MicroRNA-384 radiosensitizes human non-small cell lung cancer by impairing DNA damage response and repair signaling, which is inhibited by NF-κB.

OBJECTIVE: Radiotherapy has achieved remarkable effects in treating non-small cell lung cancer (NSCLC). However, radioresistance remains the major obstacle to achieving good outcomes. This study aims at identifying potential targets for radiosensitizing NSCLC and elucidating the underlying mechanisms. METHODS: Lentivirus-based infection and CRISPR/Cas9 technology were used to modulate the expression of microRNA-384 (miR-384). Cell clonogenic formation assays and a xenograft tumor model were used to analyze radiosensitivity in NSCLC cells. Fluorescence-activated cell sorting was used to assess the cell cycle and cell death. Immunofluorescence staining, Comet assays, and homologous recombination or non-homologous end-joining I-SceI/GFP reporter assays were used to study DNA damage and repair. Western blotting and quantitative real-time polymerase chain reaction were used to identify the targets of miR-384. Chromatin immunoprecipitation and polymerase chain reaction were performed to evaluate upstream regulators of miR-384. RESULTS: MiR-384 was downregulated in NSCLC. Overexpression of miR-384 increased the radiosensitivity of NSCLC cells in vitro and in vivo, whereas knockout of miR-384 led to radioresistance. Upregulation of miR-384 radiosensitized NSCLC cells by decreasing G2/M cell cycle arrest, inhibiting DNA damage repair, and consequently increasing cell death; miR-384 depletion had the opposite effects. Further investigation revealed that ATM, Ku70, and Ku80 were direct targets of miR-384. Moreover, miR-384 was repressed by NF-κB. CONCLUSIONS: MiR-384 is an ionizing radiation-responsive gene repressed by NF-κB. MiR-384 enhances the radiosensitivity of NSCLC cells via targeting ATM, Ku80, and Ku70, which impairs DNA damage repair. Therefore, miR-384 may serve as a novel radiosensitizer for NSCLC.

The EGR1/miR-139/NRF2 axis orchestrates radiosensitivity of non-small-cell lung cancer via ferroptosis.

Radiotherapy is one of the predominant treatment modalities for almost all kinds of malignant cancers, including non-small cell lung cancer (NSCLC). Increasing evidence shows that ionizing radiation (IR) induces reactive oxygen species (ROS) leading to lipid peroxidation and subsequently ferroptosis of cancer cells. However, cancer cells evolve multiple mechanisms against ROS biology resulting in resistance to ferroptosis and radiotherapy, of which NRF2 signaling is one of the most studied. In the current research, we identified that microRNA-139 (miR-139) could be a novel radiosensitizer for NSCLC by inhibiting NRF2 signaling. We found that miR-139 possessed great potential as a diagnostic biomarker for NSCLC and multiple other types of cancer. Overexpression of miR-139 increased radiosensitivity of NSCLC cells in vitro and in vivo. MiR-139 directly targeted cJUN and KPNA2 to impair NRF2 signaling resulting in enhanced IR-induced lipid peroxidation and cellular ferroptosis. We proved KPNA2 to be a binding partner of NRF2 that involved in nuclear translocation of NRF2. Moreover, we found that IR induced miR-139 expression through transcriptional factor EGR1. EGR1 bound to the promoter region and transactivated miR-139. Overall, our findings elucidated the effect of EGR1/miR-139/NRF2 in IR-induced ferroptosis of NSCLC cells and provided theoretical support for the potential diagnostic biomarkers and therapeutic targets for the disease.

STAT3/miR-135b/NF-κB axis confers aggressiveness and unfavorable prognosis in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is one of the most commonly diagnosed cancers worldwide but has limited effective therapies. Uncovering the underlying pathological and molecular changes, as well as mechanisms, will improve the treatment. Dysregulated microRNAs (miRNAs) have been proven to play important roles in the initiation and progression of various cancers, including NSCLC. In this manuscript, we identified microRNA-135b (miR-135b) as a tumor-promoting miRNA in NSCLC. We found that miR-135b was significantly upregulated and that its upregulation was associated with poor prognosis in NSCLC patients. miR-135b was an independent prognostic factor in NSCLC. Overexpressing miR-135b significantly promoted the aggressiveness of NSCLC, as evidenced by enhanced cell proliferation, migration, invasion, anti-apoptosis, and angiogenesis in vitro and in vivo, and knockdown of miR-135b had the opposite effects. Mechanistically, our results reveal that miR-135b directly targets the 3'-untranslated region (UTR) of the deubiquitinase CYLD, thereby modulating ubiquitination and activation of NF-κB signaling. Moreover, we found that interleukin-6 (IL-6)/STAT3 could elevate miR-135b levels and that STAT3 directly bound the promoter of miR-135b; thus, these findings highlight a new positive feedback loop of the IL-6/STAT3/miR-135b/NF-κB signaling in NSCLC and suggest that miR-135b could be a potential therapeutic target for NSCLC.

A case report of Phelan-McDermid syndrome: preliminary results of the treatment with growth hormone therapy.

BACKGROUND: Phelan-McDermid syndrome (PMS), also known as 22q13.3 deletion syndrome, is a rare neurodevelopmental syndrome resulting from a deletion of the distal long arm of chromosome 22. CASE PRESENTATION: We report a case of a 21 months old Chinese girl presenting with global developmental delay, regression of language skills, unable to understand a few words or walk independently, insomnia, and autism-like behaviors. Copy number variation (CNV) analysis showed a heterozygous loss of SHANK3 gene in the 22q13 region, consistent with a diagnosis of PMS. After treatment with recombinant human growth hormone (rhGH), this patient had an improvement in motor skills and social behaviors. No side effects from rhGH therapy were reported. CONCLUSIONS: This is the first report of using rhGH to treat a Chinese girl diagnosed with PMS. We speculate rhGH could be a reasonable alternative choice for PMS treatment with similar clinical outcomes in comparison to insulin-like growth factor-1(IGF-1). However, further clinical trials are needed to confirm this hypothesis.

Oxygen vacancy-rich hierarchical BiOBr hollow microspheres with dramatic CO2 photoreduction activity.

Conversion of carbon dioxide into useful chemicals has attracted great attention. However, the significant bottlenecks facing in the field are the poor conversion efficiency of CO2 and low selectivity of products. Herein, hierarchical BiOBr hollow microspheres are fabricated by a solvothermal method using ethylene glycol (EG) as solvent in presence of polyvinyl pyrrolidone (PVP). The hollow BiOBr microspheres prepared at 120 °C exhibit the best performance for CO2 photoreduction. The evolution rates of product CO and CH4 are up to 88.1 µmol g-1h-1 and 5.8 µmol g-1h-1, which are 8.8 times and 5.8 times higher than that of plate-like BiOBr respectively. The hollow microspheres possess larger specific area and generate multiple reflections of light in the cavity, thus enhancing the utilization efficiency of light. The modulated electronic structure by oxygen vacancy (OVs) is beneficial to the transfer of photogenerated electrons and holes. Especially, the enriched charge density of BiOBr by OVs is conductive to the adsorption and activation of CO2, which could lower the overall activation energy barrier of CO2 photoreduction. In summary, the synergistic effect of the hollow structure with OVs plays a vital role in boosting the photoreduction of CO2 for BiOBr. This work provides a new opportunity for designing the high efficiency catalyst by morphology engineering with defects at the atomic level for CO2 photoreduction.

Effect of proteins on the oxidase-like activity of CeO2 nanozymes for immunoassays.

Having the benefits of low cost, excellent stability and tolerance to extreme conditions, nanozymes are a potential alternative of horseradish peroxidase (HRP) or other enzymes for bioanalytical chemistry, especially immunoassays. CeO2 nanoparticles have oxidase-mimicking activity and can avoid the use of unstable H2O2. For robust assays, the effect of proteins on the activity of CeO2 needs to be carefully studied. Herein, we studied the adsorption and desorption of bovine serum albumin (BSA) from CeO2. The CeO2 nanoparticles exhibited a higher protein adsorption capacity compared to the other tested metal oxide nanoparticles. Although the oxidase-like activity of CeO2 was inhibited by BSA, low concentrations of phosphate and fluoride ions boosted the activity of protein-capped CeO2. CeO2 was still active under strong acidic conditions and at high temperature, while HRP lost its activity. For immunoassay development, we covered CeO2 with an amine-modified silane for covalent conjugation to antibodies. A one-step indirect competitive ELISA for fenitrothion was developed, and an IC50 value of 35.6 ng mL-1 and a limit of detection of 2.1 ng mL-1 were obtained.

Safety and Effectiveness of De-escalated Radiation Dose in T1-3 Nasopharyngeal Carcinoma: A Propensity Matched Analysis.

Backgrounds: With the excellent local control in T1 to T3 nasopharyngeal carcinoma (NPC) treated with intensity modulated radiotherapy (IMRT), the importance of toxicities is increasingly being recognised. This retrospective propensity score analysis sought to assess whether moderate dose reduction compromised long-term outcome compared with standard dose in T1-3 NPCs. Materials and Methods: A total of 266 patients (67 female, 199 male) with a median age of 50 years between June 2011 and June 2015 were analysed. All were treated with IMRT, with or without systemic chemotherapy. The prescription radiation dose to gross tumor is 70Gy/2.12Gy/33F in our institution. Results: With a median follow-up time of 50 months, the 5-year loco-regional failure-free survival (LRFS) and overall survival (OS) were 93.5% and 81.8%, respectively. 32 patients received radiation dose less than prescription dose, with a median dose of 63.6Gy (53-67Gy). Another 234 patients received exactly the prescription dose of 70Gy. Propensity scores were computed (32 patients treated with de-escalated dose and 64 patients with standard dose), there was no significant difference in 5-year LRFS and 5-year OS between the two groups (92.5% and 91.7% with standard dose; 82.1% and 85.7% with de-escalation dose; p=0.863 for LRFS and 0.869 for OS). No independent prognostic factor was associated with loco-regional failure in univariate analysis. Conclusions: T1-3 nasopharyngeal carcinoma presenting with superior locoregional control, a moderately reduced dose (about 10%) delivered with IMRT resulted in comparable prognosis to those with prescription dose of 70Gy.

Greater negative lymph node count predicts favorable survival of patients with breast cancer in the setting of neoadjuvant chemotherapy and mastectomy.

Aim: Adequate lymph node evaluation is recommended in patients with malignant tumors. However, the role of negative lymph nodes (NLNs) remains unclear in breast cancer (BC), especially in patients who have received neoadjuvant chemotherapy and mastectomy. Materials & methods: A total of 435 patients were included in the analysis. On multivariate analysis, NLN count was an independent predictor of 5 year disease-free survival and 5 year overall survival. Results: Patients with NLN count <10 showed significantly worse 5 year disease-free survival than those with NLN count ≥10 (34.8 and 78.2%; p = 0.000); the corresponding 5 year overall survival rates were also significantly different (52.0 and 82.7%; p = 0.000). Conclusion: This is the first study that confirms the relationship between NLN count and prognosis of patients in the setting of neoadjuvant chemotherapy and mastectomy. More NLNs imply better prognosis.

Depleting PTOV1 sensitizes non-small cell lung cancer cells to chemotherapy through attenuating cancer stem cell traits.

BACKGROUND: Prostate tumor over expressed gene 1 (PTOV1) has been reported as an oncogene in several human cancers. However, the clinical significance and biological role of PTOV1 remain elusive in non-small cell lung cancer (NSCLC). METHODS: The Cancer Genome Atlas (TCGA) data and NCBI/GEO data mining, western blotting analysis and immunohistochemistry were employed to characterize the expression of PTOV1 in NSCLC cell lines and tissues. The clinical significance of PTOV1 in NSCLC was studied by immunohistochemistry statistical analysis and Kaplan-Meier Plotter database mining. A series of in-vivo and in-vitro assays, including colony formation, CCK-8 assays, flow cytometry, wound healing, trans-well assay, tumor sphere formation, quantitative PCR, gene set enrichment analysis (GSEA), immunostaining and xenografts tumor model, were performed to demonstrate the effects of PTOV1 on chemosensitivity of NSCLC cells and the underlying mechanisms. RESULTS: PTOV1 is overexpressed in NSCLC cell lines and tissues. High PTOV1 level indicates a short survival time and poor response to chemotherapy of NSCLC patients. Depleting PTOV1 increased sensitivity to chemotherapy drugs cisplatin and docetaxel by increasing cell apoptosis, inhibiting cell migration and invasion. Our study verified that depleting PTOV1 attenuated cancer stem cell traits through impairing DKK1/ß-catenin signaling to enhance chemosensitivity of NSCLC cells. CONCLUSION: These results suggest that PTOV1 plays an important role in the development and progression of human NSCLC and PTOV1 may serve as a therapeutic target for NSCLC patients.

OTU deubiquitinase 4 is silenced and radiosensitizes non-small cell lung cancer cells via inhibiting DNA repair.

BACKGROUND: Radiotherapy is becoming one major therapeutics for non-small cell lung cancer (NSCLC). Identifying novel radiosensitizers will greatly increase the efficacy of radiotherapy and benefit more patients. OTU deubiquitinase 4 (OTUD4) has been reported involved in DNA damage repair pathways and could be a potential target for chemotherapy therapy. This study aimed to investigate the roles of OTUD4 in regulation of radiosensitivity of NSCLC via modulating DNA repair. METHODS: The expression of OTUD4, γ-H2Ax and ATM/CHK2/p53 pathway-related signaling molecules were detected by Western blotting and QRT-PCR. The methylation of OTUD4 promoter was investigated by 5-aza-deoxycytidine treatment, methylation-specific PCR and bisulfite genomic sequencing assays. Radiosensitivity was assessed by the clonogenic formation assay. Cell cycle, cell apoptosis were analyzed by flow cytometry. DNA damage and repair were determined by comet assay, γ-H2Ax foci staining and flow cytometry. RESULTS: OTUD4 is dramatically downregulated in NSCLC and its downregulation significantly correlates with poor prognosis of NSCLC patients. Promoter hypermethylation is responsible for the loss of OTUD4 expression in NSCLC cells. Overexpression of OTUD4 increases radiosensitivity of NSCLC cells exhibiting as impaired clonogenic formation ability, enhanced cell cycle arrest and increased cell apoptosis. Moreover, molecular mechanism study reveals that OTUD4 radiosensitizs NSCLC cells via ATM/CHK2/P53 signaling and inhibiting homology-directed repair of DNA double strand breaks induced by ionizing radiation. CONCLUSIONS: This study uncovers a tumor-suppressing role of OTUD4 and that OTUD4 is a potential radiosensitizer for NSCLC.

Moderate hypofractionated radiotherapy vs conventional fractionated radiotherapy in localized prostate cancer: a systemic review and meta-analysis from Phase III randomized trials.

PURPOSE: To determine the efficacy and late toxicities of moderate (2.5-4 Gy) hypofractionated radiotherapy (H-RT) in localized prostate cancer, a meta-analysis of published randomized clinical trials comparing moderate H-RT with conventional fractionated RT (C-RT) was performed. MATERIALS AND METHODS: Systematic search on published randomized clinical trials in English according to Cochrane review guidelines in databases of Pubmed, Embase, Cochrane, web of science, and Wiley Online Library was carried out. Outcomes of interests were biochemical and clinical disease failure (BCDF), biochemical failure (BF), overall survival (OS), and late toxicities. RESULTS: Seven of the 365 studies fulfilled inclusion criteria with 8,156 participants. Compared with C-RT, moderate H-RT showed a lower BF rate (risk ratio [RR] =0.80, 95% CI: 0.68-0.95, P=0.009), while did not improve OS (RR =0.68, 95% CI: 0.78-1.02, P=0.10). There was no significant difference in BCDF rates between H-RT and C-RT (RR =0.92, 95% CI: 0.82-1.02, P=0.12). The H-RT was deeply grouped into dose-escalated H-RT (with a higher biologically effective dose [BED1.5] than C-RT) and no dose-escalated H-RT; dose-escalated H-RT significantly decreased BCDF rate compared with C-RT (RR =0.84, 95% CI: 0.73-0.96, P=0.01). Regarding late toxicities, there is no significant difference in late gastrointestinal (GI; RR =0.97, 95% CI: 0.71-1.33, P=0.85) and genitourinary (GU) toxicities (RR =1.04, 95% CI: 0.87-1.24, P=0.69). When subgrouped into dose-escalated H-RT (with a higher BED5 compared with C-RT) and no dose-escalated H-RT, dose-escalated H-RT increased GI toxicity (RR =1.62, 95% CI: 1.26-2.09, P=0.0002) and GU toxicity (RR =1.28, 95% CI: 1.05-1.55, P=0.01), while no dose-escalated H-RT significantly lowered GI toxicity (RR =0.81, 95% CI: 0.70-0.94, P=0.005) and placed no influence on GU toxicity (RR =1.02, 95% CI: 0.88-1.20, P=0.77). CONCLUSION: This meta-analysis provides reliable evidence that moderate H-RT decreases BF rate, while does not improve OS. Compared with C-RT, H-RT with an increase in BED1.5 improved BCDF rates significantly, and accordingly, an increase in BED5 will result in elevated late GI and GU toxicities.

WT1-interacting protein inhibits cell proliferation and tumorigenicity in non-small-cell lung cancer via the AKT/FOXO1 axis.

Lung cancer is the most common cancer and the leading cause of cancer-related death worldwide; hence, it is imperative that the mechanisms underlying the malignant properties of lung cancer be uncovered in order to efficiently treat this disease. Increasing evidence has shown that WT1-interacting protein (WTIP) plays important roles both physiologically and pathologically in humans; however, the role of WTIP in cancer is unknown. Here, we investigated the role and mechanism of WTIP in cell proliferation and tumorigenesis of non-small-cell lung cancer (NSCLC). We report that WTIP is a tumor suppressor in human NSCLC. We found that WTIP expression was significantly reduced in both NSCLC cell lines and clinical specimens compared to that in normal controls; this reduction was largely attributed to promoter hypermethylation. Downregulation of WTIP significantly correlates with poor prognosis and predicts a shorter overall survival and progression-free survival among NSCLC patients. Moreover, ectopic overexpression of WTIP dramatically inhibits cell proliferation and tumorigenesis in vitro and in vivo; conversely, depletion of WTIP expression shows the opposite effects. Mechanistically, WTIP impairs AKT phosphorylation and activation, leading to enhanced expression and transcriptional activity of FOXO1, which further increases p21Cip1 and p27Kip1, and decreases cyclin D1, which consequently results in cell cycle arrest. Collectively, the results of the current study indicate that WTIP is an important proliferation-related gene and that WTIP expression may represent a novel prognostic biomarker for NSCLC.

Locoregional recurrence-associated factors and risk-adapted postmastectomy radiotherapy for breast cancer staged in cT1-2N0-1 after neoadjuvant chemotherapy.

OBJECTIVE: In order to identify risk factors associated with locoregional recurrence (LRR) and assess the role of postmastectomy radiotherapy (PMRT) in early breast cancer (BC), managed with neoadjuvant chemotherapy (NAC) and mastectomy, a retrospective analysis of BC diagnosed with clinical stage T1-2N0-1 was conducted. PATIENTS AND METHODS: A total of 217 patients were included in this analysis. The median age was 50 years (24-72 years). The clinical stage distributions were cT1 in 15 cases, cT2 in 202, cN0 in 53, and cN1 in 161 cases. All patients were treated with NAC and mastectomy, and 128 patients received PMRT. RESULTS: With a median follow-up time of 61 months, the 5-year cumulative LRR rate was 12%. Multivariate analysis demonstrated that pathological N stage, lymph-vascular invasion, and histological grade were independent prognostic factors associated with LRR. A nomogram model based on these factors was established, based on which the patients were deeply stratified into low- and high-risk group. In the low-risk group, radiotherapy did not decrease LRR (3.3% in PMRT group, 1.7% in no PMRT group, P=0.192). While in the high-risk group, PMRT significantly decreased LRR (21.8% in PMRT group, 42.2% in no PMRT group, P=0.031). CONCLUSION: Lymph-vascular invasion, histological grade, as well as pathological N stage were important prognostic factors associated with LRR in BC patients staged in cT1-2N0-1, who were managed with NAC and mastectomy. In our cohort, not only clinical and pathological stage information but also other risk factors were taken into consideration when adjuvant PMRT was recommended. In the high-risk subgroup, PMRT significantly improved the prognosis.

High PGAM5 expression induces chemoresistance by enhancing Bcl-xL-mediated anti-apoptotic signaling and predicts poor prognosis in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is the one of most common and deadly cancers, and is also highly resistant to conventional chemotherapy treatments. Mitochondrial phosphoglycerate mutase/protein phosphatase (PGAM5) regulates mitochondrial homeostasis and cell death, however, little is known about its roles in cancer. The aim of this study was to explore the clinical significance and potential biological functions of PGAM5 in hepatocellular carcinoma. For the first time, our results show that PGAM5 is significantly upregulated in HCC compared with corresponding adjacent noncancerous hepatic tissues and high PGAM5 expression is an independent predictor of reduced survival times in both univariate and multivariate analyses. Additionally, in vivo and in vitro studies showed that depleting PGAM5 expression inhibited tumor growth and increased the 5-fluorouracil sensitivity of HCC cells. Conversely, restoring PGAM5 expression in PGAM5-knockdown cells dramatically enhanced HCC cell resistance to 5-fluorouracil. Importantly, we demonstrated that the mechanism of 5-fluorouracil resistance conferred to HCC cells by PGAM5 was via inhibiting BAX- and cytochrome C-mediated apoptotic signaling by interacting and stabilizing Bcl-xL. Consistently, in the same cohorts of HCC patient tissues, Bcl-xL expression was positively correlated with PGAM5, and together predicted poor prognoses. In Conclusion, Our data highlight the molecular etiology and clinical significance of PGAM5 in HCC. Targeting the novel signaling pathway mediated by PGAM5/Bcl-xL may represent a new therapeutic strategy to improve the survival outcomes of HCC patients.

CRISPR/Cas9 Mediated GFP Knock-in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy.

Accurately identifying and quantifying cellular autophagy is very important as the significance of autophagy in physiological and pathological processes becomes increasingly evident. Ectopically expressed fluorescent-tagged microtubule-associated protein light chain 3B (MAP1LC3B, LC3) is the most widely used reporter for monitoring autophagy activity thus far. However, this approach ignores the influence of constitutively overexpressed LC3 on autophagy itself and autophagy-related processes and its accuracy in indicating autophagy is questionable. Here, we generated a knock-in GFP-LC3 reporter via the CRISPR/Cas9 system in 293FT cells to add GFP to the N-terminal of and in frame with endogenous LC3. We proved that this knock-in GFP-LC3 was expressed at biological level driven by the endogenous transcriptional regulatory elements as the wild type alleles. Compared with the ectopically expressed GFP-LC3, the endogenous knock-in reporter exhibited much higher sensitivity and signal-to-noise ratio of GFP-LC3 puncta upon the induction or inhibition of autophagy at certain step for monitoring autophagy activity. Thus, according to the previous reported concerning and the results presented here, we suggest that this knock-in GFP-LC3 reporter is better for bona fide monitoring cellular autophagy and should be employed for further study of autophagy in vitro and in vivo.

Nasal intermittent positive pressure ventilation with heliox in premature infants with respiratory distress syndrome: a randomized controlled trial.

OBJECTIVE: To assess the efficacy of nasal intermittent positive pressure ventilation with heliox in preterm infants with respiratory distress syndrome. METHODS: Premature infants with mild respiratory distress syndrome requiring non-invasive respiratory support were eligible. Infants were randomly assigned to heliox or air-oxygen group. The main outcome was the length of ventilation. RESULTS: Heliox significantly decreased the length of ventilation. The length of ventilation was positively correlated with interleukin-6 at baseline. Carbon dioxide elimination was better in the heliox group. CONCLUSION: Heliox delivered with nasal intermittent positive pressure ventilation may be effective in reducing length of ventilation and increasing carbon dioxide elimination.