Autophagy inhibition mediated by trillin promotes apoptosis in hepatocellular carcinoma cells via activation of mTOR/STAT3 signaling.

Apoptosis and autophagy have been shown to act cooperatively and antagonistically in self-elimination process. On the one side, apoptosis and autophagy can act as partners to induce cell death in a coordinated or cooperative manner; on the flip side, autophagy acts as an antagonist to block apoptotic cell death by promoting cell survival. Our previous research indicated that trillin could induce apoptosis of PLC/PRF/5 cells, but the effects of trillin on autophagy as well as its functional relationship to apoptosis have not been elucidated. Here, the running study aims to investigate the function and molecular mechanism of trillin on autophagy with hepatocellular carcinoma (HCC) cells. The objective of this study is to investigate the molecular mechanism of trillin on autophagy in HCC cells. Protein levels of autophagy markers beclin1, LC3B, and p62 were detected by western blotting. 6-Hydroxyflavone and stattic were used to test the role of trillin regulation of autophagy via serine threonine kinase (AKT)/extracellular-regulated protein kinases (ERK) 1/2/mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Flow cytometry was used to detect caspase 3 activity and apoptosis in PLC/PRF/5 cells treated with trillin for 24 h with or without rapamycin, stattic, and 6-hydroxyflavone. The protein level of autophagy marker beclin1 was decreased, whilst the protein level of p62 was significantly increased by trillin treatment, indicating trillin treatment led to inhibition of autophagy in HCC cells. Trillin treatment could reduce the protein levels of p-AKT and p-ERK1/2, but enhance the protein levels of mTOR and p-mTOR, suggesting that trillin could inhibit AKT/ERK rather than mTOR. The AKT/ERK activator 6-hydroxyflavone could reverse the loss of AKT and ERK1/2 phosphorylation induced by trillin, implying that trillin impairs autophagy through activated mTOR rather than AKT/ERK. STAT3 and p-STAT3 were significantly upregulated by the trillin treatment with an increase in dose from 0 to 50 µM, suggesting that autophagy inhibition is mediated by trillin via activation of STAT3 signaling. The STAT3 inhibitor stattic significantly reversed the increased STAT3 phosphorylation at tyrosine 705 induced by trillin. The mTOR signaling inhibitor rapamycin reversed the trillin-induced mTOR phosphorylation enhancement but exerted no effects on total mTOR levels, suggesting trillin treatment led to inhibition of autophagy in HCC cells through activating mTOR/STAT3 pathway. Furthermore, caspase 3 activities and the total rate of apoptosis were increased by trillin treatment, which was reversed by rapamycin, stattic, and 6-hydroxyflavone, proving that trillin promotes apoptosis via activation of mTOR/STAT3 signaling. Trillin induced autophagy inhibition and promoted apoptosis in PLC/PRF/5 cells via the activation of mTOR/STAT3 signaling. Trillin has the potential to be a viable therapeutic option for HCC treatment.

An ultrasensitive method for detecting mutations from short and rare cell-free DNA.

Circulating tumor DNA (ctDNA) was short and rare, making the detection performance of the current targeted sequencing methods unsatisfying. We developed the One-PrimER Amplification (OPERA) system and examined its performance in detecting mutations of low variant allelic frequency (VAF) in various samples with short-sized DNA fragments. In cell line-derived samples containing sonication-sheared DNA fragments with 50-150 bp, OPERA was capable of detecting mutations as low as 0.0025% VAF, while CAPP-Seq only detected mutations of >0.03% VAF. Both single nucleotide variant and insertion/deletion can be detected by OPERA. In synthetic fragments as short as 80 bp with low VAF (0.03%-0.1%), the detection sensitivity of OPERA was significantly higher compared to that of droplet digital polymerase chain reaction. The error rate was 5.9×10-5 errors per base after de-duplication in plasma samples collected from healthy volunteers. By suppressing "single-strand errors", the error rate can be further lowered by >5 folds in EGFR T790M hotspot. In plasma samples collected from lung cancer patients, OPERA detected mutations in 57.1% stage I patients with 100% specificity and achieved a sensitivity of 30.0% in patients with tumor volume of less than 1 cm3. OPERA can effectively detect mutations in rare and highly-fragmented DNA.

There is no fusion fascia in the abdomen and extraperitoneal fascia always surrounds the mesentery.

Toldt's fascia has always been described as a fusion fascia formed by two layers of visceral peritoneum when the mesentery attaches to the posterior abdominal wall. However, there is still no consensus about the mesentery and its surrounding fascia based on the current anatomic theories. This study aimed to determine the anatomical structures of the abdomen and provide a correct surgical plane for mesenteric-based surgery. Surgical videos of 121 patients who underwent laparoscopic operations of the digestive tract were reviewed to identify and compare the anatomical structures of the mesentery and associated fascia. Twenty-one postoperative specimens were stained with hematoxylin and eosin to indicate the histological appearance of the mesentery and its surrounding fascia. Furthermore, dynamic models had been established to explain the formation mechanism of the associated histological structures in different regions during the progression of mesenteric attachment. The fasciae surrounding the mesentery, including the submesothelial connective tissue, the subserosal connective tissue, Toldt's fascia, and "angel hair," have the same histological characteristic to extraperitoneal fascia. The general anatomical structure of the abdomen can be divided into three layers (abdominal wall, urogenital system, and digestive system) and two interlayers (transversalis fascia and extraperitoneal fascia). The extraperitoneal fascia surrounds the entire digestive system and is the natural layer separating adjacent structures from each other. Typical histological structures in the regions of posterior attachment include the fascia propria of the mesentery, mesofascial plane, extraperitoneal fascia, retrofascial plane, and anterior renal fascia. The urogenital system is surrounded by similar histological structures. There is no fusion fascia in the abdomen due to retreat of the visceral peritoneum, and all of the fasciae surrounding the mesentery are extraperitoneal fascia. This study demonstrates that the typical histological structures in the regions of attachment and mesofascial plane are the correct anatomic interface for mesenteric-based surgery.

Pilot study on demister-based dust removal methods for boiler flue gas.

This study explores the synergistic removal effect of various schemes based on demisters on boiler flue gas dust on a pilot-scale experimental bench. The results show that the dust removal efficiency is 28.43-51.30% when demisters are put into operation alone; the larger the inlet dust concentration of demisters is, the higher the dust removal efficiency will be; but it still cannot reach the 10 mg/Nm3 ultra-low emission standard. The dust removal efficiency is 93.13% when wet electro-static precipitator and demisters are put into operation simultaneously. Furthermore, the outlet dust concentration is lower than 5 mg/Nm3, and the dust removal efficiency of the demisters themselves increases to 67.28%, which has shown a significant improvement compared with operation alone. The dust removal efficiency is 70.98-78.37% when the water-washing layer and demisters are put into operation simultaneously. Moreover, the outlet dust concentration reaches the standard of 10 mg/Nm3 when the liquid-gas ratio (L/G) is more than 3.5. This research shows that when the inlet dust concentration is ≤ 35 mg/Nm3, the method of "water-washing layer + demisters" can be used as an equivalent alternative to the wet electro-static precipitator when L/G ≥ 3.5, which has reference value for reducing the construction cost of ultra-low emission reformation.

Molecular mechanisms of Baihedihuang decoction as a treatment for breast cancer related anxiety: A network pharmacology and molecular docking study.

BACKGROUND: The therapeutic effects of a combination of Chinese medicines called Baihedihuang decoction (BD) have been clinically verified, although its molecular targets in breast cancer related anxiety remain unknown. AIM: To explore the molecular mechanisms of BD for breast cancer related anxiety treatment. METHODS: We used the Traditional Chinese Medicine Systems Pharmacology database to screen the active ingredients and potential targets of BD, and constructed the "drug-ingredient-target" network map with the help of Cytoscape 3.8 software. Also, we used the Online Mendelian Inheritance in Man, DrugBank, and Gencards databases to collect the disease targets of breast cancer related anxiety, and used the STRING platform to perform protein interaction analysis and construct the protein-protein interaction network. Metascape platform was used for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of key targets. Molecular docking technology was used to verify the drug component/target disease network. RESULTS: We screened 16 active ingredients of BD for breast cancer related anxiety, with 113 target proteins. There are 931 disease targets of breast cancer related anxiety, and finally, 43 key targets and 305 Kyoto Encyclopedia of Genes and Genomes pathways were generated. The main active ingredients of BD for breast cancer related anxiety are verbascoside, ß-sitosterol, stigmasterol, catalpol, etc. CDK2, TP53, HTR2A, ESR1, etc. are its key targets, and the main involved signaling pathways may include neuroactive ligand-receptor interaction pathway, 5-hydroxytryptaminergic synapse, P53 signaling pathway, cGMP-PKG signaling pathway, the cAMP signaling pathway, etc. Finally, molecular docking was performed with Vina software to validate the key active ingredients in BD with the selected key action targets. The molecular docking results showed that verbascoside, ß-sitosterol, stigmasterol and CDK2 could stably bind and interact through amino acid residues SER249, ARG260, PRO228, ALA282, SER276, LYS273, ASN272, etc. CONCLUSION: The therapeutic effect of BD for breast cancer related anxiety is multi-level, multi-target, and multi-pathway. The findings of this study provide ideas and basis for further research.

Soot Combustion over Cu-Co Spinel Catalysts: The Intrinsic Effects of Precursors on Catalytic Activity.

In this work, a series of CuCo2O4-x (x = N, A and C) catalysts were synthesized using different metal salt precursors by urea hydrothermal method for catalytic soot combustion. The effect of CuCo2O4-x catalysts on soot conversion and CO2 selectivity in both loose and tight contact mode was investigated. The CuCo2O4-N catalyst exhibited outstanding catalytic activity with the characteristic temperatures (T10, T50 and T90) of 451 °C, 520 °C and 558 °C, respectively, while the CO2 selectivity reached 98.8% during the reaction. With the addition of NO, the soot combustion was further accelerated over all catalysts. Compared with the loose contact mode, the soot conversion was improved in the tight contact mode. The CuCo2O4-N catalysts showed better textural properties compared to the CuCo2O4-A and CuCo2O4-C, such as higher specific surface areas and pore volumes. The XRD results confirmed that the formation of a CuCo2O4 crystal phase in all catalysts. However, the CuO crystal phase only presented in CuCo2O4-N and CuCo2O4-A. The relative contents of Cu2+, Co3+ and Oads on the surface of CuCo2O4-x (x = N, A and C) catalysts were analyzed by XPS. The CuCo2O4-N catalyst displayed the highest relative content of Cu2+, Co3+ and Oads. The activity of catalytic soot combustion showed a good correlation with the order of the relative contents of Cu2+, Co3+ and Oads. Additionally, the CuCo2O4-N catalyst exhibited lower reduction temperature compared to the CuCo2O4-A and CuCo2O4-C. The cycle tests clarified that the copper-cobalt spinel catalyst obtained good stability. In addition, based on the Mars-van Krevelen mechanism, the process of catalytic soot combustion was described combined with the electron transfer process and the role of oxygen species over CuCo2O4 spinel catalysts.

Machine learning-based transcriptome analysis of lipid metabolism biomarkers for the survival prediction in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with a very high fatality rate. Our goal in this study is to find a reliable lipid metabolism-related signature associated with prognostic significance for HCC. In this study, HCC lipid metabolism-related molecular subtype analysis was conducted based on the 243 lipid metabolism genes collected from the Molecular Signatures Database. Several significant disparities in prognosis, clinicopathological characteristics, and immune and ferroptosis-related status were found across the three subtypes, especially between C1 and C3 subgroups. Differential expression analysis yielded 57 differentially expressed genes (DEGs) between C1 and C3 subtypes. GO and KEGG analysis was employed for functional annotation. Three of 21 prognostic DEGs (CXCL8, SLC10A1, and ADH4) were finally selected through machine-learning-based discovery and validation strategy. The risk score = (0.103) × expression value of CXCL8 + (-0.0333) × expression value of SLC10A1 + (-0.0812) × expression value of ADH4. We used these three to construct a HCC prognostic risk model, which stratified the patients of the validation cohort into two risk subtypes with significantly different overall survival. Our work provides possible significance of the lipid metabolism-associated model in stratifying patient prognosis and its feasibility to guide therapeutic selection.

Caryophyllene Oxide Induces Ferritinophagy by Regulating the NCOA4/FTH1/LC3 Pathway in Hepatocellular Carcinoma.

Ferritinophagy is associated with tumor occurrence, development, and therapy effects. Ferritinophagy and ferroptosis are regulated by iron metabolism and are closely connected. LC3 protein is a key protein in autophagy. Following the binding of NCOA4 to FTH1, it links to LC3Ⅱ in lysosomes, a symbol of ferritinophagy. A ferritinophagy's inducer is likely to open new avenues for anticancer medication research and development. In this study, we discovered that caryophyllene oxide has a substantial inhibitory effect on HCCLM3 and HUH7 cells, by regulating the level of cellular oxidative stress, and the levels of autophagy and iron metabolism in HCCLM3 and HUH7 cells, leading to a ferritinophagy-related phenomenon. Furthermore, the results of T-AOC, DPPH free radical scavenging rate, and hydroxyl radical inhibition indicated that caryophyllene oxide can inhibit cell anti-oxidation. The examination of the ferritinophagy-related process revealed that caryophyllene oxide promotes the production and accumulation of intracellular reactive oxygen species and lipid peroxidation. NCOA4, FTH1, and LC3Ⅱ were found to be targeted regulators of caryophyllene oxide. Caryophyllene oxide regulated NCOA4, LC3 Ⅱ, and FTH1 to promote ferritinophagy. In vivo, we discovered that caryophyllene oxide can lower tumor volume, significantly improve NCOA4 and LC3 protein levels in tumor tissue, and raise Fe2+ and malondialdehyde levels in serum. The compound can also reduce NRF2, GPX4, HO-1, and FTH1 expression levels. The reduction in the expression levels of NRF2, GPX4, HO-1, and FTH1 by caryophyllene oxide also inhibited GSH and hydroxyl radical's inhibitory capacities in serum, and promoted iron deposition in tumor tissue resulting in the inhibition of tumor growth. In summary, our study revealed that caryophyllene oxide mostly kills liver cancer cells through ferritinophagy-mediated ferroptosis mechanisms. In conclusion, caryophyllene oxide may be used as a ferritinophagy activator in the field of antitumor drug research and development.

C1R, CCL2, and TNFRSF1A Genes in Coronavirus Disease-COVID-19 Pathway Serve as Novel Molecular Biomarkers of GBM Prognosis and Immune Infiltration.

Glioblastoma multiforme (GBM) is a prevalent intracranial brain tumor associated with a high rate of recurrence and treatment difficulty. The prediction of novel molecular biomarkers through bioinformatics analysis may provide new clues into early detection and eventual treatment of GBM. Here, we used data from the GTEx and TCGA databases to identify 1923 differentially expressed genes (DEGs). GO and KEGG analyses indicated that DEGs were significantly enriched in immune response and coronavirus disease-COVID-19 pathways. Survival analyses revealed a significant correlation between high expression of C1R, CCL2, and TNFRSF1A in the coronavirus disease-COVID-19 pathway and the poor survival in GBM patients. Cell experiments indicated that the mRNA expression levels of C1R, CCL2, and TNFRSF1A in GBM cells were very high. Immune infiltration analysis revealed a significant difference in the proportion of immune cells in tumor and normal tissue, and the expression levels of C1R, CCL2, and TNFRSF1A were associated with immune cell infiltration of GBM. Additionally, the protein-protein interaction networks of C1R, CCL2, and TNFRSF1A involved a total of 65 nodes and 615 edges. These results suggest that C1R, CCL2, and TNFRSF1A may be used as molecular biomarkers of prognosis and immune infiltration in GBM patients in the future.

DJ-1/FGFR-1 Signaling Pathway Contributes to Sorafenib Resistance in Hepatocellular Carcinoma.

Sorafenib is the first-line therapeutic regimen targeting against advanced or metastatic stage of hepatocellular carcinoma (HCC). However, HCC patients at these stages will eventually fail sorafenib treatment due to the drug resistance. At present, molecular mechanisms underlying sorafenib resistance are not completely understood. Our past studies have shown that DJ-1 is upregulated in HCC, while DJ-1 knockdown inhibits HCC xenograft-induced tumor growth and regeneration, implying that DJ-1 may be a potential target in for HCC treatment. However, whether DJ-1 plays a regulatory role between tumor cells and vascular endothelial cells and whether DJ-1 contributes to sorafenib resistance in HCC cells are largely unclear. To address these questions, we have performed a series of experiments in the current study, and we found that (1) DJ-1, one of the molecules secreted from HCC cells, promoted angiogenesis and migration of vascular endothelial cells (i.e., ECDHCC-1), by inducing phosphorylation of fibroblast growth factor receptor-1 (FGFR-1), phosphorylation of mTOR, phosphorylation of ERK, and phosphorylation of STAT3; (2) downregulation of FGFR1 inhibited tube formation and migration of ECDHCC-1 cells stimulated by DJ-1; (3) FGFR1 knockdown attenuated the phosphorylation of FGFR1 and impaired the activity of Akt, ERK, and STAT3 signals induced by DJ-1 in ECDHCC-1 cells; (4) knocking down FGFR1 led to the elevated expression of proapoptotic molecules but deceased level of antiapoptotic molecules in sorafenib-resistant HCC cells; and (5) Downregulation of FGFR1 suppressed tumor growth and angiogenesis of sorafenib-resistant HCC cells in vivo. Altogether, our results hinted that DJ-1 plays vital roles in tumor microenvironment in HCC development, and DJ-1/FGFR1 signaling pathway may be a therapeutic target for overcoming sorafenib resistance in treating HCC patients at the late stage.

Berberine exerts its antineoplastic effects by reversing the Warburg effect via downregulation of the Akt/mTOR/GLUT1 signaling pathway.

Glucose transporter 1 (GLUT1) plays a primary role in the glucose metabolism of cancer cells. However, to the best of our knowledge, there are currently no anticancer drugs that inhibit GLUT1 function. The present study aimed to investigate the antineoplastic activity of berberine (BBR), the main active ingredient in numerous Traditional Chinese medicinal herbs, on HepG2 and MCF7 cells. The results of Cell Counting Kit­8 assay, colony formation assay and flow cytometry revealed that BBR effectively inhibited the proliferation of tumor cells, and induced G2/M cell cycle arrest and apoptosis. Notably, the results of luminescence ATP detection assay and glucose uptake assay showed that BBR also significantly inhibited ATP synthesis and markedly decreased the glucose uptake ability, which suggested that the antitumor effect of BBR may occur via reversal of the Warburg effect. In addition, the results of reverse transcription­quantitative PCR, western blotting and immunofluorescence staining indicated that BBR downregulated the protein expression levels of GLUT1, maintained the cytoplasmic internalization of GLUT1 and suppressed the Akt/mTOR signaling pathway in both HepG2 and MCF7 cell lines. Augmentation of Akt phosphorylation levels by the Akt activator, SC79, abolished the BBR­induced decrease in ATP synthesis, glucose uptake, GLUT1 expression and cell proliferation, and reversed the proapoptotic effect of BBR. These findings indicated that the antineoplastic effect of BBR may involve the reversal of the Warburg effect by downregulating the Akt/mTOR/GLUT1 signaling pathway. Furthermore, the results of the co­immunoprecipitation assay demonstrated that BBR increased the interaction between ubiquitin conjugating enzyme E2 I (Ubc9) and GLUT1, which suggested that Ubc9 may mediate the proteasomal degradation of GLUT1. On the other hand, BBR decreased the interaction between Gα­interacting protein­interacting protein at the C­terminus (GIPC) and GLUT1, which suggested that the retention of GLUT1 in the cytoplasm may be achieved by inhibiting the interaction between GLUT1 and GIPC, thereby suppressing the glucose transporter function of GLUT1. The results of the present study provided a theoretical basis for the application of the Traditional Chinese medicine component, BBR, for cancer treatment.

Dissecting Tumor Antigens and Immune Subtypes of Glioma to Develop mRNA Vaccine.

Background: Nowadays, researchers are leveraging the mRNA-based vaccine technology used to develop personalized immunotherapy for cancer. However, its application against glioma is still in its infancy. In this study, the applicable candidates were excavated for mRNA vaccine treatment in the perspective of immune regulation, and suitable glioma recipients with corresponding immune subtypes were further investigated. Methods: The RNA-seq data and clinical information of 702 and 325 patients were recruited from TCGA and CGGA, separately. The genetic alteration profile was visualized and compared by cBioPortal. Then, we explored prognostic outcomes and immune correlations of the selected antigens to validate their clinical relevance. The prognostic index was measured via GEPIA2, and infiltration of antigen-presenting cells (APCs) was calculated and visualized by TIMER. Based on immune-related gene expression, immune subtypes of glioma were identified using consensus clustering analysis. Moreover, the immune landscape was visualized by graph learning-based dimensionality reduction analysis. Results: Four glioma antigens, namely ANXA5, FKBP10, MSN, and PYGL, associated with superior prognoses and infiltration of APCs were selected. Three immune subtypes IS1-IS3 were identified, which fundamentally differed in molecular, cellular, and clinical signatures. Patients in subtypes IS2 and IS3 carried immunologically cold phenotypes, whereas those in IS1 carried immunologically hot phenotype. Particularly, patients in subtypes IS3 and IS2 demonstrated better outcomes than that in IS1. Expression profiles of immune checkpoints and immunogenic cell death (ICD) modulators showed a difference among IS1-IS3 tumors. Ultimately, the immune landscape of glioma elucidated considerable heterogeneity not only between individual patients but also within the same immune subtype. Conclusions: ANXA5, FKBP10, MSN, and PYGL are identified as potential antigens for anti-glioma mRNA vaccine production, specifically for patients in immune subtypes 2 and 3. In summary, this study may shed new light on the promising approaches of immunotherapy, such as devising mRNA vaccination tailored to applicable glioma recipients.

Analytical and Clinical Performance of a Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Gastrin Subtypes G34 and G17 in Serum.

BACKGROUND: Two major forms of gastrin, gastrin-17 (G17) and gastrin-34 (G34), exist in blood. However, conventional immunoassay methods can only quantify total gastrin or G17 alone. Here, we aimed to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify G17 and G34 simultaneously. METHODS: Serum samples were prepared by anion-exchange solid-phase extraction. The analytical performance of the LC-MS/MS method was validated and the method was compared to chemiluminescence immunoassay (CLIA) and radioimmunoassay (RIA). The G17 and G34 concentrations in 245 serum samples from healthy controls, individuals with gastrinoma, and individuals with other diseases were analyzed. RESULTS: The total runtime of the LC-MS/MS method was 6 min. No substantial matrix effect was observed with internal standard correction. The intraassay coefficients of variation (CVs) for G17 and G34 were 4.0%-14.2% and 4.4%-10.4%, respectively, and total CVs were 5.2%-14.1% and 4.6%-12.4%, respectively. The correlation coefficient between LC-MS/MS and CLIA was 0.87, and between LC-MS/MS and RIA was 0.84. The G17+G34 concentrations for 87.5% of individuals with gastrinoma were higher than the 95th percentile of healthy controls (18.1 pg/mL), whereas the concentrations for individuals with other diseases and gastrinoma overlapped. Based on the Youden indices calculated for G17+G34, G34, and G17, the most specific biomarker was G17 (96.9% clinical specificity at 209.8 pg/mL) for gastrinoma. CONCLUSIONS: This method should aid in the diagnosis of diseases associated with increased gastrin concentrations.

Dissecting Prognosis Modules and Biomarkers in Glioblastoma Based on Weighted Gene Co-Expression Network Analysis.

INTRODUCTION: As one of the most prevalent and malignant brain cancers, glioblastoma multiforme (GBM) presents a poor prognosis and the molecular mechanisms remain poorly understood. Consequently, molecular research, including various biomarkers, is essential to exploit the occurrence and development of glioma. METHODS: Weighted gene co-expression network analysis (WGCNA) was used to construct gene co-expression modules and networks based on the Chinese Glioma Genome Atlas (CGGA) glioblastoma specimens. Then, protein-protein interaction (PPI) and gene ontology (GO) analyses were performed to mine hub genes. RT-PCR and immunohistochemistry were employed to examine the expression level of GRPR, CXCL5, and CXCL11 in glioma patients. RESULTS: We confirmed two gene modules by protein-protein interaction networks. Functional enrichment analysis was performed to identify the significance of gene modules. Prognostic biomarkers GRPR, CXCL5, and CXCL11 related to the survival time of GBM samples were mined in The Cancer Genome Atlas (TCGA) dataset. qRT-PCR revealed that GRPR, CXCL5, and CXCL11 led to a significant increase in GBM sample compared to control. CONCLUSION: In this study, we developed and confirmed three mRNA signatures (GRPR, CXCL5, and CXCL11) for evaluating overall survival in GBM patients. Our research assists in existing understanding of GBM diagnosis and prognosis.

Immunohistochemical study of STAT3, HIF-1α and VEGF in pterygium and normal conjunctiva: Experimental research and literature review.

Purpose: Signal transducer and activator of transcription 3 (STAT3) is a DNA-binding protein that regulates various biologic processes, including cell growth, apoptosis, and malignant transformation. Abnormal activation of STAT3 is associated with many diseases, and there is currently no relevant study on the pathogenesis of pterygium. The purpose of this study was to investigate the expression and clinical significance of STAT3, HIF-1α, and VEGF in pterygium at different stages. Methods: Immunohistochemistry was used to study the expression levels of STAT3, HIF-1α, and VEGF in 50 cases of pterygium and 20 cases of control conjunctival tissue. The expression intensity of the three proteins was evaluated with Image-Pro Plus 6.0 image analysis software. Results: In the pterygium group, the positive rates for STAT3, HIF-1α, and VEGF were 82.0%, 86.0%, and 84.0%, respectively, while those in the normal conjunctiva group were 40.0%, 25.0%, and 15.0%. The expression of STAT3, HIF-1α, and VEGF in pterygium was higher than that in control conjunctiva, and the expression in advanced pterygium was statistically significantly higher than that in stationary pterygium (p < 0.01). The expression levels of STAT3 and HIF-1α in pterygium were related to the length and depth of the corneal invasion of pterygium. The expression level of VEGF in pterygium was related to the length of pterygium, but not to the depth. In addition, there was a significant positive correlation between the expression of STAT3, HIF-1α, and VEGF (p < 0.01). Conclusions: For the first time, the expression levels of the STAT3, HIF-1α, and VEGF proteins were detected simultaneously in pterygium tissue. Compared with normal conjunctiva, STAT3, HIF-1α, and VEGF were highly expressed in pterygium, and the expression in advanced pterygium tissue was more significant than in the stationary pterygium tissue. It is suggested that STAT3 may directly or through HIF-1α promote VEGF expression and participate in the growth and angiogenesis of pterygium. Targeting STAT3 may provide a new direction for the treatment of pterygium.

A novel TSC1 frameshift mutation c.1550_1551del causes tuberous sclerosis complex by aberrant splicing and nonsense-mediated mRNA degradation (NMD) simultaneously in a Chinese family.

BACKGROUND: Tuberous sclerosis complex (TSC), belongs to autosomal dominant genetic disorder, which affects multiple organ systems in the body, including the skin, brain, lungs, kidneys, liver, and eyes. Mutations in TSC1 or TSC2 was proved to be associated with these conditions. METHODS: Gene-panel Sequence of NGS was used to detect the mutation in a Chinese family. The research further investigates whether aberrant splicing and nonsense-mediated mRNA degradation (NMD) could serve as a mechanism cause by TSC1 mutation. MINI-Gene assay apply by pcMINI-TSC1wt/mut plasmids delivered in HeLa and 293T cell lines. Recombinant plasmids expressing wild-type and mutant-type EGFP-TSC1 were constructed and transiently transfected into human embryonic kidney cells 293T by lipofectamine. Real-time PCR and Western Blot were performed to analyze the expression of mRNAs and proteins of EGFP-TSC1 and NMD factor UPF1. RESULTS: The gene test verified a novel heterozygous TSC1 frameshift mutation (TSC1 c.1550_1551del) in the proband and her mother. From MINI-Gene assay, the agarose gel showed that both the mutant and wild-type mRNA possess two main bands, indicating two splicing modes, named band A and B, respectively. The mutation c.1550_1551del has not produced new splicing site, but there is a selective splicing in varying degree significantly after mutation. On the contrary, function validation assay showed that cells transfected with the mutant TSC1 plasmids expressed significantly lower TSC1 in mRNAs and proteins levels, compared with the wild-type TSC1 plasmid transfection. A translation inhibitor cycloheximide and small interfering RNA of UPF1 (siRNA-UPF1) increased mRNA or protein expression of TSC1 significantly in cells transfected with the mutant plasmids. CONCLUSION: Our study demonstrated that the novel TSC1 frameshift mutation (TSC1 c.1550_1551del) trigger aberrant splicing and NMD simultaneously, causing decrease of hamartin, then, leading to tuberous sclerosis complex formation.

Folate receptor-positive circulating tumor cells as a predictive biomarker for the efficacy of first-line pemetrexed-based chemotherapy in patients with non-squamous non-small cell lung cancer.

BACKGROUND: There is a lack of well-established biomarkers to predict the efficacy of pemetrexed-based chemotherapy. In this prospective phase II study, we investigated the correlation of folate receptor (FR)-positive circulating tumor cell (CTC) level with the clinical outcomes of patients with advanced non-squamous non-small cell lung cancer (nsNSCLC) when treated with pemetrexed-based chemotherapy. METHODS: A total of 98 nsNSCLC patients were enrolled. Peripheral blood was collected from each patient prior to initiation of treatment. FR-positive CTCs were enriched by immunomagnetic leukocyte depletion and quantified using ligand-targeted polymerase chain reaction (LT-PCR) method. RESULTS: Patients with relatively low CTC level (11-16 FU/3 mL, n=32) showed a significantly shorter progression-free survival (PFS) and overall survival (OS) compared with those in the "high CTC level group" (>16 FU/3mL, n=28; median PFS, 133 versus 320 days, P<0.001; median OS, 632 days versus "not reached", P=0.003). Patients in the "high CTC level group" also achieved superior objective response rate (ORR) and disease control rate (DCR) over those in the "low CTC level group" (ORR, 40.9% versus 9.5%, P=0.0339; DCR, 100% versus 81.0%, P=0.0485). The clinical outcomes of pemetrexed in the "negative-CTC group" (<11 FU/3mL, n=38) fell between the "high CTC level group" and the "low CTC level group" (median PFS, 290 days; median OS, 1,122 days; ORR: 21.2%, DCR: 93.9%). Further multivariate Cox proportional hazards regression analysis demonstrated that "high CTC level" was an independent factor that was significantly associated with better PFS [hazard ratio (HR) =0.26, 95% confidence interval (CI), 0.12-0.58, P=0.001] and OS (HR =0.23, 95% CI, 0.06-0.92, P=0.037). CONCLUSIONS: Our results implied that FR-positive CTC is a promising biomarker to predict the clinical outcome of pemetrexed-based chemotherapy in patients with advanced nsNSCLC.

tRFs as Potential Exosome tRNA-Derived Fragment Biomarkers for Gastric Carcinoma.

BACKGROUND: Gastric Carcinoma (GC) is one of the common diseases induced by the interaction of genes and environment. Exosomes are potential markers for several health problems, which contain lipids, proteins, long non-coding RNAs, microRNAs (miRNAs), and tRNA-derived fragments (tRFs). The roles of mRNAs and miRNAs in GC have been studied comprehensively; however, little research was focused on the function of plasma exosomal tRFs. METHODS: We collected plasma samples from fifty healthy controls and fifty GC patients, and all exosomes were isolated with a combined centrifugation and characterized by electron microscopy, western blot, and flow cytometry. The small RNA sequence was performed to detect the plasma exosomal tRFs, and tRFs markers were validated by real-time quantitative PCR. Three exosomal diagnostic tRFs were confirmed by receiver operating characteristic analyses. RESULTS: In this study, we found higher plasma exosomal tRF-25, tRF-38, tRF-18 expression in GC than in controls. Plasma exosomal tRF-25, tRF-38, and tRF-18 showed better accuracy for GC diagnosis. CONCLUSIONS: Our results suggest that plasma exosomal tRF-25, tRF-38, and tRF-18 were biomarkers for GC detection; tRF-25, tRF-38 and tRF-18 might be predictive of GC prognosis.

Trillin prevents proliferation and induces apoptosis through inhibiting STAT3 nuclear translocation in hepatoma carcinoma cells.

Trillin is a constituent of total Trillium Tschonoskii Maxim (TTM), which is extracted from TTM and displayed anti-tumor effect in many tumor cell lines. However, the anti-tumor mechanism of trillin is still unclear. This study demonstrated that trillin could dramatically inhibit hepatoma carcinoma cell proliferation, induce apoptosis and decrease migration and invasion through suppressing phosphorylated STAT3 translocated to nucleus. Trillin could down-regulate Bcl-2 and Survivin, up-regulate cleaved PRAP, leading to dramatically apoptosis; trillin could also down-regulate MMP1, MMP2, MucI and VEGF, which displayed an inhibition effect on hepatocellular tumor cells invasion and development. The results of this study indicated the potential utility of trillin as a STAT3 inhibitor for the treatment of cancers.

lncRNA THAP9-AS1 Promotes Pancreatic Ductal Adenocarcinoma Growth and Leads to a Poor Clinical Outcome via Sponging miR-484 and Interacting with YAP.

PURPOSE: Long noncoding RNAs (lncRNA) have been observed in various cancer types. Our bioinformatic analysis of existing databases demonstrated overexpression of lncRNA THAP9-AS1 in pancreatic ductal adenocarcinoma (PDAC). We aimed to investigate the roles and mechanisms of THAP9-AS1 in PDAC. EXPERIMENTAL DESIGN: The overexpression of THAP9-AS1 in samples of patients with pancreatic cancer was characterized and was associated with clinical outcomes. The nonprotein coding property of the THAP9-AS1 was verified. Various in vitro and in vivo experiments were performed to investigate the interaction between THAP9-AS1 and YAP signaling. RESULTS: We demonstrated that lncRNA THAP9-AS1 is overexpressed in PDAC in multiple patient sample sets, which is significantly associated with poor outcome of patients with PDAC. THAP9-AS1 promotes PDAC cells growth both in vitro and in vivo. THAP9-AS1 exerts its effects via enhancing YAP signaling. Ectopic YAP expression overcame the effects of THAP9-AS1 knockdown. Inversely, YAP knockdown diminished the effects of THAP9-AS1 overexpression. THAP9-AS1 acts as a competing endogenous RNA for miR-484, leading to YAP upregulation. Moreover, THAP9-AS1 binds to YAP protein and inhibits the phosphorylation-mediated inactivation of YAP by LATS1. Reciprocally, YAP/TEAD1 complex promotes THAP9-AS1 transcription to form a feed-forward circuit. Importantly, THAP9-AS1 level positively correlates with YAP expression in PDAC tissues. YAP overexpression also predicts a poor outcome in patients with PDAC. CONCLUSIONS: Our findings indicate that THAP9-AS1 plays an important role in PDAC growth via enhancing YAP signaling, which in turn also modulates THAP9-AS1 transcription. THAP9-AS1/YAP axis may serve as a potential biomarker and therapeutic target for PDAC treatment.