Strictosamide alleviates acute lung injury via regulating T helper 17 cells, regulatory T cells, and gut microbiota.

BACKGROUND: Nauclea officinalis (Pierre ex Pit.) Merr. & Chun (Rubiaceae) is widely used to treat respiratory diseases in China. Strictosamide is its main active component and has significant anti-inflammatory activity. However, the effects and molecular mechanisms of strictosamide in the treatment of acute lung injury (ALI) remain largely unknown. PURPOSE: This study aimed to examine the regulatory effects of strictosamide on T helper 17 cells (Th17 cells)/Regulatory T cells (Treg cells) and gut microbiota in ALI-affected mice. MATERIALS AND METHODS: The ALI model was induced using lipopolysaccharide (LPS) intraperitoneal injection. Hematoxylin-eosin (H&E) staining, the number of inflammatory cells in broncho-alveolar lavage fluid (BALF), the Wet/Dry (W/D) ratio, and myeloperoxidase (MPO) activity were utilized as evaluation indices for the therapeutic efficacy of strictosamide on ALI. Flow cytometry (FCM), enzyme-linked immune sorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blotting were used to determine the regulation of strictosamide on the Th17/Treg cells and the STAT3/STAT5 signaling pathway. The analysis of gut microbiota was conducted using 16S rDNA sequencing. The verification of the relationship between the gut microbiome and immune function was conducted using Spearman analysis. RESULTS: Strictosamide attenuated inflammation on ALI induced by LPS, which reduced the levels of Th17-related factors interleukin (IL)-6 and IL-17 and increased Treg-related factors IL-10 and transforming growth factor (TGF)-ß. In the spleens and whole blood, strictosamide reduced the proportion of Th17 cells and increased the proportion of Treg cells. Furthermore, strictosamide increased Forkhead/winged helix transcription factor 3 (Foxp3) and p-STAT5 protein expression while inhibiting Retinoid-related orphan nuclear receptors-γt (RORγt) and p-STAT3 expression. Moreover, strictosamide reshaped the diversity and structure of the gut microbiota, and influence the associations between immune parameters and gut microbiota in ALI mice. CONCLUSIONS: In summary, the results of the current investigation showed that strictosamide has a therapeutic impact on LPS-induced ALI. The mechanism of action of this effect may be associated with the modulation of Th17 and Treg cells differentiation via the SATA signaling pathway, as well as the impact of the gut microbiota.

TRIM29 modulates proteins involved in PTEN/AKT/mTOR and JAK2/STAT3 signaling pathway and suppresses the progression of hepatocellular carcinoma.

Tripartite motif-containing 29 (TRIM29), also known as the ataxia telangiectasia group D-complementing (ATDC) gene, has been reported to play an oncogenic or tumor suppressive role in developing different tumors. So far, its expression and biological functions in hepatocellular carcinoma (HCC) remain unclear. We investigated TRIM29 expression pattern in human HCC samples using quantitative RT-PCR and immunohistochemistry. Relationships between TRIM29 expression level, clinical prognostic indicators, overall survival (OS), and disease-free survival (DFS) were evaluated by Kaplan-Meier analysis and Cox proportional hazards model. A series of in vitro experiments and a xenograft tumor model were conducted to detect the functions of TRIM29 in HCC cells. RNA sequencing, western blotting, and immunochemical staining were performed to assess the molecular regulation of TRIM29 in HCC. We found that the mRNA and protein levels of TRIM29 were significantly reduced in HCC samples, compared with adjacent noncancerous tissues, and were negatively correlated with poor differentiation of HCC tissues. Survival analysis confirmed that lower TRIM29 expression significantly correlated with shorter OS and DFS of HCC patients. TRIM29 overexpression remarkably inhibited cell proliferation, migration, and EMT in HCC cells, whereas knockdown of TRIM29 reversed these effects. Moreover, deactivation of the PTEN/AKT/mTOR and JAK2/STAT3 pathways might be involved in the tumor suppressive role of TRIM29 in HCC. Our findings indicate that TRIM29 in HCC exerts its tumor suppressive effects through inhibition of the PTEN/AKT/mTOR and JAK2/STAT3 signaling pathways and may be used as a potential biomarker for survival in patients with HCC.

Two somatic mutations in the androgen receptor N-terminal domain are oncogenic drivers in hepatocellular carcinoma.

The androgen receptor (AR) plays an important role in male-dominant hepatocellular carcinoma, and specific acquired somatic mutations of AR have been observed in HCC patients. Our previous research have established the role of AR wild type as one of the key oncogenes in hepatocarcinogenesis. However, the role of hepatic acquired somatic mutations of AR remains unknown. In this study, we identify two crucial acquired somatic mutations, Q62L and E81Q, situated close to the N-terminal activation function domain-1 of AR. These mutations lead to constitutive activation of AR, both independently and synergistically with androgens, making them potent driver oncogene mutations. Mechanistically, these N-terminal AR somatic mutations enhance de novo lipogenesis by activating sterol regulatory element-binding protein-1 and promote glycogen accumulation through glycogen phosphorylase, brain form, thereby disrupting the AMPK pathway and contributing to tumorigenesis. Moreover, the AR mutations show sensitivity to the AMPK activator A769662. Overall, this study establishes the role of these N- terminal hepatic mutations of AR as highly malignant oncogenic drivers in hepatocarcinogenesis and highlights their potential as therapeutic targets for patients harboring these somatic mutations.

Evaluation of the efficacy of a modified method of treating the incisions of the single-port video-assisted thoracoscopic surgery using V-Loc™ barbed sutures.

This paper describes a modified method of applying unidirectional barbed sutures to treat the incisions of the single-port video-assisted thoracoscopic surgery (VATS) and discusses its safety and feasibility. This was a retrospective analysis of 108 patients who underwent single-port VATS admitted to the Department of Thoracic Surgery, the China-Japan Union Hospital of Jilin University, from April 2019 to April 2020. The experimental group (65 patients) was given unidirectional barbed sutures (V-Loc™ sutures) to treat the incision, and the control group (43 patients) had a skin stapler to treat the incision. The complications related to the incisions of the two groups were compared. There was no statistically significant difference between the experimental and control groups regarding incisional infection, incisional splitting, fat liquefaction, and incisional resewing. The pleural fluid outflow from the drainage orifice after removal of the chest tube (0 cases in the experimental group and 7 cases in the control group, P = 0.001) was significantly lower in the experimental group than in the control group. The scores of the scars showed that the experimental group was significantly better than the control group. The modified method of treating the incisions of the single-port VATS with V-Loc™ sutures has good efficacy and safety. It reduces the incidence of pleural fluid outflow from the drainage orifice after removal of the chest tube compared with the traditional stapler suture method, and it has superior cosmetic outcomes.

Phosphorylation of androgen receptor by mTORC1 promotes liver steatosis and tumorigenesis.

BACKGROUND AND AIMS: Androgen receptor (AR) has been reported to play an important role in the development and progression of man's prostate cancer. Hepatocellular carcinoma (HCC) is also male-dominant, but the role of AR in HCC remains poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) also has been reported to be highly activated in HCC. In this study, we aimed to explore the role of AR phosphorylation and its relationship with mTORC1 in hepatocarcinogenesis. APPROACH AND RESULTS: In vitro experiment, we observed that mTORC1 interacts with hepatic AR and phosphorylates it at S96 in response to nutrient and mitogenic stimuli in HCC cells. S96 phosphorylation promotes the stability, nuclear localization, and transcriptional activity of AR, which enhances de novo lipogenesis and proliferation in hepatocytes and induces liver steatosis and hepatocarcinogenesis in mice independently and cooperatively with androgen. Furthermore, high ARS96 phosphorylation is observed in human liver steatotic and HCC tissues and is associated with overall survival and disease-free survival, which has been proven as an independent survival predictor for patients with HCC. CONCLUSIONS: AR S96 phosphorylation by mTORC1 drives liver steatosis and HCC development and progression independently and cooperatively with androgen, which not only explains why HCC is man-biased but also provides a target molecule for prevention and treatment of HCC and a potential survival predictor in patients with HCC.

Combination Treatment With Inhibitors of ERK and Autophagy Enhances Antitumor Activity of Betulinic Acid in Non-small-Cell Lung Cancer In Vivo and In Vitro.

Aberrant activation of the Ras-ERK signaling pathway drives many important cancer phenotypes, and several inhibitors targeting such pathways are under investigation and/or approved by the FDA as single- or multi-agent therapy for patients with melanoma and non-small-cell lung cancer (NSCLC). Here, we show that betulinic acid (BA), a natural pentacyclic triterpenoid, inhibits cell proliferation, and induces apoptosis and protective autophagy in NSCLC cells. Thus, the cancer cell killing activity of BA is enhanced by autophagy inhibition. Mitogen-activated protein kinases, and especially ERK that facilitates cancer cell survival, are also activated by BA treatment. As such, in the presence of ERK inhibitors (ERKi), lung cancer cells are much more sensitive to BA. However, the dual treatment of BA and ERKi results in increased protective autophagy and AKT phosphorylation. Accordingly, inhibition of AKT has a highly synergistic anticancer effect with co-treatment of BA and ERKi. Notably, autophagy inhibition by hydroxychloroquine (HCQ) increases the response of lung cancer cells to BA in combination with ERKi. In vivo, the three-drug combination (BA, ERKi, and HCQ), resulted in superior therapeutic efficacy than single or dual treatments in the xenograft mouse model. Thus, our study provides a combined therapy strategy that is a highly effective treatment for patients with NSCLC.

Lnc-GAN1 expression is associated with good survival and suppresses tumor progression by sponging mir-26a-5p to activate PTEN signaling in non-small cell lung cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) play vital roles in the development and progression of non-small-cell lung cancer (NSCLC); however, the role of most lncRNAs in NSCLC remains unknown. This study explored the clinical significance, biological function and underlying mechanism of lnc-GAN1 in NSCLC. METHODS: With a custom lncRNA microarray we found that lnc-GAN1 is markedly downregulated in NSCLC tissues. Then lnc-GAN1 expression level was measured using qRT-PCR in NSCLC tissues and cell lines. Survival was assessed using the Kaplan-Meier method. The biological functions of lnc-GAN1 in lung cancer cells were evaluated in vitro and in vivo. RNA fluorescence in situ hybridization and subcellular localization assays revealed the subcellular distribution of lnc-GAN1 in cells. Bioinformatic analysis was adopted to predict miRNAs and signaling pathways regulated by lnc-GAN1. RNA immunoprecipitation and Dual-luciferase reporter assays were used to assess the interaction between lnc-GAN1 and miR-26a-5p in lung cancer cells. RESULTS: lnc-GAN1 is downregulated in HCC tissues and associated with larger tumor size and poor overall survival and disease-free survival; its ectopic expression suppresses cell proliferation, colony formation, and cell cycle progression and induces apoptosis in NSCLC cells; it also inhibits tumor growth in the NSCLC xenograft model. We further proved that lnc-GAN1 is localized in cytoplasm and transcribed independently from its parental gene GAN. Mechanistically, lnc-GAN1 acts as a sponge for miR-26a-5p by two seed sequences, and the two non-coding RNAs have a negative relationship in NSCLC tissues; we further prove that PTEN is a direct target of miR-26a-5p and lnc-GAN1 inhibits cell cycle signaling pathway by activating PTEN, whose expression level correlated negatively with miR-26a-5p level but positively with lnc-GAN1 level in NSCLC samples. CONCLUSIONS: Lnc-GAN1 is downregulated and associated with poor survival of NSCLC patients, and mechanistically acts as a tumor suppressor via sponging and inhibiting miR-26a-5p to upregulate PTEN. This study provides a potential prognostic biomarker and treatment target for NSCLC.

A new indole alkaloid with HUVEC proliferation activities from Nauclea officinalis.

A new indole alkaloid, namely naucleofficine H (1), was obtained from the aqueous extract of Nauclea officinalis, together with four known alkaloids, vincosamide (2), strictosamide (3), angustoline (4) and pumiloside (5). Their structures were characterized by analyzing their physicochemical data including NMR, and HRMS. In addition, five compounds were tested for their proliferation activities. The expression of vascular endothelial growth factor (VEGF), extra-cellular signal-regulated protein kinase 1 and 2 (ERK) and phosphorylation of ERK 1/2 (p-ERK) were also detected in HUVEC treated withbioactive compounds using western blotting. The result showed that these compounds could promote HUVEC cell proliferation. Compounds 3 and 5 could up-regulate VEGF and p-ERK in HUVEC.

Body composition and sarcopenia in patients with stable chronic obstructive pulmonary disease (COPD) and its influencing factors / 中华全科医师杂志

Objective:To investigate the body composition and sarcopenia in patients with stable chronic obstructive pulmonary disease (COPD) and to analyze the influencing factors.Methods:A total of 220 patients with stable COPD were enrolled in the study from China-Japan Friendship Hospital during July 2018 to December 2019; 220 age and sex-matched healthy subjects (control group 1) and 220 healthy young adults aged 20-40 years (control group 2) were enrolled from the community. The body composition was measured by bioelectrical impedance method. The demographic characteristics, disease conditions, living background and other related factors were collected by questionnaire. T test or one-way ANOVA were used for comparison between groups, and non parametric test was used for non normal distribution data.Results:Compared with the control group 1 and control group 2, the body fat rate ( Z=-10.037, t=-8.411), the fat free mass index ( Z=-8.165, t=-7.856), and appendicular skeletal muscle index (ASMI) ( t=-7.158, t=-11.989) were significantly lower in stable COPD patients ( P<0.05). Among 220 patients with stable COPD, 51 (23.2%) were diagnosed as sarcopenia; the prevalence of sarcopenia was 18.3% (24/131) in men and 30.3% (27/89) in women (χ2=4.297, P=0.038). The decreased ASMI and grip strength in COPD patients with sarcopenia were significantly associated with age≥75 (χ2= 15.746, F= 14.048), female sex ( Z=5.805, t=2.672), low income ( Z=-4.291, t=-4.789), Global Initiative for Chronic Obstructive Lung Disease (GOLD) grade Ⅳ (χ2=22.644, F=3.905), Modified British Medical Research Council (mMRC) Dyspnea Scale grade 4 (χ2=12.475, F=4.369), not receiving systematic health education ( Z=-4.239, Z=-2.474), no exercise (χ2=14.786, F=3.402), insufficient nutrition intake (χ2=40.531, F=10.529). The range of 6-min walking distance was (110-268) m, that was even shorter for patients with mMRC dyspnea scale grade 4 ( F=3.468, P<0.05). Conclusion:The impairment of body composition is common in COPD patients, which will further affect the body function. It is suggested that the routine evaluation of COPD should include the measurement of body composition.

Identification of a 4-lncRNA signature predicting prognosis of patients with non-small cell lung cancer: a multicenter study in China.

BACKGROUND: Previous findings have indicated that the tumor, nodes, and metastases (TNM) staging system is not sufficient to accurately predict survival outcomes in patients with non-small lung carcinoma (NSCLC). Thus, this study aims to identify a long non-coding RNA (lncRNA) signature for predicting survival in patients with NSCLC and to provide additional prognostic information to TNM staging system. METHODS: Patients with NSCLC were recruited from a hospital and divided into a discovery cohort (n = 194) and validation cohort (n = 172), and detected using a custom lncRNA microarray. Another 73 NSCLC cases obtained from a different hospital (an independent validation cohort) were examined with qRT-PCR. Differentially expressed lncRNAs were determined with the Significance Analysis of Microarrays program, from which lncRNAs associated with survival were identified using Cox regression in the discovery cohort. These prognostic lncRNAs were employed to construct a prognostic signature with a risk-score method. Then, the utility of the prognostic signature was confirmed using the validation cohort and the independent cohort. RESULTS: In the discovery cohort, we identified 305 lncRNAs that were differentially expressed between the NSCLC tissues and matched, adjacent normal lung tissues, of which 15 are associated with survival; a 4-lncRNA prognostic signature was identified from the 15 survival lncRNAs, which was significantly correlated with survivals of NSCLC patients. This signature was further validated in the validation cohort and independent validation cohort. Moreover, multivariate Cox analysis demonstrates that the 4-lncRNA signature is an independent survival predictor. Then we established a new risk-score model by combining 4-lncRNA signature and TNM staging stage. The receiver operating characteristics (ROC) curve indicates that the prognostic value of the combined model is significantly higher than that of the TNM stage alone, in all the cohorts. CONCLUSIONS: In this study, we identified a 4-lncRNA signature that may be a powerful prognosis biomarker and can provide additional survival information to the TNM staging system.

LncRNA CSMD1-1 promotes the progression of Hepatocellular Carcinoma by activating MYC signaling.

Emerging evidence suggests that long non-coding RNAs (lncRNA) play critical roles in the development and progression of diverse cancers including hepatocellular carcinoma (HCC), but the underlying molecular mechanisms of lncRNAs that are involved in hepatocarcinogenesis have not been fully explored. Methods: In this study, we profiled lncRNA expression in 127 pairs of HCC and nontumor liver tissues (a Discovery Cohort) using a custom microarray. The expression and clinical significance of lncCSMD1-1 were then validated with qRT-PCR and COX regression analysis in a Validation Cohort (n=260) and two External Validation Cohorts (n=92 and n=124, respectively). In vitro and in vivo assays were performed to explore the biological effects of lncCSMD1-1 on HCC cells. The interaction of lncCSMD1-1 with MYC was identified by RNA pull-down and RNA immunoprecipitation. The role of LncCSMD1-1 in the degradation of MYC protein was also investigated. Results: With microarray, we identified a highly upregulated lncRNA, lncCSMD1-1, which was associated with tumor progression and poor prognosis in the Discovery Cohort, and validated in another 3 HCC cohorts. Consistently, ectopic expression of lncCSMD1-1 notably promotes cell proliferation, migration, invasion, tumor growth and metastasis of HCC cells in in vitro and in vivo experiments. Gene expression profiling on HCC cells and gene sets enrichment analysis indicated that the MYC target gene set was significantly enriched in HCC cells overexpressing lncCSMD1-1, and lncCSMD1-1 was found to directly bind to MYC protein in the nucleus of HCC cells, which resulted in the elevation of MYC protein. Mechanistically, lncCSMD1-1 interacted with MYC protein to block its ubiquitin-proteasome degradation pathway, leading to activation of its downstream target genes. Conclusion: lncCSMD1-1 is upregulated in HCC and promotes progression of HCC by activating the MYC signaling pathway. These results provide the evidence that lncCSMD1-1 may serve as a novel prognostic marker and potential therapeutic target for HCC.

Identification of immunological subtypes of hepatocellular carcinoma with expression profiling of immune-modulating genes.

Recent studies demonstrate that immune checkpoint inhibitor (ICI) therapy has achieved success in many types of advanced cancers including advanced hepatocellular carcinoma (HCC). However, ICI therapy is beneficial in only some HCC patients, suggesting that immune-responses are highly variable in HCCs. Therefore, understanding the immune status in HCC microenvironment will facilitate ICI immunotherapy and guide patient selection for the therapy. In this study, we first analyzed the expression profile of immune-modulating genes and their relationship with survival of HCC patients using the data downloaded from The Cancer Genome Atlas - Liver Hepatocellular Carcinoma (TCGA-LIHC) database, and found that the higher expressions of CD276 (B7-H3) and CD47 were significantly associated with poor survival. Then we identified 4 immune subtypes of HCCs with different survivals by using the combination expression of B7-H3 (or CD47) and CD8. Patients with B7-H3low/CD8high or CD47low/CD8high have the best survival while ones with B7-H3high/CD8low or CD47high/CD8low have the worst survival. The 4 immune subtypes were validated in another 72 HCC patients obtained from South China. In conclusion, our findings suggest that HCC patient prognosis is associated with immunophenotypes by T cell infiltration (CD8 expression) and the expression of the adaptive immune resistance gene (B7-H3 or CD47), and this immune classification system will facilitate HCC patient selection for ICI immunotherapy.

The prognostic value of a seven-lncRNA signature in patients with esophageal squamous cell carcinoma: a lncRNA expression analysis.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been reported to be prognostic biomarkers in many types of cancer. We aimed to identify a lncRNA signature that can predict the prognosis in patients with esophageal squamous cell carcinoma (ESCC). METHODS: Using a custom microarray, we retrospectively analyzed lncRNA expression profiles in 141 samples of ESCC and 81 paired non-cancer specimens from Sun Yat-Sen University Cancer Center (Guangzhou, China), which were used as a training cohort to identify a signature associated with clinical outcomes. Then we conducted quantitative RT-PCR in another 103 samples of ESCC from the same cancer center as an independent cohort to verify the signature. RESULTS: Microarray analysis showed that there were 338 lncRNAs significantly differentially expressed between ESCC and non-cancer esophagus tissues in the training cohort. From these differentially expressed lncRNAs, we found 16 lncRNAs associated with overall survival (OS) of ESCC patients using Cox regression analysis. Then a 7-lncRNA signature for predicting survival was identified from the 16 lncRNAs, which classified ESCC patients into high-risk and low-risk groups. Patients with high-risk have shorter OS (HR: 3.555, 95% CI 2.195-5.757, p < 0.001) and disease-free survival (DFS) (HR: 2.537, 95% CI 1.646-3.909, p < 0.001) when compared with patients with low-risk in the training cohort. In the independent cohort, the 7 lncRNAs were detected by qRT-PCR and used to compute risk score for the patients. The result indicates that patients with high risk also have significantly worse OS (HR = 2.662, 95% CI 1.588-4.464, p < 0.001) and DFS (HR 2.389, 95% CI 1.447-3.946, p < 0.001). The univariate and multivariate Cox regression analyses indicate that the signature is an independent factor for predicting survival of patients with ESCC. Combination of the signature and TNM staging was more powerful in predicting OS than TNM staging alone in both the training (AUC: 0.772 vs 0.681, p = 0.002) and independent cohorts (AUC: 0.772 vs 0.660, p = 0.003). CONCLUSIONS: The 7-lncRNA signature is a potential prognostic biomarker in patients with ESCC and may help in treatment decision when combined with the TNM staging system.

miR-665 expression predicts poor survival and promotes tumor metastasis by targeting NR4A3 in breast cancer.

Cancer metastasis is the main cause of death in breast cancer (BC) patients. Therefore, prediction and treatment of metastasis is critical for enhancing the survival of BC patients. In this study, we aimed to identify biomarkers that can predict metastasis of BC and elucidate the underlying mechanism of the functional involvement of such markers in metastasis. miRNA expression profile was analyzed using a custom microarray system in 422 BC tissues. The relationship between the upregulated miR-665, metastasis and survival of BC was analyzed and verified in another set of 161 BC samples. The biological function of miR-665 in BC carcinogenesis was explored with in vitro and in vivo methods. The target gene of miR-665 and its signaling cascade were also analyzed. There are 399 differentially expressed miRNAs between BC and noncancerous tissues, of which miR-665 is the most upregulated miRNA in the BC tissues compared with non-tumor breast tissues (P < 0.001). The expression of miR-665 predicts metastasis and poor survival in 422 BC patients, which is verified in another 161 BC patients and 2323 BC cases from online databases. Ectopic miR-665 expression promotes epithelial-mesenchymal transition (EMT), proliferation, migration and invasion of BC cells, and increases tumor growth and metastasis of BC in mice. Bioinformatics, luciferase assay and other methods showed that nuclear receptor subfamily 4 group A member 3 (NR4A3) is a target of miR-665 in BC. Mechanistically, we demonstrated that miR-665 promotes EMT, invasion and metastasis of BC via inhibiting NR4A3 to activate MAPK/ERK kinase (MEK) signaling pathway. Our study demonstrates that miR-665 upregulation is associated with metastasis and poor survival in BC patients, and mechanistically, miR-665 enhances progression of BC via NR4A3/MEK signaling pathway. This study provides a new potential prognostic biomarker and therapeutic target for BC patients.

Identification of two microRNA signatures in whole blood as novel biomarkers for diagnosis of nasopharyngeal carcinoma.

BACKGROUND: Early diagnosis is critical to reduce the mortality caused by nasopharyngeal carcinoma (NPC). MicroRNAs (miRNAs) are dysregulated and play important roles in carcinogenesis. Therefore, this study aimed to identify diagnostically relevant circulating miRNA signatures in patients with NPC. METHODS: Total RNA was extracted from whole blood samples obtained from 120 patients with NPC, 30 patients with head-neck tumors (HNT), and 30 healthy subjects (HSs), and examined by using a custom microarray. The expression levels of four miRNAs identified by using the microarray were validated with quantitative real-time reverse transcription polymerase chain reaction. The 120 patients with NPC and 30 HSs were randomly assigned to training group-1 and validation group-1, respectively. By using significance analysis of microarray (SAM), the specific miRNA expression profiles in whole blood from patients with NPC are obtained. By using lasso regression and adaptive boosting, a diagnostic signature was identified in training group-1, and its accuracy was verified in validation group-1. By using the same methods, another signature to distinguish patients with NPC from those with HNT and HSs was identified in training group-2 and confirmed in validation group-2. RESULTS: There were 117 differentially expressed miRNAs (upregulated and downregulated fold change ≥ 1.5) between the patients with NPC and HSs, among which an 8-miRNA signature was identified with 96.43% sensitivity and 100% specificity [area under the curve (AUC) = 0.995] to diagnose NPC in training group-1 and 86.11% sensitivity and 88.89% specificity (AUC = 0.941) in validation group-1. Compared with traditional Epstein-Barr virus (EBV) seromarkers, this signature was more specific for NPC. Furthermore, a 16-miRNA signature to differentiate NPC from HNT and HS (HNT-HS) was established from 164 differentially expressed miRNAs, which diagnosed NPC and HNT-HS with 100% accuracy (AUC = 1.000) in training group-2 and 87.04% (AUC = 0.924) in validation group-2. CONCLUSIONS: The present study identified two miRNA signatures for the highly accurate diagnosis and differential diagnosis of patients with NPC from HSs and patients with HNT. The identified miRNAs might represent novel serological biomarkers and potential therapeutic targets for NPC.

Measurement of pharmacokinetics and tissue distribution of three bioactive constituents from Zanthoxylum armatum DC in rat plasma and tissues through UFLC-MS/MS.

The compounds of N-Methylanhydrotetrahydroberberrubine A, dictamnine and eudesmin were the primary bioactive components in the roots of Zanthoxylum armatum DC (Z. armatum). To clarify the pharmacokinetics and distribution of these three compounds, an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was employed to determine the contents of these three compounds in rat plasma and seven tissues. The separation was achieved on a Kinetex XB-C18 100A column (2.1 × 50 mm, 2.6 µm, Phenomenex). The optimized mobile phase system was set with 0.1‰ formic acid aqueous solution (A) and acetonitrile (containing 0.1‰ formic acid) (B) with a programmed elution of 0.00 to 0.50 min, 2% B; 0.51-4.00 min, 30%-60% B; and 4.01-5.00 min, 2% B. All analytes were measured with optimized multiple reaction monitoring (MRM) in the positive ion ESI mode. Berberine hydrochloride was selected as the internal standard (IS). The MS/MS transitions of N-Methylanhydrotetrahydroberberrubine A, dictamnine, eudesmin and IS were 339.9135.1, 200.1 → 129.1, 387.4 → 369.0 and 337.1 → 321.1, respectively. The lower limits quantification (LLOQ) of the three analytes was 0.5-20 ng/ml. The linear ranges were 0.5-400 ng/ml for N-Methylanhydrotetrahydroberberrubine A and dictamnine and 20-4000 ng/ml for eudesmin. The present analysis showed that the two alkaloids were quickly absorbed, with Tmax in 0.167-0.292 h, and eudesmin was absorbed in 2.5 h. Moreover, all compounds were found at high concentrations in the gastrointestinal track. These results are helpful for further investigation of the clinical application of Z. armatum.

Epithelial cell adhesion molecule overexpression regulates epithelial-mesenchymal transition, stemness and metastasis of nasopharyngeal carcinoma cells via the PTEN/AKT/mTOR pathway.

Epithelial cell adhesion molecule (EpCAM) is known to be highly expressed in a variety of epithelial carcinomas, and it is involved in cell adhesion and proliferation. However, its expression profile and biological function in nasopharyngeal carcinoma (NPC) remains unclear. In this study, higher expression of EpCAM was found in NPC samples compared with non-cancer nasopharyngeal mucosa by qRT-PCR. Additionally, immunohistochemistry (IHC) analysis of NPC specimens from 64 cases showed that high EpCAM expression was associated with metastasis and shorter survival. Multivariate survival analysis identified high EpCAM expression as an independent prognostic factor. Ectopic EpCAM expression in NPC cells promoted epithelial-mesenchymal transition (EMT), induced a cancer stem cell (CSC)-like phenotype, and enhanced metastasis in vitro and in vivo without an effect on cell proliferation. Notably, EpCAM overexpression reduced PTEN expression and increased the level of AKT, mTOR, p70S6K and 4EBP1 phosphorylation. Correspondingly, an AKT inhibitor and rapamycin blocked the effect of EpCAM on NPC cell invasion and stem-like phenotypes, and siRNA targeting PTEN rescued the oncogenic activities in EpCAM knockdown NPC cells. Our data demonstrate that EpCAM regulates EMT, stemness and metastasis of NPC cells via the PTEN/AKT/mTOR pathway.