior ganho de peso e maior frequência de obesidade central. Pacientes com ensino superior apresentaram escores mais baixos de sonolência excessiva diurna, escores mais altos no teste de função olfatória e menores taxas de obesidade central. Discussão: Pacientes com narcolepsia tipo 1 apresentaram maior ganho de peso e obesidade central quando comparados aos com narcolepsia tipo 2. Os pacientes com maior escolaridade apresentaram menores escores de sonolência diurna, de obesidade central e melhores escores no teste da função olfatória. Conclusão: Nos indivíduos com narcolepsia tipo 1 e tipo 2, os dados indicaram que o envelhecimento e a deficiência de hipocretina estão associados à obesidade central, enquanto anos de estudo é a variável que mais influencia na função olfatória.

Apoplexy in sporadic pituitary adenomas: a single referral center experience and AIP mutation analysis

ABSTRACT Objective: To analyze the clinical, laboratory, and radiological findings and management of patients with clinical pituitary apoplexy and to screen for aryl hydrocarbon receptor-interacting protein (AIP) mutations. Subjects and methods: The clinical findings were collected from the medical records of consecutive sporadic pituitary adenoma patients with clinical apoplexy. Possible precipitating factors, laboratory data, magnetic resonance imaging (MRI) findings and treatment were also analyzed. Peripheral blood samples were obtained for DNA extraction from leukocytes, and the entire AIP coding region was sequenced. Results: Thirty-five patients with pituitary adenoma were included, and 23 (67%) had non-functioning pituitary adenomas. Headache was observed in 31 (89%) patients. No clear precipitating factor was identified. Hypopituitarism was observed in 14 (40%) patients. MRI from 20 patients was analyzed, and 10 (50%) maintained a hyperintense signal in MRI performed more than three weeks after pituitary apoplexy (PA). Surgery was performed in ten (28%) patients, and 25 (72%) were treated conservatively with good outcomes. No AIP mutation was found in this cohort. Conclusion: Patients with stable neuroophthalmological impairments can be treated conservatively if no significant visual loss is present. Our radiological findings suggest that hematoma absorption lasts more than that observed in other parts of the brain. Additionally, our study suggests no benefits of AIP mutation screening in sporadic patients with apoplexy.

Prognostic and clinicopathological value of zwint expression levels in patients with lung adenocarcinoma: a systematic review and meta-analysis

The current study found that high Zeste White 10 interactor (ZWINT) expression is related to the poor prognosis of patients with a variety of cancers. This study mainly explored the relationship between the expression level of ZWINT and the prognosis of patients with lung adenocarcinoma (LUAD). Briefly, four English databases and two high-throughput sequencing databases were searched and relevant data for meta-analysis were extracted. Pooled mean difference and 95% confidence interval (CI) were used to assess the relationships between clinical features and the expression of ZWINT. Pooled hazard ratio and 95% CI were also used to assess the relationships between clinical features and the expression level of ZWINT. This meta-analysis was registered in PROSPERO (CRD42021249475). A total of 16 high-quality datasets comprising 2,847 LUAD patients were included in this study. Higher ZWINT expression levels were found in patients younger than 65 years, males, and smokers, and were correlated with advanced TNM stages and poor prognosis. Notably, there was no publication bias in this meta-analysis. Overall, our findings indicate that ZWINT is a potential biomarker for poor prognosis and clinicopathological outcomes of patients with LUAD.

Identification and genetic analysis of new mutations in EYA1 gene of BOS syndrome / 中华耳鼻咽喉头颈外科杂志

Objective: To analyze the clinical manifestations of a patient with branchiootic syndrome(BOS) and her families and to carry out genetic testing in order to specify the biological pathogenesis. Methods: Clinical data of the patient and her families were collected. Genomic DNA in the peripheral blood of the proband and her family members was extracted. All exons of 406 deafness-related susceptible genes as well as their flanking regions were sequenced by high-throughput sequencing, and the mutation sites of the proband and her parents were validated by Sanger sequencing. Results: There were nine members in three generations, of whom four presented with hearing loss, preauricular fistula and branchial fistula which met the diagnostic criteria of BOS. Proband and her mother presented with auricle malformation and inner ear malformation. And no one had abnormalities in the kidneys of all the patients. Pedigree analysis revealed that the mode of inheritance in the family was consistent with the autosomal dominant pattern. Mutational analysis showed that all the affected patients detected a heterozygous frameshift variation c.1255delT in the EYA1 gene, which had not been reported. Genotype and phenotype were co-isolated in this family. Such a frameshift variation produced a premature termination codon, thereby causing premature termination of translation (p.C419VFS*12). ACMG identified that the mutation was pathogenic. This mutation was novel and not detected in controls. A heterozygous missense variation mutation c.403G>A(p.G135S) in EYA1 gene was also detected in three members of this family. ACMG identified that the mutation clinical significance was uncertain. However, two of whom were normal, which seemed the disease was not caused by this mutation in this family. Conclusions: A novel frameshift mutation in EYA1(c.1255delT) is the main molecular etiology of BOS in the Chinese family. This study expands the mutational spectrum of EYA1 gene. The clinical manifestations are heterogeneous among patients in this family. The diagnosis of BOS should combine gene tests with clinical phenotypes analysis.

Effect of the regulator of G-protein signaling 2 on the proliferation and invasion of oral squamous cell carcinoma cells and its molecular mechanism / 华西口腔医学杂志

OBJECTIVES@#This study aims to investigate the effect of the regulator of G-protein signaling 2 (RGS2) on the proliferation and invasion of oral squamous cell carcinoma (OSCC) cells and its potential molecular mechanism. Metho⁃ds The expression status and clinical significance of RGS2 in head and neck squamous cell carcinomas and matched adjacent normal tissues were evaluated using TCGA database. Three OSCC cell lines (i.e., SCC-9, Cal27, and Fadu) were overexpressed with RGS2, and the effect of RGS2 on cell proliferation and invasion was determined using the Transwell, clone formation, and cell counting kit (CCK)-8 assays. Moreover, the yeast two-hybrid scree-ning and co-immunoprecipitation (Co-IP) assays were conducted to detect the correlation of RGS2, four and a half LIM domains protein 1 (FHL1), and damage DNA-binding protein 1 (DDB1).@*RESULTS@#The expression level of RGS2 in OSCC was significantly lower than that in matched adjacent normal tissues (@*CONCLUSIONS@#RGS2 plays an important role in the inhibition of OSCC proliferation and invasion. The structure stability of RGS2 is competitively regulated by FHL1 and DDB1.

KIF2C: a novel link between Wnt/β-catenin and mTORC1 signaling in the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is the fourth-leading cause of cancer-related deaths worldwide. HCC is refractory to many standard cancer treatments and the prognosis is often poor, highlighting a pressing need to identify biomarkers of aggressiveness and potential targets for future treatments. Kinesin family member 2C (KIF2C) is reported to be highly expressed in several human tumors. Nevertheless, the molecular mechanisms underlying the role of KIF2C in tumor development and progression have not been investigated. In this study, we found that KIF2C expression was significantly upregulated in HCC, and that KIF2C up-regulation was associated with a poor prognosis. Utilizing both gain and loss of function assays, we showed that KIF2C promoted HCC cell proliferation, migration, invasion, and metastasis both in vitro and in vivo. Mechanistically, we identified TBC1D7 as a binding partner of KIF2C, and this interaction disrupts the formation of the TSC complex, resulting in the enhancement of mammalian target of rapamycin complex1 (mTORC1) signal transduction. Additionally, we found that KIF2C is a direct target of the Wnt/β-catenin pathway, and acts as a key factor in mediating the crosstalk between Wnt/β-catenin and mTORC1 signaling. Thus, the results of our study establish a link between Wnt/β-catenin and mTORC1 signaling, which highlights the potential of KIF2C as a therapeutic target for the treatment of HCC.

Xinfeng Capsules promotes apoptosis of synovial fibroblasts and attenuates inflammation in rheumatoid arthritis by regulating lncRNA MAPKAPK5-AS1 / 中国中药杂志

To explore the regulatory effects of Xinfeng Capsules(XFC) on the apoptosis of synovial fibroblasts(FLS) and inflammation in rheumatoid arthritis(RA) via lncRNA MAPKAPK5-AS1(MK5-AS1). Thirty healthy people and 30 patients with RA due to spleen deficiency and dampness exuberance were collected for extracting the peripheral blood mononuclear cells(PBMCs) before and after XFC treatment, which were used to observe the correlation between MK5-AS1 and clinical indicators as well as MK5-AS1 expression before and after XFC treatment. Following the establishment of RA-FLS cell line and the preparation of XFC-containing serum, MK5-AS1-overexpression plasmid was constructed and transfected into RA-FLS for investigating the efficacy of XFC-containing serum in regulating inflammation and apoptosis of RA-FLS via MK5-AS1. The expression of MK5-AS1 in PBMCs of patients with RA due to spleen deficiency and dampness exuberance was decreased(P<0.001). The ROC curve analysis revealed the AUC of 83.9%. Correlation analysis showed that MK5-AS1 was negatively correlated with ESR, CRP, RF, CCP, and spleen deficiency and dampness exuberance syndrome score. The expression of MK5-AS1 increased significantly after XFC treatment(P<0.001). As demonstrated by association analysis, XFC decreased MK5-AS1, ESR, CRP, RF, and spleen deficiency and dampness exuberance syndrome score, with the degree of support all greater than 83%, confidence greater than 80%, and lift greater than 1. The results of RT-qPCR showed that the MK5-AS1 RNA expression significantly decreased after TNF-α stimulation(P<0.01), which, however, increased significantly after the intervention with XFC-containing serum(P<0.05). Such expression rose again after the transfection of pcDNA3.1-MK5-AS1(P<0.01). ELISA results showed that TNF-α stimulation elevated the expression of pro-inflammatory factor IL-17 but lowered the expression of anti-inflammatory factor IL-4(P<0.01). After intervention with XFC-containing serum, the expression of IL-17 decreased while that of IL-4 increased(P<0.01). The transfection of pcDNA3.1-MK5-AS1 contributed to the reduction in IL-17 expression but the elevation in IL-4 expression(P<0.01). The immunofluorescence(IF) findings demonstrated that the expression of pro-apoptotic protein Bax was down-regulated, whereas that of the anti-apoptotic protein Bcl-2 was up-regulated after TNF-α stimulation(P<0.01). After the intervention with XFC-containing serum, the Bax expression was increased, while Bcl-2 expression was decreased(P<0.01), which were remarkably collaborated by the transfection of pcDNA3.1-MK5-AS1(P<0.05). The expression of MK5-AS1 is significantly decreased in both RA-PBMCs and RA-FLS, implying that XFC inhibits inflammatory reaction and promotes the apoptosis in RA by regulating the expression of MK5-AS1.

Two naturally derived small molecules disrupt the sineoculis homeobox homolog 1-eyes absent homolog 1 (SIX1-EYA1) interaction to inhibit colorectal cancer cell growth / 中华医学杂志(英文版)

BACKGROUND@#Emerging evidence indicates that the sineoculis homeobox homolog 1-eyes absent homolog 1 (SIX1-EYA1) transcriptional complex significantly contributes to the pathogenesis of multiple cancers by mediating the expression of genes involved in different biological processes, such as cell-cycle progression and metastasis. However, the roles of the SIX1-EYA1 transcriptional complex and its targets in colorectal cancer (CRC) are still being investigated. This study aimed to investigate the roles of SIX1-EYA1 in the pathogenesis of CRC, to screen inhibitors disrupting the SIX1-EYA1 interaction and to evaluate the efficiency of small molecules in the inhibition of CRC cell growth.@*METHODS@#Real-time quantitative polymerase chain reaction and western blotting were performed to examine gene and protein levels in CRC cells and clinical tissues (collected from CRC patients who underwent surgery in the Department of Integrated Traditional and Western Medicine, West China Hospital of Sichuan University, between 2016 and 2018, n = 24). In vivo immunoprecipitation and in vitro pulldown assays were carried out to determine SIX1-EYA1 interaction. Cell proliferation, cell survival, and cell invasion were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic assay, and Boyden chamber assay, respectively. The Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen) method was used to obtain small molecules that specifically disrupted SIX1-EYA1 interaction. CRC cells harboring different levels of SIX1/EYA1 were injected into nude mice to establish tumor xenografts, and small molecules were also injected into mice to evaluate their efficiency to inhibit tumor growth.@*RESULTS@#Both SIX1 and EYA1 were overexpressed in CRC cancerous tissues (for SIX1, 7.47 ± 3.54 vs.1.88 ± 0.35, t = 4.92, P = 0.008; for EYA1, 7.61 ± 2.03 vs. 2.22 ± 0.45, t = 6.73, P = 0.005). The SIX1/EYA1 complex could mediate the expression of two important genes including cyclin A1 (CCNA1) and transforming growth factor beta 1 (TGFB1) by binding to the myocyte enhancer factor 3 consensus. Knockdown of both SIX1 and EYA1 could decrease cell proliferation, cell invasion, tumor growth, and in vivo tumor growth (all P < 0.01). Two small molecules, NSC0191 and NSC0933, were obtained using AlphaScreen and they could significantly inhibit the SIX1-EYA1 interaction with a half-maximal inhibitory concentration (IC50) of 12.60 ± 1.15 μmol/L and 83.43 ± 7.24 μmol/L, respectively. Administration of these two compounds could significantly repress the expression of CCNA1 and TGFB1 and inhibit the growth of CRC cells in vitro and in vivo.@*CONCLUSIONS@#Overexpression of the SIX1/EYA1 complex transactivated the expression of CCNA1 and TGFB1, causing the pathogenesis of CRC. Pharmacological inhibition of the SIX1-EYA1 interaction with NSC0191 and NSC0933 significantly inhibited CRC cell growth by affecting cell-cycle progression and metastasis.

Association of serum levels of secreted frizzled-related protein 5 and Wnt member 5a with glomerular filtration rate in patients with type 2 diabetes mellitus and chronic renal disease: a cross-sectional study

ABSTRACT BACKGROUND: Diabetic nephropathy is a common complication of chronic kidney disease (CKD). ­Inflammation in the kidneys is crucial for promoting development and progression of this complication. Wnt member 5a (Wnt5a) and secreted frizzled-related protein 5 (Sfrp5) are proinflammatory proteins associated with insulin resistance and chronic low-grade adipose tissue inflammation. OBJECTIVE: To determine the correlation between serum Sfrp5 and Wnt5a concentrations and glomerular filtration rate in patients with type 2 diabetes mellitus and CKD. DESIGN AND SETTING: Cross-sectional, comparative and observational study in the Department of Endocrinology, Civil Hospital, Culiacán, Sinaloa, Mexico. METHODS: Eighty individuals with chronic kidney disease were recruited. Their serum Sfrp5 and Wnt5a concentrations were quantified using the enzyme-linked immunosorbent assay (ELISA) test. The statistical analysis consisted of the Mann-Whitney U test for independent samples and Spearman correlation, with statistical significance of P < 0.05. RESULTS: Serum Sfrp5 concentration continually increased through the stages of CKD progression, whereas serum Wnt5a concentration presented its highest levels in stage 3 CKD. Negative correlations between estimated glomerular filtration rate (eGFR) and serum concentrations of Sfrp5 (r = -0434, P = 0.001) and Wnt5a (r = -0481, P = 0.001) were found. CONCLUSIONS: There were negative correlations between serum Sfrp5 and Wnt5a concentrations and eGFR at each stage of CKD, with higher levels in female patients. This phenomenon suggests that Sfrp5 and Wnt5a might be involved in development and evolution towards end-stage renal disease.

Spinal protein kinase A and phosphorylated extracellular signal-regulated kinase signaling are involved in the antinociceptive effect of phytohormone abscisic acid in rats / A proteína quinase A da medula espinhal e a sinalização da quinase fosforilada regulada por sinal extracelular estão envolvidas no efeito antinociceptivo do ácido fito-hormônio abscísico em ratos

Abstract Objective: The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats. Methods: The animals were cannulated intrathecally and divided into different experimental groups (n=6‒7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting. Results: Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group. Conclusions: Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.

Resumo Objetivo: O ácido fito-hormônio abscísico (ABA) existe como molécula sinalizadora em vários tipos de organismos, de multicelulares a células e tecidos animais. Foi demonstrado que o ABA tem efeito antinociceptivo em roedores. O presente estudo foi desenhado para avaliar o possível papel da PKA e da ERK fosforilada (p-ERK) nos efeitos antinociceptivos do ABA intratecal (i.t.) em ratos Wistar machos. Métodos: Os animais foram canulados por via i.t. e divididos em diferentes grupos experimentais (n=6‒7): controle (sem cirurgia), veículo (veículo ABA recebido), grupos tratados com ABA (recebeu ABA em doses de 10 ou 20 µg/rato), grupo tratado com ABA mais H.89 (inibidor de PKA) que recebeu o inibidor 15 minutos antes da injeção de ABA. Os testes de movimento da cauda e placa quente foram utilizados como estimuladores nociceptivos agudos para avaliar os efeitos analgésicos da ABA. A p-ERK foi avaliada na porção dorsal da medula espinhal por imunotransferência. Resultados: A microinjeção de ABA (10 e 20 µg/rato, i.t.) aumentou significativamente o limiar nociceptivo nos testes de movimento da cauda e placa quente. A aplicação de inibidor de PKA (H.89, 100 nM/rato) inibiu significativamente os efeitos analgésicos induzidos por ABA. A expressão de p-ERK diminuiu significativamente em animais injetados com ABA que não foram observados no grupo tratado com ABA+H.89. Conclusões: No geral, a administração i.t. de ABA (10 µg/rato) induziu a analgesia e expressão negativa de p-ERK provavelmente envolvendo mecanismo dependente de PKA.

RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.