Chemokine C-C motif ligand 2 suppressed the growth of human brain astrocytes under Ischemic/hypoxic conditions via regulating ERK1/2 pathway.

PRIMARY OBJECTIVE: Chemokine C-C motif ligand 2 (CCL2) plays a critical role in inflammation-related diseases in the central nervous system (CNS). However, the role of CCL2 in ischemic stroke remains unclear. RESEARCH DESIGN: To investigate the role of CCL2 in ischemic stroke, we performed oxygen-glucose deprivation (OGD) on human brain astrocytes. METHODS AND PROCEDURES: To assess cell proliferation, the CCK-8 assay was performed. Cell apoptosis was determined using flow cytometry. qRT-PCR and western blotting were utilized to measure gene expression. MAIN OUTCOMES AND RESULTS: Our results suggest that CCL2 and its receptor CCR2 are upregulated in OGD cells. Moreover, a CCL2 antibody significantly alleviated the ischemic/hypoxic-induced suppression of growth in human brain astrocytes. Human recombinant protein, CCL2, inhibited the growth of human brain astrocytes under normoxia conditions. These results demonstrate that CCL2 upregulation suppresses the recovery of human brain astrocytes under ischemic/hypoxic conditions. This effect was abolished by the ERK inhibitor PD98059. Therefore, CCL2/CCR2 activation may suppress the growth of human brain astrocytes through enhancing the activity of ERK1/2. CONCLUSIONS: Our results not only developed a deeper understanding of the role of CCL2 in human brain astrocytes but also provided novel insight into potential treatments for ischemic stroke.

MiR-320 inhibits the growth of glioma cells through downregulating PBX3.

BACKGROUND: MiR-320 is downregulated in multiple cancers, including glioma and acts as tumor suppressor through inhibiting tumor cells proliferation and inducing apoptosis. PBX3 (Pre-B cell leukemia homeobox 3), a putative target gene of miR-320, has been reported to be upregulated in various tumors and promote tumor cell growth through regulating MAKP/ERK pathway. This study aimed to verify whether miR-320 influences glioma cells growth through regulating PBX3. METHODS: Twenty-four human glioma and paired adjacent nontumorous tissues were collected for determination of miR-320 and PBX3 expression using RT-qPCR and western blot assays. Luciferase reporter assay was performed to verify the interaction between miR-320 and its targeting sequence in the 3' UTR of PBX3 in glioma cells U87 and U251. Increased miR-320 level in U87 and U251 cells was achieved through miR-320 mimic transfection and the effect of which on glioma cells growth, proliferation, cell cycle, apoptosis and activation of Raf-1/MAPK pathway was determined using MTT, colony formation, flow cytometry and western blot assays. PBX3 knockdown was performed using shPBX3 and the influence on MAPK pathway activation was evaluated. RESULTS: MiR-320 downregulation and PBX3 upregulation was found in glioma tissues. Luciferase reporter assays identified miR-320 directly blinds to the 3' UTR of PBX3 in glioma cells. MiR-320 mimic transfection suppressed glioma cells proliferation, and induced cell cycle arrest and apoptosis. Both miR-320 overexpression and PBX3 knockdown inhibited Raf-1/MAPK activation. CONCLUSION: MiR-320 may suppress glioma cells growth and induced apoptosis through the PBX3/Raf-1/MAPK axis, and miR-320 oligonucleotides may be a potential cancer therapeutic for glioma.

Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism.

Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

Effect of the Total Extract of Averrhoacarambola (Oxalidaceae) Root on the Expression Levels of TLR4 and NF-κB in Streptozotocin-Induced Diabetic Mice.

BACKGROUND: Averrhoacarambola L., which is a folk medicine used in diabetes mellitus (DM) in ancient China, has been reported to have anti-diabetic efficacy. AIMS: The aim of this study was to evaluate the hypoglycemic effect of the extract of Averrhoacarambola L. root (EACR) on the regulation of the Toll-like receptor 4 (TLR4)-Nuclear-factor kappa B (NF-κB) pathway in B) pathway in streptozotocin (STZ)-induced diabetic mice. METHODS: the mice were injected with STZ (120 mg/kg body weight) via a tail vein. After 72 h, the mice with FBG ≥ 11.1 mmol/L were confirmed as having diabetes. Subsequently, the mice were treated intragastrically with EACR (300, 600, 1200 mg/kg body weight/d) and metformin (320 mg/kg body weight/d) for 14 days. RESULTS: As a result the serum fasting blood glucose (FBG), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were decreased following EACR administration. Immunohistochemical analysis revealed that the pancreatic tissue expression levels of TLR4 and NF-κB were downregulated after EACR administration. EACR suppressed pancreatic mRNA expression level of TLR4 and blocked the downstream NF-κB pathway in the pancreas. According to Western blot analysis EACR suppressed pancreatic TLR4 and NF-κB protein expression levels. Histopathological examination of the pancreas showed that STZ-induced pancreas lesions were alleviated by the EACR treatment. CONCLUSION: These findings suggest that the modulation of the IL-6 and TNF-α inflammatory cytokines and the suppression of the TLR4-NF-κB pathway are most likely involved in the anti-hyperglycemic effect of EACR in STZ-induced diabetic mice.

Regulatory effect of Ganoderma lucidum and its active components on gut flora in diseases.

Driven by the good developmental potential and favorable environment at this stage, Ganoderma lucidum is recognized as a precious large fungus with medicinal and nutritional health care values. Among them, polysaccharides, triterpenoids, oligosaccharides, trace elements, etc. are important bioactive components in G. lucidum. These bioactive components will have an impact on gut flora, thus alleviating diseases such as hyperglycemia, hyperlipidemia and obesity caused by gut flora disorder. While numerous studies have demonstrated the ability of G. lucidum and its active components to regulate gut flora, a systematic review of this mechanism is currently lacking. The purpose of this paper is to summarize the regulatory effects of G. lucidum and its active components on gut flora in cardiovascular, gastrointestinal and renal metabolic diseases, and summarize the research progress of G. lucidum active components in improving related diseases by regulating gut flora. Additionally, review delves into the principle by which G. lucidum and its active components can treat or assist treat diseases by regulating gut flora. The research progress of G. lucidum in intestinal tract and its potential in medicine, health food and clinical application were fully explored for researchers.

PBX3 as a biomarker for the early diagnosis and prediction of prognosis of glioma.

BACKGROUND: Increasing evidence have elucidated that PBX3 played a crucial role in cancer initiation and progression. PBX3 was differentially expressed in many cancer types. However, PBX3 potential involvement in gliomas remains to be explored. METHODS: The expression level of PBX3 in glioma tissues and glioma cells, and its correlation with clinical features were analyzed by data from TCGA, GEPIA, CGGA and CCLE. Univariable survival and Multivariate Cox analysis was used to compare several clinical characteristics with survival. We also analyzed the correlation between PBX3 expression level and survival outcome and survival time of LGG and GBM patients by using linear regression equation. GSEA was used to generate an ordered list of all genes related to PBX3 expression and screening of genes co-expressed with PBX3 mRNA by "limma" package. RESULTS: The results showed that PBX3 was highly expressed in gliomas and its expression increased with the increase of malignancy. Survival analysis found that PBX3 is more valuable in predicting the OS and PFI of LGG patients than that of GBM. For further study, TCGA and CGGA data were downloaded for univariate Cox analysis and multivariate Cox analysis which showed that the expression of PBX3 was independent influencing factors for poor prognosis of LGG patients. Meanwhile, Receiver operating characteristic (ROC) curve showed that PBX3 was a predictor of overall survival rate and progression-free survival rate of LGG. Linear regression model analysis indicated that the higher expression of PBX3 the higher the risk of death of LGG patients, and the higher expression of PBX3 the higher the risk of disease progression of LGG patients. Next, TCGA data were downloaded for GSEA and Co-expression analyses, which was performed to study the function of PBX3. CONCLUSION: PBX3 may be involved in the occurrence and development of glioma, and has potential reference value for the early diagnosis and prediction of prognosis of glioma.

Inhibition of Alzheimer's disease by 4-octyl itaconate revealed by RNA-seq transcriptome analysis.

AIMS: This study aimed to examine the therapeutic effects and response mechanisms of 4-OI in Alzheimer's disease (AD). METHODS: In this study, network pharmacology was employed to analyze potential targets for AD drug therapy. Immunofluorescence and quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques were utilized to detect inflammatory phenotypes in a 4-OI-resistant mouse microglia cell line (BV2). We conducted four classical behavioral experiments, namely the open field test, new object recognition test, Y maze test, and Morris water maze, to assess the emotional state and cognitive level of APPswe/PS1dE9 (referred to as APP/PS1) mice after 4-OI treatment. Hematoxylin and eosin (HE) staining, along with immunofluorescence staining, were performed to detect amyloid (Aß) deposition in mouse brain tissue. To explore the potential molecular mechanisms regulating the effects of 4-OI treatment, we performed RNA-SEQ and transcription factor prediction analyses. Additionally, mouse BV2 cells underwent Western blotting analysis to elucidate potential molecular mechanisms underlying the observed effects. RESULTS: We discovered that 4-OI exerts an inhibitory effect on neuroinflammation by promoting autophagy. This effect is attributed to the activation of the AMPK/mTOR/ULK1 pathway, achieved through enhanced phosphorylation of AMPK and ULK1, coupled with a reduction in mTOR phosphorylation. Furthermore, 4-OI significantly enhances neuronal recovery in the hippocampus and diminishes Aß plaque deposition in APP/PS1 mice, improved anxiety in mice, and ultimately led to improved cognitive function. CONCLUSIONS: Overall, the results of this study demonstrated that 4-OI improved cognitive deficits in AD mice, confirming the therapeutic effect of 4-OI on AD.

A composition of ursolic acid derivatives from Ludwigia hyssopifolia induces apoptosis in throat cancer cells via the Akt/mTOR and mitochondrial signaling pathways and by modulating endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Ludwigia hyssopifolia (LH), an ethnopharmacological herb used in Guangxi Zhuang medicine, is known for its extensive therapeutic use in treating throat disorders. The anti-laryngeal-cancer benefits of the ethyl acetate and petroleum ether fractions of the ethanolic extracts of LH have been shown in our prior cell-based research. Nevertheless, the specific impacts and underlying processes by which LH combats throat cancer effects have not been fully understood. AIM OF THE STUDY: This study involved the extraction of a composition containing two derivatives of ursolic acid from LH (LH-CUAD). The present study aimed to assess the anti-throat-cancer effects of these derivatives and the underlying mechanisms through in vitro and in vivo experiments. MATERIALS AND METHODS: Solvent extraction, fractionation, chromatography, and semipreparative high-performance liquid chromatography were used for the extraction, purification, and analysis of LH-CUAD. The in vitro and in vivo anti-throat-cancer effects of LH-CUAD were investigated using the throat cancer cell lines Hep-2 and FaDu as well as Hep-2 tumor-bearing nude mice. RESULTS: LH-CUAD significantly inhibited the proliferation and migration of throat cancer cells without any prominent toxicity. The Hoechst 33258 staining, Annexin V-FITC/PI double-staining assays, and flow cytometry confirmed that LH-CUAD could induce throat cancer cell death from early to late apoptosis in vitro. LH-CUAD exhibited significant antitumor activity and low toxicity in a xenograft model, and induced throat cancer cells apoptosis in vivo. The apoptotic effects of LH-CUAD therapy were validated using Western blotting, which demonstrated the activation of a caspase cascade response triggered by an imbalance between the endoplasmic reticulum and mitochondria. In addition, it was observed that LH-CUAD exhibited inhibitory effects on Akt and mTOR phosphorylation, hence promoting apoptosis. CONCLUSIONS: LH-CUAD induces apoptosis in both in vivo and in vitro models of throat cancer. This effect is achieved by activating the mitochondrial pathway, inhibiting the Akt/mTOR pathway and initiating endoplasmic reticulum stress. The findings of this study suggest that LH-CUAD has the potential to offer a novel approach to the clinical management of throat cancer.

[Study on HPLC fingerprint of Taxus chinensis var. mairei].

OBJECTIVE: To establish the HPLC fingerprint of Taxus chinensis var. mairei collected from different parts during different seasons and provide scientific basis for its comprehensive utilization. METHODS: Supercritical CO2 extraction was used to extract the effective fraction, HPLC method to establish the fingerprint and similarity evaluation software to analyze the fingerprint chromatogram. RESULTS: 12 batches of Taxus chinensis var. mairei medicinal materials from different parts collected in different seasons were analyzed and HPLC fingerprint of Taxus chinensis var. mairei was established. CONCLUSION: The HPLC fingerprint can be used to evaluate the quality of Taxus chinensis var. mairei medicinal materials.

Phenolic and lignan glycosides from the butanol extract of Averrhoa carambola L. root.

Fifteen compounds, which included six chiral lignans and nine phenolic glycosides, were separated from the butanol fraction of Averrhoa carambola L. root and identified. All of the compounds, namely 3,4,5-trimethoxyphenol-1-O-ß-D-glucopyranoside, benzyl-1-O-ß-D-glucopyranoside, (+)-5'-methoxyisolariciresinol 3α-O-ß-D-gluco-pyranoside, (+)-isolariciresinol 3α-O-ß-D-glucopyranoside, koaburaside, (+)-lyoniresinol 3α-O-ß-D-glucopyranoside, (-)-lyoniresinol 3α-O-ß-D-glucopyranoside, (-)-5'-methoxyisolariciresinol 3α-O-ß-D-glucopyranoside, (-)-isolariciresinol 3α-O-ß-D-glucopyranoside, 3,5-dimethoxy-4-hydroxyphenyl 1-O-ß-apiofuranosyl (1''→6')-O-ß-D-glucopyranoside, 3,4,5-trimethoxyphenyl 1-O-ß-apiofuranosyl (1''→6')-ß-gluco-pyranoside, methoxyhydroquinone-4-ß-D-glucopyranoside, (2S)-2-O-ß-D-gluco-pyranosyl-2-hydroxyphenylacetic acid, 3-hydroxy-4-methoxyphenol 1-O-ß-D-apio-furanosyl-(1''→6')-O-ß-D-glucopyranoside and 4-hydroxy-3-methoxyphenol 1-O-ß-D-apiofuranosyl-(1''→6')-O-ß-D-glucopyranoside were isolated from this plant for the first time.

[Research on the influence of LED temperature shifts on differential optical absorption spectroscopy for measuring NO2].

Influences of LEDs (without etalon structure and center wavelengths are respectively 370 nm (near-UV), 452 nm (blue) and 660 nm(red)) temperature shifts on differential optical absorption spectroscopy(DOAS) for measuring NO2 were studied. NO2 absorption spectra were formed using LED emitting spectra at 10 degrees C. The measured LED spectra at other temperatures were used as reference spectra of DOAS. Thus, NO2 differential optical densities under different LED temperature shifts were acquired and then NO2 differential cross-sections were fitted to the acquired differential optical densities. From fitting results, the linear relations of 0.995, 0.945 and 0.989 correlation between delta of fitting residual and near-UV, blue and red LEDs temperature shifts were found and their slopes are respectively 1.12 x 10(-3), 5.25 x 10(-5) and 7.45 x 10(-4) degrees C(-1). The fitting results show that the influence of temperature shifts of blue LED on DOAS retrieval is negligible and the temperature shifts of near-UV and red LED are impressible to DOAS measurement resulting in degradation of detection sensitivity. The retrieval results of blue LED with and without etalon with similar temperature properties were compared and showed that etalon of LED will greatly increase the influence of temperature shifts of LED on DOAS retrieval.

S100A9­containing serum exosomes obtained from patients with burn injuries promote myocardial cell pyroptosis through NLRP3.

S100 calcium-binding protein A9 (S100A9) is highly expressed in the serum exosomes of patients with burn injuries. The present study aimed to investigate the underlying mechanisms of burn injury-associated exosomes in the regulation of myocardial cell pyroptosis. Reverse transcription-quantitative PCR and western blotting were used to examine relative mRNA and protein levels. The morphology of exosomes was visualized using transmission electron microscopy. The expression levels of IL-1ß and IL-18 in cells were examined using ELISA kits. Finally, cell pyroptosis was examined using flow cytometry. When AC16 cells were treated with the serum exosomes obtained from patients with burn injuries, pyroptosis was significantly promoted and the expression levels of IL-1ß and IL-18 were increased. NLR family pyrin domain containing 3 (NLRP3), S100A9, caspase-1 and Gasdermin D (GSDMD)-N expression levels were also upregulated. However, these were significantly reversed by anti-S100A9 antibodies. Thereafter, CY-09, an NLRP3 inhibitor, was revealed to restore the increase in pyroptosis and IL-18, IL-1ß, caspase-1, NLRP3 and GSDMD-N expression levels caused by recombinant S100A9 to be similar to the control. These findings suggested that burn injury-associated exosomes containing S100A9 can affect AC16 cell pyroptosis through NLRP3.

Role of TGM2 in T­cell lymphoblastic lymphoma via regulation of IL­6/JAK/STAT3 signalling.

Transglutaminase 2 (TGM2) is a Ca2+­dependent enzyme that is closely associated with cancer progression; however, the function of TGM2 in T­cell lymphoma remains unclear. In the present study, TGM2 was identified as an upregulated gene by bioinformatics analysis of the microarray datasets GSE132550 and GSE143382 from the Gene Expression Omnibus database. The effects and mechanisms of TGM2 on T­cell lymphoma cells were evaluated using the Cell Counting Kit­8, colony formation assay, 5­ethynyl­2'­deoxyuridine (EdU) assay, flow cytometry, reverse transcription­quantitative polymerase chain reaction, western blotting and gene set enrichment analysis (GSEA). TGM2 expression was shown to be elevated in formalin­fixed paraffin­embedded skin biopsies from patients with T­cell lymphoma relative to skin tissue from healthy cases. TGM2 expression was also increased in T­cell lymphoma cell lines compared with that in CD4+ T cells. Transfection with TGM2 small interfering RNAs (siRNAs) decreased the number of EdU­positive cells, and the viability and colony formation of T­cell lymphoma cells. Furthermore, TGM2 siRNAs enhanced the apoptosis of T­cell lymphoma cells potentially via cleavage of caspase­3 and poly ADP­ribose polymerase. GSEA identified the IL­6/JAK/STAT3 pathway as a potential downstream signalling pathway of TGM2. Notably, the effects of TGM2 siRNAs on T­cell lymphoma cells were attenuated by IL­6 and accelerated by IL­6/JAK/STAT3 inhibitor AG490. These findings indicated that TGM2 siRNAs inhibited the proliferation of T­cell lymphoma cells by regulating the IL­6/JAK/STAT3 signalling pathway; therefore, TGM2 may function as a potential therapeutic target for T­cell lymphoma.

Identification and Validation of METTL3-Related Molecules for Predicting Prognosis and Efficacy of Immunotherapy in Gastric Cancer Based on m6A Methylome and Transcriptome Sequencing Analysis.

Abnormal N6-methyladenosine (m6A) modification levels caused by METTL3 have been identified to be a critical regulator in human cancers, and its roles in the immune microenvironment and the relationship between targeted therapy and immunotherapy sensitivity in gastric cancer (GC) remain poorly understood. In this study, we assessed the transcriptome-wide m6A methylation profile after METTL3 overexpression by m6A sequencing and RNA sequencing in BGC-823 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to analyze the function of core targets of METTL3. Eighteen methylation core molecules were identified in GC patients by combining transcriptome and methylome sequencing. GC patients can be separated into two subtypes based on the expression of 18 methylation core molecules. Furthermore, subgroup analysis showed that patients with different subtypes had a different OS, PFS, stage, grade, and TMB. Gene set enrichment analysis (GSEA) showed that immune-related pathways were enriched among subtype A. The ESTIMATE analysis suggested that the extent of infiltration of immune cells was different in two subtypes of GC patients. Tumor Immune Dysfunction and Exclusion (TIDE) and The Cancer Immunome Atlas (TCIA) database also showed that there were significant differences in the efficacy of immunotherapy among different types of GC patients. Altogether, our results reveal that METTL3-mediated m6A methylation modification is associated with the immune microenvironment and the effects of immunotherapy in GC patients. Our findings provide novel insights for clinicians in the diagnosis and optimal treatment of GC patients.

m7G-Related DNA Damage Repair Genes are Potential Biomarkers for Predicting Prognosis and Immunotherapy Effectiveness in Colon Cancer Patients.

Objective: m7G is a post-transcriptional modification modality, however, limited research has been conducted on its role in colon cancer. DNA damage repair (DDR) is an important factor that contributes to colon cancer development, growth and chemoresistance. This study aimed to explore whether m7G-related DNA damage repair genes may be used as biomarkers to predict the prognosis of colon cancer patients. Methods: We use non-negative matrix factorization (NMF) to type CRC patients into. Risk models were constructed using different expression genes in two clusters. We assessed the reliability of risk models with DCA curves, and a Nomogram. Meanwhile, The receiver operating characteristic and C-index curves were used to compare the predictive significance of the constructed risk models with other studies. In additional, we examined the significance of risk models on patients' immunity microenvironment and response to immune therapy. Finally, we used a series of cellular experiments to validate the effect of model genes on the malignant progression of CRC cells. Results: Twenty-eight m7G genes were obtained from the GSEA database. Multivariate Cox and LASSO Cox regression analysis was performed and eleven m7G-related DDR genes were identified for constructing the risk model. Survival and stage of CRC patients were worser in the high-risk group than in the low-risk group for both the training and test sets. Additionally, the different immune microenvironment status of patients in the high- and low-risk groups, suggesting that patients in the low-risk group may be more sensitive to immunotherapy, particularly immune checkpoint inhibitors. Finally, we found that depletion of ATP2A1, one of the risk genes in our model, influence the biologic behaviour of CRC cells significantly. Conclusion: The m7G-related DDR genes can be used as important markers for predicting patient prognosis and immunotherapy response. Our data suggest that ATP2A1 may promote the proliferation of colon cancer cells. These findings may provide new therapeutic targets for the treatment of colon cancer.

Models for Predicting Response to Immunotherapy and Prognosis in Patients with Gastric Cancer: DNA Damage Response Genes.

Objective: DNA damage response (DDR) is a complex system that maintains genetic integrity and the stable replication and transmission of genetic material. m6A modifies DDR-related gene expression and affects the balance of DNA damage response in tumor cells. In this study, a risk model based on m6A-modified DDR-related gene was established to evaluate its role in patients with gastric cancer. Methods: We downloaded 639 DNA damage response genes from the Gene Set Enrichment Analysis (GSEA) database and constructed risk score models using typed differential genes. We used Kaplan-Meier curves and risk curves to verify the clinical relevance of the model, which was then validated with the univariate and multifactorial Cox analysis, ROC, C-index, and nomogram, and finally this model was used to evaluate the correlation of the risk score model with immune microenvironment, microsatellite instability (MSI), tumor mutational burden (TMB), and immune checkpoints. Results: In this study, 337 samples in The Cancer Genome Atlas (TCGA) database were used as training set to construct a DDR-related gene model, and GSE84437 was used as external data set for verification. We found that the prognosis and immunotherapy effect of gastric cancer patients in the low-risk group were significantly better than those in the high-risk group. Conclusion: We screened eight DDR-related genes (ZBTB7A, POLQ, CHEK1, NPDC1, RAMP1, AXIN2, SFRP2, and APOD) to establish a risk model, which can predict the prognosis of gastric cancer patients and guide the clinical implementation of immunotherapy.

Integrative proteomics and m6A microarray analyses of the signatures induced by METTL3 reveals prognostically significant in gastric cancer by affecting cellular metabolism.

METTL3-mediated RNA N6-methyladenosine (m6A) is the most prevalent modification that participates in tumor initiation and progression via governing the expression of their target genes in cancers. However, its role in tumor cell metabolism remains poorly characterized. In this study, m6A microarray and quantitative proteomics were employed to explore the potential effect and mechanism of METTL3 on the metabolism in GC cells. Our results showed that METTL3 induced significant alterations in the protein and m6A modification profile in GC cells. Gene Ontology (GO) enrichment indicated that down-regulated proteins were significantly enriched in intracellular mitochondrial oxidative phosphorylation (OXPHOS). Moreover, the protein-protein Interaction (PPI) network analysis found that these differentially expressed proteins were significantly associated with OXPHOS. A prognostic model was subsequently constructed based on the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases, and the high-risk group exhibited a worse prognosis in GC patients. Meanwhile, Gene Set Enrichment Analysis (GSEA) demonstrated significant enrichment in the energy metabolism signaling pathway. Then, combined with the results of the m6A microarray analysis, the intersection molecules of DEPs and differential methylation genes (DMGs) were significantly correlated with the molecules of OXPHOS. Besides, there were significant differences in prognosis and GSEA enrichment between the two clusters of GC patients classified according to the consensus clustering algorithm. Finally, highly expressed and highly methylated molecules regulated by METTL3 were analyzed and three (AVEN, DAZAP2, DNAJB1) genes were identified to be significantly associated with poor prognosis in GC patients. These results signified that METTL3-regulated DEPs in GC cells were significantly associated with OXPHOS. After combined with m6A microarray analysis, the results suggested that these proteins might be implicated in cell energy metabolism through m6A modifications thus influencing the prognosis of GC patients. Overall, our study revealed that METTL3 is involved in cell metabolism through an m6A-dependent mechanism in GC cells, and indicated a potential biomarker for prognostic prediction in GC.

Human leukocyte antigen-G is closely associated with tumor immune escape in gastric cancer by increasing local regulatory T cells.

Human leukocyte antigen-G (HLA-G) plays an important role in tumor cell escape. We investigated HLA-G expression and regulatory T cells (Tregs) infiltrates in patients with gastric cancer (GC), analyzed their relationship with clinicopathologic features, and characterized their role in tumor immune escape. We also investigated the plasma soluble HLA-G level and its potential in the diagnosis of GC. Effect of HLA-G on Tregs was further assessed by coculture experiments in vitro. Most interestingly, HLA-G positive expression was detected in GC tissues and it was significantly correlated with the presence of tumor-infiltrating Tregs. Patients with HLA-G positive expression or high Tregs had significantly poorer survival at 5 years after operation. Multivariate analysis indicated that HLA-G positive expression was an independent prognostic factor of GC. The coculture experiment showed overexpression of HLA-G in GC cell lines significantly enhanced the frequency of Tregs when GC cells were directly cocultured with human peripheral blood mononuclear cell. However, this effect disappeared when the indirect coculture system was applied. Some cytokines such as interleukin-6, interleukin-10, and tumor necrosis factor-α significantly changed in the coculture system. Moreover, plasma soluble HLA-G level in GC patients was higher than that in normal controls. Taken together, our results indicated that HLA-G expression was closely associated with tumor progression and involved in tumor evasion by increasing the frequency of infiltrating Tregs locally. Thus, HLA-G might be a promising predictor for disease prognosis and a possible novel target for immunotherapy in GC patients.

RNA interference of human papillomavirus type 16 E7 increases HLA class I antigen expression in HaCaT-E7 cells.

BACKGROUND: High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer. The E7 protein of high-risk HPV disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting an ideal target for development of the immunotherapy in HPV-positive cervical cancers. We previously reported that HPV16 E7 could down-regulate cell-surface HLA class I antigen accompanying decreased expression of transporter associated with antigen processing 1 (TAP-1). The purpose of this study was to determine whether knockdown of HPV16 E7 could up-regulate surface HLA class I antigen expression in HPV16 E7 expressing HaCaT cells (HaCaT-E7). METHODS: An E7-specific small interfering RNA (siRNA) was transfected into the HaCaT-E7 cells, and the expression of HPV16 E7 was measured by real-time reverse transcriptase polymerase chain reaction and Western blot. With the use of flow cytometry analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 expression were detected. RESULTS: It was found that transfection of HPV16 E7-siRNA reduced HPV16 E7 expression as measured on messenger RNA and protein levels. The flow cytometry analysis showed that, compared with mock transfection, a statistically significant increase of approximately 75% in surface HLA class I levels was observed in HaCaT-E7 cells at 72 hours after transfection of E7 siRNA. Moreover, he knockdown of E7 in HaCaT-E7 cells could result in an increase of intracellular TAP-1 expression, which is essential for the expression of HLA class I at cell surface. CONCLUSIONS: Our study showed that the knockdown of HPV16 E7 could increase cell surface HLA class I antigen expression in HaCaT-E7 cells. In addition, for HPV-positive human cervical cancer, our observations indicate that the HPV E7 gene is a target of choice.

Effect of (E)-2-isopropyl-5-methylcyclohexyl octadec-9-enoate on transdermal delivery of Aconitum alkaloids.

OBJECTIVE: The aim of this work was to evaluate the percutaneous absorption of Aconitum alkaloids using (E)-2-isopropyl-5-methylcyclohexyl octadec-9-enoate (M-OA) as an enhancer as well as to investigate the effect of M-OA in isopropyl palmitate (IPP) solution (5% ethanol in IPP, w/v), with or without an enhancer, on the stratum corneum (SC) barrier properties in vitro. METHODS: The in vitro permeation studies of Aconitum alkaloids were conducted in isopropyl myristate (IPM) solution in side-by-side diffusion cells. In addition, scanning electron microscopy (SEM) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy were used to evaluate the M-OA biophysical changes in SC barrier function in vitro. RESULTS: The in vitro permeation studies indicated that M-OA had significant enhancing effect on the permeation of mesaconitine (MA) and hypaconitine (HA); however, aconitine (AC) was too low to be detected on the receiver side, and L-menthol had no effect on the penetration of all the Aconitum alkaloids. Morphological changes in the skin after enhancer treatment demonstrated that the extraction of the SC lipids by the enhancers led to disruption of the SC and the desquamation of SC flake. ATR-FTIR spectra of C-H asymmetric/symmetric stretching peak shifts and amide II stretching vibrations were indicative of SC lipid fluidization and changes in protein conformation, respectively. CONCLUSION: The results showed that M-OA was worthy of further investigation as a potential candidate for inclusion in transdermal formulations as a penetration enhancer.