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.

Study on gene therapy for DPOAE and ABR threshold changes in adult Otof-/- mice / 临床耳鼻咽喉头颈外科杂志

Objective:This study aims to analyze the threshold changes in distortion product otoacoustic emissions（DPOAE） and auditory brainstem response（ABR） in adult Otof-/- mice before and after gene therapy, evaluating its effectiveness and exploring methods for assessing hearing recovery post-treatment. Methods:At the age of 4 weeks, adult Otof-/- mice received an inner ear injection of a therapeutic agent containing intein-mediated recombination of the OTOF gene, delivered via dual AAV vectors through the round window membrane（RWM）. Immunofluorescence staining assessed the proportion of inner ear hair cells with restored otoferlin expression and the number of synapses.Statistical analysis was performed to compare the DPOAE and ABR thresholds before and after the treatment. Results:AAV-PHP. eB demonstrates high transduction efficiency in inner ear hair cells. The therapeutic regimen corrected hearing loss in adult Otof-/- mice without impacting auditory function in wild-type mice. The changes in DPOAE and ABR thresholds after gene therapy are significantly correlated at 16 kHz. Post-treatment,a slight increase in DPOAE was observeds,followed by a recovery trend at 2 months post-treatment. Conclusion:Gene therapy significantly restored hearing in adult Otof-/- mice, though the surgical delivery may cause transient hearing damage. Precise and gentle surgical techniques are essential to maximize gene therapy's efficacy.

Early occurrence of primary angle-closure glaucoma in a patient with retinitis pigmentosa and CRB1 gene variations / Manifestação precoce de glaucoma de ângulo fechado em paciente com retinopatia por mutação no gene CRB1

ABSTRACT We describe the case of a 15-year-old girl with decreased visual acuity associated with elevated intraocular pressure in both eyes and angle closure on gonioscopy. She also presented attenuation of retinal vessels and optic disc pallor with large excavation in the left eye. Ultrasound biomicroscopy revealed an anteriorly positioned ciliary body and absence of ciliary sulcus, confirming the plateau iris configuration. Spectral-domain optical coherence tomography revealed a bilateral cystoid macular edema. Genetic screening revealed heterozygous variants of the Crumbs homolog 1 (CRB1) gene (c.2843G>A and c.2506C>A). The patient underwent trabeculectomy for intraocular pressure control and topical treatment for macular edema. This case highlights the importance of performing gonioscopy and evaluating intraocular pressure in patients with a shallow anterior chamber despite young age. In addition, it also shows the importance of genetic screening, when available, in elucidating the diagnosis and providing patients and their families' information on the patient's prognosis and possible therapeutic options.

RESUMO Nós descrevemos um caso de uma paciente de 15 anos com queda de acuidade visual e aumento da pressão intraocular em ambos os olhos, juntamente com fechamento angular no exame de gonioscopia. Na fundoscopia a paciente apresentava atenuação dos vasos retinianos, palidez de disco e aumento de escavação em olho esquerdo. Ao exame da biomicroscopia ultrassônica, foi evidenciado corpo ciliar anteriorizado e ausência de sulco ciliar em ambos os olhos, relevando presença de íris em plateau. Ao exame de tomografia de coerência óptica, visualizamos presença de edema macular cistoide bilateral. O screening genético revelou heterozigose no gene CRB1 (c.2843G>A and c.2506C>A), confirmando o diagnóstico de retinose pigmentar. Este caso reforça a importância do exame de gonioscopia e da avaliação da pressão intraocular em pacientes em câmara rasa, mesmo em pacientes jovens. Além disso, mostra a importância do screening genético como ferramenta útil para elucidação diagnóstica.

Phase separation in cGAS-STING signaling / 医学前沿

Biomolecular condensates formed by phase separation are widespread and play critical roles in many physiological and pathological processes. cGAS-STING signaling functions to detect aberrant DNA signals to initiate anti-infection defense and antitumor immunity. At the same time, cGAS-STING signaling must be carefully regulated to maintain immune homeostasis. Interestingly, exciting recent studies have reported that biomolecular phase separation exists and plays important roles in different steps of cGAS-STING signaling, including cGAS condensates, STING condensates, and IRF3 condensates. In addition, several intracellular and extracellular factors have been proposed to modulate the condensates in cGAS-STING signaling. These studies reveal novel activation and regulation mechanisms of cGAS-STING signaling and provide new opportunities for drug discovery. Here, we summarize recent advances in the phase separation of cGAS-STING signaling and the development of potential drugs targeting these innate immune condensates.

The I226R protein of African swine fever virus inhibits the cGAS-STING-mediated innate immune response / 生物工程学报

This study aimed to explore the mechanism of how African swine fever virus (ASFV) I226R protein inhibits the cGAS-STING signaling pathway. We observed that I226R protein (pI226R) significantly inhibited the cGAS-STING-mediated type Ⅰ interferons and the interferon-stimulated genes production by dual-luciferase reporter assay system and real-time quantitative PCR. The results of co-immunoprecipitation assay and confocal microscopy showed that pI226R interacted with cGAS. Furthermore, pI226R promoted cGAS degradation through autophagy-lysosome pathway. Moreover, we found that pI226R decreased the binding of cGAS to E3 ligase tripartite motif protein 56 (TRIM56), resulting in the weakened monoubiquitination of cGAS, thus inhibiting the activation of cGAS and cGAS-STING signaling. In conclusion, ASFV pI226R suppresses the antiviral innate immune response by antagonizing cGAS, which contributes to an in-depth understanding of the immune escape mechanism of ASFV and provides a theoretical basis for the development of vaccines.

Production and antigenicity analysis of a recombinant insulinoma associated protein-2 in HEK293 cells / 生物工程学报

Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.

An atlas of immune cell transcriptomes in human immunodeficiency virus-infected immunological non-responders identified marker genes that control viral replication / 中华医学杂志(英文版)

BACKGROUND@#Previous studies have examined the bulk transcriptome of peripheral blood immune cells in acquired immunodeficiency syndrome patients experiencing immunological non-responsiveness. This study aimed to investigate the characteristics of specific immune cell subtypes in acquired immunodeficiency syndrome patients who exhibit immunological non-responsiveness.@*METHODS@#A single-cell transcriptome sequencing of peripheral blood mononuclear cells obtained from both immunological responders (IRs) (CD4 + T-cell count >500) and immunological non-responders (INRs) (CD4 + T-cell count <300) was conducted. The transcriptomic profiles were used to identify distinct cell subpopulations, marker genes, and differentially expressed genes aiming to uncover potential genetic factors associated with immunological non-responsiveness.@*RESULTS@#Among the cellular subpopulations analyzed, the ratios of monocytes, CD16 + monocytes, and exhausted B cells demonstrated the most substantial differences between INRs and IRs, with fold changes of 39.79, 11.08, and 2.71, respectively. In contrast, the CD4 + T cell ratio was significantly decreased (0.39-fold change) in INRs compared with that in IRs. Similarly, the ratios of natural killer cells and terminal effector CD8 + T cells were also lower (0.37-fold and 0.27-fold, respectively) in the INRs group. In addition to several well-characterized immune cell-specific markers, we identified a set of 181 marker genes that were enriched in biological pathways associated with human immunodeficiency virus (HIV) replication. Notably, ISG15 , IFITM3 , PLSCR1 , HLA-DQB1 , CCL3L1 , and DDX5 , which have been demonstrated to influence HIV replication through their interaction with viral proteins, emerged as significant monocyte marker genes. Furthermore, the differentially expressed genes in natural killer cells were also enriched in biological pathways associated with HIV replication.@*CONCLUSIONS@#We generated an atlas of immune cell transcriptomes in HIV-infected IRs and INRs. Host genes associated with HIV replication were identified as markers of, and were found to be differentially expressed in, different types of immune cells.

ATAD3A gene variations in a family with Harel-Yoon syndrome / 浙江大学学报·医学版

An 11-day-old female neonate was admitted for cough with mouth foaming and feeding difficulties. The laboratory results indicated hyperlactatemia, elevated markers of myocardial injury and inflammation, and high levels of acylcarnitine octanoylcarnitine and decanoylcarnitine in tandem mass spectrometry. Ultrasonography and MRI suggested cardiac insufficiency and hypertrophic cardiomyopathy. Whole exome sequencing showed that both the proband and her elderly sister had a compound heterozygous variant of c.1492dup (p.T498Nfs*13) and c.1376T>C (p.F459S) in the ATAD3A gene, inherited from their father and mother, respectively. The diagnosis of Harel-Yoon syndrome was confirmed. The proband and her sister were born with clinical manifestations of metabolic acidosis, hyperlactatemia, feeding difficulties, elevated markers of myocardial injury as well as cardiac insufficiency, and both died in early infancy.

Analysis of a Chinese pedigree affected with Meckel syndrome due to variants of TMEM67 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic etiology for a Chinese pedigree affected with Meckel syndrome.@*METHODS@#A pedigree with a history of three consecutive adverse pregnancies which presented at the First Affiliated Hospital of Zhengzhou University on August 31, 2017 was selected as the study subject. Clinical data of the pedigree were collected. High-throughput sequencing was carried out to screen for variants of ciliopathy-related genes in the third fetus following induced abortion, and candidate variant was verified by Sanger sequencing.@*RESULTS@#The first pregnancy of the couple had ended as spontaneous abortion, whilst the fetus of the second pregnancy was suspected for having ciliopathy, though no genetic testing was carried out following elected abortion. The fetus of the third pregnancy was suspected for having ciliopathy, and high-throughput sequencing and Sanger sequencing had shown that the fetus had harbored compound heterozygous variants of the TMEM67 gene, including c.978+1G>A from the father and c.1288G>C (p.D430H) from the mother. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.978+1G>A was classified as a pathogenic variant (PVS1+PM2_Supporting+PP5), whilst the newly discovered c.1288G>C (p.D430H) was classified as a likely pathogenic variant (PM2_Supporting+PM3+PM5+PP3).@*CONCLUSION@#The c.978+1G>A and c.1288G>C (p.D430H) compound heterozygous variants of the TMEM67 gene probably underlay the three consecutive adverse pregnancies suspected for ciliopathy in this pedigree. The discovery of c.1288G>C (p.D430H) has also expanded the mutational spectrum of the TMEM67 gene.

Analysis of a patient with early-onset retinitis pigmentosa due to novel variants of CRB1 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a patient with early-onset retinitis pigmentosa (RP).@*METHODS@#A patient who had presented at the West China Hospital of Sichuan University on March 10, 2020 was selected as the study subject. The patient and his parents were subjected to whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing and in silico analysis.@*RESULTS@#The patient has featured substantial loss of binocular vision field. Funduscopy revealed characteristic bone spicule-type pigment deposits, as well as attenuated retinal arterioles and pale-appearing optic discs. WES revealed that he has harbored compound missense variants of a RP-associated CRB1 gene, including c.2969T>C (p.Leu990Ser) and c.1816T>C (p.Cys606Arg), which were respectively inherited from his father and mother. Homozygous c.1816T>C (p.Cys606Arg) variant has been identified among RP patients, whilst the c.2969T>C (p.Leu990Ser) variant was unreported previously. Both variants were predicted as likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG).@*CONCLUSION@#The novel compound heterozygous variants of the CRB1 gene probably underlay the early-onset RP in this patient. Above finding has enriched the mutational spectrum of the CRB1 gene.

Mechanism of Buyang Huanwu Decoction in protecting ischemic myocardium by regulating platelet autophagy in rats with acute myocardial infarction / 中国中药杂志

This study explored the effects of Buyang Huanwu Decoction(BYHWD) on platelet activation and differential gene expression after acute myocardial infarction(AMI). SD rats were randomly divided into a sham-operated group, a model group, a positive drug(aspirin) group, and a BYHWD group. Pre-treatment was conducted for 14 days with a daily oral dose of 1.6 g·kg~(-1) BYHWD and 0.1 g·kg~(-1) aspirin. The AMI model was established using the high ligation of the left anterior descending coronary artery method. The detection indicators included myocardial infarct size, heart function, myocardial tissue pathology, peripheral blood flow perfusion, platelet aggregation rate, platelet membrane glycoprotein CD62p expression, platelet transcriptomics, and differential gene expression. The results showed that compared with the sham-operated group, the model group showed reduced ejection fraction and cardiac output, decreased peripheral blood flow, and increased platelet aggregation rate and CD62p expression, and activated platelets. At the same time, TXB_2 content increased and 6-keto-PGF1α content decreased in serum. Compared with the model group, BYHWD increased ejection fraction and cardiac output, improved blood circulation in the foot and tail regions and cardiomyocytes arrangement, reduced myocardial infarct size and inflammatory infiltration, down-regulated platelet aggregation rate and CD62p expression, reduced serum TXB_2 content, and increased 6-keto-PGF1α content. Platelet transcriptome sequencing results revealed that BYHWD regulated mTOR-autophagy pathway-related genes in platelets. The differential gene expression levels were detected using real-time quantitative PCR. BYHWD up-regulated mTOR, down-regulated autophagy-related FUNDC1 and PINK genes, and up-regulated p62 gene expression. The results demonstrated that BYHWD could regulate platelet activation, improve blood circulation, and protect ischemic myocardium in AMI rats, and its mechanism is related to the regulation of the mTOR-autophagy pathway in platelets.

NDFIP1 limits cellular TAZ accumulation via exosomal sorting to inhibit NSCLC proliferation

NDFIP1 has been previously reported as a tumor suppressor in multiple solid tumors, but the function of NDFIP1 in NSCLC and the underlying mechanism are still unknown. Besides, the WW domain containing proteins can be recognized by NDFIP1, resulted in the loading of the target proteins into exosomes. However, whether WW domain-containing transcription regulator 1 (WWTR1, also known as TAZ) can be packaged into exosomes by NDFIP1 and if so, whether the release of this oncogenic protein via exosomes has an effect on tumor development has not been investigated to any extent. Here, we first found that NDFIP1 was low expressed in NSCLC samples and cell lines, which is associated with shorter OS. Then, we confirmed the interaction between TAZ and NDFIP1, and the existence of TAZ in exosomes, which requires NDFIP1. Critically, knockout of NDFIP1 led to TAZ accumulation with no change in its mRNA level and degradation rate. And the cellular TAZ level could be altered by exosome secretion. Furthermore, NDFIP1 inhibited proliferation in vitro and in vivo, and silencing TAZ eliminated the increase of proliferation caused by NDFIP1 knockout. Moreover, TAZ was negatively correlated with NDFIP1 in subcutaneous xenograft model and clinical samples, and the serum exosomal TAZ level was lower in NSCLC patients. In summary, our data uncover a new tumor suppressor, NDFIP1 in NSCLC, and a new exosome-related regulatory mechanism of TAZ.

Tinker, tailor, soldier, cell: the role of C-type lectins in the defense and promotion of disease

C-type lectins (CTLs) represent a large family of soluble and membrane-bound proteins which bind calcium dependently via carbohydrate recognition domains (CRDs) to glycan residues presented on the surface of a variety of pathogens. The deconvolution of a cell's glycan code by CTLs underpins several important physiological processes in mammals such as pathogen neutralization and opsonization, leukocyte trafficking, and the inflammatory response. However, as our knowledge of CTLs has developed it has become apparent that the role of this innate immune family of proteins can be double-edged, where some pathogens have developed approaches to subvert and exploit CTL interactions to promote infection and sustain the pathological state. Equally, CTL interactions with host glycoproteins can contribute to inflammatory diseases such as arthritis and cancer whereby, in certain contexts, they exacerbate inflammation and drive malignant progression. This review discusses the 'dual agent' roles of some of the major mammalian CTLs in both resolving and promoting infection, inflammation and inflammatory disease and highlights opportunities and emerging approaches for their therapeutic modulation.

Lnc-TMEM132D-AS1 overexpression reduces sensitivity of non-small cell lung cancer cells to osimertinib / 南方医科大学学报

OBJECTIVE@#To screen the differentially expressed long non-coding RNAs (lncRNAs) in non-small cell lung cancer (NSCLC) cells with acquired resistance to osimertinib and explore their roles in drug resistance of the cells.@*METHODS@#The cell lines H1975_OR and HCC827_OR with acquired osimertinib resistance were derived from their osimertinib-sensitive parental NSCLC cell lines H1975 and HCC827, respectively, and their sensitivity to osimertinib was assessed with CCK-8 assay, clone formation assay and flow cytometry. RNA sequencing (RNA-seq) and real-time quantitative PCR (qPCR) were used to screen the differentially expressed lncRNAs in osimertinib-resistant cells. The role of the identified lncRNA in osimertinib resistance was explored using CCK-8, clone formation and Transwell assays, and its subcellular localization and downstream targets were analyzed by nucleoplasmic separation, bioinformatics analysis and qPCR.@*RESULTS@#The resistance index of H1975_OR and HCC827_OR cells to osimertinib was 598.70 and 428.82, respectively (P < 0.001), and the two cell lines showed significantly increased proliferation and colony-forming abilities with decreased apoptosis (P < 0.01). RNA-seq identified 34 differentially expressed lncRNAs in osimertinib-resistant cells, and among them lnc-TMEM132D-AS1 showed the highest increase of expression after acquired osimertinib resistance (P < 0.01). Analysis of the TCGA database suggested that the level of lnc-TMEM132D-AS1 was significantly higher in NSCLC than in adjacent tissues (P < 0.001), and its high expression was associated with a poor prognosis of the patients. In osimertinib-sensitive cells, overexpression of Lnc-TMEM132D-AS1 obviously promoted cell proliferation, colony formation and migration (P < 0.05), while Lnc-TMEM132D-AS1 knockdown partially restored osimertinib sensitivity of the resistant cells (P < 0.01). Lnc-TMEM132D-AS1 was localized mainly in the cytoplasm, and bioinformatics analysis suggested that hsa-miR-766-5p was its candidate target, and their expression levels were inversely correlated. The target mRNAs of hsa-miR-766-5p were mainly enriched in the Ras signaling pathway.@*CONCLUSION@#The expression of lnc-TMEM132D-AS1 is significantly upregulated in NSCLC cells with acquired osimertinib resistance, and may serve as a potential biomarker and therapeutic target for osimertinibresistant NSCLC.

LASS2/TMSG1 overexpression inhibits proliferation and promotes apoptosis of human lung cancer A549 cells possibly by upregulating ceramide and p38 MAPK to activate a signaling cascade / 南方医科大学学报

OBJECTIVE@#To investigate the effects of LASS2/TMSG1 gene overexpression on proliferation and apoptosis of human lung cancer A549 cells and explore the possible mechanism.@*METHODS@#We examined LASS2/TMSG1 expression level in a previously constructed A549 cell line overexpressing LASS2/TMSG1 using Western blotting. The proliferation and apoptosis of the cells were detected using colony-forming assay, CCK-8 assay, Hoechst/PI double staining and flow cytometry. Fourteen nude mice were randomized into 2 groups (n=7) to receive subcutaneous injection of A549 cells with or without LASS2/TMSG1 overexpression on the back of the neck, and the cell proliferation in vivo was observed. The expression levels of p38 MAPK protein and p-p38 MAPK protein in the xenografts were detected with Western blotting. ELISA was used to detect the levels of ceramide and p38 MAPK protein in cultured A549 cell supernatants and the xenografts in nude mice.@*RESULTS@#Compared with the negative control cells, A549 cells with LASS2/TMSG1 overexpression had significantly lowered proliferation ability in vitro with increased early apoptosis rate (P < 0.05), and showed obvious growth inhibition after inoculation in nude mice(P < 0.05). Western blotting showed that in both cultured A549 cells and the xenografts in nude mice, LASS2/TMSG1 gene overexpression significantly increased the expression levels of p38 MAPK protein and p-p38 MAPK protein (P < 0.05); the results of ELISA also revealed significantly increased levels of ceramide and p38 MAPK protein in the cell supernatant andxenografts as well (P < 0.05).@*CONCLUSION@#Overexpression of LASS2/TMSG1 gene can significantly inhibit the proliferation and promote early apoptosis of human lung cancer A549 cells both in vitro and in vivo possibly by upregulating the expressions of ceramide and p38 MAPK protein to activate a signal transduction cascade.

Silenced ANP32A inhibits the growth, invasion and migration of colorectal cancer in vitro via the inactivation of AKT pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of ANP32A silencing on invasion and migration of colon cancer cells and the influence of the activity of AKT signaling pathway on this effect.@*METHODS@#Colorectal cancer HCT116 and SW480 were transfected with a small interfering RNA targeting ANP32A via a lentiviral vector. At 24, 48 and 72 h after the transfection, the changes in cell proliferation and AKT activity in the cells were detected using MTT assay and Western blotting, respectively. HCT116 and SW480 cells were treated with the AKT agonist SC79 or its inhibitor MK2206 for 24, 48, 72 and 96 h, and the changes in cell migration and invasion ability were analyzed using Transwell chamber assay and cell proliferation was assessed using MTT assay. The effects of SC79 and MK2206 on migration and invasion abilities of HCT116 and SW480 cells with or without ANP32A silencing were examined using wound healing and Transwell chamber assays, and the changes in the expression of metadherin (MTDH), a factor associated with cells invasion and migration, was detected with Western blotting.@*RESULTS@#Lentivirus-mediated ANP32A silencing significantly down-regulated the activity of AKT and inhibited the proliferation of both HCT116 and SW480 cells (P < 0.01). The application of AKT inhibitor MK2206 obviously inhibited the proliferation, invasion and migration of the colorectal cancer cells (P < 0.05), while the AKT agonist SC79 significantly promoted the invasion and migration of the cells (P < 0.01). In HCT116 and SW480 cells with ANP32A silencing, treatment with MK2206 strongly enhanced the inhibitory effects of ANP32A silencing on cell invasion and migration (P < 0.05) and the expression of MTDH, while SC79 partially reversed these inhibitory effects (P < 0.01).@*CONCLUSION@#ANP32A silencing inhibits invasion and migration of colorectal cancer cells possibly by inhibiting the activation of the AKT signaling pathway.

Bloodletting Acupuncture at Jing-Well Points Alleviates Myocardial Injury in Acute Altitude Hypoxic Rats by Activating HIF-1α/BNIP3 Signaling-Mediated Mitochondrial Autophagy and Decreasing Oxidative Stress / 中国结合医学杂志

OBJECTIVE@#To explore the protective effect and possible mechanisms of bloodletting acupuncture at Jing-well points (BAJP) pre-treatment on acute hypobaric hypoxia (AHH)-induced myocardium injury rat.@*METHODS@#Seventy-five rats were randomly divided into 5 groups by a random number table: a control group (n=15), a model group (n=15), a BAJP group (n=15), a BAJP+3-methyladenine (3-MA) group (n=15), and a BANA (bloodletting at nonacupoint; tail bleeding, n=15) group. Except for the control group, the AHH rat model was established in the other groups, and the corresponding treatment methods were adopted. Enzyme-linked immunosorbent assay (ELISA) was used to detect creatine kinase isoenzyme MB (CK-MB) and cardiac troponins I (CTnI) levels in serum and superoxide dismutase (SOD) and malondialdehyde (MDA) levels in myocardial tissue. Hematoxylin-eosin (HE) staining was used to observe myocardial injury, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to observe cell apoptosis. Transmission electron microscopy detection was used to observe mitochondrial damage and autophagosomes in the myocardium. The mitochondrial membrane potential of the myocardium was analyzed with the fluorescent dye JC-1. Mitochondrial respiratory chain complex (complex I, III, and IV) activities and ATPase in the myocardium were detected by mitochondrial respiratory chain complex assay kits. Western blot analysis was used to detect the autophagy index and hypoxia inducible factor-1α (HIF-1α)/Bcl-2 and adenovirus E1B 19k Da-interacting protein 3 (BNIP3) signaling.@*RESULTS@#BAJP reduced myocardial injury and inhibited myocardial cell apoptosis in AHH rats. BAJP pretreatment decreased MDA levels and increased SOD levels in AHH rats (all P<0.01). Moreover, BAJP pretreatment increased the mitochondrial membrane potential (P<0.01), mitochondrial respiratory chain complex (complexes I, III, and IV) activities (P<0.01), and mitochondrial ATPase activity in AHH rats (P<0.05). The results from electron microscopy demonstrated that BAJP pretreatment improved mitochondrial swelling and increased the autophagosome number in the myocardium of AHH rats. In addition, BAJP pretreatment activated the HIF-1α/BNIP3 pathway and autophagy. Finally, the results of using 3-MA to inhibit autophagy in BAJP-treated AHH rats showed that suppression of autophagy attenuated the treatment effects of BAJP in AHH rats, further proving that autophagy constitutes a potential target for BAJP treatment of AHH.@*CONCLUSION@#BAJP is an effective treatment for AHH-induced myocardial injury, and the mechanism might involve increasing HIF-1α/BNIP3 signaling-mediated autophagy and decreasing oxidative stress.

Treadmill exercise alleviates neuropathic pain by regulating mitophagy of the anterior cingulate cortex in rats / 生理学报

This study aimed to investigate the effect of treadmill exercise on neuropathic pain and to determine whether mitophagy of the anterior cingulate cortex (ACC) contributes to exercise-mediated amelioration of neuropathic pain. Chronic constriction injury of the sciatic nerve (CCI) was used to establish a neuropathic pain model in Sprague-Dawley (SD) rats. Von-Frey filaments were used to assess the mechanical paw withdrawal threshold (PWT), and a thermal radiation meter was used to assess the thermal paw withdrawal latency (PWL) in rats. qPCR was used to evaluate the mRNA levels of Pink1, Parkin, Fundc1, and Bnip3. Western blot was used to evaluate the protein levels of PINK1 and PARKIN. To determine the impact of the mitophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP) on pain behaviors in CCI rats, 24 SD rats were randomly divided into CCI drug control group (CCI+Veh group), CCI+CCCP low-dose group (CCI+CCCP0.25), CCI+CCCP medium-dose group (CCI+CCCP2.5), and CCI+CCCP high-dose group (CCI+CCCP5). Pain behaviors were assessed on 0, 1, 3, 5, and 7 days after modeling. To explore whether exercise regulates pain through mitophagy, 24 SD rats were divided into sham, CCI, and CCI+Exercise (CCI+Exe) groups. The rats in the CCI+Exe group underwent 4-week low-moderate treadmill training one week after modeling. The mechanical pain and thermal pain behaviors of the rats in each group were assessed on 0, 7, 14, 21, and 35 days after modeling. Western blot was used to detect the levels of the mitophagy-related proteins PINK1, PARKIN, LC3 II/LC3 I, and P62 in ACC tissues. Transmission electron microscopy was used to observe the ultrastructure of mitochondrial morphology in the ACC. The results showed that: (1) Compared with the sham group, the pain thresholds of the ipsilateral side of the CCI group decreased significantly (P < 0.001). Meanwhile, the mRNA and protein levels of Pink1 were significantly higher, and those of Parkin were lower in the CCI group (P < 0.05). (2) Compared with the CCI+Veh group, each CCCP-dose group showed higher mechanical and thermal pain thresholds, and the levels of PINK1 and LC3 II/LC3 I were elevated significantly (P < 0.05, P < 0.01). (3) The pain thresholds of the CCI+Exe group increased significantly compared with those of the CCI group after treadmill intervention (P < 0.001, P < 0.01). Compared with the CCI group, the protein levels of PINK1 and P62 were decreased (P < 0.001, P < 0.01), and the protein levels of PARKIN and LC3 II/LC3 I were increased in the CCI+Exe group (P < 0.01, P < 0.05). Rod-shaped mitochondria were observed in the ACC of CCI+Exe group, and there were little mitochondrial fragmentation, swelling, or vacuoles. The results suggest that the mitochondrial PINK1/PARKIN autophagy pathway is blocked in the ACC of neuropathic pain model rats. Treadmill exercise could restore mitochondrial homeostasis and relieve neuropathic pain via the PINK1/PARKIN pathway.

Reverse effect of Semaphorin-3F on rituximab resistance in diffuse large B-cell lymphoma via the Hippo pathway / 中华医学杂志(英文版)

BACKGROUND@#Exploring the underlying mechanism of rituximab resistance is critical to improve the outcomes of patients with diffuse large B-cell lymphoma (DLBCL). Here, we tried to identify the effects of the axon guidance factor semaphorin-3F (SEMA3F) on rituximab resistance as well as its therapeutic value in DLBCL.@*METHODS@#The effects of SEMA3F on the treatment response to rituximab were investigated by gain- or loss-of-function experiments. The role of the Hippo pathway in SEMA3F-mediated activity was explored. A xenograft mouse model generated by SEMA3F knockdown in cells was used to evaluate rituximab sensitivity and combined therapeutic effects. The prognostic value of SEMA3F and TAZ (WW domain-containing transcription regulator protein 1) was examined in the Gene Expression Omnibus (GEO) database and human DLBCL specimens.@*RESULTS@#We found that loss of SEMA3F was related to a poor prognosis in patients who received rituximab-based immunochemotherapy instead of chemotherapy regimen. Knockdown of SEMA3F significantly repressed the expression of CD20 and reduced the proapoptotic activity and complement-dependent cytotoxicity (CDC) activity induced by rituximab. We further demonstrated that the Hippo pathway was involved in the SEMA3F-mediated regulation of CD20. Knockdown of SEMA3F expression induced the nuclear accumulation of TAZ and inhibited CD20 transcriptional levels via direct binding of the transcription factor TEAD2 and the CD20 promoter. Moreover, in patients with DLBCL, SEMA3F expression was negatively correlated with TAZ, and patients with SEMA3F low TAZ high had a limited benefit from a rituximab-based strategy. Specifically, treatment of DLBCL cells with rituximab and a YAP/TAZ inhibitor showed promising therapeutic effects in vitro and in vivo .@*CONCLUSION@#Our study thus defined a previously unknown mechanism of SEMA3F-mediated rituximab resistance through TAZ activation in DLBCL and identified potential therapeutic targets in patients.

Polygalacin D inhibits the growth of hepatocellular carcinoma cells through BNIP3L-mediated mitophagy and endogenous apoptosis pathways / 中国天然药物

Platycodon grandiflorum (Jacq.) A. DC. is a famous medicinal plant commonly used in East Asia. Triterpene saponins isolated from P. grandiflorum are the main biologically active compounds, among which polygalacin D (PGD) has been reported to be an anti-tumor agent. However, its anti-tumor mechanism against hepatocellular carcinoma is unknown. This study aimed to explore the inhibitory effect of PGD in hepatocellular carcinoma cells and related mechanisms of action. We found that PGD exerted significant inhibitory effect on hepatocellular carcinoma cells through apoptosis and autophagy. Analysis of the expression of apoptosis-related proteins and autophagy-related proteins revealed that this phenomenon was attributed to the mitochondrial apoptosis and mitophagy pathways. Subsequently, using specific inhibitors, we found that apoptosis and autophagy had mutually reinforcing effects. In addition, further analysis of autophagy showed that PGD induced mitophagy by increasing BCL2 interacting protein 3 like (BNIP3L) levels.In vivo experiments demonstrated that PGD significantly inhibited tumor growth and increased the levels of apoptosis and autophagy in tumors. Overall, our findings showed that PGD induced cell death of hepatocellular carcinoma cells primarily through mitochondrial apoptosis and mitophagy pathways. Therefore, PGD can be used as an apoptosis and autophagy agonist in the research and development of antitumor agents.