NSCLC extracellular vesicles containing miR-374a-5p promote leptomeningeal metastasis by influencing blood‒brain barrier permeability.

Leptomeningeal metastasis (LM) is a devastating complication of advanced non-small cell lung cancer (NSCLC). Diagnosis and monitoring of LM can be challenging. Extracellular vesicles (EVs) microRNAs (miRNAs) have become a new noninvasive diagnostic biomarker. The purpose of this study was to examine the clinical value and role of EVs miRNAs in NSCLC-LM. According to next-generation sequencing (NGS), miRNAs with differential expression of EVs in serum of NSCLC patients with LM and non-LM were detected to find biological markers for the diagnosis of LM. Cellular and in vivo experiments were conducted to explore the pathogenesis of EVs miRNA promoting LM in NSCLC. In the present study, we first demonstrated the serum level of EV-associated miR-374a-5p in patients with LM of lung cancer was much higher than that in patients without LM and was correlated with the survival time of patients with LM. Further studies showed that EVs miR-374a-5p efficiently destroys tight junctions and the integrity of the cerebral microvascular endothelial cell barrier, resulting in increased blood-brain barrier (BBB) permeability. Mechanistically, miR-374a-5p regulates the distribution of ZO-1 and occludin in endothelial cells by targeting ADD3, increasing vascular permeability and promoting LM. Implications: These results suggest that serum NSCLC-derived EVs miR-374a-5p is involved in premetastatic niche formation by regulating the permeability of BBB to promote NSCLC-LM, and can be used as a blood biomarker for the diagnosis and prognosis of NSCLC-LM.

Application of enhanced recovery after surgery during the perioperative period in children with Meckel's diverticulum-a single-center prospective clinical trial.

Background: Enhanced recovery after surgery (ERAS) has been widely used in adult surgery. However, few studies have reported the efficacy of ERAS in paediatric patients with Meckel's diverticulum (MD), the aim of the study was to prospectively evaluate the safety and efficacy of ERAS in treating MD. Methods: A prospective randomised controlled study of children with MD admitted to our hospital from Jan 1, 2021 to Dec 31, 2023 were conducted, we developed and implemented an ERAS program for this patients. All cases were strictly selected according to the inclusion and exclusion criteria. Among these patients, they were randomly assigned to the ERAS group or the traditional (TRAD) group with random number table row randomization. The main observational indicators were operation time, intraoperative hemorrhage, FLACC pain scale results on 2 h, 6 h, 12 h, 24 h after surgery, length of postoperative stay (LOPS), time to first defecation, time to first eating after surgery, time to discontinuation of intravenous infusion, total treatment cost, incidence of postoperative complications, 30-day readmission rate and parental satisfaction rate. Results: A total of 50 patients underwent Meckel's diverticulectomy during this period, 7 patients were excluded, 23 patients were assigned to the ERAS group and 20 patients were assigned to the TRAD group. There were no significant differences in demographic data and operation time, intraoperative hemorrhage. The FLACC pain scale results on 2 h, 6 h, 12 h, 24 h after surgery were significantly lower in the ERAS group. The LOPS was 6.17 ± 0.89 days in the ERAS group and 8.30 ± 1.26 days in the TRAD group, resulting in a significantly shorter LOPS in ERAS group. ERAS could also reduce the first postoperative defecation time, the time to first eating after surgery and the time to discontinuation of intravenous infusion. The treatment cost was decreased in the ERAS group. The rate of complications and 30-day readmission were not significantly different between the two groups. Conclusions: In this single-center study, the ERAS protocol for patients with MD requiring surgery was safe and effective.

Detection of pTDP-43 via routine muscle biopsy: A promising diagnostic biomarker for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, pathologically characterized by TDP-43 aggregates. Recent evidence has been indicated that phosphorylated TDP-43 (pTDP-43) is present not only in motor neurons but also in muscle tissues. However, it is unclear whether testing pTDP-43 aggregation in muscle tissue would assist in the diagnosis of ALS. We propose three key questions: (i) Is aggregation of pTDP-43 detectable in routine biopsied muscles? (ii) Can detection of pTDP-43 aggregation discriminate between ALS and non-ALS patients? (iii) Can pTDP-43 aggregation be observed in the early stages of ALS? We conducted a diagnostic study comprising 2 groups: an ALS group in which 18 cases underwent muscle biopsy screened from a registered ALS cohort consisting of 802 patients and a non-ALS control group, in which we randomly selected 54 muscle samples from a biospecimen bank of 684 patients. Among the 18 ALS patients, 3 patients carried pathological GGGGCC repeats in the C9ORF72 gene, 2 patients carried SOD1 mutations, and 7 patients were at an early stage with only one body region clinically affected. The pTDP-43 accumulation could be detected in routine biopsied muscles, including biceps brachii, deltoid, tibialis anterior, and quadriceps. Abnormal aggregation of pTDP-43 was present in 94.4% of ALS patients (17/18) compared to 29.6% of non-ALS controls (16/54; p < 0.001). The pTDP-43 aggregates were mainly close to the sarcolemma. Using a semi-quantified pTDP-43 aggregates score, we applied a cut-off value of 3 as a diagnostic biomarker, resulting in a sensitivity of 94.4% and a specificity of 83.3%. Moreover, we observed that accumulation of pTDP-43 occurred in muscle tissues prior to clinical symptoms and electromyographic lesions. Our study provides proof-of-concept for the detection of pTDP-43 accumulation via routine muscle biopsy which may serve as a novel biomarker for diagnosis of ALS.

Glycol Monomethyl Ether-Substituted Carbazolyl Hole-Transporting Material for Stable Inverted Perovskite Solar Cells with Efficiency of 25.52.

Organic self-assembled molecules (OSAMs) based hole transporting materials play a pivotal role in achieving highly efficient and stable inverted perovskite solar cells (IPSCs). However, the reported carbazol-based OSAMs have serious drawbacks, such as poor solubility in alcohol solution, worse matched energy arrangement with perovskite, and limited molecular species, which greatly limit the device performance. To address above problems, a novel OSAM 4-(3,6-glycol monomethyl ether-9H-carbazol-9-yl) butyl]phosphonic acid (GM-4PACz) was synthesized as hole-transporting material by introducing glycol monomethyl ether (GM) side chains at carbazolyl unit. GM groups enhance the surface energy of Indium Tin Oxide (ITO)/SAM substrate to facilitate the nucleation and growth of up perovskite film, suppress cation defects, release the residual stress at SAM/perovskite interface, and evaluate energy level for matching with perovskite. Consequently, the GM-4PACz based IPSC achieves a champion PCE of 25.52%, a respectable open-circuit voltage (VOC) of 1.21 V, a high stability, possessing 93.29% and 91.75% of their initial efficiency after aging in air for 2000 h or tracking at maximum power point for 1000 h, respectively.

CircHERC1 promotes non-small cell lung cancer cell progression by sequestering FOXO1 in the cytoplasm and regulating the miR-142-3p-HMGB1 axis.

BACKGROUND: Noncoding RNAs such as circular RNAs (circRNAs) are abundant in the human body and influence the occurrence and development of various diseases. Non-small cell lung cancer (NSCLC) is one of the most common malignant cancers. Information on the functions and mechanism of circRNAs in lung cancer is limited; thus, the topic needs more exploration. The purpose of this study was to identify aberrantly expressed circRNAs in lung cancer, unravel their roles in NSCLC progression, and provide new targets for lung cancer diagnosis and therapy. METHODS: High-throughput sequencing was used to analyze differential circRNA expression in patients with lung cancer. qRT‒PCR was used to determine the level of circHERC1 in lung cancer tissues and plasma samples. Gain- and loss-of-function experiments were implemented to observe the impacts of circHERC1 on the growth, invasion, and metastasis of lung cancer cells in vitro and in vivo. Mechanistically, dual luciferase reporter assays, fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down experiments were performed to confirm the underlying mechanisms of circHERC1. Nucleocytoplasmic localization of FOXO1 was determined by nucleocytoplasmic isolation and immunofluorescence. The interaction of circHERC1 with FOXO1 was verified by RNA pull-down, RNA immunoprecipitation (RIP) and western blot assays. The proliferation and migration of circHERC1 in vivo were verified by subcutaneous and tail vein injection in nude mice. RESULTS: CircHERC1 was significantly upregulated in lung cancer tissues and cells, ectopic expression of circHERC1 strikingly facilitated the proliferation, invasion and metastasis, and inhibited the apoptosis of lung cancer cells in vitro and in vivo. However, knockdown of circHERC1 exerted the opposite effects. CircHERC1 was mainly distributed in the cytoplasm. Further mechanistic research indicated that circHERC1 acted as a competing endogenous RNA of miR-142-3p to relieve the repressive effect of miR-142-3p on its target HMGB1, activating the MAPK/ERK and NF-κB pathways and promoting cell migration and invasion. More importantly, we found that circHERC1 could bind FOXO1 and sequester it in the cytoplasm, adjusting the feedback AKT pathway. The accumulation of FOXO1 in the cytosol and nuclear exclusion promoted cell proliferation and inhibited apoptosis. CircHERC1 is a new circRNA that promotes tumor function in NSCLC and may serve as a potential prognostic biomarker and therapeutic target for NSCLC. CONCLUSIONS: CircHERC1 is a new circRNA that promotes tumor function in NSCLC and may serve as a potential diagnosis biomarker and therapeutic target for NSCLC. Our findings indicate that circHERC1 facilitates the invasion and metastasis of NSCLC cells by regulating the miR-142-3p/HMGB1 axis and activating the MAPK/ERK and NF-κB pathways. In addition, circHERC1 can promote cell proliferation and inhibit apoptosis by sequestering FOXO1 in the cytoplasm to regulate AKT activity and BIM transcription.

Plasma exosomal hsa_circ_0079439 as a novel biomarker for early detection of gastric cancer.

BACKGROUND: Due to the poor prognosis of gastric cancer (GC), early detection methods are urgently needed. Plasma exosomal circular RNAs (circRNAs) have been suggested as novel biomarkers for GC. AIM: To identify a novel biomarker for early detection of GC. METHODS: Healthy donors (HDs) and GC patients diagnosed by pathology were recruited. Nine GC patients and three HDs were selected for exosomal whole-transcriptome RNA sequencing. The expression profiles of circRNAs were analyzed by bioinformatics methods and validated by droplet digital polymerase chain reaction. The expression levels and area under receiver operating characteristic curve values of plasma exosomal circRNAs and standard serum biomarkers were used to compare their diagnostic efficiency. RESULTS: There were 303 participants, including 240 GC patients and 63 HDs, involved in the study. The expression levels of exosomal hsa_circ_0079439 were significantly higher in GC patients than in HDs (P < 0.0001). However, the levels of standard serum biomarkers were similar between the two groups. The area under the curve value of exosomal hsa_circ_0079439 was higher than those of standard biomarkers, including carcinoembryonic antigen, carbohydrate antigen (CA)19-9, CA72-4, alpha-fetoprotein, and CA125 (0.8595 vs 0.5862, 0.5660, 0.5360, 0.5082, and 0.5018, respectively). The expression levels of exosomal hsa_circ_0079439 were significantly decreased after treatment (P < 0.05). Moreover, the expression levels of exosomal hsa_circ_0079439 were obviously higher in early GC (EGC) patients than in HDs (P < 0.0001). CONCLUSION: Our results suggest that plasma exosomal hsa_circ_0079439 is upregulated in GC patients. Moreover, the levels of exosomal hsa_circ_0079439 could distinguish EGC and advanced GC patients from HDs. Therefore, plasma exosomal hsa_circ_0079439 might be a potential biomarker for the diagnosis of GC during both the early and late stages.

Generation and characterization of an inducible renal proximal tubule-specific CreERT2 mouse.

Protein reabsorption in renal proximal tubules is essential for maintaining nutrient homeostasis. Renal proximal tubule-specific gene knockout is a powerful method to assess the function of genes involved in renal proximal tubule protein reabsorption. However, the lack of inducible renal proximal tubule-specific Cre recombinase-expressing mouse strains hinders the study of gene function in renal proximal tubules. To facilitate the functional study of genes in renal proximal tubules, we developed an AMN CreERT2 knock-in mouse strain expressing a Cre recombinase-estrogen receptor fusion protein under the control of the promoter of the amnionless (AMN) gene, a protein reabsorption receptor in renal proximal tubules. AMN CreERT2 knock-in mice were generated using the CRISPR/Cas9 strategy, and the tissue specificity of Cre activity was investigated using the Cre/loxP reporter system. We showed that the expression pattern of CreERT2-mEGFP in AMN CreERT2 mice was consistent with that of the endogenous AMN gene. Furthermore, we showed that the Cre activity in AMN CreERT2 knock-in mice was only detected in renal proximal tubules with high tamoxifen induction efficiency. As a proof-of-principle study, we demonstrated that renal proximal tubule-specific knockout of Exoc4 using AMNCreERT2 led to albumin accumulation in renal proximal tubular epithelial cells. The AMN CreERT2 mouse is a powerful tool for conditional gene knockout in renal proximal tubules and should offer useful insight into the physiological function of genes expressed in renal proximal tubules.

Alpha-kinase1 promotes tubular injury and interstitial inflammation in diabetic nephropathy by canonical pyroptosis pathway.

BACKGROUND: Alpha-kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1ß in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1ß and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined. METHODS: Immunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1ß, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1ß and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD. RESULTS: The heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68+ macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1ß in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1ß, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA. CONCLUSIONS: Our data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.

circTAB2 inhibits lung cancer proliferation, migration and invasion by sponging miR-3142 to upregulate GLIS2.

Circular RNAs (circRNAs) are a specialized circular structure, are deregulated in cancers and play essential roles in biological processes involved in tumor progression. However, the mechanism by which circRNAs affect lung tumorigenesis and progression remains largely unexplored. To investigate the role of circRNA in lung cancer, circRNA expression profile was screened by bioinformatics analysis. The levels of circTAB2, miR-3142, and GLIS family zinc finger 2 (GLIS2) were measured by quantitate real-time (qRT-PCR) or western blot. Cell proliferation, apoptosis, migration and invasion were detected by EdU, flow cytometry, and transwell assays, respectively. Bioinformatics, western blot, RIP, pull down, dual luciferase reporter and rescue experiments were used to verify the direct relationship between miR-3142 and circTAB2 or GLIS2. The xenograft assays were used to assess the role of circTAB2 in vivo.CircTAB2 exhibited low expression in cancer tissues. Gain and loss-of-function assays indicated that circTAB2 could inhibit cell proliferation, migration and invasion. Functional studies revealed that circTAB2 acted as a miRNA sponge, directly interacted with miR-3142 and consequently regulated GLIS2 /AKT. Taken together, circTAB2 serves as an inhibitory role in lung cancer through a novel circTAB2 /miR-3142 /GLIS2 /AKT pathway and could be exploited a novel marker in lung cancer.

Novel staging for gastric neuroendocrine neoplasms by incorporating the WHO grading into the TNM staging system.

BACKGROUND: The 8th tumor-node-metastasis (TNM) classification of the American Joint Committee on Cancer (AJCC) can be used to estimate the prognosis of gastric neuroendocrine tumor (gNET) and gastric neuroendocrine carcinoma (gNEC) patients but not gastric neuroendocrine neoplasms (gNENs). METHODS: First, in the SEER (training) dataset, a TNMG system was built by combining the WHO G grade (G1-4; NEC grouped into G4) with the 8th AJCC T (T1-4), N (N0-1), and M (M0-1) stage, which was then validated in a Chinese (validation) cohort. RESULTS: In all, 2245 gNENs cases from the training dataset and 280 cases from the validation dataset were eligible. The T stage, M stage, and G grade were independent prognostic factors for OS in both datasets (all p < 0.05). The TNMG staging system demonstrated better C-index for predicting OS than the 8th AJCC TNM staging system in both the training (0.87, 95%CI: 0.86-0.88 vs. 0.79, 95%CI: 0.77-0.81) and validation (0.77, 95%CI: 0.73-0.80 vs. 0.75, 95%CI: 0.71-0.79) datasets. The AUC of the 3-year OS for the TNMG staging system was 0.936 and 0.817 in the SEER and validation dataset, respectively; higher than those of the 8th AJCC system (vs. 0.843 and 0.779, respectively). DCA revealed that compared with the 8th AJCC TNM staging system, the TNMG staging system demonstrated superior net prognostic benefit in both the training and validation datasets. CONCLUSIONS: The proposed TNMG staging system could more accurately predict the 3- and 5-year OS rate of gNENs patients than the 8th AJCC TNM staging system.

Alpha-kinase1 promotes tubular injury and interstitial inflammation in diabetic nephropathy by canonical pyroptosis pathway

BACKGROUND: Alpha-kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1ß in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1ß and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined. METHODS: Immunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1ß, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1ß and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD. RESULTS: The heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68+ macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1ß in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1ß, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA. CONCLUSIONS: Our data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.

Gasdermin D is involved in switching from apoptosis to pyroptosis in TLR4-mediated renal tubular epithelial cells injury in diabetic kidney disease.

BACKGROUND: Toll-like receptor 4 (TLR4) mediated pathway plays a pivotal role in promoting tubulointerstitial inflammation and contributes to the progression in type 2 diabetic kidney disease (T2DKD). As the first identified key pyroptosis executor, gasdermin D (GSDMD) is activated by caspases and might be the key protein to switch apoptosis to pyroptosis. It remains unclear that role of TLR4 on canonical pyroptosis pathway, and whether GSDMD is involved in switching from apoptosis to pyroptosis in the TLR4-related tubular injury in T2DKD. METHODS: Immunohistochemistry staining was used to detect the expression of pyroptosis-related proteins in renal tissues of T2DKD patients. T2DKD models was induced in TLR4 knockout (TLR4-/-) mice through a high-fat diet combined with streptozotocin. Pyroptosis (caspase-1, GSDMD, interleukin 18(IL-18), interleukin 1ß(IL-1ß)) and apoptosis levels (caspase-3, Bax and Bcl-2) were detected by Western blot. HK-2 cells were cultured under high-glucose (HG) conditions as an in vitro model and then challenged with a TLR4-specific antagonist (TAK-242). GSDMD small interfering RNA (siRNA) and overexpression plasmid were transfected into HK-2 cells to down- or up-regulate GSDMD. The pyroptosis and apoptosis rates were determined by flow cytometry. RESULTS: The expression levels of caspase-1, GSDMD, IL-18 and IL-1ß were increased in renal biopsy tissues of T2DKD patients and GSDMD expression was positively correlated with tubular injury. Silencing GSDMD attenuated HG-induced IL-18, IL-1ß, FN and α-SMA, and reduced pyroptotic cells rate in HK-2 cells. Up-regulation of GSDMD inhibited HG-induced expression of Bax and cleaved caspase-3 and reduced apoptosis rate. TLR4 knockout alleviated tubular injury and interstitial macrophages infiltration, improved impaired renal dysfunction, and decreased the expressions of active N-terminal of GSDMD(GSDMD-N), cleaved caspase-1(cl-caspase-1) and cleaved caspase-3(cl-caspase-3) in T2DKD mice. TLR4 inhibition reduced HG-induced pyroptosis and apoptosis level in HK-2 cells, while GSDMD up-regulation increased pyroptosis rate and decreased apoptosis rate. CONCLUSIONS: TLR4 could exacerbate tubular injury and fibrosis via GSDMD-mediated canonical pyroptosis pathway in T2DKD. Activation of GSDMD could inhibit apoptosis and activate pyroptosis, which may involve the potential switch mechanism between TLR4-mediated pyroptosis and apoptosis in T2DKD.

Tumor Endothelial Marker 8 Promotes Proliferation and Metastasis via the Wnt/ß-Catenin Signaling Pathway in Lung Adenocarcinoma.

Tumor endothelial marker 8 (TEM8), also known as ANTXR1, was highly expressed in cancers, and was identified as a biomarker for early diagnosis and prognosis in some cancers. However, the clinical role and molecular mechanisms of TEM8 in lung adenocarcinoma (LUAD) are still unclear. The present study aimed to explore its clinical value and the molecular mechanisms of TEM8 underlying the progression of LUAD. Our study found the elevation of TEM8 in LUAD cell lines and tissues. What's more, we observed that the TEM8 expression level was associated with tumor size, primary tumor, and AJCC stage, and LUAD patients with high TEM8 expression usually have a poor prognosis. Then, we conducted a series of experiments by the strategy of loss-of-function and gain-of-function, and our results suggested that the knockdown of TEM8 suppressed proliferation, migration, and invasion and induced apoptosis in LUAD whereas overexpression of TEM8 had the opposite effect. Molecular mechanistic investigation showed that TEM8 exerted its promoting effects mainly through activating the Wnt/ß-catenin signaling pathway. In short, our findings suggested that TEM8 played a crucial role in the progression of LUAD by activating the Wnt/ß-catenin signaling pathway and could serve as a potential therapeutic target for LUAD.

Development of a Four-mRNA Expression-Based Prognostic Signature for Cutaneous Melanoma.

We aim to find a biomarker that can effectively predict the prognosis of patients with cutaneous melanoma (CM). The RNA sequencing data of CM was downloaded from The Cancer Genome Atlas (TCGA) database and randomly divided into training group and test group. Survival statistical analysis and machine-learning approaches were performed on the RNA sequencing data of CM to develop a prognostic signature. Using univariable Cox proportional hazards regression, random survival forest algorithm, and receiver operating characteristic (ROC) in the training group, the four-mRNA signature including CD276, UQCRFS1, HAPLN3, and PIP4P1 was screened out. The four-mRNA signature could divide patients into low-risk and high-risk groups with different survival outcomes (log-rank p < 0.001). The predictive efficacy of the four-mRNA signature was confirmed in the test group, the whole TCGA group, and the independent GSE65904 (log-rank p < 0.05). The independence of the four-mRNA signature in prognostic prediction was demonstrated by multivariate Cox analysis. ROC and timeROC analyses showed that the efficiency of the signature in survival prediction was better than other clinical variables such as melanoma Clark level and tumor stage. This study highlights that the four-mRNA model could be used as a prognostic signature for CM patients with potential clinical application value.

LncRNA MYCNOS promotes glioblastoma cell proliferation by regulating miR-216b/FOXM1 axis.

MYCNOS is an oncogenic lncRNA in liver cancer, but its role in glioblastoma (GBM) is unknown. We predicted that MYCNOS might interact with miR-216b, which targets FOXM1 to perform tumor suppressive roles. This study was performed to analyze the role of MYCNOS in GBM and explore its potential interactions with miR-216b and FOXM1. MYCNOS expression in paired GBM and non-tumor tissues from 62 GBM patients was analyzed by RT-qPCR. The interaction between MYCNOS and miR-216b was predicted by IntaRNA 2.0 and confirmed by dual luciferase activity assay. Overexpression of MYCNOS, miR-216b, and FOXM1 was achieved in GBM cells, followed by performing RT-qPCR and Western blot to explore the relationship among them. CCK-8 assay was performed to explore the role of MYCNOS, miR-216b, and FOXM1 in regulating GBM cell proliferation. MYCNOS was upregulated in GBM tissues compared to the paired non-tumor tissues. MYCNOS is predicted to interact with miR-216b, but overexpression of MYCNOS and miR-216b failed to affect each other's expression significantly. Dual luciferase activity assay showed that MYCNOS and miR-216b could directly interact with each other. MYCNOS overexpression increased the expression of FOXM1, which is a direct target of miR-216b. Cell proliferation assay showed that MYCNOS and FOXM1 overexpression resulted in an increased proliferation rate of GBM cells, while miR-216b overexpression suppressed cell proliferation. Moreover, MYCNOS overexpression suppressed the role of miR-216b. MYCNOS may regulate FOXM1 expression of by serving as an endogenous sponge of miR-216b axis to promote the proliferation of GBM cells.

Genetic variability of human papillomavirus type 39 based on E6, E7 and L1 genes in Southwest China.

BACKGROUND: Human papillomavirus type 39 associated with genital intraepithelial neoplasia and invasive cancers, has a high prevalence in Southwest China. HPV E6, E7 are two main papillomavirus oncoproteins, closely relate to the function of HPV immortalization, cell transformation, and carcinogenesis. L1 is the major capsid protein, can reflect the replication status of the virus in cells and the progression of cervical lesions. The purpose of this study is to reveal the prevalence of HPV 39 and the genetic polymorphisms of HPV39 based on E6, E7 and L1 gene in southwest China. METHODS: Cell samples were collected by cervical scraped for HPV detecting and typing, and HPV39 positive samples were selected out. Important E6, E7 and L1 genes of HPV39 were sequenced and analyzed for the study of HPV39 genetic polymorphisms. Phylogenetic trees were constructed by Maximum-likelihood and Kimura 2-parameters methods in Molecular Evolutionary Genetics Analysis version 6.0. The selection pressures of E6, E7 and L1 genes were estimated by Datamonkey web server. The secondary and three-dimensional structure of HPV39 E6, E7 proteins were created by sopma server and SWISS-MODEL software. RESULTS: 344 HPV39 positive samples were selected from 5718 HPV positive cell samples. Among HPV39 E6-E7 sequences, 20 single nucleotide mutations were detected, including 10 non-synonymous and 10 synonymous mutations; 26 single nucleotide mutations were detected in HPV39 L1 sequences, including 7 non-synonymous and 19 synonymous mutations respectively. 11 novel variants of HPV39 E6-E7 (5 in E6 and 6 in E7) and 14 novel variants of HPV39 L1 were identified in this study. A-branch was the most frequent HPV39 lineage in southwest China during our investigation. Selective pressure analysis showed that codon sites 26, 87, 151 in E6 and 75, 180, 222, 272, 284, 346, 356 in L1 were positively selected sites, as well as codon sites 45, 138, 309, 381 were negative selection sites in L1 gene, E7 has neither positive selection sites nor negative selection sites. A certain degree of secondary and three-dimensional structure dislocation was existed due to the non-synonymous mutations. CONCLUSIONS: Amino acid substitution affected the secondary and three-dimensional structure of HPV39, and resulting in the differences of carcinogenic potential and biological functions as well as the immune response due to the antigen epitopes difference, the antigen epitopes with stronger adaptability in Southwest will be screened out based on the above research results for the later vaccine development. And gene polymorphism of HPV39 in Southwest China may improve the effectiveness of clinical test and vaccine design, specifically for women in Southwest China.

Feedback activation of STAT3 limits the response to PI3K/AKT/mTOR inhibitors in PTEN-deficient cancer cells.

The PI3K/AKT/mTOR signaling pathway is constitutively active in PTEN-deficient cancer cells, and its targeted inhibition has significant anti-tumor effects. However, the efficacy of targeted therapies is often limited due to drug resistance. The relevant signaling pathways in PTEN-deficient cancer cells treated with the PI3K/mTOR inhibitor BEZ235 were screened using a phosphokinase array, and further validated following treatment with multiple PI3K/AKT/mTOR inhibitors or AKT knockdown. The correlation between PTEN expression levels and STAT3 kinase phosphorylation in the tissue microarrays of gastric cancer patients was analyzed by immunohistochemistry. Cell proliferation and clonogenic assays were performed on the suitably treated PTEN-deficient cancer cells. Cytokine arrays, small molecule inhibition and knockdown assays were performed to identify related factors. PTEN-deficient tumor xenografts were established in nude mice that were treated with PI3K/AKT/mTOR and/or STAT3 inhibitors. PTEN deficiency was positively correlated with low STAT3 activity. PI3K/mTOR inhibitors increased the expression and secretion of macrophage migration inhibitory factor (MIF) and activated the JAK1/STAT3 signaling pathway. Both cancer cells and in vivo tumor xenografts showed that the combined inhibition of PI3K/AKT/mTOR and STAT3 activity enhanced the inhibitory effect of BEZ235 on the proliferation of PTEN-deficient cancer cells. Our findings provide a scientific basis for a novel treatment strategy in cancer patients with PTEN deficiency.

PRSS contributes to cetuximab resistance in colorectal cancer.

Cetuximab improves the survival of patients with metastatic colorectal cancer. The main limitation is primary and secondary resistance, the underlying mechanism of which requires extensive investigation. We proved that PRSS expression levels are significantly negatively associated with the sensitivity of cancer cells to cetuximab. Detailed mechanistic analysis indicated that PRSS can cleave cetuximab, leading to resistance. Cetuximab or bevacizumab combined with SPINK1, a PRSS inhibitor, inhibited cell growth more efficiently than cetuximab or bevacizumab alone in xenograft models. PRSS levels in the serum of 156 patients with mCRC were analyzed, and poor efficacy of cetuximab therapy was observed in patients with aberrant PRSS expression. PRSS expression in monoclonal antibody (mAb)-treated patients with cancer from The Cancer Genome Atlas database was also evaluated to determine whether patients with higher PRSS expression have significantly reduced progression-free survival. Our work provides a strong scientific rationale for targeting PRSS in combination with cetuximab therapy.

miR-200c-3p Suppresses the Proliferative, Migratory, and Invasive Capacities of Nephroblastoma Cells via Targeting FRS2.

Objectives: miR-200c-3p has been shown to serve as a tumor suppressor in various tumor types. However, the biological function of miR-200c-3p in nephroblastoma remains unknown. This study aims to investigate the biological function and regulatory mechanisms of miR-200c-3p in nephroblastoma development. Methods: The expression of miR-200c-3p in nephroblastoma tissues and cells was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of miR-200c-3p on the proliferation and cell cycle of SK-NEP-1 nephroblastoma cell line were evaluated by CCK-8 assay, colony formation assay, and flow cytometry. The effects of miR-200c-3p on the migratory and invasive capacities of SK-NEP-1 cells were measured by wound healing assay and transwell assay. The ability of miR-200c-3p to target fibroblast growth factor receptor substrate 2 (FRS2) was detected by quantitative PCR, western blot, and luciferase reporter assay. Results: The expression of miR-200c-3p was significantly downregulated in nephroblastoma tissues and cells compared with that in normal renal tissues and cells. miR-200c-3p inhibited the proliferative, migratory, and invasive capacities of nephroblastoma cells by targeting FRS2. Conclusions: miR-200c-3p suppresses the malignant behaviors of nephroblastoma cells by downregulating the expression of FRS2.

TEM8 functions as a receptor for uPA and mediates uPA-stimulated EGFR phosphorylation.

BACKGROUND: TEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown. RESULTS: Here we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections. CONCLUSIONS: Taken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.