BRD4 plays an antiaging role in the senescence of renal tubular epithelial cells.

Background: Age-related kidney failure is often induced by a decrease in the bioavailability of tubular epithelial cells in elderly chronic kidney disease (CKD) patients. BRD4, an epigenetic regulator and a member of the bromodomain and extraterminal (BET) protein family, acts as a super-enhancer (SE) organizing and regulating genes expression during embryogenesis and cancer development. But the physiological function of BRD4 in normal cells has been less studied. This study aimed to research certain biological roles of BRD4 in the process of normal cell aging and discuss the potential mechanisms. Methods: In this study, we investigated the biological functions of BRD4 proteins in the aging of renal tubular cells. At first, we used a D-galactose (D-gal) and BRD4 inhibitor (Abbv-075) to replicate kidney senescence in vivo. D-gal and Abbv-075 were then used to measure the aging-related changes, such as changes in cell cycle, ß-galactosidase activity, cell migration, and p16 protein expression in vitro. At last, we knocked down and over-expressed BRD4 to investigate the aging-related physiological phenomena in renal tubular cells. Results: In vitro, D-gal treatment induced noticeable aging-related changes such as inducing cell apoptosis and cell cycle arrest, increasing ß-galactosidase activity as well as up-regulating p16 protein expression in primary human tubular epithelial cells. In the aging mice model, D-gal significantly induced renal function impairment and attenuated BRD4 protein expression. At the same time, the BRD4 inhibitor (Abbv-075) was able to mimic D-gal-induced cell senescence. In vivo, Abbv-075 also decreased kidney function and up-regulated p21 protein expression. When we knocked down the expression of BRD4, the senescence-associated ß-galactosidase (SA-ß-gal) activity increased dramatically, cell migration was inhibited, and the proportion of cells in the G0/G1 phase increased. Additionally, the knockdown also promoted the expression of the senescence-related proteins p16. When the renal tubular cells were overexpressed with BRD4, cell aging-related indicators were reversed in the D-gal-induced cell aging model. Conclusions: BRD4 appears to have an active role in the aging of renal tubular cells in vivo and in vitro. The findings also suggest that BRD4 inhibitors have potential nephrotoxic effects for oncology treatment. BRD4 may be a potential therapeutic biomarker and drug target for aging-related kidney diseases, which warrants additional studies.

Effect of multicomponent intervention on malnutrition in older adults: A multicenter randomized clinical trial.

BACKGROUND & AIMS: Malnutrition is a significant geriatric syndrome (GS) prevalent in older adults and seriously affects patient prognosis and quality of life. We assessed the impact of the multicomponent intervention of health education, dietary advice, and exercise with oral nutritional supplementation (ONS) on nutritional status, body composition, physical functions, and quality of life. METHODS: This multicenter randomized clinical trial (RCT) was performed from April 2021 to April 2022. The intervention lasted for 12 weeks, and 99 older adults with malnutrition or at risk of malnutrition were enrolled in six nursing homes. All participants were randomly assigned to the control (health education plus standard diet plus exercise) or research (health education plus standard diet plus exercise plus ONS) group. The research group consumed ONS (244 kcal, 9.8g protein, and 9.6g fat per time) twice a day between meals. The primary outcomes were changes in the nutritional status and body composition from baseline to 12 weeks. The secondary outcomes were changes in physical function, quality of life and nutritional associated other blood markers. RESULTS: For primary outcomes, after 12 weeks, body weight increased similarly in both treatment arms (time × treatment effect, P > 0.05). There were no between-group differences in body mass index (BMI) or mini nutritional assessment tool-short form (MNA-SF) scores (time × treatment effects, P > 0.05). The MNA-SF score from 11.0 (10.5, 12.0) to 13.0 (11.0, 13.0) in the research group and from 11.0 (10.0, 12.0) to 12.0 (11.0, 13.0) in the control group (both P < 0.05). There were no between-group differences in the skeletal muscle mass index (SMI), fat-free mass index (FFMI), appendicular skeletal muscle mass (ASMM), fat mass (FAT), or leg muscle mass (LMM) (time × treatment effects, P > 0.05). Both groups showed similar and highly significant increases in SMI, FFMI, and LMM after (P < 0.05). The research group showed an increase in fat-free mass (FFM) and ASMM and a decrease in the percent of body fat (PBF) and waist circumference (WC) (P < 0.05). For secondary outcomes, There were no between-group differences in grip strength, short physical performance battery (SPPB), 6-min walking distance (6MWD), activities of daily living (ADL), instrumental activities of daily living (IADL), frailty status (FRAIL), mini-mental state examination (MMSE), Tinetti, geriatric depression scale-15 (GDS-15), or 12-item short form survey (SF-12) (time × treatment effects, P > 0.05). Although there was no significant difference, the 6MWD changed differentially between the two treatment arms during the study period in favor of the research group. Although not significant, SF-12 scores improved after 12 weeks in both groups. No between-group differences were observed in prealbumin (PRE), c-reactive protein (CRP), vitamin D (VIT-D), insulin-like growth factor 1 (IGF-1), alanine transaminase (ALT), aspartate aminotransferase (AST), serum creatinine (Scr), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), insulin, and adiponectin levels (time × treatment effects, P > 0.05). Insulin and adiponectin levels were significantly higher in the control group (P < 0.05). CONCLUSION: The twelve-week multicomponent intervention improved the nutritional status of older people in China at risk of malnutrition. ONS may enhance the effects of exercise on muscle mass. This clinical trial was registered (https://www. CLINICALTRIALS: gov). The trial number is ChiCTR2000040343.

Identification of cucumber S-adenosylmethionine decarboxylase genes and functional analysis of CsSAMDC3 in salt tolerance.

As one of the key enzymes in the biosynthesis of polyamines, S-adenosylmethionine decarboxylase (SAMDC) plays an important role in plant stress resistance. In this study, four SAMDC genes (CsSAMDC1-4) were identified in cucumber (Cucumis sativus L.) and divided into three groups (I, II, and III) by phylogenetic analysis. Motif analysis suggested the existence of many conserved motifs, which is compatible with SAMDC protein classification. Gene structure analysis revealed that CsSAMDC2 and CsSAMDC3 in group I have no intron, which showed a similar response to salt stress by gene expression analysis. CsSAMDC3 responded differently to hormone and stress treatments, and was more susceptible to salt stress. Compared with wild-type (WT) tobacco, the activities of superoxide dismutase, peroxidase, and catalase were increased in CsSAMDC3-overexpressing tobacco under salt stress, but the content of electrolyte leakage, malondialdehyde, and hydrogen peroxide were decreased, which alleviated the inhibition of growth induced by salt stress. Under salt stress, overexpression of CsSAMDC3 in transgenic tobacco plants exhibited salt tolerance, mainly in the form of a significant increase in dry and fresh weight, the maximal quantum yield of PSII photochemistry, the net photosynthetic rate and the content of spermidine and spermine, while the content of putrescine was reduced. In addition, the expression levels of antioxidase-related coding genes (NtSOD, NtPOD, NtCAT) and PAs metabolism-related coding genes (NtSAMS, NtSPDS, NtSPMS, NtPAO) in transgentic plants was lower than WT under salt stress, which suggested that overexpression of CsSAMDC3 affected the expression of these genes. In summary, our results showed that CsSAMDC3 could be used as a potential candidate gene to improve salt tolerance of cucumber by regulating polyamine and antioxidant metabolism.

Association of frailty with clinical outcomes in chronic obstructive pulmonary disease: A retrospective longitudinal cohort study.

Background: Frailty is a clinical syndrome and common phenomenon in the elderly, particularly when it coexists with chronic obstructive pulmonary disease (COPD). However, the relationship between frailty and its prognosis in COPD patients has not been clearly elucidated. Methods: We collected electronic data of inpatients who were diagnosed with COPD in the First Affiliated Hospital with Nanjing Medical University (NJMU) from January 2018 to December 2020. In further, we divided them into different groups based on Frailty Index Common Laboratory Tests (FI-LAB). Binary logistic regression was performed to analyze the risk factors associated with COPD. The receiver operating characteristic (ROC) curve and area under the curve (AUC) were applied to validate FI-LAB's value in prognosis. Primary clinical outcomes contained 30-day mortality and readmission. Moreover, we also compared the prognositic value of FI-LAB with Hospital Frailty Risk Score (HRS) by ROC curve, significance was set at P < 0·05. Findings: The final study included 826 COPD patients, among of them, 30-day mortality and readmission of frailty group was 11·2%, 25·9%, the robust group was 4·3%, 16·0%, and p value was 0·001, 0·004 respectively. Multivariate analysis revealed that smoking, CCI≥3, oral drug≥5, pneumonia, abnormal lymphocyte, abnormal haemoglobin were independent risk factors with frailty. As for the prediction of FI-LAB about frailty in 30-day mortality, the AUC was 0·832, and 30-day readmission was 0·661. As for the prognositic value, FI-LAB and HRS showed no difference in predicting clinical outcomes. Interpretation: COPD individuals have a higher rate of frailty and pre-frailty. There exists a strong correlation between frailty and 30-day mortality in COPD patients, and FI-LAB has good prognostic value in clinical outcomes of patients with COPD.

High-Dosage NMN Promotes Ferroptosis to Suppress Lung Adenocarcinoma Growth through the NAM-Mediated SIRT1-AMPK-ACC Pathway.

BACKGROUND: Nicotinamide mononucleotide (NMN) is the physiological circulating NAD precursor thought to elevate the cellular level of NAD+ and to ameliorate various age-related diseases. An inseparable link exists between aging and tumorigenesis, especially involving aberrant energetic metabolism and cell fate regulation in cancer cells. However, few studies have directly investigated the effects of NMN on another major ageing-related disease: tumors. METHODS: We conducted a series of cell and mouse models to evaluate the anti-tumor effect of high-dose NMN. Transmission electron microscopy and a Mito-FerroGreen-labeled immunofluorescence assay (Fe2+) were utilized to demonstrate ferroptosis. The metabolites of NAM were detected via ELISA. The expression of the proteins involved in the SIRT1-AMPK-ACC signaling were detected using a Western blot assay. RESULTS: The results showed that high-dose NMN inhibits lung adenocarcinoma growth in vitro and in vivo. Excess NAM is produced through the metabolism of high-dose NMN, whereas the overexpression of NAMPT significantly decreases intracellular NAM content, which, in turn, boosts cell proliferation. Mechanistically, high-dose NMN promotes ferroptosis through NAM-mediated SIRT1-AMPK-ACC signaling. CONCLUSIONS: This study highlights the tumor influence of NMN at high doses in the manipulation of cancer cell metabolism, providing a new perspective on clinical therapy in patients with lung adenocarcinoma.

Prognostic significance of frailty status in patients with primary lung cancer.

Lung cancer has one of the highest morbidity and mortality rates in the world. Frailty is common in many countries and is a major cause of premature functional decline and premature death in older adults, and may affect the treatment and prognosis of lung cancer patients. To investigate the predictive value of frailty at diagnosis on all-cause mortality in lung cancer patients, this study retrospectively collected and analysed clinical information on lung cancer patients from 2015-2018. A total of 1667 patients with primary lung cancer were finally included in this study. The median follow-up time of patients was 650 (493, 1001.5) days. A total of 297(17.8%) patients had FI-LAB(the frailty index based on laboratory test) status of frail at the moment of diagnosis and the all-cause mortality rate for all patients was 61.1% (1018/1667). In a univariate model, we found a higher total all-cause mortality risk in frail patients (frail vs. robust, HR(hazard ratio) = 1.616, 95% CI(confidence interval) = 1.349,1.936), after balancing other variables combined into model 1 to model 6. The results were analyzed visually using ROC(Receiver operating characteristic) curves with nomogram and the AUC values ranged from 0.866-0.874. The final inclusion of age, TNM stage, CCI(Charlson comorbidity index) score, surgery history and chemotherapy into a multifactorial model balanced the predictive power of frailty grading on all-cause mortality. The study showed that for lung cancer patients, the higher the level of frailty at diagnosis, the higher the risk of all-cause mortality. In the context of widespread electronic medical records in hospitals, it is convenient and feasible to use FI-LAB to assess the prognosis of lung cancer patients.

Single-cell transcriptomics highlights immunological dysregulations of monocytes in the pathobiology of COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common respiratory disease, whose pathogenetic complexity was strongly associated with aging/smoking and poorly understood. METHODS: Here we performed single-cell RNA sequencing (scRNA-seq) analysis of 66,610 cells from COPD and age-stratified control lung tissues of donors with different smoking histories to prioritize cell types most perturbed in COPD lungs in aging/smoking dependent or independent manner. By performing an array of advanced bioinformatic analyses, such as gene set enrichment analysis, trajectory analysis, cell-cell interactions analysis, regulatory potential analysis, weighted correlation network analysis, functional interaction analysis, and gene set variation analysis, we integrated cell-type-level alterations into a system-level malfunction and provided a more clarified COPD pathological model containing specific mechanisms by which aging and smoking facilitate COPD development. Finally, we integrated the publicly available scRNA-seq data of 9 individuals, resulting in a total of 110,931 cells, and replicated the analyses to enhance the credibility of our findings. RESULTS: Our study pointed to enrichment of COPD molecular alteration in monocytes, which further induced a previously unrecognized pro-inflammatory effect on alveolar epithelial cells. In addition, aged monocytes and club cells facilitated COPD development via maintaining an autoimmune airway niche. Unexpectedly, macrophages, whose defect to resolve inflammation was long-recognized in COPD pathogenesis, primarily induced an imbalance of sphingolipids rheostat in a smoking-dependent way. These findings were validated in a meta-analysis including other public single-cell transcriptomic data. CONCLUSIONS: In sum, our study provided a clarified view of COPD pathogenesis and demonstrated the potential of targeting monocytes in COPD diagnosis and treatment.

Preventive measures significantly reduced the risk of nosocomial infection in elderly inpatients during the COVID-19 pandemic.

In December 2019, there was an outbreak of pneumonia of unknown causes in Wuhan, China. The etiological pathogen was identified to be a novel coronavirus, named severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19). The number of infected patients has markedly increased since the 2019 outbreak and COVID-19 has also proven to be highly contagious. In particular, the elderly are among the group of patients who are the most susceptible to succumbing to COVID-19 within the general population. Cross-infection in the hospital is one important route of SARS-CoV-2 transmission, where elderly patients are more susceptible to nosocomial infections due to reduced immunity. Therefore, the present study was conducted to search for ways to improve the medical management workflow in geriatric departments to ultimately reduce the risk of nosocomial infection in elderly inpatients. The present observational retrospective cohort study analysed elderly patients who were hospitalised in the Geriatric Department of the First Affiliated Hospital with Nanjing Medical University (Nanjing, China). A total of 4,066 elderly patients, who were admitted between January and March in 2019 and 2020 and then hospitalised for >48 h were selected. Among them, 3,073 (75.58%) patients hospitalised from January 2019 to March 2019 were allocated into the non-intervention group, whereas the remaining 933 (24.42%) patients hospitalised from January 2020 to March 2020 after the COVID-19 outbreak were allocated into the intervention group. Following multivariate logistic regression analysis, the risk of nosocomial infections was found to be lower in the intervention group compared with that in the non-intervention group. After age stratification and adjustment for sex, chronic disease, presence of malignant tumour and trauma, both inverse probability treatment weighting and standardised mortality ratio revealed a lower risk of nosocomial infections in the intervention group compared with that in the non-intervention group. To rule out interference caused by changes in the community floating population and social environment during this 1-year study, 93 long-stay patients in stable condition were selected as a subgroup based on 4,066 patients. The so-called floating population refers to patients who have been in hospital for <2 years. Patients aged ≥65 years were included in the geriatrics program. The incidence of nosocomial infections during the epidemic prevention and control period (24 January 2020 to 24 March 2020) and the previous period of hospitalisation (24 January 2019 to 24 March 2019) was also analysed. In the subgroup analysis, a multivariate analysis was also performed on 93 elderly patients who experienced long-term hospitalisation. The risk of nosocomial and pulmonary infections was found to be lower in the intervention group compared with that in the non-intervention group. During the pandemic, the geriatric department took active preventative measures. However, whether these measures can be normalised to reduce the risk of nosocomial infections among elderly inpatients remain unclear. In addition, the present study found that the use of an indwelling gastric tube is an independent risk factor of nosocomial pulmonary infection in elderly inpatients. However, nutritional interventions are indispensable for the long-term wellbeing of patients, especially for those with dysphagia in whom an indwelling gastric tube is the most viable method of providing enteral nutrition. To conclude, the present retrospective analysis of the selected cases showed that enacting preventative and control measures resulted in the effective control of the incidence of nosocomial infections.

Identification of TRP-Related Subtypes, Development of a Prognostic Model, and Characterization of Tumor Microenvironment Infiltration in Lung Adenocarcinoma.

The TRP (transient receptor potential) superfamily, as cation channels, is a critical chemosensor for potentially harmful irritants. Their activation is closely related not only to tumor progression and prognosis but also to tumor therapy response. Nevertheless, the TRP-related immune gene (TRIG) expression of the tumor microenvironment (TME) and the associations with prognosis remain unclear. First, we represented the transcriptional and genetic variations in TRIGs in 535 lung adenocarcinoma (LUAD) samples as well as their expression patterns. LUAD samples were divided into two distinct subtypes based on the TRIG variations. Significant differences had been found in prognosis, clinical features, and TME cell-infiltration features between the two subtypes of patients. Second, we framed a TRIG score for predicting overall survival (OS) and validated the predictive capability of the TRIG score in LUAD patients. Accordingly, to enhance the clinical applicability of TRIG score, we developed a considerable nomogram. A low TRIG score, characterized by increased immunity activation, indicated favorable advantages of OS compared with a high TRIG score. Furthermore, the TRIG score was found to have a significant connection with the TME cell-infiltration and immune checkpoint expressions. Our analysis of TRIGs in LUAD showed their potential roles in prognosis, clinical features, and tumor-immune microenvironments. These results may advance our knowledge of TRP genes in LUAD and show a new light on prognosis estimation and the improvement of immunotherapy strategies.

SHANK1 facilitates non-small cell lung cancer processes through modulating the ubiquitination of Klotho by interacting with MDM2.

SH3 and multiple ankyrin repeat domains 1 (SHANK1) is a scaffold protein, plays an important role in the normal function of neuron system. It has recently been shown to be a potential oncogene. In the present study, we report that the expression of SHANK1 is upregulated in non-small cell lung cancer (NSCLC), and is correlated with clinic pathological characteristics of NSCLC. Moreover, SHANK1 overexpression enhances the proliferation, migration and invasion of NSCLC cells. Mouse cell-derived xenograft model also confirmed the effects of SHANK1 on tumor growth in vivo. Furthermore, we found that SHANK1 increases the protein degradation of Klotho (KL), an important tumor suppressor, through ubiquitination-dependent pathway. In particular, we report discovery of KL as a SHANK1-interacting protein that acts as a new substate of the E3 ubiquitin ligase MDM2. SHANK1 can form a complex with KL and MDM2 and enhance the interaction between KL and MDM2. Our findings reveal an important oncogenic role and mechanism of SHANK1, suggesting SHANK1 can be a potential therapeutic target in NSCLC.

Activation of peracetic acid via Co3O4 with double-layered hollow structures for the highly efficient removal of sulfonamides: Kinetics insights and assessment of practical applications.

Sulfonamides (SAs) have been of ecotoxicological concern for ambient ecosystems due to their widespread application in the veterinary industry. Herein, we developed a powerful advanced oxidation peracetic acid (PAA) activation process for the remediation of SAs by Co3O4 with double-layered hollow structures (Co3O4 DLHSs). Systematic characterization results revealed that the polyporous hollow hierarchical structure endows Co3O4 DLHSs with abundant active reaction sites and enhanced mass transfer rate, which were conducive for improving the PAA activation efficiency. Laser flash photolysis experiment and mechanism studies indicated that organic radical species were dominant reactive species for SAs removal. The present system is also highly effective under natural water matrices and trace SAs concentration (20 µg/L) condition. More importantly, the chlorella acute toxicity of the SAs solution was eliminated during mineralization process, supporting this catalytic system may be efficaciously applied for the remediation of SAs contamination in ambient waterways.

Interleukin-19 Aggravates Pulmonary Fibrosis via Activating Fibroblast through TGF-ß/Smad Pathway.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial pneumonia disease with no cure. Communication between injured cells is triggered and maintained by a complicated network of cytokines and their receptors. IL-19 is supported by increasing evidences for a deleterious role in respiratory diseases. However, its potential role in lung fibrosis has never been explored. Methods: Bioinformatic, immunohistochemistry and western blot analysis were used to assess the expression of IL-19 in human and mouse fibrosis lung tissues. CCK-8, transwell and flow cytometry assay were utilized to analyze the effect of IL-19 on biological behaviors of lung fibroblasts. Histopathology was used to elucidate profibrotic effect of IL-19 in vivo. Results: IL-19 was upregulated in fibrosis lung tissues. IL-19 promoted lung fibroblasts proliferation and invasion, inhibited cell apoptosis, and induced differentiation of fibroblasts to the myofibroblast phenotype, which could be revised by LY2109761, a TGF-ß/Smad signaling pathway inhibitor. Furthermore, we found that IL-19 aggravated lung fibrosis in murine bleomycin-induced lung fibrosis. Conclusions: Our results imply the profibrotic role for IL-19 through direct effects on lung fibroblasts and the potential of targeting IL-19 for therapeutic intervention in pulmonary fibrosis.

Analysis of Genetic Variants and the ceRNA Network of miR-9 in Non-Small Cell Lung Cancer.

Objective: To explore the role of single-nucleotide polymorphisms (SNPs) in hsa-miR-9 in non-small cell lung cancer (NSCLC). Methods: Log-rank and Cox regression analyses were conducted to assess the association of four functional SNPs of miR-9 with overall survival (OS) of Chinese patients with NSCLC. A reporter luciferase assay was performed to examine the relationship between the SNPs and transcriptional activity of miR-9. The expression of miR-9 in cells was detected by quantitative real-time PCR assay. Xenograft model was established in nude mice, which were treated with Lv-MiR-9-mimics or Lv-miR-9-inhibitor. A long noncoding RNA (lncRNA)-miR-9-messenger RNA (mRNA) competing endogenous RNA (ceRNA) network was established based on bioinformatics analyses. Results: We found that rs1501672 was associated with the prognosis of 1001 Chinese NSCLC patients (A>G, additive model: adjusted hazard ratio = 0.89, 95% confidence interval = 0.79-1.00, p = 0.056). Luciferase reporter assay showed higher luciferase activity with wild A allele than that with mutant G allele in 293T, SPC-A1, and A549 cell lines. The miR-9 level was significantly higher in lung cancer cells than normal lung cells. miR-9 was also over expressed in lung cancer tissue according to The Cancer Genome Atlas and gene expression omnibus databases. Xenograft models based on H1299 cells showed that lv-miR-9-inhibitor significantly decreased tumor growth compared with the lv-miR-9-NC group (p < 0.001). Bioinformatics analysis showed that one target gene leukemia inhibitory factor receptor and two lncRNAs (KIAA0087 and GVINP1) were associated with OS of NSCLC patients. Conclusion: The rs1501672 of miR-9 was associated with the prognosis of NSCLC patients in the Chinese population. The lncRNA-miR-9-mRNA ceRNA network revealed potential molecular biological regulation pathways and prognostic biomarkers for NSCLC.

Efficacy of YAP1-gene Knockdown to Inhibit Alveolar-Epithelial-Cell Senescence and Alleviate Idiopathic Pulmonary Fibrosis (IPF).

BACKGROUND/AIM: The prevalence of idiopathic pulmonary fibrosis (IPF) increases with age and is associated with senescence of alveolar epithelial cells (AECs). AEC senescence in pulmonary cells mediates IPF. We herein aimed to determine if YAP1 gene knockdown, a member of the Hippo/YAP signal pathway, in the bleomycin (BLM)-induced mouse model of IPF, inhibits onset of senescence of AECs and alleviates IPF. MATERIALS AND METHODS: Adeno-associated viruses (AAVs) expressing Yes-associated protein 1 (YAP1) short hairpin RNA (shRNA) were delivered into the lung of BLM-induced IPF mice via intratracheal injection, to knockdown the YAP1 gene in AECs. The mice were assigned to 4 groups: G1: control (normal mice); G2: IPF mice; G3: IPF + AAV/YAP1; G4: IPF + AAV/scramble. After 28 days, AECs were examined for senescence using H&E staining, Masson's trichrome Staining, senescence-associated ß-galactosidase (SA-ß-gal) staining, western blotting and co-immunofluorescence staining, to determine the expression of YAP1, Smad-3 and p21, in order to determine the induction of senescence of ACEs. RESULTS: The severity of IPF determined by H&E staining, Masson's staining and immunofluorescence (IF) staining was positively correlated with the senescence of AECs. Down-regulation of YAP1 expression of the Hippo-signaling pathway, determined by western blotting in AECs, alleviated pulmonary fibrosis as determined by Masson's staining. Down regulation of YAP1 expression reduced the senescence of AECs as determined by ß-galactosidase (SA-ß-gal) staining, which alleviated the clinical symptoms of IPF mice, as determined by body weight and lung index. CONCLUSION: Down-regulation of YAP1 expression in AECs inhibited AEC senescence which is thought to be the cause of IPF. Therefore, future studies can focus on inhibiting YAP1 to effectively treat IPF.

YAP manipulates proliferation via PTEN/AKT/mTOR-mediated autophagy in lung adenocarcinomas.

BACKGROUND: Autophagy is a double-edged sword during the initiation and progression of multiple tumors. The Hippo pathway effector YAP has been proved to be involved in autophagy processes. The present study aimed to investigate how YAP regulates cell proliferation via autophagy in lung adenocarcinomas (LUAD). METHODS: Data of LUAD chip GSE43458 was obtained from Gene Expression Omnibus (GEO). RT-qPCR and Western blot were performed to assess YAP expression in LUAD cell lines. CCK-8 assay, xenograft tumor model, immunochemistry and GFP-mRFP-LC3 fusion proteins were utilized to evaluate the effect of YAP on autophagy of LUAD cells in vitro and in vivo. Autophagy inhibitor treatment and rescue experiments were carried out to elucidate the mechanism by which YAP manipulates autophagy in LUAD cells. RESULTS: YAP was significantly overexpressed in samples of LUAD patients and its expression level is related to 5-year survival. YAP manipulated the proliferation and autophagy in A549 and H1299 LUAD cells. YAP could induce activation of Akt/mTOR signaling pathway via suppressing PTEN in a Hippo-pathway-dependent manner. 3-Methyladenine impeded autophagy flux and promoted the proliferation in vitro and in vivo. CONCLUSIONS: Hippo pathway critical transcriptional coactivators YAP manipulates the proliferation of lung adenocarcinoma, which is regulated by PTEN/AKT/mTOR autophagic signaling.

[Auxiliary Diagnostic Value of Tumor Markers in the Cerebrospinal Fluid and Blood for Leptomeningeal Metastasis from Non-small Cell Lung Cancer].

BACKGROUND: Leptomeningeal metastasis (LM) is one of the most common causes of death in patients with advanced non-small cell lung cancer (NSCLC), which is defined as malignant cells spreading to meninges and cerebrospinal fluid (CSF). Therefore, early diagnosis and timely treatment are essential. CSF cytology is the gold standard for LM diagnosis, however, it has a low sensitivity for diagnosis and can't be used to evaluate the treatment effect. The aim of this study was to assess the clinical value of serum and CSF tumor markers (TM) in the diagnosis and treatment of NSCLC patients with LM. METHODS: Nineteen patients with NSCLC-LM and 27 patients with nonmalignant neurological diseases (NMNDs) were included. We tested the levels and positive rates of carbohydrate antigen (CEA), carbohydrate antigen-125 (CA125), cytokeratin 19 fragments (CYFRA21-1) and neurone specific enolase (NSE) in CSF and serum, compared the sensitivity and specificity in the diagnosis of LM between different groups, and analyzed the correlation of detection between serum and CSF. Finally, we measured serum and CSF TM dynamically in 2 patients with NSCLC developing LM in an attempt to correlate these with the treatment response of extracranial and intracranial, respectively. RESULTS: The levels and positive rates of TM in CSF and serum in LM group were higher than those in NMNDs (P<0.05). In LM group, the levels of CEA, CYFRA21-1 and CEA were significantly higher in CSF than that in the serum (P<0.05), whereas, there was no statistical significance in positive rates of TM between CSF and serum (P>0.05). In CSF, CYFRA21-1 has the highest sensitivity (88.2%) and CEA has the best specificity (92.3%) to distinguish patients between LM and NMNDs. For combined detection of CEA, CA125, CYFRA 21-1 and NSE in CSF, when at least CEA or NSE was positive in patients with LM, the sensitivity and negative predictive value were 100.0%, and the specificity was 74.1%. When both CYFRA21-1 and NSE were positive, the specificity and positive predictive value were 100.0%, and the sensitivity was 78.9%. Furthermore, subgroup analysis showed that the detection rates of TM in CSF cytology positive population was higher than that in typical abnormalities magnetic resonance imaging population, but there was no statistical difference (P>0.05). The detection of TM between serum and CSF in LM patients had no significant correlation. Moreover, biochemical properties of CSF from ventricle and lumbar puncture are similar, therefore evaluating the levels of TM in serum and CSF dynamically can be used to assess the extracranial and intracranial treatment effect, respectively. CONCLUSIONS: Our study demonstrates that Serum and CSF TM can work as an auxiliary clinical diagnostic tool, which has a potential value in early diagnosis of NSCLC patients with LM. Serial measurement of TM may play an important role in the clinical management of NSCLC patients with LM, which is worthy of further promotion and clinical application.

Klotho antagonizes pulmonary fibrosis through suppressing pulmonary fibroblasts activation, migration, and extracellular matrix production: a therapeutic implication for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) has been widely accepted as an aging-related fatal lung disease with a therapeutic impasse, largely a consequence of the complex and polygenic gene architecture underlying the molecular pathology of IPF. Here, by conducting an integrative network analysis on the largest IPF case-control RNA-seq dataset to date, we attributed the systems-level alteration in IPF to disruptions in a handful of biological processes including cell migration, transforming growth factor-ß (TGF-ß) signaling and extracellular matrix (ECM), and identified klotho (KL), a typical anti-aging molecule, as a potential master regulator of those disease-relevant processes. Following experiments showed reduced Kl in isolated pulmonary fibroblasts from bleomycin-exposed mice, and demonstrated that recombinant KL effectively mitigated pulmonary fibrosis in an ex vivo model and alleviated TGF-ß-induced pulmonary fibroblasts activation, migration, and ECM production in vitro, which was partially ascribed to FOXF1 and CAV1, two highly co-expressed genes of KL in the IPF. Overall, KL appears to be a vital regulator during pulmonary fibrosis. Given that administration of exogenous KL is a feasible treatment strategy, our work highlighted a promising target gene that could be easily manipulated, leaving the field well placed to further explore the therapeutic potential of KL for IPF.

Rab8 GTPase regulates Klotho-mediated inhibition of cell growth and progression by directly modulating its surface expression in human non-small cell lung cancer.

BACKGROUND: The klotho (KL) gene is an anti-aging gene that has recently been shown to also function as a general tumor suppressor. However, there is currently only limited information regarding the potential molecular signals for regulation of Klotho without identifying precise molecular mechanisms or interactions. METHODS: We performed a mass spectrometry (MS) assay to screen candidate proteins complexed with Klotho derived from immunoprecipitation in human non-small cell lung cancer (NSCLC) cells, and identified Rab8 to be the protein that most prominently interacts with Klotho. We further investigated whether Rab8 can regulate trafficking of Klotho and which process it would modulate using surface biotinylation assay, immunofluorescence and fluorescence ratio microscopy. Furthermore, we explored whether Rab8 is involved in Klotho-mediated function in NSCLC, and verified the results which we found in vivo using xenograft mouse model. FINDINGS: We report discovery of Rab8 as a Klotho-interacting protein that acts as a critical modulator of Klotho surface expression in human NSCLC. In particular, we report that Rab8 is co-localized and associated with Klotho, and Klotho trafficking is regulated by Rab8. Moreover, we found that Rab8 modulates surface levels of Klotho via a post-biosynthetic pathway, as opposed to an endocytic pathway. Furthermore, we demonstrate that Rab8 is involved in Klotho-mediated regulation of cell proliferation, migration, invasiveness, epithelial-mesenchymal transition (EMT), and Wnt-ß-catenin signaling in NSCLC. Additionally, Rab8 overexpression was also found to increase Klotho-mediated inhibition of NSCLC tumorigenesis in vivo. INTERPRETATION: Overall, our findings suggest that Rab8 GTPase can regulate Klotho-mediated inhibition of Wnt signaling activity by modulating translocation of Klotho onto the cell surface, which in turn affects Klotho-mediated inhibition of cell proliferation, migration and invasiveness in NSCLC. These results have important implications for the development of new therapeutic targets, Klotho-related research in the context of NSCLC as well as other areas, and provide a working model for Rab8 function in the context of cancer and cancer biology.

Prognostic Value of Survival of MicroRNAs Signatures in Non-small Cell Lung Cancer.

Introduction: Accumulating evidence showed that a large number of microRNAs (miRNAs) are abnormally expressed in lung cancer tissues and play critical roles in cancer development and progression. The aim of this study is to identify the differentially expressed miRNAs (DEMs) between non-small cell lung cancer (NSCLC) and normal lung tissues, and evaluate the prognostic value and potential target gene functional enrichment of the DEMs. Materials and Methods: We first downloaded the high-throughput miRNA data from The Cancer Genome Atlas Project (TCGA) database, and subsequently analyzed the data using bioinformatics analysis including limma package in R, Kaplan-Meier curve and Log-rank method, and several online analysis tools. Results: A total of 125 DEMs and 138 DEMs were respectively identified in lung adenocarcinoma (LUAD) tissues and lung squamous cell carcinoma (LUSC) tissues compared with their matched normal tissues. Moreover, we found that the prognostic function of the eight miRNAs (miR-375, miR-148a, miR-29b-1 and miR-584 for LUAD; miR-4746, miR-326, miR-93 and miR-671 for LUSC). Furthermore, the two four-miRNA signatures were constructed and found to be an independent prognostic factor for LUAD and LUSC patients, respectively. Additionally, our results indicated that the target genes of eight miRNAs may be involved in various pathways related to NSCLC, including PI3K-Akt, TGF-beta, FoxO, Ras, GPI-anchor biosynthesis and metabolic, Rap1, HIF-1 and proteasome. Conclusion: Overall, eight miRNAs were closely correlated with survival of NSCLC patients, and the constructed two four-miRNA signatures could be respectively used as prognostic markers in LUAD and LUSC patients.

Genetic variations in miR-125 family and the survival of non-small cell lung cancer in Chinese population.

To investigate the associations between the functional single nucleotide polymorphisms (SNPs) in the miR-125 family and the survival of non-small cell lung cancer (NSCLC) patients, we systematically selected six functional SNPs located in three pre-miRNAs (miR-125a, miR-125b-1, miR-125b-2). Cox proportional hazard regression analyses were conducted to estimate the crude and adjusted hazard ratios (HRs) and their 95% confidence intervals (CIs). Reporter gene luciferase assay was performed to examine the relationship between the SNPs and transcriptive activity of the miRNAs. The expression of miRNAs in different cells was detected using quantitative real-time PCR assay. We found that rs2241490 (upstream of miR-125b-1, G > A, adjusted HR = 1.24, 95%CI = 1.05-1.48, P = 0.014, in dominant model; adjusted HR = 1.18, 95%CI = 1.03-1.35, P = 0.014, in additive model), rs512932 (upstream of miR-125b-1, A > G, dominant model: adjusted HR = 1.25, 95%CI = 1.05-1.48, P = 0.013) and rs8111742 (upstream of miR-125a, G > A, dominant model: adjusted HR = 0.84, 95%CI = 0.71-1.00, P = 0.047) were associated with the prognosis of 1001 Chinese NSCLC patients. The combined analysis of the three SNPs related the number of risk alleles (rs2241490-A, rs512932-G and rs8111742-G) to death risk of NSCLC in a locus-dosage mode (P for trend <0.001). Furthermore, luciferase reporter gene assay showed significantly higher levels of luciferase activity with rs512932 variant G than that with A allele in 293T, SPC-A1 and A549 cell lines. Besides, miR-125b was highly expressed in lung cancer cells than the normal lung cell. Our study indicated that genetic variations in miR-125 family were implicated in the survival of NSCLC patients. Larger population-based and functional studies are needed to verify these findings.