Nucleoporin 93, a new substrate of the E3 ubiquitin protein ligase HECTD1, promotes esophageal squamous cell carcinoma progression.

Nucleoporin 93 (NUP93) is an important component of the nuclear pore complex, exhibiting pro-tumorigenic properties in some cancers. However, its function in esophageal squamous cell carcinoma (ESCC) has not been elucidated. This study aimed to investigate the effects of NUP93 in ESCC and the underlying mechanisms involved. Through analysis of public human cancer datasets, we observed higher expression of NUP93 in esophageal cancer tissues than in normal tissues. Stable ESCC cell lines with NUP93 overexpression or knockdown were established by lentiviral vector transduction and puromycin selection. NUP93 knockdown suppressed the proliferation, colony formation, cell cycle transition, migration, and invasion of ESCC cells, while the overexpression of NUP93 displayed opposite effects. NUP93 positively regulated epithelial-mesenchymal transition and AKT signaling transduction in ESCC cells. In addition, NUP93 increased the expression of programmed death ligand 1 (PD-L1) in ESCC cells and attenuated NK cell-mediated lysis of ESCC cells. In vivo experiments demonstrated that NUP93 promotes the growth of ESCC in nude mice, enhances Ki67 and PD-L1 expression, and promotes AKT signaling transduction in xenografts. Mechanistically, we demonstrated that the HECT domain E3 ubiquitin protein ligase 1 (HECTD1) contributes to the ubiquitination and degradation of NUP93 and acts as a tumor suppressor in ESCC. To conclude, this study has shown that NUP93 has pro-tumor properties in ESCC and that HECTD1 functions as an upstream regulator of NUP93 in ESCC. These findings may contribute to the investigation of potential therapeutic targets in ESCC.

Analysis of multiple programmed cell death-related prognostic genes and functional validations of necroptosis-associated genes in oesophageal squamous cell carcinoma.

BACKGROUND: Oesophageal squamous cell carcinoma (ESCC) is a lethal malignancy. Immune checkpoint inhibitors (ICIs) showed great clinical benefits for patients with ESCC. We aimed to construct a model predicting prognosis and response to ICIs by integrating diverse programmed cell death (PCD) forms. METHODS: Genes related to 14 PCDs were collected to generate multi-gene signatures, including apoptosis, necroptosis, pyroptosis, ferroptosis, and cuproptosis. Bulk and single-cell RNA transcriptome datasets were used to develop and validate the model. We assessed the functions of two necroptosis-related genes in ESCC cells by Western blot, co-immunoprecipitation (Co-IP), LDH release assay, CCK-8, and migration assay, followed by immunohistochemistry (IHC) staining on samples of patients with ESCC (n = 67). FINDINGS: We built and validated a 16-gene prognostic combined cell death index (CCDI) by combining immunogenic cell death (ICD) and necroptosis signatures. The CCDI could also predict response to ICIs in cancer, as shown by Tumour Immune Dysfunction and Exclusion (TIDE) analysis, confirmed in four independent ICI clinical trials. Trajectory analysis revealed that HOOK1 and CUL4A might affect ESCC cell fate. We found that HOOK1 induced necroptosis and inhibited the proliferation and migration of ESCC cells, while CUL4A exhibited the opposite effects. Co-IP assay confirmed that HOOK1 and CUL4A promoted and reduced necrosome formation in ESCC cells. Data from patients with ESCC further supported that HOOK1 and CUL4A might be a tumour suppressor and oncogene, respectively. INTERPRETATION: We constructed a CCDI model with potential in predicting prognosis and response to ICIs in cancer. HOOK1 and CUL4A in the CCDI model are crucial prognostic biomarkers in ESCC. FUNDING: The Natural Science Foundation of China [82172786], The National Cancer Center Climbing Fund of China [NCC201908B06], The Natural Science Foundation of Heilongjiang Province [LH2021H077].

Neoadjuvant camrelizumab combined with paclitaxel and nedaplatin for locally advanced esophageal squamous cell carcinoma: A single-arm phase 2 study (cohort study).

BACKGROUND: Neoadjuvant administration of immune checkpoint inhibitors (ICIs) combined with chemotherapy demonstrated promising efficacy and manageable safety in locally advanced esophageal squamous cell carcinoma (ESCC). This prospective, single-arm, phase 2 study evaluated the efficacy and safety of neoadjuvant therapy with camrelizumab plus paclitaxel and nedaplatin for 2-4 cycles in ESCC. METHODS: Patients with locally advanced stage IIa-IIIb ESCC were enrolled in the study and received camrelizumab (200 mg), paclitaxel (155 mg/m2), and nedaplatin (80 mg/m2) intravenously on day one every three weeks. Patients underwent surgery after 2-4 cycles of treatment regimes. The primary endpoint was the pathological complete response (pCR) rate. Secondary endpoints included the major pathological response (MPR) rate, R0 resection rate, tumor regression, objective response rate (ORR), and disease-free survival (DFS). Programmed cell death 1 ligand 1 (PD-L1) expression in tumor tissues was measured and quantified using immunohistochemistry staining and combined positive score (CPS), respectively. RESULTS: In total, 75 patients were enrolled and received neoadjuvant treatment. Of them, 45 (60%) received two cycles, 18 (24%) received three cycles, and 10 patients (13.3%) received four cycles of neoadjuvant therapy. Ultimately, 62 (82.7%) patients underwent surgery. Patients achieved a pCR of 27.4% (95% CI 16.9-40.2), an MPR of 45.2% (95% CI 33.1-59.2), and an ORR of 48.4% (95% CI 35.5-61.4); all patients had an R0 resection. T and N downstaging occurred in 55 (88.7%) and 27 patients (43.5%). Moreover, ESCC patients with CPS ≥ 10 tended to have enhanced ORR, pCR, and MPR compared to those with CPS < 10. Treatment-related adverse events (TRAEs) of grade 1-2 occurred in 59 (78.7%) patients, grade 3 TRAEs in four (5.3%), and one patient (1.3%) experienced a grade 4 TRAE. CONCLUSIONS: Neoadjuvant camrelizumab combined with chemotherapy showed promising efficacy in locally advanced ESCC, with a manageable safety profile, when administered flexibly in two to four cycles.

Methyltransferase-like 3 facilitates the stem cell properties of esophageal cancer by upregulating patched homolog 1 via N6-methyladenosine methylation.

Patched homolog 1 (PTCH1) has been proven to facilitate cell proliferation and self-renewal in esophageal cancer (EC). The present study intended to exploit the influence of PTCH1 on EC cells and the potential mechanisms. PTCH1 and methyltransferase-like 3 (METTL3) expression were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in EC cell lines. Following the loss- and gain-of-function assays, cell proliferation was examined by cell counting kit (CCK)-8 and clone formation assays, invasion and migration by Transwell and scratch assays, and the sphere-forming ability of stem cells by cell sphere-forming assay. The expression of stemness genes NANOG homeobox protein (NANOG), octamer-binding transcription factor 4 (Oct4), and sex-determining region Y-box 2 (SOX2) was detected by Western blot. Methylated RNA immunoprecipitation (Me-RIP) assay was performed to test N6-methyladenosine (m6A) modification levels of PTCH1 mRNA, RIP and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) assays to assess the binding of METTL3 to PTCH1, and actinomycin D treatment to examine PTCH1 mRNA stability. A xenograft tumor model in nude mice was established for further in vivo verification. PTCH1 and METTL3 expression was high in EC cells. Knockdown of METTL3 reduced m6A level and stability of PTCH1 mRNA. Knockdown of PTCH1 or METTL3 declined invasion, proliferation, migration, and NANOG, Oct4, and SOX2 levels in EC cells, and reduced sphere-forming abilities of EC stem cells. Overexpression of PTCH1 abolished the suppressive effect of METTL3 knockdown on EC cells in vitro. METTL3 knockdown repressed tumor growth in nude mice, which was negated by further overexpressing PTCH1. METTL3 facilitated growth and stemness of EC cells via upregulation of PTCH1 expression by enhancing PTCH1 m6A modification.NEW & NOTEWORTHY PTCH1 has been proved to facilitate cell proliferation and self-renewal in esophageal cancer. We studied the upstream regulation mechanism of PTCH1 by METTL3 through m6A modification. Our results provide a new target and theoretical basis for the treatment of esophageal cancer.

Development of a cuproptosis-related signature for prognosis prediction in lung adenocarcinoma based on WGCNA.

Background: Cuproptosis is a novel mitochondrial respiration-dependent cell death mechanism induced by copper that can kill cancer cells via copper carriers in cancer therapy. However, the clinical significance and prognostic value of cuproptosis in lung adenocarcinoma (LUAD) remains unclear. Methods: We performed a comprehensive bioinformatics analysis of the cuproptosis gene set, including copy number aberration, single-nucleotide variation, clinical characteristics, survival analysis, etc. Cuproptosis-related gene set enrichment scores (cuproptosis Z-scores) were calculated in The Cancer Genome Atlas (TCGA)-LUAD cohort using single-sample gene set enrichment analysis (ssGSEA). Modules significantly associated with cuproptosis Z-scores were screened by weighted gene co-expression network analysis (WGCNA). The hub genes of the module were then further screened by survival analysis and least absolute shrinkage and selection operator (LASSO) analysis, in which TCGA-LUAD (497 samples) and GSE72094 (442 samples) were used as the training and validation cohorts, respectively. Finally, we analyzed the tumor characteristics, immune cell infiltration levels, and potential therapeutic agents. Results: Missense mutation and copy number variant (CNV) events were general in the cuproptosis gene set. We identified 32 modules, of which the MEpurple (107 genes) and MEpink (131 genes) modules significantly positively and negatively correlated with cuproptosis Z-scores, respectively. We identified 35 hub genes significantly related to overall survival and constructed a prognostic model consisting of 7 cuproptosis-related genes in patients with LUAD. Compared with the low-risk group, patients in the high-risk group had a worse overall survival and gene mutation frequency, as well as significantly higher tumor purity. In addition, infiltration of immune cells was also significantly different between the 2 groups. Furthermore, the correlation between the risk scores and half-maximum inhibitory concentration (IC50) of antitumor drugs in the Genomics of Drug Sensitivity in Cancer (GDSC) v. 2 database was explored, revealing differences in drug sensitivity across the 2 risk groups. Conclusions: Our study provided a valid prognostic risk model for LUAD and improved understanding of its heterogeneity, which may aid in the development of personalized treatment strategies.

Response to neoadjuvant immune checkpoint inhibitors and chemotherapy in Chinese patients with esophageal squamous cell carcinoma: the role of tumor immune microenvironment.

BACKGROUND: Immune checkpoint inhibitors (ICIs) through programmed cell death 1 blockade improve the survival outcomes of patients with advanced esophageal squamous cell carcinoma (ESCC). Recently, the use of neoadjuvant immunotherapy for the treatment of ESCC has been gradually increasing. We aimed to evaluate the efficacy of neoadjuvant treatment of ICIs with chemotherapy and explore tumor microenvironment (TME) immune profiles of ESCC samples during neoadjuvant therapy. METHODS: Patients with previously untreated, resectable, locally advanced ESCC (stage II or III) in Harbin Medical University Cancer Hospital were enrolled. Each patient received two to four cycles of neoadjuvant ICIs combined with chemotherapy before surgical resection. The TME immune profiles of formalin-fixed paraffin-embedded tumor samples at baseline and after surgery were evaluated by multiplex staining and multispectral imaging. RESULTS: In all, 18 patients were enrolled, and all patients received surgery with R0 resection. The postoperative pathological evaluation indicated that 7 (38.9%) patients had a pathological complete response (pCR) and 11 (61.1%) patients had a partial response. The neoadjuvant therapeutic regimens had acceptable side effect profiles. The TME immune profiles at baseline observed higher densities of stroma CD3 + , PD-1 + , and PD-1 + CD3 + cells in pCR patients than in non-pCR patients. Comparing TME immune profiles before and after neoadjuvant treatment, an increase in CD8 + T cells and a decrease in CD163 + CD68 + M2-like macrophage cells were observed after neoadjuvant treatment. CONCLUSIONS: Neoadjuvant ICIs combined with chemotherapy produced a satisfactory treatment response, demonstrating its anti-tumor efficacy in locally advanced ESCC. Further large-scale studies are required to understand the role of tumor immunities and ICIs underlying ESCC.

Long Non-coding RNA SNHG16 Facilitates Esophageal Cancer Cell Proliferation and Self-renewal through the microRNA-802/PTCH1 Axis.

OBJECTIVE: This research sought to explore the effect and mechanism of long non-coding RNA SNHG16 on esophageal cancer (EC) cell proliferation and self-renewal. METHODS: SNHG16 expression was measured in EC9706 and KYSE150 cells. EC9706 and KYSE150 cells were transfected with Lenti-SNHG16, sh-SNHG16, Lenti-protein patched homolog 1 (PTCH1), miR-802 mimic, or miR-802 inhibitor. Flow cytometry was used to sort cancer stem cells (CSCs) in EC9706 and KYSE150 cells. Cell proliferation in EC cells was measured, in addition to colony and tumorsphere numbers. The possible interactions among SNHG16, PTCH1, and miR-802 were identified by dual luciferase reporter and RNA pull-down assays. The expression of the genes in the Hedgehog pathway was detected. Nude mice were injected with SNHG16-silenced EC9706 cells to observe the tumorigenicity of EC9706 cells. RESULTS: Upregulated SNHG16 expression was found in CSCs, whose expression was decreased during the differentiation of CSCs. SNHG16 or PTCH1 overexpression or miR-802 inhibition promoted the proliferation, colony formation, and tumorsphere formation of EC9706 and KYSE150 cells as well as SOX2, OCT4, Bmi-1, and PTCH1 expression. Consistently, SNHG16 knockdown or miR-802 overexpression inhibited EC progression. Moreover, SNHG16 and PTCH1 were competitively bound to miR-802, and SNHG16 orchestrated the miR-802/PTCH1 axis to activate the Hedgehog pathway. SNHG16 silencing repressed the tumorigenicity of EC9706 in nude mice. CONCLUSION: Conclusively, SNHG16 acts as a sponge of miR-802 to upregulate PTCH1 and activate the Hedgehog pathway, thus promoting EC cell proliferation and selfrenewal.

Knock-down of microRNA miR-556-5p increases cisplatin-sensitivity in non-small cell lung cancer (NSCLC) via activating NLR family pyrin domain containing 3 (NLRP3)-mediated pyroptotic cell death.

MicroRNAs (miRNAs) are small non-coding RNAs that are closely associated with cancer progression and drug resistance, however, up until now, the involvement of miR-556-5p in regulating cisplatin-sensitivity in non-small cell lung cancer (NSCLC) has not been studied. In the present study, we found that miR-556-5p was significantly upregulated in the cisplatin-resistant NSCLC (CR-NSCLC) patients' tissues and cells, instead of the corresponding cisplatin-sensitive NSCLC (CS-NSCLC) tissues and cells. Further experiments validated that knock-down of miR-556-5p suppressed cell viability and tumorigenesis, and induced cell apoptosis in the cisplatin-treated CR-NSCLC cells, and conversely, upregulation of miR-556-5p increased cisplatin-resistance in CS-NSCLC cells. Interestingly, miR-556-5p ablation triggered pyroptotic cell death in cisplatin-treated CR-NSCLC cells via upregulating NLRP3, and the promoting effects of miR-556-5p silence on cisplatin-sensitivity in CR-NSCLC cells were abrogated by both cell pyroptosis inhibitor NSA and NLRP3 downregulation. Taken together, this study firstly evidenced that induction of NLRP3-mediated cell pyroptosis by miR-556-5p downregulation was effective to increase cisplatin-sensitivity in NSCLC, which provided new therapy strategies to overcome chemo-resistance for NSCLC patients in clinic.

Combination of Danshen and ligustrazine has dual anti-inflammatory effect on macrophages and endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia Miltiorrhiza Radix et Rhizoma (Danshen) and Chuanxiong Rhizoma (Chuanxiong) are both traditional Chinese medicines with vascular protective effects, and their combination is widely used in China to treat occlusive or ischemic diseases of the cerebrovascular or cardiovascular system. Although it is widely accepted that these diseases have high relevance to inflammation, little is known about the anti-inflammatory effect of Danshen, Chuanxiong, and their combination. AIM OF STUDY: We aimed to investigate the complex mode of action of Danshen, Chuanxiong, and their combination and the molecular mechanisms underlying their anti-inflammatory activity. Specifically, toll-like receptor (TLR1/2, 3, and 4)-triggered macrophages and endothelial cells, the two major cell players in atherosclerosis as well as in related cardiovascular and cerebrovascular injuries, were emphasized. METHODS: TLR1/2-, TLR3-, and TLR4-induced bone marrow macrophages (BMMs) and human umbilical vein endothelial cells (HUVECs) were treated with Danshen extract (S. miltiorrhiza extract, SME), ligustrazine (2, 3, 5, 6-tetramethylpyrazine, TMP), and their combination (S. miltiorrhiza and TMP injection, SLI), respectively. The proinflammatory cytokines interleukin 6 (IL-6), IL-12, and tumor necrosis factor alpha (TNF-α) were detected as the preliminary indicators of inflammation. In addition, RNA sequencing (RNA-seq)-based transcriptional profiling analyses were conducted for TLR2-activated BMMs to determine the molecular mode of action of SLI as well as the contribution of SME to SLI activity. RESULTS: SLI mitigated inflammation in both BMMs and HUVECs. Refer to the combination, SME had pronounced anti-inflammatory effect on BMMs but had only a slight effect on HUVECs. In contrast, TMP had considerable anti-inflammatory effect on HUVECs but not on BMMs. Bioinformatic analysis identified a broad spectrum of regulatory genes, in addition to IL-6 gene, and C-X-C motif chemokine ligand 10 (CXCL10) appeared to be another key molecule involved in the mechanism underlying SLI and SME effects. At the molecular level, SME was a major contributor of the anti-inflammatory activity of SLI. CONCLUSIONS: In TLR-activated inflammation, SLI exhibits a "multiple ingredient-multiple target" effect, with SME primarily affecting macrophages and TMP affecting HUVECs. Our study provides evidence for the clinical application of SLI in treating complex diseases involving inflammation-induced injury of both macrophages and epithelial cells. Further bioinformatics studies are required to reveal the entire molecular network involved in TMP, SME, and SLI activity.

A HPLC-DAD-MS/MS Method for Simultaneous Determination of Six Active Ingredients of Salviae Miltiorrhizae and Ligustrazine Hydrochloride Injection in Rat Plasma and its Application to Pharmacokinetic Studies.

AIMS: This is a pharmacokinetic study of Salviae miltiorrhizae and ligustrazine hydrochloride injection. The study aimed to evaluate the mechanism of action, safety and rational clinical use of Salviae miltiorrhizae and ligustrazine hydrochloride injection. BACKGROUND: Salviae miltiorrhizae and ligustrazine hydrochloride injection is a compound preparation consisted of Salvia miltiorrhiza extract and ligustrazine hydrochloride for the treatment of cardiovascular and cerebrovascular diseases in China. OBJECTIVE: The study aimed to develop a rapid and sensitive high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) method for simultaneous determination of six major active ingredients of Salviae miltiorrhizae and ligustrazine hydrochloride injection, namely danshensu, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, salvianolic acid A, and ligustrazine hydrochloride, in rat plasma. METHODS: Plasma samples were precipitated with methanol, which was spiked with ascorbic acid and the supernatant was separated on a Waters Cortecs C18 column, by using a gradient mobile phase system of acetonitrile-water containing 0.05% formic acid (v/v). For internal standards, puerarin was selected for the five salvianolic acids, while isofraxidin was used for ligustrazine hydrochloride. Besides, electrospray ionization in negative mode and multiplereaction monitoring were used to identify and quantify the five salvianolic acids, whereas ligustrazine hydrochloride was quantified at 310 nm using the diode array detector. RESULTS: Noticeably, all calibration curves showed good linearity (R2>0.99) over the concentration range, with a lower limit of quantification between 0.00411 and 0.0369 µg/mL for salvianolic acids, and 1.74 µg/mL for ligustrazine hydrochloride. Next, the precision of the developed method was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation was within 10%. Although the extraction efficiency of some salvianolic acids was not very satisfactory, the sensitivity of the analytical method met the analysis requirements of rat plasma samples. Moreover, the validated method was successfully applied to a pharmacokinetic study of Salviae miltiorrhizae and ligustrazine hydrochloride injection in the rat model. CONCLUSION: Linear pharmacokinetic characteristics were observed for the six active ingredients after intravenous infusion administration in rats within the dose range examined here. In summary, our study proposed a HPLC-DADMS/ MS method with the simultaneous determination of multiple ingredients, and demonstrated its applicability in pharmacokinetic studies.

Cucurbitacin E inhibits esophageal carcinoma cell proliferation, migration, and invasion by suppressing Rac1 expression through PI3K/AKT/mTOR pathway.

As an oxygenated tetracyclic triterpenoid, Cucurbitacin E (CuE) possesses potential antitumor properties in sorts of malignancies. However, its effect on human esophageal carcinoma cells has not been previously unearthed, and the mechanism underlying its anticarcinoma activity remains vague. Hence, this study was arranged to probe the function of CuE on esophageal carcinoma cells and its specific mechanism. Human esophageal carcinoma cells (ECA109 and EC9706) and human normal esophageal epithelial cells (Het-1A) were selected for subsequent experiments. The expression levels of Rac1 in esophageal carcinoma cells were measured. After transfection of sh-Rac1 or pCDNA3.1-Rac1, esophageal carcinoma cells were exposed to CuE. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-ethynyl-2'-deoxyuridine staining were utilized for measurement of cell proliferation ability, cell scratch assay for inspection of cell migration rate, and Transwell for detection of cell invasion ability. The phosphorylation levels of protein kinase B and mTOR were analyzed by Western blot. Rac1 was highly expressed in esophageal carcinoma cells. Transfection of sh-Rac1 in esophageal carcinoma cells resulted in suppression on cell proliferation, migration, and invasion, as well as downregulated phosphorylation levels of AKT and mammalian target of rapamycin (mTOR) in esophageal carcinoma cells, while transfection of pCDNA3.1-Rac1 had an opposite effect, implicating that Rac1 can promote the viability of esophageal carcinoma cells. Esophageal carcinoma cells subjected to CuE treatment had decreased expression of Rac1, suppressed cell viability, and decreased phosphorylation levels of AKT and mTOR. Transfection of pCDNA3.1-Rac1 and CuE treatment in esophageal carcinoma cells enhanced viability of esophageal carcinoma cells and promoted the phosphorylation levels of AKT and mTOR in comparison with cells treated with CuE alone. CuE inhibits proliferation, invasion, and migration of esophageal carcinoma cells via downregulating Rac1 to block the phosphoinositide 3-kinase/AKT/mTOR pathway.

Effect of Danshen on TLR2-triggered inflammation in macrophages.

BACKGROUND: Danshen (Salvia Miltiorrhiza Radix et Rhizoma) is a valued herbal plant widely used to treat cardiovascular diseases in Asian countries. In modern medicine, innate immunity-induced inflammation is considered a risk factor for cardiovascular diseases. However, little is known about the anti-inflammatory effects and molecular mechanism of Danshen. PURPOSE: To evaluate the molecular mechanisms of Danshen on Toll-like receptor (TLR) 2-triggered inflammation in macrophages and identify its bioactive components. METHODS: Pam3CSK4-stimulated bone marrow-derived macrophages (BMMs) were treated with Danshen water extract (DSE), and the levels of proinflammatory cytokines (interleukin (IL)-6, IL-12 and tumor necrosis factor (TNF)-α) were measured by both real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). RNA sequencing (RNA-seq)-based bioinformatics analyses were applied to reveal the novel molecular mechanisms of DSE, followed by western blotting for verification. Additionally, HPLC-UV analysis along with bioassays was performed to identify the bioactive ingredients of DSE. RESULTS: The results of RT-qPCR and ELISA showed that DSE significantly inhibited proinflammatory cytokine expression in a dose-dependent manner. Transcriptome analyses revealed that a wider panel of inflammatory cytokines responded to the regulatory effect of DSE, and that the TNF signaling pathway might have played a vital role. Western blotting data confirmed the involvement of extracellular signal-regulated protein kinases (ERK) and Jun N-terminal Kinase (JNK) related singling pathway. Among the seven components identified in DSE, Danshensu (DSS) and protocatechuic aldehyde (PA) were confirmed as bioactive ones with anti-inflammatory effects. CONCLUSION: DSE showed a promising effect against TLR2-triggered inflammation associated with the inhibition of the TNF cascade down-streamed mitogen-activated protein kinase (MAPK) signaling pathway, in which IL-6 acts as the key effective molecule, and ERK and JNK phosphorylation was inhibited. Notably, DSS and PA were considered bioactive components with anti-inflammatory bioactivity.

Inhibition of nuclear factor kappa B as a mechanism of Danshensu during Toll-like receptor 2-triggered inflammation in macrophages.

Danshensu (DSS) is a water-soluble phenolic compound in Danshen (Salvia Miltiorrhiza Radix et Rhizoma). Although various pharmacological activities have been recognized, little is known regarding its anti-inflammatory effect and related molecular mode of action. In the current study, bone marrow-derived macrophages (BMMs) were activated by a Toll-like receptor 2 (TLR2) agonist Pam3CSK4 with or without DSS intervention. Production of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-12 (IL-12) was detected by both enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (RT-qPCR). Activation of signaling pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) was assessed by Western blot. Additionally, RNA sequencing (RNA-seq) combined with bioinformatics analyses was applied to investigate the molecular mechanisms of DSS. Emphasis was placed on the construction of the protein-protein interaction (PPI) network and transcription factor (TF) enrichment analysis of data including co-regulated differentially expressed genes (DEGs) in the Pam3CSK4 vs. control and DSS vs. Pam3CSK4 groups. The RT-qPCR and ELISA results showed that DSS effectively inhibited the expressions of IL-6 and IL-12, indicating a significant anti-inflammatory effect. Western blot verified that DSS suppressed the phosphorylation of p65, which was in accordance with the results of the TF enrichment analysis. Additionally, the PPI network analysis showed several key molecules, including lactoferrin (Ltf), CC-chemokine receptor 7 (Ccr7), interferon-gamma (IFN-γ) and C-X-C motif chemokine ligand 9 (Cxcl9), to be regulatory genes that responded to DSS treatment. Overall, our study revealed that DSS has a pronounced anti-inflammatory effect involving TLR2 and macrophages through the NF-κB signaling pathway, which supports the novel application of DSS in the treatment of relevant diseases including atherosclerosis and ischemic or ischemic/perfusion injury of the heart and brain.

The construction and analysis of the aberrant lncRNA-miRNA-mRNA network in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common cancer and the pathogenesis remain unclear. According to the competing endogenous RNA (ceRNA) theory, long noncoding RNA (lncRNA) have a competition with mRNAs for the connecting with miRNAs that affecting the level of mRNA. In this work, the ceRNA network and the important genes to predict the survival prognosis were explored. METHODS: In the study, we recognized differently expressed genes (mRNAs, lncRNAs and miRNAs) between NSCLC and normal tissues from The Cancer Genome Atlas database (fold change >2, P<0.01) using edgeR. Then, the interaction between lncRNA and miRNA or mRNA and miRNA was explored by miRcode, miRDB, TargetScan, and miRanda. Furthermore, the functions and KEGG pathway were analyzed with DAVID and KOBAS. The connections of these mRNAs were explored by STRING online database. The relation between genes in the network and survival time were further explored by survival package in R. RESULTS: By bioinformatics tools, we explored 155 lncRNAs, 30 miRNAs and 68 mRNAs and constructed ceRNA network. The functions and KEGG pathway of 68 mRNAs were further analyzed. AQP2, EGF, SLC12A1, TRPV5 and AVPR2 was in the center of network and may play key roles in the development of NSCLC. And mRNA (CCNB1, COL1A1, E2F7, EGLN3, FOXG1 and PFKP), miRNA (miR-31, miR-144 and miR-192) and lncRNA (AC080129.1, AC100791.1, AL163952.1, AP000525.1, AP003064.2, C2orf48, C10orf91, FGF12-AS2, HOTAIR, LINC00518, LNX1-AS1, MED4-AS1, MIG31HG, MUC2, TTTY16 and UCA1) were closely related with overall survival (OS). CONCLUSIONS: In summary, the present study provides a deeper understanding of the lncRNA-related ceRNA network in NSCLC and some genes may be new target to treat for NSCLC patients.

Overexpression of Shp-2 attenuates apoptosis in neonatal rat cardiac myocytes through the ERK pathway.

During cardiac ischemia and end-stage heart disease, a large number of cardiac cells are apoptotic, and therefore, heart function is impaired. Although the role of Shp-2 in cell survival has been reported, its regulation of cardiac apoptosis is still undetermined. To better understand the potential role of Shp-2 in apoptosis, cell death was determined in serum-depleted cardiomyocytes. Shp-2 was inhibited by NSC87877, and apoptosis, Cyt C release and caspase 3 activation were determined. To evaluate the notion that Shp-2 plays a role in survival stimulation, wild-type and gain-of-function mutant Shp-2 adenoviruses were infected into neonatal cardiomyocytes, and ERK activation was examined. Finally, the MEK inhibitor U0126 was utilized to block the ERK pathway and determine the role of Shp-2 in this pathway. We found that Shp-2 inhibition enhanced apoptosis via regulation of mitochondrial Cyt C release and activation of caspase 3. Overexpression of Shp-2 inhibited apoptosis through activation of ERK. The MEK inhibitor U0126 abolished Shp-2's effect on apoptosis in cardiomyocytes. Our results have revealed that Shp-2 functions as an intracellular inhibitor of apoptosis. These data provide insight into the pathogenesis and the therapeutic strategies of heart diseases.