LY294002 ameliorates psoriatic skin inflammation in mice via blocking the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop.

(IL)-17A, the effective factor of Th17 cells, acts an important pathological role in the pathogenesis of psoriasis. Notch1/hairy and split 1 (Hes1) and PI3K/AKT signaling pathways are interpenetrated and involved in Th17 cell differentiation and IL-17A production. In this present study, we used imiquimod (IMQ)-induced mouse psoriatic skin inflammation to explore the possible mechanism of Notch1/Hes1-PTEN/AKT/IL-17A feedback loop in psoriasis by employing AKT inhibitor LY294002 as an intervention with the methods of flow cytometry analysis, reverse transcription-quantitative polymerase chain reaction, western blot, co-immunoprecipitation, and immunofluorescence. First, LY294002 inhibition can obviously alleviate the mouse psoriatic skin inflammation both in skin structural and histopathological characteristics, which is similar to the changes found in IL-17A antibody-treated mice. Additionally, the interaction between Notch1 intracellular domain (NICD1) and nuclear factor kappa B (NF-κB) activator 1 (Act1) was demonstrated. LY294002 interruption resulted in consistent changes in expression levels of key signaling molecules both in Notch1/Hes1 and PI3K/AKT signaling pathways in a time-dependent manner. Moreover, chloroquine (CQ) can partly reverse the inhibitory effects of LY294002 on the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop by affecting Notch1 ubiquitination and lysosomal degradation. The present study showed that LY294002 can exert the inhibitory effect on Notch1/Hes1-PTEN/AKT/IL-17A feedback loop to regulate Th17 cell differentiation and IL-17A function in the process of psoriasis, which provides a new possible therapeutic strategy for psoriasis.

Exploring the association between circRNA expression and pediatric obesity based on a case-control study and related bioinformatics analysis.

OBJECTIVE: Our present study utilized case-control research to explore the relationship between specific circRNAs and pediatric obesity through a literature review and bioinformatics and to predict their possible biological functions, providing ideas for epigenetic mechanism studies of pediatric obesity. METHODS: CircRNAs related to pediatric obesity were preliminarily screened by a literature review and qRT-PCR. CircRNA expression in children with obesity (n = 75) and control individuals (n = 75) was confirmed with qRT-PCR in a case-control study. This was followed by bioinformatics analyses, such as GO analysis, KEGG pathway analysis, and ceRNA network construction. Multivariate logistic regression was utilized to analyze the effects of circRNAs on obesity. A receiver operating characteristic (ROC) curve was also drawn to explore the clinical application value of circRNAs in pediatric obesity. RESULTS: Has_circ_0046367 and hsa_circ_0000284 were separately validated to be statistically downregulated and upregulated, respectively, in the peripheral blood mononuclear cells of children with obesity and revealed as independent indicators of increased CHD risk [hsa_circ_0046367 (OR = 0.681, 95% CI: 0.480 ~ 0.967) and hsa_circ_0000284 (OR = 1.218, 95% CI: 1.041 ~ 1.424)]. The area under the ROC curve in the combined analysis of hsa_circ_0046367 and hsa_circ_0000284 was 0.706 (95% CI: 0.623 ~ 0.789). Enrichment analyses revealed that these circRNAs were actively involved in neural plasticity mechanisms, cell secretion and signal regulation. CONCLUSION: The present research revealed that low expression of hsa_circ_0046367 and high expression of hsa_circ_0000284 are risk factors for pediatric obesity and that neural plasticity mechanisms are closely related to obesity.

Genome-Wide Characterization and Gene Expression Analyses of Malate Dehydrogenase (MDH) Genes in Low-Phosphorus Stress Tolerance of Chinese Fir (Cunninghamia lanceolata).

Malate dehydrogenase (MDH) genes play vital roles in developmental control and environmental stress tolerance in sessile plants by modulating the organic acid-malic acid level. However, MDH genes have not yet been characterized in gymnosperm, and their roles in nutrient deficiency are largely unexplored. In this study, 12 MDH genes were identified in Chinese fir (Cunninghamia lanceolata), namely, ClMDH-1, -2, -3, …, and -12. Chinese fir is one of the most abundant commercial timber trees in China, and low phosphorus has limited its growth and production due to the acidic soil of southern China. According to the phylogenetic analysis, MDH genes were classified into five groups, and Group 2 genes (ClMDH-7, -8, -9, and 10) were only found to be present in Chinese fir but not in Arabidopsis thaliana and Populus trichocarpa. In particular, the Group 2 MDHs also had specific functional domains-Ldh_1_N (malidase NAD-binding functional domain) and Ldh_1_C (malate enzyme C-terminal functional domain)-indicating a specific function of ClMDHs in the accumulation of malate. All ClMDH genes contained the conserved MDH gene characteristic functional domains Ldh_1_N and Ldh_1_C, and all ClMDH proteins exhibited similar structures. Twelve ClMDH genes were identified from eight chromosomes, involving fifteen ClMDH homologous gene pairs, each with a Ka/Ks ratio of <1. The analysis of cis-elements, protein interactions, and transcription factor interactions of MDHs showed that the ClMDH gene might play a role in plant growth and development, and in response to stress mechanisms. The results of transcriptome data and qRT-PCR validation based on low-phosphorus stress showed that ClMDH1, ClMDH6, ClMDH7, ClMDH2, ClMDH4, ClMDH5, ClMDH10 and ClMDH11 were upregulated under low-phosphorus stress and played a role in the response of fir to low-phosphorus stress. In conclusion, these findings lay a foundation for further improving the genetic mechanism of the ClMDH gene family in response to low-phosphorus stress, exploring the potential function of this gene, promoting the improvement of fir genetics and breeding, and improving production efficiency.

A cross-sectional analysis of registry data of severe mental disorders in Fuzhou, China: current status and prospects.

OBJECTIVE: To investigate the proportion of registered cases relative to size, distribution characteristics, medication status, and management status of patients diagnosed with severe mental disorders (SMD) in Fuzhou. The medication status and management status were compared between patients in urban and non-urban areas to provide scientific evidence for improving SMD care, control, and treatment in primary health care institutions. METHODS: Data (case types, demographic data, distribution data, medication status, and management status, etc.) of patients diagnosed with SMD in 12 districts, counties, and prefectures in the urban and non-urban areas of Fuzhou City were collected from October 2017 to September 2018. Three distributions (population, local, and districts/counties) were used to describe the proportion of registered cases relative to size and clinical characteristics of diagnosed SMD. Chi squared (χ2) test was used to compare the severity in urban and non-urban areas. RESULTS: A total of 30,362 registered SMD patients were identified in Fuzhou City of which schizophrenia accounted for the highest number of cases (26,204, 86.31%), and paranoid psychosis had the least number of cases (47, 0.15%). Moreover, approximately half of SMD patients were 18 to 44 years old (45.38%). Close to one third of patients were farmers (30.23%), had a primary school or lower education level (54.17%), were poor, with most below the poverty line (55.35%). The proportion of diagnosed SMD relative to size was highest in Minqing County (0.53%) and lowest in Mawei District (0.38%). A total of 22,989 (75.72%) of the patients were taking medications, and only 17,509 (57.67%) were taking medications regularly. Moreover, the percentage of cases taking medications and those taking medications regularly were higher in urban areas than in non-urban areas (P<0.05). A total of 3065 patients were registered for management (10.09%). The managed proportion of SMD cases was higher in the urban areas than in the non-urban areas (P < 0.05). CONCLUSION: Schizophrenia is a key disease for comprehensive care and control of severe mental disorders in Fuzhou. The management of severe mental disorders should focus on poor groups with low educational backgrounds. Drug usage and management are better in urban areas than in non-urban areas, and thus management should be enhanced in non-urban areas. The medication management and case management of patients with severe mental disorders in Fuzhou need further improvements.

WNK4 kinase is a physiological intracellular chloride sensor.

With-no-lysine (WNK) kinases regulate renal sodium-chloride cotransporter (NCC) to maintain body sodium and potassium homeostasis. Gain-of-function mutations of WNK1 and WNK4 in humans lead to a Mendelian hypertensive and hyperkalemic disease pseudohypoaldosteronism type II (PHAII). X-ray crystal structure and in vitro studies reveal chloride ion (Cl-) binds to a hydrophobic pocket within the kinase domain of WNKs to inhibit its activity. The mechanism is thought to be important for physiological regulation of NCC by extracellular potassium. To test the hypothesis that WNK4 senses the intracellular concentration of Cl- physiologically, we generated knockin mice carrying Cl--insensitive mutant WNK4. These mice displayed hypertension, hyperkalemia, hyperactive NCC, and other features fully recapitulating human and mouse models of PHAII caused by gain-of-function WNK4. Lowering plasma potassium levels by dietary potassium restriction increased NCC activity in wild-type, but not in knockin, mice. NCC activity in knockin mice can be further enhanced by the administration of norepinephrine, a known activator of NCC. Raising plasma potassium by oral gavage of potassium inactivated NCC within 1 hour in wild-type mice, but had no effect in knockin mice. The results provide compelling support for the notion that WNK4 is a bona fide physiological intracellular Cl- sensor and that Cl- regulation of WNK4 underlies the mechanism of regulation of NCC by extracellular potassium.

Association between greenness and dyslipidemia in patients with coronary heart disease: A proteomic approach.

BACKGROUND: Residential surrounding greenness may be protective of dyslipidemia are often theorized but remain poorly quantified. In particular, the underlying biological mechanisms of blood lipid changes with green spaces remain unclear. METHODS: Our observational epidemiology study included a residentially stable sample of 1035 coronary heart disease patients, and proteomics study included 16 participants. Normalized Difference Vegetation Index (NDVI) was used to evaluate residential greenness exposures. Proteomics technology was used to identify plasma greenness-related proteome disturbance, and the pathway analysis was employed to evaluate the potential biological mechanisms of greenness decreasing dyslipidemia risk. RESULT: Higher residential surrounding greenness in the 500-m area was associated with lower risks of dyslipidemia (odds ratio (OR) = 0.871, 95% confidence interval (CI): 0.763, 0.994 for per one-quartile NDVI increase). Lymphocytes mediated 18.7% of the association between greenness and dyslipidemia. Greenness related proteins (including PLXDC1, IGFBP2 and LY6D) may regulate the biological functions of lipid metabolism and transport-related proteins (including ADIPOQ and CES1) through a series of biological processes. CONCLUSION: People in greener surroundings have a lower risk of dyslipidemia, which may be due to their lower inflammation, stronger lipid transporter activity, and normal cholesterol metabolism.

[Effect of a Simple Home-Based Oral Care Regimen on Oral Mucositis in Patients With Oral Cavity and Nasopharyngeal Cancer: A Quasi-Experimental Study].

BACKGROUND: The majority of patients with oral cavity and nasopharyngeal cancer experience severe oral mucositis during concurrent radiochemotherapy. The effectiveness of routine nursing education remains limited. PURPOSE: To evaluate the effect of a simple home-based oral care regimen on oral mucositis. METHODS: A double-group quasi-experimental design was adopted in this study. The participants were all newly diagnosed patients with oral cavity and nasopharyngeal cancer who were scheduled to receive concurrent radiochemotherapy in a northern medical center. A total of 31 patients in the experimental group and 32 patients in the control group were enrolled as participants. The control group received routine care, while the experimental group received an additional six- to seven-week two-way interactive home-based oral care regimen. The measurement tools included a plaque record and oral assessment guide (OAG) implemented twice during the study period. Study data were collected at 8 time points, including before treatment, at 1-5 weeks of treatment, at the end of treatment, and at one-month post-treatment. Data analysis was performed using two-way repeated measures ANCOVA. RESULTS: After controlling for OAG score, nutrition, age, living habits, and oral hygiene, the development of mucositis was found to be significantly slower in the experimental group than in the control group during the traumatic phase (effect of group: F = 11.1, p < .01; effect of group x time: F = 3.5, p = .01). However, both groups reported a statistically similar rate of improvement during the repair phase (effect of group and group x time: F = 0.19, p = .67). CONCLUSIONS / IMPLICATIONS FOR PRACTICE: The simple home-based oral care regimen introduced in this study may be used to improve traumatic oral mucositis in patients with oral cavity and nasopharyngeal cancer. It is recommended that even after the completion of radiotherapy, medical staffs should continue to strengthen patients' execution of proper oral care to maintain the positive effect until the mucositis has abated.

The Ci3+3 design for dual-agent combination dose-finding clinical trials.

We propose a rule-based statistical design for combination dose-finding trials with two agents. The Ci3 + 3 design is an extension of the i3 + 3 design with simple up-and-down decision rules comparing the observed toxicity rates and equivalence intervals that define the maximum tolerated dose combination. Ci3 + 3 consists of two stages to allow fast and efficient exploration of the dose-combination space. Statistical inference is restricted to a beta-binomial model for dose evaluation, and the entire design is built upon a set of fixed rules. We show via simulation studies that the Ci3 + 3 design exhibits similar and comparable operating characteristics to more complex designs utilizing model-based inferences. Implementation of Ci3 + 3 for practical trials is simple for the first stage, where the up-and-down decisions may be carried out using a decision table based on the preselected escalation path and i3 + 3. The second stage is not simpler than model-based designs, however, since it also requires computation of posterior probabilities based on a Bayesian model. We believe that the Ci3 + 3 design may provide an alternative choice to help simplify the design and conduct of combination dose-finding trials in practice.

Assessment of Internet Hospitals in China During the COVID-19 Pandemic: National Cross-Sectional Data Analysis Study.

BACKGROUND: Internet hospitals in China are being rapidly developed as an innovative approach to providing health services. The ongoing COVID-19 pandemic has triggered the development of internet hospitals that promote outpatient service delivery to the public via internet technologies. To date, no studies have assessed China's internet hospitals during the COVID-19 pandemic. OBJECTIVE: This study aimed to elucidate the characteristics of China's internet hospitals and assess the health service capacity of these hospitals. METHODS: Data on 711 internet hospitals were collected from official websites, the WeChat (Tencent Inc) platform, smartphone apps, and the Baidu search engine until July 16, 2020. RESULTS: As of July 16, 2020, 711 internet hospitals were developed in mainland China. More than half of these internet hospitals (421/711, 59.2%) were established during 2019 (206/711, 29%) and 2020 (215/711, 30.2%). Furthermore, about one-third (215/711, 30.2%) of internet hospitals were established at the beginning of 2020 as an emergency response to the COVID-19 epidemic. The 711 internet hospitals consisted of the following 3 types of hospitals: government-oriented (42/711, 5.91%), hospital-oriented (143/711, 20.11%), and enterprise-oriented internet hospitals (526/711, 73.98%). The vast majority of internet hospitals were traditional hospitals (526/711, 74%). Nearly 46.1% (221/711) of internet hospitals requested doctors to provide health services at a specific web clinic. Most patients (224/639, 35.1%) accessed outpatient services via WeChat. Internet hospitals' consulting methods included SMS text messaging consultations involving the use of graphics (552/570, 96.8%), video consultations (248/570, 43.5%), and telephone consultations (238/570, 41.8%). The median number of available web-based doctors was 43, and the median consultation fees of fever clinics and other outpatient clinics were ¥0 (US $0) per consultation and ¥6 (US $0.93) per consultation, respectively. Internet hospitals have provided various services during the COVID-19 pandemic, including medical prescription, drug delivery, and medical insurance services. CONCLUSIONS: The dramatic increase of internet hospitals in China has played an important role in the prevention and control of COVID-19. Internet hospitals provide different and convenient medical services for people in need.

The pharmaceutical practice of mask distribution by pharmacists in Taiwan's community pharmacies under the Mask Real-Name System, in response to the COVID-19 outbreak.

BACKGROUND: Pharmacists hold to their promise to foster, implement and promote the health of the population and to prevent disease, given their knowledge, skills, and proximity to the locals. The objective of this study was to foster equality and cost-effectiveness in the distribution and sale of masks to all Taiwanese citizens, in response to the COVID-19 pandemic. METHODS: All 6336 special community pharmacies participating in the NHI (National Health Insurance) served as mask-selling sites. Access to masks by citizens was determined and controlled, based on the weekly rationing of the number of purchasable masks per citizen and the last digit of their NHI card number. Masks were available on different weekdays for holders of cards ending with odd and even numbers, except on Sundays, when everyone was eligible to buy a mask. RESULTS: Implementing the program has provided equal access to masks for all citizens across Taiwan. It has stabilized the pricing of masks and mitigated the public's anxiety of a perceived likely market shortage. CONCLUSION: The community pharmacy-based approach to the distribution of prevention face masks to citizens represents a new and innovative engagement of pharmacists in public health promotion and protection initiatives. Community pharmacies can greatly improve the efficiency, reliability, and cost-saving of the distribution of public health resources to local communities, especially in the face of an epidemic.

Epigenetic Silencing of LMX1A Contributes to Cancer Progression in Lung Cancer Cells.

Epigenetic modification is considered a major mechanism of the inactivation of tumor suppressor genes that finally contributes to carcinogenesis. LIM homeobox transcription factor 1α (LMX1A) is one of the LIM-homeobox-containing genes that is a critical regulator of growth and differentiation. Recently, LMX1A was shown to be hypermethylated and functioned as a tumor suppressor in cervical cancer, ovarian cancer, and gastric cancer. However, its role in lung cancer has not yet been clarified. In this study, we used public databases, methylation-specific PCR (MSP), reverse transcription PCR (RT-PCR), and bisulfite genomic sequencing to show that LMX1A was downregulated or silenced due to promoter hypermethylation in lung cancers. Treatment of lung cancer cells with the demethylating agent 5-aza-2'-deoxycytidine restored LMX1A expression. In the lung cancer cell lines H23 and H1299, overexpression of LMX1A did not affect cell proliferation but suppressed colony formation and invasion. These suppressive effects were reversed after inhibition of LMX1A expression in an inducible expression system in H23 cells. The quantitative RT-PCR (qRT-PCR) data showed that LMX1A could modulate epithelial mesenchymal transition (EMT) through E-cadherin (CDH1) and fibronectin (FN1). NanoString gene expression analysis revealed that all aberrantly expressed genes were associated with processes related to cancer progression, including angiogenesis, extracellular matrix (ECM) remodeling, EMT, cancer metastasis, and hypoxia-related gene expression. Taken together, these data demonstrated that LMX1A is inactivated through promoter hypermethylation and functions as a tumor suppressor. Furthermore, LMX1A inhibits non-small cell lung cancer (NSCLC) cell invasion partly through modulation of EMT, angiogenesis, and ECM remodeling.

NKX6.1 Represses Tumorigenesis, Metastasis, and Chemoresistance in Colorectal Cancer.

Accumulating evidence suggests that NKX6.1 (NK homeobox 1) plays a role in various types of cancer. In our previous studies, we identified NKX6.1 hypermethylation as a promising marker and demonstrated that the NKX6.1 gene functions as a metastasis suppressor through the epigenetic regulation of the epithelial-to-mesenchymal transition (EMT) in cervical cancer. More recently, we have demonstrated that NKX6.1 methylation is related to the chemotherapy response in colorectal cancer (CRC). Nevertheless, the biological function of NKX6.1 in the tumorigenesis of CRC remains unclear. In this study, we showed that NKX6.1 suppresses tumorigenic and metastatic ability both in vitro and in vivo. NKX6.1 represses cell invasion partly through the modulation of EMT. The overexpression of NKX6.1 enhances chemosensitivity in CRC cells. To further explore how NKX6.1 exerts its tumor-suppressive function, we used RNA sequencing technology for comprehensive analysis. The results showed that differentially expressed genes (DEGs) were mainly related to cell migration, response to drug, transcription factor activity, and growth factor activity, suggesting that these DEGs are involved in the function of NKX6.1 suppressing cancer invasion and metastasis. Our results demonstrated that NKX6.1 functions as a tumor suppressor partly by repressing EMT and enhancing chemosensitivity in CRC, making it a potential therapeutic target.

Porous reduced graphene oxide/nickel foam for highly efficient solar steam generation.

Solar-driven water evaporation is considered to be an effective method for seawater desalination and wastewater purification. Here, we report a novel solar steam generation (SSG) system based on reduced graphene oxide (rGO)/nickel foam. Porous rGO foam acting as a photothermal conversion layer is fabricated by coating the rGO microsheets on the metallic nickel foam. The porous structure shows a rough surface, which can improve the harvest of light by scattering effect. On the other hand, the porous structure ensures the rapid flow of steam in the evaporation process. This SSG system based on rGO/nickel foam converts the absorbed solar energy into heat energy at the water-air interface and can effectively evaporate (∼83.4%) under low irradiation of 1 sun (1 kw m-2). The system shows great potential for the practical applications of water treatment at large-scale because of the high efficiency, simple preparation method and low cost.

Copper nanoparticles with near-unity, omnidirectional, and broadband optical absorption for highly efficient solar steam generation.

Solar steam generation provides a renewable and environmentally friendly approach to solve the water shortage issue. The pursuit of efficient, stable, and cheap photothermal agents is thus of great significance. In this work, Cu nanoparticles (NPs) fabricated simply by a substitution reaction, exhibit a near-unity (∼97.7%) light absorption, covering a broad incident angle and wavelength range (200-1300 nm). Thereby, a high photothermal conversion efficiency of 93% is achieved. The excellent photothermal performance offers a unique opportunity for the development of solar steam generation. By coating the Cu NPs on a cellulose membrane, a solar steam generation efficiency up to 73% is acquired at a low irradiation power density of 2 kW m-2 (1 kW m-2 = 1 sun). Moreover, the Cu NPs are recyclable with the high stability being resistant to heat, photoirradiation and corrosion of brine.

Knockdown of POLDIP2 suppresses tumor growth and invasion capacity and is linked to unfavorable transformation ability and metastatic feature in non-small cell lung cancer.

The main problem in the treatment of non-small cell lung cancer (NSCLC) is metastasis. Epithelial-mesenchymal transition (EMT) is known as the critical signaling in tumor progression, metastasis, and also the drug resistance. In this study, we reported a novel gene Polymerase delta-interacting protein 2 (POLDIP2) was downregulated in NSCLC tissues and first demonstrated that overexpression of POLDIP2 increased the anchorage-independent growth (AIG) and invasiveness of H1299 cells. In addition, we examined that knockdown of POLDIP2 in H1299 and A549 cells reduced tumorigenicity and metastatic capacity in vitro and also in vivo. Moreover, downregulation of the cell proliferation marker cyclin D1 and EMT markers CDH2, Slug, and Twist was showed in H1299 cells by POLDIP2 knockdown, suggesting that the inhibition of malignancy was affected by modulating key genes for tumor growth and invasiveness. Taken together, our study is the first study that demonstrated that POLDIP2 gene was function as an oncogene in NSCLC and implied the oncogenic ability might be through promoting cell proliferation or EMT.

Emetine Synergizes with Cisplatin to Enhance Anti-Cancer Efficacy against Lung Cancer Cells.

Cisplatin is still the primary therapeutic choice for advanced lung cancers without driver mutations. The occurrence of cisplatin resistance is a major clinical problem in lung cancer treatment. The natural extracted agent emetine reportedly has anticancer effects. This study aimed to explore the possible role of emetine in cisplatin resistance. We used cell viability, Western blot, and Wnt reporter assays to show that emetine suppresses proliferation, ß-catenin expression, and Wnt/ß-catenin signaling in non-small cell lung cancer (NSCLC). The synergism of emetine and cisplatin was assessed by constructing isobolograms and calculating combination index (CI) values using the Chou-Talalay method. Emetine effectively synergized with cisplatin to suppress the proliferation of cancer cells. Furthermore, nuclear ß-catenin and cancer stem cell-related markers were upregulated in the cisplatin-resistant subpopulation of CL1-0 cells. Emetine enhanced the anticancer efficacy of cisplatin and synergized with cisplatin in the cisplatin-resistant subpopulation of CL1-0 cells. Taken together, these data suggest that emetine could suppress the growth of NSCLC cells through the Wnt/ß-catenin pathway and contribute to a synergistic effect in combination with cisplatin.

A Novel Prognostic DNA Methylation Panel for Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common cancers and the second leading cause of cancer-related deaths. Discrepancies in clinical outcomes are observed even among patients with same-stage CRC due to molecular heterogeneity. Thus, biomarkers for predicting prognosis in CRC patients are urgently needed. We previously demonstrated that stage II CRC patients with NKX6.1 methylation had poor 5-year overall survival. However, the methylation frequency of NKX6.1 was only 23% in 151 pairs of CRC tissues. Thus, we aimed to develop a more robust prognostic panel for CRC using NKX6.1 in combination with three genes: LIM homeobox transcription factor 1α (LMX1A), sex-determining region Y-box 1 (SOX1), and zinc finger protein 177 (ZNF177). Through quantitative methylation analysis, we found that LMX1A, SOX1, and ZNF177 were hypermethylated in CRC tissues. LMX1A methylation was significantly associated with poor 5-year overall, and disease-free survivals in stage I and II CRC patients. Sensitivity and specificity analyses of the four-gene combination revealed the best sensitivity and optimal specificity. Moreover, patients with the four-gene methylation profile exhibited poorer disease-free survival than those without methylation. A significant effect of the four-gene methylation status on overall survival and disease-free survival was observed in early stage I and II CRC patients (p = 0.0016 and p = 0.0230, respectively). Taken together, these results demonstrate that the combination of the methylation statuses of NKX6.1, LMX1A, SOX1, and ZNF177 creates a novel prognostic panel that could be considered a molecular marker for outcomes in CRC patients.

NKX6.1 hypermethylation predicts the outcome of stage II colorectal cancer patients undergoing chemotherapy.

Colorectal cancer (CRC) is a common malignancy worldwide. CRC patients in the same stage often present with dramatically different clinical scenarios. Thus, robust prognostic biomarkers are urgently needed to guide therapies and improve treatment outcomes. The NKX6.1 gene has been identified as a hypermethylation marker in cervical cancer, functioning as a metastasis suppressor by regulating epithelial-mesenchymal transition. Here, we investigated whether hypermethylation of NKX6.1 might be a prognostic biomarker for CRC. By analyzing the methylation and expression of NKX6.1 in CRC tissues and CRC cell lines. We quantitatively examined the NKX6.1 methylation levels in 151 pairs of CRC tissues by using methylation-specific polymerase chain reaction analysis and found that NKX6.1 was hypermethylated in 35 of 151 CRC tissues (23%). NKX6.1 gene expression was inversely correlated with the DNA methylation level in CRC cell lines in vitro. Then, we analyzed the association of NKX6.1 methylation with clinical characteristics of these CRC patients. Our data demonstrated that patients with NKX6.1 methylation presented poorer 5-year overall survival (P = 0.0167) and disease-free survival (P = 0.0083) than patients without NKX6.1 methylation after receiving adjuvant chemotherapy. Most importantly, these data revealed that stage II CRC patients with NKX6.1 methylation had poorer 5-year disease-free survival (P = 0.0322) than patients without NKX6.1 methylation after adjuvant chemotherapy. Our results demonstrate that methylation of NKX6.1 is a novel prognostic biomarker in CRC and that it may be used as a predictor of the response to chemotherapy.

Concordance analysis of methylation biomarkers detection in self-collected and physician-collected samples in cervical neoplasm.

BACKGROUND: Non-attendance at gynecological clinics is a major limitation of cervical cancer screening and self-collection of samples may improve this situation. Although HPV testing of self-collected vaginal samples is acceptable, the specificity is inadequate. The current focus is increasing self-collection of vaginal samples to minimize clinic visits. In this study, we analyzed the concordance and clinical performance of DNA methylation biomarker (PAX1, SOX1, and ZNF582) detection in self-collected vaginal samples and physician-collected cervical samples for the identification of cervical neoplasm. METHODS: We enrolled 136 cases with paired methylation data identified from abnormal Pap smears (n = 126) and normal controls (n = 10) regardless of HPV status at gynecological clinics. The study group comprised 37 cervical intraepithelial neoplasm I (CIN1), 23 cervical intraepithelial neoplasm II (CIN2), 16 cervical intraepithelial neoplasm III (CIN3), 30 carcinoma in situ (CIS), 13 squamous cell carcinomas (SCCs) and seven adenocarcinomas (ACs)/adenosquamous carcinomas (ASCs). PAX1, SOX1 and ZNF582 methylation in study samples was assessed by real-time quantitative methylation-specific polymerase chain reaction analysis. We generated methylation index cutoff values for the detection of CIN3+ in physician-collected cervical samples for analysis of the self-collected group. Concordance between the physician-collected and self-collected groups was evaluated by Cohen's Kappa. Sensitivity, specificity and area under curve (AUC) were calculated for detection of CIN3+ lesions. Finally, we produced an optimal cutoff value with the best sensitivity from the self-collected groups. RESULTS: We generated a methylation index cutoff value from physician-collected samples for detection of CIN3+. There were no significant differences in sensitivity, specificity of PAX1, SOX1 and ZNF582 between the self-collected and physician-collected groups. The methylation status of all three genes in the normal control samples, and the CIN 1, CIN2, CIN3, CIS, ACs/ASCs and SCC samples showed reasonable to good concordance between the two groups (κ = 0.443, 0.427, and 0.609 for PAX1, SOX1, and ZNF582, respectively). In determining the optimal cutoff values from the self-collected group, ZNF582 showed the highest sensitivity (0.77; 95%CI, 0.65-0.87) using a cutoff value of 0.0204. CONCLUSIONS: Methylation biomarker analysis of the three genes for detection of CIN3+ lesions shows reasonable to good concordance between the self-collected and physician-collected samples. Therefore, self-collection of samples could be adopted to decrease non-attendance and improve cervical screening.

The role of Sp1 and EZH2 in the regulation of LMX1A in cervical cancer cells.

We have reported previously that LIM homeobox transcription factor 1α (LMX1A) is hypermethylated and functions as a metastasis suppressor in cervical cancer cells. However, the regulation of LMX1A in carcinogenesis has not been reported. We aim to clarify whether specificity protein 1 (Sp1) and enhancer of zeste homolog 2 (EZH2) are involved in the regulation of LMX1A in cervical cancer. First we characterized the LMX1A promoter and used overexpression, knockdown, and reporter assays to show that Sp1 increased LMX1A promoter activity. Next, we used site-directed mutagenesis and electrophoresis mobility shift assays (EMSAs) to demonstrate that Sp1-binding sites were important for Sp1-mediated activation of the LMX1A promoter. Chromatin immunoprecipitation data demonstrated that Sp1 could bind directly to the LMX1A promoter and activate endogenous LMX1A expression in cells pretreated with 5-aza-2'-deoxycytidine (5-aza-dC). Knockdown of EZH2 decreased H3K27me3 histone modification but was insufficient to restore LMX1A expression. To explore the effect of EZH2 on the endogenous LMX1A promoter, we treated EZH2-knockdown cells with 5-aza-dC and trichostatin A (TSA) and then depleted the cells of drugs for 3days. H3K14ac was enriched at the LMX1A promoter in EZH2-knockdown cells and LMX1A mRNA was still expressed. Taken together, these data imply that Sp1 may activate LMX1A expression upon oncogenic stress during cervical cancer development. Moreover, suppression of EZH2 may delay resilencing of LMX1A after the removal of 5-aza-dC and TSA.