Carbon sinks/sources' spatiotemporal heterogeneity and their spatial response to economic-social factors in relatively developed regions: A case of China's coastal area.

With the global climate change, carbon reduction in economically active regions has gradually become a focus of attention and its underlying drivers were essential for understanding alterations in ecosystems in response to human behavior. However, the exploration of Carbon Sinks/Sources Patterns (CSSP) in an Economic-Social context was lacking. Distinguished from traditional Net Ecosystem Productivity (NEP) estimation methods, we optimized model parameters, adjusted estimation logic, and revealed CSSP more reasonably. Moreover, spatial econometric model was used to reveal the spatial effects mechanism of Economic-Social Development on CSSP. Over the past 20 years, we revealed that: (a) The pattern of NEP exhibited distinct spatial heterogeneity, with higher sinks observed in the north and offshore regions. It demonstrated regular cyclic fluctuations, averaging a 3-4-year cycle, featuring a gradual ascent followed by a rapid descent; (b) The Carbon Sequestration Capacity (CSC) of vegetation significantly increased. Based on the carbon sink properties, the study area was distinctly divided into three clusters; (c) CSSP have been profoundly affected by economic-social factors. Economic growth and industrial structure optimization contributed to the enhancement of CSC, but population aggregation and urban expansion had negative impacts. The direct effect of innovation capacity and the spatial spillover effect of industrial structure optimization were negative. Overall, exploring CSSP against the backdrop of economic-social factors not only provides a new perspective for understanding the regularities of change and the underlying mechanisms driven by human factors but also offers valuable insights for achieving sustainable development and green growth in other coastal regions globally.

Highly sensitive and rapid determination of Mycobacterium leprae based on real-time multiple cross displacement amplification.

BACKGROUND: Mycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed. RESULTS: In this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor. CONCLUSION: The E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings.

STK24 Promotes Progression of LUAD and Modulates the Immune Microenvironment.

Objective: Recent studies have shown that serine/threonine-protein kinase 24 (STK24) plays an important role in cancer development. However, the significance of STK24 in lung adenocarcinoma (LUAD) remains to be determined. This study is aimed at investigating the significance of STK24 in LUAD. Methods: STK24 was silenced and overexpressed by siRNAs and lentivirus, respectively. Cellular function was assessed by CCK8, colony formation, transwell, apoptosis, and cell cycle. mRNA and protein abundance was checked by qRT-PCR and WB assay, respectively. Luciferase reporter activity was evaluated to examine the regulation of KLF5 on STK24. Various public databases and tools were applied to investigate the immune function and clinical significance of STK24 in LUAD. Results: We found that STK24 was overexpressed in lung adenocarcinoma (LUAD) tissues. High expression of STK24 predicted poor survival of LUAD patients. In vitro, STK24 enhanced the proliferation and colony growth ability of A549 and H1299 cells. STK24 knockdown induced apoptosis and cell cycle arrest at G0/G1 phase. Furthermore, Krüppel-like factor 5 (KLF5) activated STK24 in lung cancer cells and tissues. Enhanced lung cancer cell growth and migration triggered by KLF5 could be reversed by silencing of STK24. Finally, the bioinformatics results showed that STK24 may be involved in the regulation of the immunoregulatory process of LUAD. Conclusion: KLF5 upregulation of STK24 contributes to cell proliferation and migration in LUAD. Moreover, STK24 may participate in the immunomodulatory process of LUAD. Targeting KLF5/STK24 axis may be a potential therapeutic strategy for LUAD.

Assessment of a computerized quantitative quality control tool for whole slide images of kidney biopsies.

Inconsistencies in the preparation of histology slides and whole-slide images (WSIs) may lead to challenges with subsequent image analysis and machine learning approaches for interrogating the WSI. These variabilities are especially pronounced in multicenter cohorts, where batch effects (i.e. systematic technical artifacts unrelated to biological variability) may introduce biases to machine learning algorithms. To date, manual quality control (QC) has been the de facto standard for dataset curation, but remains highly subjective and is too laborious in light of the increasing scale of tissue slide digitization efforts. This study aimed to evaluate a computer-aided QC pipeline for facilitating a reproducible QC process of WSI datasets. An open source tool, HistoQC, was employed to identify image artifacts and compute quantitative metrics describing visual attributes of WSIs to the Nephrotic Syndrome Study Network (NEPTUNE) digital pathology repository. A comparison in inter-reader concordance between HistoQC aided and unaided curation was performed to quantify improvements in curation reproducibility. HistoQC metrics were additionally employed to quantify the presence of batch effects within NEPTUNE WSIs. Of the 1814 WSIs (458 H&E, 470 PAS, 438 silver, 448 trichrome) from n = 512 cases considered in this study, approximately 9% (163) were identified as unsuitable for subsequent computational analysis. The concordance in the identification of these WSIs among computational pathologists rose from moderate (Gwet's AC1 range 0.43 to 0.59 across stains) to excellent (Gwet's AC1 range 0.79 to 0.93 across stains) agreement when aided by HistoQC. Furthermore, statistically significant batch effects (p < 0.001) in the NEPTUNE WSI dataset were discovered. Taken together, our findings strongly suggest that quantitative QC is a necessary step in the curation of digital pathology cohorts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Development and evaluation of deep learning-based segmentation of histologic structures in the kidney cortex with multiple histologic stains.

The application of deep learning for automated segmentation (delineation of boundaries) of histologic primitives (structures) from whole slide images can facilitate the establishment of novel protocols for kidney biopsy assessment. Here, we developed and validated deep learning networks for the segmentation of histologic structures on kidney biopsies and nephrectomies. For development, we examined 125 biopsies for Minimal Change Disease collected across 29 NEPTUNE enrolling centers along with 459 whole slide images stained with Hematoxylin & Eosin (125), Periodic Acid Schiff (125), Silver (102), and Trichrome (107) divided into training, validation and testing sets (ratio 6:1:3). Histologic structures were manually segmented (30048 total annotations) by five nephropathologists. Twenty deep learning models were trained with optimal digital magnification across the structures and stains. Periodic Acid Schiff-stained whole slide images yielded the best concordance between pathologists and deep learning segmentation across all structures (F-scores: 0.93 for glomerular tufts, 0.94 for glomerular tuft plus Bowman's capsule, 0.91 for proximal tubules, 0.93 for distal tubular segments, 0.81 for peritubular capillaries, and 0.85 for arteries and afferent arterioles). Optimal digital magnifications were 5X for glomerular tuft/tuft plus Bowman's capsule, 10X for proximal/distal tubule, arteries and afferent arterioles, and 40X for peritubular capillaries. Silver stained whole slide images yielded the worst deep learning performance. Thus, this largest study to date adapted deep learning for the segmentation of kidney histologic structures across multiple stains and pathology laboratories. All data used for training and testing and a detailed online tutorial will be publicly available.

Two target genes based multiple cross displacement amplification combined with a lateral flow biosensor for the detection of Mycobacterium tuberculosis complex.

BACKGROUND: Tuberculosis (TB) is a serious chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC). Hence, the development of a novel, simple, rapid and sensitive method to detect MTBC is of great significance for the prevention and treatment of TB. RESULTS: In this study, multiple cross displacement amplification (MCDA) combined with a nanoparticle-based lateral flow biosensor (LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC (MCDA-LFB). One suite of specific MCDA primers designed for the IS6110 and mpb64 genes was validated using genomic DNA extracted from the reference strain H37Rv. The MCDA amplicons were analyzed using a real-time turbidimeter, colorimetric indicator (malachite green, MG) and LFBs. The optimal amplification temperature and time were confirmed, and the MCDA-LFB method established in the current report was evaluated by detecting various pathogens (i.e., reference strains, isolates and clinical sputum samples). The results showed that the two sets of MCDA primers targeting the IS6110 and mpb64 genes could effectively detect MTBC strains. The optimal reaction conditions for the MCDA assay were determined to be 67 °C for 35 min. The MCDA assay limit of detection (LoD) was 100 fg per reaction for pure genomic DNA. The specificity of the MCDA-LFB assay was 100%, and there were no cross-reactions for non-MTBC strains. For sputum samples and MTBC strain detection, the positive rate of MCDA-LFB for the detection of MTBC strains was consistent with seminested automatic real-time PCR (Xpert MTB/RIF) and higher than acid-fast staining (AFS) and culture assays when used for sputum samples. The MCDA-LFB assay was a rapid tool, and the whole procedure for MCDA-LFB, including DNA template preparation, MCDA reaction and amplification product analysis, was completed within 70 min. CONCLUSION: The MCDA-LFB assay targeting the IS6110 and mpb64 genes is a simple, rapid, sensitive and reliable detection method, and it has potential significance for the prevention and treatment of TB.

Circular RNA LPAR3 sponges microRNA-198 to facilitate esophageal cancer migration, invasion, and metastasis.

In this study, we explored expression and functions of circular RNA LPAR3 (circLPAR3) in esophageal squamous cell carcinoma (ESCC). The differential expression of circular RNAs (circRNAs) in 10 ESCC and corresponding paracarcinoma tissues was analyzed through circRNA microarray, then the candidate circRNAs were detected and verified through quantitative RT-PCR, and a novel circRNA was screened, which was circLPAR3. Circular RNA LPAR3 showed apparently high expression in ESCC tissues and cells, which was closely correlated with the clinical stage and lymph node metastasis of ESCC patients. Circular RNA LPAR3 was mainly located in the cytoplasm of ESCC cells, which was more stable than the baseline gene. Circular RNA LPAR3 upregulated MET gene expression through sponge adsorption of microRNA (miR)-198, activated the RAS/MAPK and the PI3K/Akt pathways, and promoted ESCC cell migration, invasion, and metastasis in vivo and in vitro. However, it had no effect on ESCC cell proliferation. Circular RNA LPAR3 can regulate the miR-198-MET signal axis to promote the migration, invasion, and metastasis of esophageal cancer cells, which can thereby serve as a potential diagnostic and therapeutic target of esophageal cancer.

MicroRNA-1204 promotes cell proliferation by regulating PITX1 in non-small-cell lung cancer.

MicroRNA-1204 (miR-1204), a member of the PVT1 region, may improve B cell differentiation and metastasis in breast cancer. However, the role of miR-1204 in non-small-cell lung cancer (NSCLC) and its mechanism remain unclear. The GEO public database was first employed to find differentially expressed genes. The expression level of miR-1204 in patient tissues and NSCLC cell lines was determined using qRT-PCR. Cell proliferation assays were performed to investigate the impact of miR-1204 on cell growth. Bioinformatics analysis and dual-luciferase reporter assays were conducted to find potential target genes. Finally, we performed in vivo experiments to identify the effect of miR-1204 on tumor formation in nude mice. It was first found that miR-1204 was overexpressed in NSCLC tissues and cells. miR-1204 increased the proliferation of NSCLC cells and reduced cell cycle arrest in vitro. PITX1 (paired like homeodomain 1) was found as a potential target gene. In addition, PITX1 was also found to be low in expression in NSCLC tissues and cells. To show that PITX1 reversed the function of miR-1204 in promoting proliferation, confirmatory experiments were performed. Moreover, high miR-1204 and low PITX1 expression was highly correlated with tumor size, lymph node metastasis, and the TNM stage in patients diagnosed with NSCLC. Our results suggested that upregulated miR-1204 in NSCLC is associated with NSCLC progression and promotes NSCLC cell proliferation by downregulating PITX1. miR-1204 may act as a poor prognostic factor and a potential therapeutic target for NSCLC.

Functional promoter -1816C>G variant of RANKL predicts risk and prognosis of lone atrial fibrillation.

Receptor activator of nuclear factor-κB ligand (RANKL) had been confirmed contributing to the development and progression of AF by regulating atrial structural remodeling. But the involved genetic mechanism is unknown. We intended to explore the association between the polymorphism RANKL -1816C>G (rs7984870) and susceptibility and prognosis of lone AF. RANKL rs7984870 was genotyped in a case-control study of 828 patients and 834 controls in Chinese population. The CG and/or CC genotypes had an increased lone AF risk [adjusted odds ratio (OR) 1.20 for CG, OR 2.16 for CC, and OR 1.55 for CG/CC], compared with the GG genotype. Moreover, patients carrying CG/CC genotypes showed a higher possibility of AF recurrence after catheter ablation, compared with patients carrying GG genotype. In a genotype-phenotype correlation analysis using 24 normal left atrial appendage samples, increasing gradients of atrial RANKL expression levels positively correlated with atrial collagen volume fraction were identified in samples with CC, CG and GG genotypes. The in vitro luciferase assays also showed a higher luciferase activity of the -1816 C/C allele than that of the -1816 G/G allele. These results suggested that RANKL rs7984870 is involved in the etiology of lone AF and thus may be a marker for genetic susceptibility to lone AF and predicting prognosis after catheter ablation in Chinese populations. Therefore, we provide new information about treatment strategies and our understanding of RANKL in AF.

The long non-coding RNA AK001796 contributes to tumor growth via regulating expression of p53 in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the prevalent and deadly cancers worldwide, especially in China. Considering the poor prognosis of ESCC, the aim of this study is to dissect the effects of long non-coding RNA (lncRNA) AK001796 on cell proliferation and cell cycle in vitro and tumorigenicity in vivo, providing therapeutic targets for ESCC. METHODS: We conducted quantitative real time PCR to detect the expression level of lncRNA AK001796 in human ESCC tumor and adjacent non-tumor tissues, and analyzed the correlation between lncRNA AK001796 expression and clinicopathologic feature of ESCC patients. Then we knocked down the expression of lncRNA AK001796 in human ESCC cell lines Eca-109 and TE-1, and next inspected cell cycle and apoptosis condition in these cells using flow cytometry. Subsequently, we used CCK-8 assay to test proliferation ability of the lncRNA AK001796-silenced ESCC cells, and the MDM2/p53 signaling pathway in these cells was analyzed by western blot analysis. At last, the ESCC xenograft models were established to verify the role of lncRNA AK001796 on the occurrence and development of ESCC. RESULTS: In this study, we demonstrated that lncRNA AK001796 was significantly upregulated in ESCC tumor tissues compared to adjacent non-tumor tissues. Knockdown of lncRNA AK001796 inhibited ESCC cell growth, cell cycle, and tumor growth in a xenograft mouse model via regulating MDM2/p53 signal pathway. The expression of lncRNA AK001796 was positively correlated with MDM2 levels in human ESCC samples. CONCLUSIONS: Overall, lncRNA AK001796 regulates cell proliferation and cell cycle via modulating MDM2/p53 signaling in ESCC, which provides a new insight into the treatment targets for ESCC.Trial registration This study was registrated in the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (Trial registration: 2012-SR-127, Registered 20 January 2012).

MiR-106b-5p Promotes Proliferation and Inhibits Apoptosis by Regulating BTG3 in Non-Small Cell Lung Cancer.

BACKGROUND/AIMS: MicroRNAs have been validated to play a crucial role in tumorigenesis of non-small cell lung cancer (NSCLC). Although miR-106b-5p has been reported to play a vital role in various malignancies the physiological function of miR-106b-5p in NSCLC still remain unknown. In this study, we investigated the role of miR-106b-5p in NSCLC. METHODS: Quantitative real-time polymerase chain reaction was conducted to estimate the expression of miR-106b-5p and BTG3 in both NSCLC tissues and cell lines. The effects of miR-106b-5p on proliferation were determined in vitro using CCK-8 proliferation assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, colony formation assays and cell-cycle assays and the in vivo effects were evaluated by a mouse tumorigenicity model. Cell apoptosis and cell cycle was investigated by flow cytometric analysis in vitro. The molecular mechanism underlying the relevance between miR-106b-5p and BTG3 was confirmed by luciferase assay and western blot. RESULTS: In current study, we found a relatively higher miR-106b-5p and lower BTG3 expression in NSCLC specimens and cell lines. BTG3 was verified as a direct target of miR-106b-5p by luciferase assay. In vitro, over-expression of miR-106b-5p promoted proliferation and inhibited apoptosis by down-regulating BTG3 expression. In vivo, miR-106b-5p promoted xenograft tumor formation. CONCLUSION: Our findings revealed for the first time that miR-106b-5p plays a tumorigenesis role in NSCLC progression by down-regulating BTG3 expression, which may lead to a novel insight to the potential biomarker and novel therapeutic strategies for NSCLC patients.

Long Noncoding RNA FAL1 Promotes Cell Proliferation, Invasion and Epithelial-Mesenchymal Transition Through the PTEN/AKT Signaling Axis in Non-Small Cell Lung Cancer.

BACKGROUND/AIMS: Recently, long non-coding RNAs (lncRNAs) have been found to have many biological effects in different cancer stages. Several studies have revealed that focally amplified lncRNA on chromosome 1 (FAL1) regulates cancer progression via p21. However, the expression and mechanism of FAL1 in non-small cell lung cancer (NSCLC) still remain unclear. METHODS: We detected the FAL1 level in NSCLC tissues and in established cell lines using quantitative real-time PCR and evaluated the clinical significance. FAL1 was silenced or overexpressed using siRNA or lentivirus to study whether FAL1 affected cell proliferation, invasion and migration. Xenograft growth and pulmonary metastasis were observed using nude mouse models. The mechanisms were explored with western blotting and immunohistochemistry. RESULTS: FAL1 was significantly overexpressed in NSCLC compared with adjacent normal tissues, and a high level of FAL1 correlated with poor histological grade, increased lymph node metastasis and advanced TNM stage. Loss- and gain-of-function experiments in vitro verified that knockdown of FAL1 inhibited cell proliferation, invasion, migration and EMT via the PTEN/AKT pathway. Furthermore, an in vivo assay confirmed that overexpression of FAL1 facilitated tumor growth and metastasis. CONCLUSION: FAL1 may promote tumorigenesis and progression of NSCLC through the PTEN/AKT axis, which could lead to lncRNA-related diagnostics and therapeutics in NSCLC.

Either at left or right, both high and low frequency rTMS of dorsolateral prefrontal cortex decreases cue induced craving for methamphetamine.

BACKGROUND AND OBJECTIVES: The study is to investigate if left or right dorsolateral prefrontal cortex is important in modulating the craving to drug related cues. METHODS: The present investigation examined different combinations between left/right and high/low frequency rTMS on cue induced craving to methamphetamine, with active sham rTMS on P3 region on a total of 50 subjects. RESULTS: The results showed that either at left or right side, both high and low frequency rTMS are effective in decreasing the cue induced craving to methamphetamine. DISCUSSION AND CONCLUSIONS: In conclusion, bilateral frontal hemispheres are effective in modulating the cue induced craving to methamphetamine with rTMS. SCIENTIFIC SIGNIFICANCE: TMS targeting prefrontal cortex is effective for craving modulation; however the mechanism is more than potentiation or depression of single brain region. (Am J Addict 2017;26:776-779).

FGF2 regulates proliferation, migration, and invasion of ECA109 cells through PI3K/Akt signalling pathway in vitro.

Esophageal cancer is one of the most common malignant cancers that arise from esophagus tissues. Fibroblast growth factor 2 (FGF2) has been implicated in multiple biological functions and was considered as an oncogenic factor in tumorigenesis. However, the effects of FGF2 in esophageal carcinoma are yet to be fully elucidated. To better understand the function of FGF2 in esophageal cancer, we used the esophageal cancer cell line ECA109 as a cell model and downregulated FGF2 expression using RNAi; the results showed that insufficient expression of FGF2 inhibited cells proliferation, migration, and invasion of ECA109 cells. Meanwhile, the proliferation, migration, and invasion abilities were stimulated after treatment of exogenous FGF2. In addition, a PI3K/Akt signalling pathway inhibitor (LY294002) alleviated the tumorigenic effects of FGF2. These findings implied that the oncogenic effects of FGF2 was mediated, at least in part, through the PI3K/Akt signalling pathway and FGF2 may be a potential therapeutic target to constrain the tumorigenesis of esophageal cancer.

Genome-wide association study identifies a novel susceptibility locus at 12q23.1 for lung squamous cell carcinoma in han chinese.

Adenocarcinoma (AC) and squamous cell carcinoma (SqCC) are two major histological subtypes of lung cancer. Genome-wide association studies (GWAS) have made considerable advances in the understanding of lung cancer susceptibility. Obvious heterogeneity has been observed between different histological subtypes of lung cancer, but genetic determinants in specific to lung SqCC have not been systematically investigated. Here, we performed the GWAS analysis specifically for lung SqCC in 833 SqCC cases and 3,094 controls followed by a two-stage replication in additional 2,223 lung SqCC cases and 6,409 controls from Chinese populations. We found that rs12296850 in SLC17A8-NR1H4 gene region at12q23.1 was significantly associated with risk of lung SqCC at genome-wide significance level [additive model: odds ratio (OR) = 0.78, 95% confidence interval (CI) = 0.72-0.84, P = 1.19×10(-10)]. Subjects carrying AG or GG genotype had a 26% (OR = 0.74, 95% CI = 0.67-0.81) or 32% (OR = 0.68, 95% CI = 0.56-0.83) decreased risk of lung SqCC, respectively, as compared with AA genotype. However, we did not observe significant association between rs12296850 and risk of lung AC in a total of 4,368 cases with lung AC and 9,486 controls (OR = 0.96, 95% CI = 0.90-1.02, P = 0.173). These results indicate that genetic variations on chromosome 12q23.1 may specifically contribute to lung SqCC susceptibility in Chinese population.

Genetic variants at 6p21.1 and 7p15.3 are associated with risk of multiple cancers in Han Chinese.

Cancer susceptibility loci identified in reported genome-wide association studies (GWAS) are often tumor-specific; however, evidence of pleiotropy of some genes/loci has also been observed and biologically plausible. We hypothesized that there are important regions in the genome harboring genetic variants associated with risk of multiple types of cancer. In the current study, we attempted to map genetic variants that have consistent effects on risk of multiple cancers using our existing genome-wide scan data of lung cancer, noncardia gastric cancer, and esophageal squamous-cell carcinoma with overall 5,368 cases and 4,006 controls (GWAS stage), followed by a further evaluation in additional 9,001 cases with one of these cancer types and 11,436 controls (replication stage). Five variants satisfying the criteria of pleiotropy with p values from 1.10 × 10(-8) to 8.96 × 10(-6) for genome-wide scans of three cancer types were further evaluated in the replication stage. We found consistent associations of rs2494938 at 6p21.1 and rs2285947 at 7p15.3 with these three cancers in both GWAS and replication stages. In combined samples of GWAS and replication stages, the minor alleles of rs2494938 and rs2285947 were significantly associated with an increased risk of the cancers (odds ratio [OR] = 1.15, 95% confidence interval [CI], 1.10-1.19 and OR = 1.17, 95% CI, 1.12-1.21), with the p values being 1.20 × 10(-12) and 1.26 × 10(-16), respectively, which are at a genome-wide significance level. Our findings highlight the potential importance of variants at 6p21.1 and 7p15.3 in the susceptibility to multiple cancers.

Up-Regulation of MiR-452 Inhibits Metastasis of Non-Small Cell Lung Cancer by Regulating BMI1.

BACKGROUND/AIMS: MicroRNAs (miRNAs) have been regarded as a new class of regulators in cellular processes in non-small cell lung cancer (NSCLC). However, the relationship between miR-452 and the development of NSCLC remains unclear. METHODS: qRT-PCR was used to detect the expression of miR-452 and its target gene in NSCLC samples (n=60). The transwell assay was used to test the cell invasion capability. The regulation mechanism was confirmed by luciferase reporter assay and western blot assay. RESULTS: In the current study, a relatively lower miR-452 and higher BMI1 expression levels were confirmed to be associated with advanced tumor stage and more extent of lymph nodes metastasis. In vitro, down-regulated miR-452 could enhance cell invasion capability. Furthermore, miR-452 modulated BMI1 expression by binding to its 3'-UTR. The enhancement of cell invasion capability induced by down-regulated miR-452 was eliminated by repression of BMI1. CONCLUSIONS: Our results suggest that miR-452 plays a vital role in development of NSCLC, and this miR-452-BMI1 pathway might generate a novel insight into the treatment of NSCLC.

WT1 enhances proliferation and impedes apoptosis in KRAS mutant NSCLC via targeting cMyc.

BACKGROUND: A novel link between oncogenic KRAS signalling and WT1 was recently identified. We sought to investigate the role of WT1 and KRAS in proliferation and apoptosis. METHODS: KRAS mutations and WT1 (cMyc) expression were detected using Sanger sequencing and real-time PCR in 77 patients with non-small cell lung cancer (NSCLC). Overexpression and knockdown of WT1 were generated with plasmid and siRNA via transient transfection technology in H1299 and H1568 cells. MTT assay for detection of cell proliferation, and TUNEL assay and proteomic profiler assay for apoptosis evaluation were carried out. Dual luciferase reporter assay and ChIP-PCR were performed to validate the effect of WT1 on the cMyc promoter. RESULTS: KRAS mutations showed a negative impact on overall survival (OS). High expressions of WT1 and cMyc were associated with poor OS in KRAS mutant subgroup. The potential mechanisms that WT1 promotes proliferation and impedes apoptosis through affecting multiple apoptosis-related regulators in KRAS mutant NSCLC cells were identified. WT1 could activate cMyc promoter directly in KRAS mutant cells. CONCLUSION: The results suggest that WT1 and c-MYC expression is important for survival in KRAS mutant tumors as opposed to KRAS wild-type tumors. For treatment of KRAS mutant NSCLC, targeting WT1 and cMyc may provide alternative therapeutic strategies.

Genetic variants reducing MTR gene expression increase the risk of congenital heart disease in Han Chinese populations.

AIMS: Elevated homocysteine levels are known to be a risk factor for congenital heart disease (CHD), but the mechanism underlying this effect is unknown. During early embryonic development, homocysteine removal is dictated exclusively by the MTR activity. To examine the role of MTR in CHD risk, we identified genetic variants in MTR and investigated the mechanisms that affect its expression levels and that increase the risk of CHD in Chinese populations. METHODS AND RESULTS: The association between regulatory variants of the MTR gene and CHD was examined in three independent case-control studies in a total of 2340 patients with CHD and 2270 controls. The functional consequences of these variants were demonstrated using dual-luciferase assays, real-time polymerase chain reaction (PCR), electrophoretic mobility shift assays, surface plasma resonance, chromatin immunoprecipitation, and bisulfite sequencing, as well as by a group of predicted microRNAs using a gene reporter system. Two regulatory variants of MTR, -186T>G and +905G>A, were associated with an increased risk of CHD in both the separate and combined case-control studies (-186GG P = 1.32 × 10(-9); +905AA P = 6.35 × 10(-14)). Compared with the major allele, the -186G allele exhibited significantly lower promoter activity, decreased hnRNA and mRNA levels, reduced transcription factor binding affinity, and a more highly methylated promoter. The +905A allele exhibited a statistically stronger binding affinity to functional microRNAs that down regulate MTR expression at the translational level. Both of the minor alleles were correlated with elevated plasma homocysteine concentrations, indicating a genetic component for hyperhomocysteinaemia. CONCLUSIONS: Regulatory variants of the MTR gene increase CHD risk by reducing MTR expression and inducing the homocysteine accumulation and elevation.

MiR-16 induced the suppression of cell apoptosis while promote proliferation in esophageal squamous cell carcinoma.

BACKGROUND/AIMS: MicroRNAs (miRNAs) are non-coding small RNAs that regulate cell proliferation and functions by interfering with the translation of target mRNAs. Altered expression of miRNA is known to induce various human malignancies, but little is known about the role of miRNAs in esophageal squamous cell carcinoma (ESCC). METHODS: RT-PCR and Western blot were used to examine the expression of miRNAs and candidate genes in 40 pairs of squamous cell carcinoma of human. MiR-16 mimics and inhibitor were transfected in human TE-1 and Eca-109 cells before detecting the cell migration, proliferation, apoptosis and cell cycle. The regulation mechanism was confirmed by luciferase reporter assay. Caspase-3 and 9 were detected by RT-PCR and Western blot. RESULTS: Aberrant increased level of miR-16 was detected in the ESCC tissues compared with the corresponding adjacent tumor tissues. MiR-16 could inhibit cell apoptosis while promote cell proliferation by down-regulating RECK and SOX6 in TE-1 and Eca-109 cell lines through binding the 3'UTR of both RECK and SOX6 mRNA. CONCLUSIONS: Aberrant expression level of miR-16 could suppress cell apoptosis while promote growth by regulating RECK and SOX6 which play important roles in the pathogenesis of ESCC.