Identification of oxidative stress signatures of lung adenocarcinoma and prediction of patient prognosis or treatment response with single-cell RNA sequencing and bulk RNA sequencing data.

Lung adenocarcinoma (LUAD), the most common subtype of lung cancer globally, has seen improved prognosis with advancements in diagnostic, surgical, radiotherapy, and molecular therapy techniques, while its 5-year survival rate remains low. Molecular biomarkers provide prognostic value. Oxidative stress factors, such as reactive nitrogen species and ROS, are crucial in various stages of tumor progression, influencing cell transformation, proliferation, angiogenesis, and metastasis. ROS demonstrate dual roles, affecting tumor cells, hypoxia sensitivity, and the microenvironment. Comprehensive analysis of oxidative stress in LUAD has not been conducted to date. Therefore, we systematically investigated the regulatory patterns of oxidative stress in LUAD based on oxidative stress-related genes and correlated these patterns with cellular infiltration characteristics of the tumor immune microenvironment. The model utilizes single-factor Cox analysis to screen key differential genes with prognostic value and employs least absolute shrinkage and selection operator (LASSO) penalized Cox regression analysis to construct a prognostic-related prediction model. Ten candidate genes were selected based on this model. The risk score was constructed using the coefficients and expression levels of these ten genes. Furthermore, the impact of this risk score on overall survival (OS) was determined. Two genes with the most significant differential expression, SFTPB and S100P, were selected through qRT-PCR. Cell experiments including CCK-8, Edu, transwell assays confirmed their effects on lung cancer cells growth, consistent with the results of bioinformatics analysis. These findings suggested that this model held potential clinical value for evaluating the prognosis of lung adenocarcinoma.

Identification of the key miRNA-mRNA regulatory network in lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is the most common type of lung cancer, has a high incidence rate and is a serious threat to human health. However, the pathogenesis of LUAD is still unclear. Further research on the pathogenesis of LUAD may provide targets for the early diagnosis and treatment of LUAD. Methods: First, a transcriptome analysis was conducted to sequence the messenger RNA (mRNA) and micro RNA (miRNA) of the LUAD and adjacent control tissues. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were then performed for the functional annotation. Next, a differential miRNA-differential mRNA regulatory network was then constructed, and the function of the mRNAs in the network was analyzed and the key regulatory molecules (the hub molecules) were identified. Cytohubba was then used to analyze the top 20 hub molecules in the total miRNA-mRNA network, and the miRNAs regulating the top 20 hub genes (of which 2 were upregulated and 18 were downregulated). Finally, the key molecules were identified. Results: By analyzing the function of the mRNA molecules in the regulatory network, we found that the immune response was inhibited, as were the movement and adhesion of immune-related cells; however, cell tumorigenesis, body death, and tumor cell proliferation were activated. The functions of the 20 hub molecules were mainly related to cytotoxicity, cell exosmosis, and cell adhesion mediated by immune cells. Further, we found that miR-5698, miR224-5p, and miR4709-3P regulate multiple key genes (e.g., PECAM1, CX3CR1, KLRD1, and CXCL12), and may be the key miRNAs regulating LUAD. Conclusions: Immune response, cell tumorigenesis, and tumor cell proliferation play central roles in the overall regulatory network. miR-5698, miR224-5p, and miR4709-3p may be important biomarkers for the occurrence and development of LUAD and have great potential in the prognosis of LUAD patients and the development of new therapeutic targets.

Transcription factor-target gene regulatory network analysis in human lung adenocarcinoma.

Background: Transcription factors (TFs) play a crucial role in the occurrence and progression of lung adenocarcinoma (LUAD), and targeting TFs is an important direction for treating LUAD. However, targeting a single TF often fails to achieve satisfactory therapeutic outcomes. Furthermore, the regulatory TF-target gene networks involved in the development of LUAD is complex and not yet fully understood. Methods: In this study, we performed RNA sequencing (RNA-seq) to analyze the transcriptome profile of human LUAD tissues and matched adjacent nontumor tissues. We selected the differentially expressed TFs, performed enrichment analysis and survival curve analysis, and predicted the regulatory networks of the top differential TFs with their target genes. Finally, alternative splicing analyses were also performed. Results: We found that TFs GRHL3, SIX1, SIX2, SPDEF, and ETV4 were upregulated, while TAL1, EPAS1, SOX17, NR4A1, and EGR3 were significantly downregulated in LUAD tissues compared to normal tissues. We propose a potential GRHL3-CDH15-Wnt-ß-catenin pro-oncogenic signaling axis and a potential TAL1-ADAMTS1-vascular antioncogenic signaling axis. In addition, we found that alternative splicing of intron retention (IR), approximate IR (XIR), multi-IR (MIR), approximate MIR (XMIR), and approximate alternative exon ends (XAE) showed abnormally increased frequencies in LUAD tissues. Conclusions: These findings revealed a novel TF-target gene regulatory axis related to tumorigenesis and provided potential therapeutic targets and mechanisms for LUAD.

The molecular mechanism of sepsis-induced diaphragm dysfunction.

Background: No effective drugs for the treatment of sepsis-induced diaphragm dysfunction are currently available. Therefore, it is particularly important to clarify the molecular regulatory mechanism of this condition and subsequently implement effective treatment and prevention of sepsis-induced diaphragm dysfunction. Methods: A mouse model of diaphragm dysfunction was established via injection of lipopolysaccharide (LPS). An RNA-sequencing (RNA-seq) technique was used to detect the differentially expressed genes (DEGs) in the diaphragms of mice. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed for functional analysis of DEGs. The protein-protein interaction network obtained from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) website was imported into Cytoscape, the key molecular regulatory network was constructed with CytoNCA, the ClueGo plugin was further used to analyze the core regulatory pathways of key molecular, and finally, the iRegulon plugin was used to the identify key transcription factors. Results: The genes upregulated after LPS treatment were involved in biological processes and pathways related to immune response; the genes downregulated after LPS treatment were mainly correlated with the muscle contraction. The expressions of several inflammation-related genes were upregulated after LPS treatment, of which tumor necrosis factor (Tnf), interleukin (Il)-1ß, and Il-6 assumed a core regulatory role in the network; meanwhile, the downregulated key genes included Col1a1, Uqcrfs1, Sdhb, and ATP5a1, among others. These key regulatory factors participated in the activation of Toll-like receptor (TLR) signaling pathway, nuclear factor (NF)-κB signaling pathway, and TNF signaling pathway as well as the inhibition of oxidative phosphorylation pathway, cardiac muscle contraction pathway, and citrate cycle pathway. Finally, RelA, IRF1, and STAT3, were identified as the key regulators in the early stage of diaphragmatic inflammatory response. Conclusions: Sepsis-induced diaphragm dysfunction in mice is closely correlated with the activation of TLR signaling pathway, NF-κB signaling pathway, and TNF signaling pathway and the inhibition of oxidative phosphorylation pathway, cardiac muscle contraction pathway, and citrate cycle pathway. Our findings provide insight into the molecular mechanism of sepsis-induced diaphragm dysfunction in mice and provide a promising new strategy for targeted treatment of diaphragm dysfunction.

MiR-202-3p inhibits the proliferation and metastasis of lung adenocarcinoma cells by targeting RRM2.

Background: Lung adenocarcinoma (LUAD) is the most common type of lung cancer, and its pathogenesis is still unclear. The present study aimed to investigate the role of miR-202-3p and its downstream target gene, ribonucleotide reductase regulatory subunit M2 (RRM2), in the occurrence and development of LUAD and elucidate the correlation between RRM2 and the clinicopathological stage and prognosis of LUAD. Methods: The expression of miR-202-3p was analyzed using the CancerMIRNome database and quantitative polymerase chain reaction (qPCR). The effects of miR-202-3p and RRM2 on the proliferation, migration, and invasion of A549 cells were analyzed. A dual luciferase reporter assay was used to verify the targeting of miR-202-3p and RRM2. Additionally, the correlation between RRM2 expression and clinicopathology was analyzed. Results: (I) MiR-202-3p was lowly expressed in LUAD and the LUAD cell lines. qPCR confirmed that microRNA (miRNA) transfection was effective and sufficient for subsequent experiments. (II) MiR-202-3p inhibited the proliferation, invasion, and migration of LUAD cells. (III) There was a targeting relationship between miR-202-3p and RRM2, and miR-202-3p affected the expression of the RRM2 protein. RRM2 was highly expressed in lung cancer tissue. (IV) RRM2 was associated with the clinicopathological staging of lung cancer. The prognosis of patients with low RRM2 expression was better, and the prognostic sensitivity of RRM2 to lung cancer was high. RRM2 may exert its effects via the Notch pathway. (V) Si-RRM2 inhibited the expression of the RRM2 protein. RRM2 promoted the proliferation, migration, and invasion of LUAD cells. A miR-202-3p inhibitor restored the inhibitory effect of si-RRM2 on LUAD cells. Conclusions: MiR-202-3p was lowly expressed in lung cancer tissue. MiR-202-3p overexpression inhibited the proliferation and metastasis of lung cancer cells. RRM2 was highly expressed in lung cancer tissue and promoted the proliferation and metastasis of lung cancer cells. MiR-202-3p targeted and inhibited RRM2, thereby reducing the proliferation and metastasis of LUAD cells. LUAD patients with low RRM2 expression had a better prognosis, and the expression level of RRM2 was correlated with the clinical characteristics of lung cancer patients.

Expert consensus on thermal ablation therapy of pulmonary subsolid nodules (2021 Edition).

The Expert Consensus reviews current literatures and provides clinical practice guidelines for thermal ablation of pulmonary subsolid nodules or ground-glass nodule (GGN). The main contents include the following: (1) clinical evaluation of GGN; (2) procedures, indications, contraindications, outcomes evaluation, and related complications of thermal ablation for GGN; and (3) future development directions.

The predictive prognostic values of CBFA2T3, STX3, DENR, EGLN1, FUT4, and PCDH7 in lung cancer.

BACKGROUND: Lung cancer is one of the most malignant tumors. However, neither the pathogenesis of lung cancer nor the prognosis markers are completely clear. The purpose of this study is to screen the diagnostic or prognostic markers of lung cancer. METHODS: TCGA and GEO datasets were used to analyze the relationship between lung cancer-related genes and lung cancer samples. Common differential genes were screened, and a univariate Cox regression analysis was used to screen survival related genes. A univariable Cox proportional hazards regression analysis was used to verify the genes and construct risk model. The key factors affecting the prognosis of lung cancer were determined by univariate and multivariate regression analyses. The ROC curve, AUC and the survival of each risk gene was analyzed. Finally, the biological functions of high- and low-risk patients were explored by GSEA and an immune-infiltration analysis. RESULTS: Based on the common differential genes, 13 genes significantly related to lung cancer survival were identified. Eight risk genes (CBFA2T3, DENR, EGLN1, FUT2, FUT4, PCDH7, PHF14, and STX3) were screened out. The results showed that risk status may be an independent prognostic factor, and the risk score predicted the prognosis of lung cancer. CBFA2T3 and STX3 are protective genes, while DENR, EGLN1, FUT4 and PCDH7 are dangerous genes. These 6 genes can be used as independent lung cancer prognosis markers. The corresponding biological functions of genes expressed in high-risk patients were mostly related to tumor proliferation and inflammatory infiltration. Neutrophil, CD8+T, Macrophage M0, Macrophage M1- and mDC-activated cells were high in high-risk status samples. CONCLUSIONS: CBFA2T3, STX3, DENR, EGLN1, FUT4, and PCDH7 are important participants in the occurrence and development of lung cancer. High-risk patients display serious inflammatory infiltration. This study not only provides insight into the mechanism of occurrence and development of lung cancer, but also provides potential targets for targeted therapy of lung cancer.

[Expert Consensus for Thermal Ablation of Pulmonary Subsolid Nodules (2021 Edition)].

"The Expert Group on Tumor Ablation Therapy of Chinese Medical Doctor Association, The Tumor Ablation Committee of Chinese College of Interventionalists, The Society of Tumor Ablation Therapy of Chinese Anti-Cancer Association and The Ablation Expert Committee of the Chinese Society of Clinical Oncology" have organized multidisciplinary experts to formulate the consensus for thermal ablation of pulmonary subsolid nodules or ground-glass nodule (GGN). The expert consensus reviews current literatures and provides clinical practices for thermal ablation of GGN. The main contents include: (1) clinical evaluation of GGN, (2) procedures, indications, contraindications, outcomes evaluation and related complications of thermal ablation for GGN and (3) future development directions.
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Pentacyclic Triterpenes from the resin of Liquidambar formosana have anti-angiogenic properties.

Phytochemical investigation of the resin of Liquidambar formosana Hanc led to the separation and identification of five undescribed pentacyclic triterpenoids, including two lupane type, one taraxerane type, and two oleanane type triterpenoids, in addition to ten known analogues. Structures and relative or absolute configurations were elucidated by intensive spectroscopic methods, and single-crystal X-ray diffraction analysis. All isolated compounds were evaluated for their anti-angiogenic effects in vitro against VEGF-induced endothelial cell proliferation and migration in HUVECs. Among them, (5R, 8R, 9R, 10R, 13S, 14R, 17R, 18R, 19S)-17,18-epoxy-17,18-seco-28-norlupa-17- hydroxy-20 (29) -ene-3-one, (5R, 8R, 9R, 10R, 13S, 14R, 17S, 18S, 19S, 20S)-17, 20-peroxy-28- norlupa -29 -hydroxy- 3-one, 11α,12α:13ß,28-diepoxyoleanane- 3-one, 28-norlup-20 (29)-ene- 3ß,17ß-diol, liquidambaric lactone and 13,28-epoxy-11- oleanene- 3-one significantly inhibited VEGF-induced HUVECs proliferation with IC50 values ranging from 1.64 ± 0.36 to 7.06 ± 0.28 µM. In addition, they also effectively decreased VEGF-induced cell migration with IC50 values ranging from 1.57 ± 0.60 to 4.77 ± 0.62 µM. The structure-activity relationship of these compounds is discussed. The anti-angiogenic property of (5R, 8R, 9R, 10R, 13S, 14R, 17R, 18R, 19S)-17,18-epoxy-17,18-seco-28-norlupa-17- hydroxy-20 (29) -ene-3-one is mediated by the VEGFR2 - AKT signaling pathway.

Inhibition of IL-6/JAK/STAT3 pathway rescues denervation-induced skeletal muscle atrophy.

BACKGROUND: The molecular mechanisms underlying denervated skeletal muscle atrophy with concomitant muscle mass loss have not been fully elucidated. Therefore, this study aimed to attain a deeper understanding of the molecular mechanisms underlying denervated skeletal muscle atrophy as a critical step to developing targeted therapy and retarding the concomitant loss of skeletal muscle mass. METHODS: We employed microarray analysis to reveal the potential molecular mechanisms underlying denervated skeletal muscle atrophy. We used in vitro and in vivo atrophy models to explore the roles of the interleukin 6 (IL-6), Janus kinase (JAK), and signal transducers and activators of transcription 3 (STAT3) in muscle atrophy. RESULTS: In this study, microarray analysis of the differentially expressed genes demonstrated that inflammation-related cytokines were markedly triggered and IL-6/JAK/STAT3 signaling pathway was strongly activated during denervated skeletal muscle atrophy. The high level of IL-6 enhanced C2C12 myotube atrophy through the activation of JAK/STAT3, while inhibiting JAK/STAT3 pathway by ruxolitinib (a JAK1/2 inhibitor) or C188-9 (a STAT3 inhibitor) significantly attenuated C2C12 myotube atrophy induced by IL-6. Pharmacological blocking of IL-6 by tocilizumab (antibody against IL-6 receptor) and pharmacological/genetic inhibition of JAK/STAT3 pathway by ruxolitinib/C188-9 (JAK/STAT3 inhibitor) and STAT3 short hairpin RNA (shRNA) lentivirus in tibialis anterior muscles could suppress muscle atrophy and inhibit mitophagy, and was accompanied by the decreased expression of atrophic genes (MuRF1 and MAFbx) and autophagy-related genes (PINK1, BNIP3, Beclin 1, ATG7, and LC3B). CONCLUSIONS: Taken together, the results suggest that IL-6/JAK/STAT3 pathway may be a principal mediator in denervated skeletal muscle atrophy, meaning targeted therapy against IL-6/JAK/STAT3 pathway might have potential as a therapeutic strategy for prevention of skeletal muscle atrophy.

Protective effect of gastrodin on myocardial ischemia-reperfusion injury and the expression of Bax and Bcl-2.

The protective effects of gastrodin on myocardial ischemia-reperfusion injury in rats and the underlying mechanism were investigated. Sprague Dawley (SD) rats were randomly divided into three groups, of which the gastrodin group was treated with gastrodin, and the other two groups were treated with normal saline. In the myocardial ischemia-reperfusion model group, myocardial ischemia was induced by ligation of the left anterior descending coronary artery, and myocardial reperfusion was performed by ligature removal. Only thread without ligation for the sham operation group was conducted. The rats were euthanized 8 days after surgery. Heart tissues were harvested and used for measurement of apoptotic rate and expression levels of apoptosis-related proteins. Serum levels of cytokines were measured also using blood samples. Compared with the myocardial ischemia-reperfusion model group, significant reduction of cardiomyocyte apoptosis was observed in the gastrodin group (P<0.05). In the gastrodin group, the protein and mRNA expression levels for Bax and activated caspase-3 decreased, while for Bcl-2 increased (P<0.05). Gastrodin can downregulate inflammatory cytokines (P<0.05) and upregulate anti-inflammatory cytokines such as IL-10 (P<0.05) in serum of SD rats. Therefore, gastrodin played a protective role in myocardial ischemia-reperfusion injury by regulating the expression levels of apoptosis-related signaling proteins and inflammatory cytokines.

Long-term sky islands generate highly divergent lineages of a narrowly distributed stream salamander (Pachyhynobius shangchengensis) in mid-latitude mountains of East Asia.

BACKGROUND: Climate oscillation may have a profound effect on species distributions, gene flow patterns and population demography. In response to environmental change, those species restricted to montane habitats experienced expansions and contractions along elevation gradients, which can drive differentiation among sky islands. RESULTS: The Shangcheng stout salamander (Pachyhynobius shangchengensis) is a cool stream amphibian restricted to high-elevation areas in the Dabie Mountains, East China. In the present study, we used mtDNA genes (Cyt b and ND2) of 193 individuals and 12 nuclear microsatellite loci genotyped on 370 individuals, representing 6 populations (JTX, KHJ, MW, TTZ, BYM and KJY) across the taxon's distribution area, to investigate their genetic variation and evolutionary history of P. shangchengensis. Most populations showed unusually high levels of genetic diversity. Phylogenetic analyses revealed five monophyletic clades with divergence times ranging from 3.96 to 1.4 Mya. Accordingly, significant genetic differentiation was present between these populations. Bayesian skyline plot analyses provided that all populations underwent long-term population expansions since the last inter-glacial (0.13 Mya ~ 0.12 Mya). Msvar analyses found recent signals of population decline for two northern populations (JTX and KHJ) reflecting a strong bottleneck (approximately 15-fold decrease) during the mid-Holocene (about 6000 years ago). Ecological niche modelling has shown a discontinuity in suitable habitats for P. shangchengensis under different historical climatic conditions. CONCLUSIONS: Our results suggest that the niche conservatism of P. shangchengensis and sky island effects may have led to long-term isolation between populations. In sky island refuges, the mid-latitude Dabie Mountains have provided a long-term stable environment for P. shangchengensis, which has led to the accumulation of genetic diversity and has promoted genetic divergence.

Isoquercitrin promotes peripheral nerve regeneration through inhibiting oxidative stress following sciatic crush injury in mice.

BACKGROUND: Oxidative stress has been recognized to play a crucial role in the pathogenesis of peripheral nerve injury. Isoquercitrin (quercetin-3-glucoside) is a flavonoid that exhibited many biological activities, including anti-oxidative effect. However, it is unclear whether isoquercitrin has protective effects on peripheral nerve injury. METHODS: Mice treated by isoquercitrin were used as a case group, and mice injected with saline was the control group. Sciatic behavioral function was assessed using SFI and CMAPs were measured by electrophysiology. Schwann cells proliferation and migration were tested using EdU staining and Transwell migration chambers respectively. The expression of oxidative stress related factors were detected by qRT-PCR and Western blotting. RESULTS: In present study, our results demonstrated that isoquercitrin (20 mg/kg/day) treatment achieved significantly higher SFI and higher amplitude of CMAP, promoted the nerve regeneration and remyelination, increased the production of GAP43, NF200, MAG and PMP22, alleviated target muscle atrophy and autophagy, and suppressed the expression of ATG7, PINK1 and Beclin1 in soleus muscles after sciatic nerve crush. In vitro studies found that isoquercitrin promoted the axonal regeneration of DRGs neurons, the proliferation and migration of Schwann cells, and the expression of proliferating cell nuclear antigen (PCNA) in Schwann cells. The administration of isoquercitrin at 40 and 320 µM showed a dose dependent, and high doses of isoquercitrin (160 and 320 µM) showed better performance in promoting axonal regeneration of DRGs neurons, and the proliferation and migration of Schwann cells than low dose of isoquercitrin (40 µM). Furthermore, isoquercitrin significantly inhibited oxidative stress through reducing the production of Nox4 and Duox1, and promoting the expression of Nrf2 and SOD2 in soleus muscles after sciatic nerve crush. CONCLUSIONS: Isoquercitrin may promote motor functional recovery and nerve regeneration following peripheral nerve injury though inhibition of oxidative stress, which highlighted the therapeutic values of isoquercitrin as a neuroprotective drug for peripheral nerve repair applications.

Histone deacetylase 5 promotes the proliferation and invasion of lung cancer cells.

Histone deacetylase 5 (HDAC5), as a member of the class IIa family of HDACs, is frequently dysregulated in human malignancies. However, little is known regarding the specific role of HDAC5 in lung cancer. We aimed to evaluate HDAC5 expression in human lung cancer and to determine the effects of HDAC5 on lung cancer cells. First, the expression levels of both HDAC5 protein and mRNA were evaluated in lung cancer tissues and cell lines by western blot analysis and RT­qPCR, and the results suggested that HDAC5 was significantly upregulated in human lung cancer tissues and cell lines. To address the effects of HDAC5 on the biological behavior of human lung adenocarcinoma cells, we generated human lung cancer A549 cell lines in which HDAC5 was either overexpressed or depleted. The results indicated that overexpression of HDAC5 significantly promoted the proliferation and invasion, and inhibited the apoptosis of A549 cells. On the contrary, HDAC5 knockdown largely decreased the proliferation and invasion and enhanced the apoptosis of A549 cells. Furthermore, we demonstrated that HDAC5 overexpression promoted the expression of DLL4, Six1, Notch 1 and Twist 1 in A549 cells. Downregulation of HDAC5 caused a significant inhibition of the expression of DLL4, Six1, Notch 1 and Twist 1 in A549 cells. Taken together, our data demonstrated that HDAC5 displayed a significant upregulation in lung cancer, and elevated HDAC5 might be involved in the potentiation of proliferation and invasion of lung cancer cells, as well as the inhibition of lung cancer cell apoptosis by the upregulation of DLL4, Six1, Notch 1 and Twist 1. The present study may provide an evidence for the potential application of HDAC5 inhibitors in the therapy of lung cancer.

MicroRNA-125a-5p plays a role as a tumor suppressor in lung carcinoma cells by directly targeting STAT3.

Increasing evidence supports that the dysregulation of microRNA expression plays an important role in the process of tumor occurrence and development. Studies have found that mir-125a-5p expression was downregulated in a variety of tumors, but the effects and mechanism of mir-125a-5p in lung cancer are still unclear. The aim of this study is to detect the expression of mir-125a-5p in lung cancer tissues and lung cancer cell lines and to explore the effects of mir-125a-5p on the biological characteristics of lung cancer cells; thus, this study aims to provide new methods and new strategies for the treatment of lung cancer. The result from quantitative reverse transcription polymerase chain reaction showed that the expression of miR-125a-5p was significantly lower in lung cancer tissues and lung cancer cell lines (95-D, A549, HCC827, and NCI-H1299) than that in normal tissue adjacent to lung cancer or normal human bronchial epithelial cells. In order to explore the function and mechanism of mir-125a-5p in lung cancer cells, miR-125a-5p mimic or mir-125a-5p inhibitor was transfected into A549 cells. Mir-125a-5p displayed an obvious upregulation in A549 cells transfected with miR-125a-5p and an obvious downregulation in A549 cells transfected with mir-125a-5p inhibitor compared to that in A549 cells transfected with control miRNA. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, BrdU staining, flow cytometry, and Transwell assay showed that the upregulation of miR-125a-5p could significantly decrease the cell viability, proliferation, and invasion of lung cancer cells and increase apoptosis of lung cancer cells. The downregulation of miR-125a-5p provided very contrasting results. Computational algorithms predicted that the STAT3 is a target of miR-125a-5p. Here, we validated that miR-125a-5p could directly bind to the 3'-untranslated region of STAT3, and miR-125a-5p overexpression could significantly inhibit the protein expression of STAT3. These results suggested that mir-125a-5p can regulate the expression of STAT3 in lung cancer cells. To further verify whether mir-125a-5p can play a biological role through regulating STAT3, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometry, and Transwell analysis demonstrated that overexpression of STAT3 can reverse the cells' biological effects induced by mir-125a-5p overexpression. Mir-125a-5p downregulated in lung cancer tissue and cell lines can negatively regulate STAT3 protein expression. Taken together, mir-125a-5p inhibited the proliferation and invasion of lung cancer cells and facilitated lung cancer cell apoptosis through suppressing STAT3. Enhancing the expression of miR-125a-5p is expected to benefit the therapy for the patients with lung cancer.

Clinical analysis on 113 patients with lung cancer treated by percutaneous CT-guided microwave ablation.

BACKGROUND: Lung Cancer is a primary tumor with poor prognosis. For early stage lung cancer, treatment options include surgical resection and microwave techniques. Percutaneous ablative techniques emerged as best therapeutic options for nonsurgical patients. METHODS: The aim of this study was to retrospectively analyze the clinical effect of CT-guided microwave ablation (MWA) treatment for patients with lung cancer who were not eligible for surgical resection. MWA was used to treat the tumor lesion of 113 patients with lung cancer who were in our hospital from Jan, 2013 to Jun, 2015. The median diameter of tumors was 3.1 cm (0.7-6 cm). Follow-up were paid to all the patients who received MWA therapy. The average follow-up was 22.1±8.6 months, and the median follow-up was 18 (7-40) months. RESULTS: All of 113 cases of patients with lung cancer experienced MWA therapy, and their tumors were found to have vacuolization, lower density and much smaller distinct shrinkage of tumor size with varying degrees. The local progression rate or relapse rate of the whole group was 15.9%. The counterpart of patients in the early-stage group was 5.7% (2/35), and the diameter of tumors in the two patients was more than 3 cm. The local progression rate or relapse rate of patients in advanced-stage group was 20.5%, wherein, 81.3% of local progression or relapse occurred to the patients with a tumorous diameter of more than 3 cm. The results indicated that the patients in advanced-stage group were vulnerable to local progression or relapse, the tumorous with greater diameter had higher incidence of local relapse. No mortality occurred within 30 days after surgery, the survival rate of patients in early-stage group at first, second, third year was respectively 97.1%, 94.1% and 84.7%. The counterpart of patients in advanced-stage group was respectively 93.6%, 87.7% and 71.7%. The difference of survival rate between both groups was not statistically significant (P=0.576). No perioperative deaths occurred, and the main complications i.e., fever, pneumothorax, pleural effusion, hemoptysis, pneumonia, and pain were slight and tolerable. CONCLUSIONS: MWA is an effective, safe and minimally invasive treatment for the patients with lung cancer who cannot be tolerated by surgical resection.

Prognostic value of decreased GRK6 expression in lung adenocarcinoma.

BACKGROUND: In recent years, G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through phosphorylation of the activated form of G protein-coupled receptors. However, little is known of GRK6 expression in lung adenocarcinoma (LADC) and its potential prognostic value in LADC. METHODS: In this study, protein expression of GRK6 was determined in LADC tissues (n = 122) and normal lung tissues (n = 45) by immunohistochemistry (IHC) analysis on tissue microarray (TMA). In addition, mRNA level of GRK6 in matched pairs of cancerous and non-cancerous fresh frozen tissues from 20 LADC patients was investigated using real-time quantitative PCR (qPCR). Furthermore, protein expression level of GRK6 was evaluated in matched pairs of cancerous and non-cancerous fresh frozen tissues from another 18 LADC patients. Univariate and multivariate analyses based on Cox proportional hazards regression models were performed to investigate the correlation between GRK6 expression and overall survival of LADC patients. RESULTS: According to the IHC analysis on TMA, GRK6 expression was significantly down-regulated in LADC patients, but high in normal lung tissue (P < 0.001). Besides, our qPCR and western blot results confirmed GRK6 down-regulation in both mRNA and protein levels in LADC tissues as compared to matched adjacent non-cancerous tissues (all P < 0.001). Additionally, For TMA slides, protein expression of GRK6 was significantly associated with staging (P = 0.030), pathology grade (P = 0.036). Consistent with the associated poor clinicopathologic features, patients with GRK6 low expression tumors had a worse overall survival compared to patients with GRK6 high expression tumors. Further multivariate analysis using the Cox proportional hazards model revealed that GRK6 expression level (P = 0.004) was an independent prognostic factor for overall survival. CONCLUSION: These findings indicate for the first time that decreased expression of GRK6 may serve as an independent predictor of overall survival in LADC patients.

PI3K-mediated glioprotective effect of epidermal growth factor under oxidative stress conditions.

AIM: To determine the effects of epidermal growth factor (EGF) on the proliferation and migration of Müller cell line Moorfields/Institute of Ophthalmology-Müller 1 (MIO-M1), and its related molecular mechanisms under normal and oxidative stress conditions. METHODS: Müller cells were cultured with different concentrations of EGF in the presence or absence of varied amounts of H2O2 and glucose oxidase (GO) which induced oxidative stress. The proliferation and migration of Müller cells were examined by 5-Bromo-2-deoxyUridine (BrdU), MTT assay, Transwell assay and scratch wound healing assays. The cell viability was determined with the MTT assay. The secretion of EGF by Müller cells was evaluated by ELISA. Western blot was performed to detect the activation of extracellular regulated protein kinases (ERK)1/2 and Akt signal pathways. RESULTS: EGF stimulated the proliferation and migration of Müller cells in a concentration-dependent manner in vitro. Under oxidative damage condition, 2h of pretreatment with 10-100 ng/mL EGF can mostly inhibit 50% lethal dose of 0.08 mmol/L H2O2-induced cell damage. The Western blot results showed that after Müller cells were exposed to varying EGF for 24h, Akt and ERK1/2 were phosphorylated in a dose-dependent manner. In the presence of the LY294002, the potent PI3K inhibitor, the p-Akt was significantly attenuated. CONCLUSION: EGF may induce the proliferation and migration of human Müller cells through the Akt and the ERK1/2 signal pathways, and induce PI3K-mediated glioprotective effect under oxidative stress.

Effect of TRAF6 on the biological behavior of human lung adenocarcinoma cell.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a unique adaptor protein of the tumor necrosis factor receptor-associated factor family that mediates both tumor necrosis factor receptor and interleukin-1 receptor/Toll-like receptor signaling. A recent study showed that TRAF6 played an important role in tumorigenesis and invasion through activation of nuclear factor kappa B (NF-κB). However, the biological role of TRAF6 remains unknown in lung cancer up to now. To address the expression of TRAF6 in lung cancer cells, four lung cancer cell lines (A549, HCC827, NCI-H292, and 95-D) and human bronchial epithelial cells were used to detect the expression of TRAF6 protein by western blotting. Results indicated that TRAF6 displayed an upregulation in human lung cancer cell lines. To investigate the effects of TRAF6 on the biological behavior of human lung adenocarcinoma cell, we generated human lung adenocarcinoma A549 cell line in which TRAF6 was depleted. The results showed that downregulation of TRAF6 could decrease cell viability, suppress cell proliferation and invasion, and promote cell apoptosis. At the same time, we explored the effects of TRAF6 on the expression of the following proteins: phosphor-NF-κB (p-p65), cyclin D1, caspase-3, and matrix metalloproteinase 9 (MMP9). Downregulation of TRAF6 could decrease the expression of p-p65, cyclin D1, and MMP9 and increase the expression of caspase-3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, and invasion of A549 cell line, as well as the inhibition of A549 cell apoptosis by the activation of NF-κB. To make a long story short, the overexpression of TRAF6 might be related to the tumorigenesis and invasion of lung cancer.

Protective effect of epidermal growth factor on proliferation and migration of the human Müller cell from oxidative damage / 中华实验眼科杂志

Background Oxidative damage plays an important role in pathogenesis of age-related macular degeneration( AMD ),and its mechanism is the destroy of blood-retinal barrier.Müller cells is a primary component to stabilize the inner barrier of the blood-retina.Researches showed that epidermal growth factor(EGF) can promote the proliferation and migration of animal Müller cells,but less study was found in the effect of EGF on human Müller cells. Objective The present study was to investigate the effects of EGF on the proliferation and migration of human Müller cells and its molecular mechanism. Methods Human Müller cell line MIO-M1 cells were cultured and incubated,and cultured cells were identified using glial fibrillory acidic protein (GFAP),factor Ⅷ,α-smooth muscle actin( α-SMA ),keratin and S-100.Different concentrations of EGF( 0,1,10,30,100 mg/L)was added in freeserum DMEM,and the positive rate of the cells was calculated using 5-bromo-2-deoxyuridine(BrdU) method.The cells were divided into EGF group,H2 O2 group,EGF + H2 O2 group,glucose oxidase ( GO ) group,GO + EGF group,EGF + LY294002+H2O2 group according to the different intervention,and the effects of LY294002 on the proliferation of Müller cells (A590 )were detected by colorimetric assay for cellular growth and survival( MTT assay).The scratch test of Müller cells was used to assess the influence of EGF(0,1,10,30,100 mg/L)on H2 O2-induced damage of human Müller cell.Western blot was used to detect the cell proliferation under the protection of EGF on co-cultured cells using LY294002 and H2O2 and the activation of Akt signal pathways. Results The proliferative rates of the cells were 28.0％,32.9％,39.0％ in 10,30,100 mg/L EGF groups respectively and obviously higher than those in 0,1 mg/L EGF groups (24.5 ％,26.2 ％ ).Under the H2O2 culture,GO culture,respectively,the A570 value of the Müller cell in high concentrations of EGF groups was significantly increased in comparison with lower concentrations EGF groups with the statistical significance among the groups( F=23.582,P=0.000).Compared with EGF+H2O2 group,the A570value of the Müller cells was lowed in EGF+LY294002+H2O2 group.The maximum migration rate of Müller cells was found in 10 mg/L EGF group.Western blot revealed that the presence of H2O2 reinforced the expression of Akt in Müller cells,however,pretreatment with 100 mg/L EGF antagonized the harmful effect of H2O2 on Müller cells.Meanwhile,pretreatment with EGF and LY294002 reduced the expression of Akt in Müller cells. Conclusions EGF can induce the proliferation and migration of human Müller cells with the strongest effect in 10 mg/L.100 mg/L exogenous EGF has a stronger protection to the Müiller cells against H2O2-induced cell damage by activating the PI3KAkt cell survival pathway.