Identification of C2H2 zinc finger genes through genome-wide association study and functional analyses of LkZFPs in response to stresses in Larix kaempferi.

BACKGROUND: C2H2 zinc finger proteins (C2H2-ZFPs), one of the largest transcription factors, play a variety of roles in plant development and growth as well as stress response. While, the evolutionary history and expression profile of the C2H2-ZFP genes in Larix kaempferi (LkZFPs) have not been reported so far. RESULTS: In this study, the whole genome of the LkZFPs was identified and characterized, including physicochemical properties, phylogenetic relationships, conservative motifs, the promoter cis-elements and Gene Ontology (GO) annotation. We identified 47 LkZFPs and divided them into four subfamilies based on phylogenetic analysis and conserved motifs. Subcellular localization prediction showed that most of the LkZFPs were located in the nucleus. Promoter cis-element analysis suggested that the LkZFPs may be involved in the regulation of stress responses. Moreover, Real-time quantitative PCR (RT-qPCR) results showed that Q-type LkZFP genes were involved in the response to abiotic stress, such as salt, drought and hormone stresses. Subcellular localization results showed that LkZFP7 and LkZFP37 were located in the nucleus, LkZFP32 was located in both cytoplasm and nucleus. CONCLUSION: The identification and functional analysis of LkZFPs suggested that some LkZFP genes might play important roles in coping with both biological and abiotic stresses. These results could further increase understanding of the function of the LkZFPs, and provide some research direction and theoretical support.

Downregulation of miR­7 and miR­153 is involved in Helicobacter pylori CagA induced gastric carcinogenesis and progression.

Helicobacter pylori (H. pylori) infection plays a pivotal role in the development of gastric cancer (GC). However, the association between aberrant microRNAs (miRNAs/miRs) expression and H. pylori­induced GC remains poorly understood. The present study reported that repeated infection of H. pylori caused the oncogenicity of GES­1 cells in BALB/c Nude mice. miRNA sequencing revealed that both miR­7 and miR­153 were significantly decreased in the cytotoxin­associated gene A (CagA) positive GC tissues and this was further confirmed in a chronic infection model of GES­1/HP cells. Further biological function experiments and in vivo experiments validated that miR­7 and miR­153 can promote apoptosis and autophagy, inhibit proliferation and inflammatory response in GES­1/HP cells. All the associations between miR­7/miR­153 and their potential targets were revealed via bioinformatics prediction and dual­luciferase reporter assay. Particularly, downregulation of both miR­7 and miR­153 obtained an improved sensitivity and specificity in diagnosing H. pylori (CagA+)­induced GC. The present study identified that the combination of miR­7 and miR­153 may be regarded as novel therapeutic targets in H. pylori CagA (+)­associated GC.

Comprehensive Analysis of the NF-YB Gene Family and Expression under Abiotic Stress and Hormone Treatment in Larix kaempferi.

NF-YB, a subfamily of Nuclear Factor Y (NF-Y) transcription factor, play crucial role in many biological processes of plant growth and development and abiotic stress responses, and they can therefore be good candidate factors for breeding stress-resistant plants. However, the NF-YB proteins have not yet been explored in Larix kaempferi, a tree species with high economic and ecological values in northeast China and other regions, limiting the breeding of anti-stress L. kaempferi. In order to explore the roles of NF-YB transcription factors in L. kaempferi, we identified 20 LkNF-YB family genes from L. kaempferi full-length transcriptome data and carried out preliminary characterization of them through series of analyses on their phylogenetic relationships, conserved motif structure, subcellular localization prediction, GO annotation, promoter cis-acting elements as well as expression profiles under treatment of phytohormones (ABA, SA, MeJA) and abiotic stresses (salt and drought). The LkNF-YB genes were classified into three clades through phylogenetic analysis and belong to non-LEC1 type NF-YB transcription factors. They have 10 conserved motifs; all genes contain a common motif, and their promoters have various phytohormones and abiotic stress related cis-acting elements. Quantitative real time reverse transcription PCR (RT-qPCR) analysis showed that the sensitivity of the LkNF-YB genes to drought and salt stresses was higher in leaves than roots. The sensitivity of LKNF-YB genes to ABA, MeJA, SA stresses was much lower than that to abiotic stress. Among the LkNF-YBs, LkNF-YB3 showed the strongest responses to drought and ABA treatments. Further protein interaction prediction analysis for LkNF-YB3 revealed that LkNF-YB3 interacts with various factors associated with stress responses and epigenetic regulation as well as NF-YA/NF-YC factors. Taken together, these results unveiled novel L. kaempferi NF-YB family genes and their characteristics, providing the basic knowledge for further in-depth studies on their roles in abiotic stress responses of L. kaempferi.

Characterization of the early gene expression profile in Populus ussuriensis under cold stress using PacBio SMRT sequencing integrated with RNA-seq reads.

Populus ussuriensis is an important and fast-growing afforestation plant species in north-eastern China. The whole-genome sequencing of P. ussuriensis has not been completed. Also, the transcriptional network of P. ussuriensis response to cold stress remains unknown. To unravel the early response of P. ussuriensis to chilling (3 °C) stress and freezing (-3 °C) stresses at the transcriptional level, we performed single-molecule real-time (SMRT) and Illumina RNA sequencing for P. ussuriensis. The SMRT long-read isoform sequencing led to the identification of 29,243,277 subreads and 575,481 circular consensus sequencing reads. Approximately 50,910 high-quality isoforms were generated, and 2272 simple sequence repeats and 8086 long non-coding RNAs were identified. The Ca2+ content and abscisic acid (ABA) content in P. ussuriensis were significantly increased under cold stresses, while the value in the freezing stress treatment group was significantly higher than the chilling stress treatment group. A total of 49 genes that are involved in the signal transduction pathways related to perception and transmission of cold stress signals, such as the Ca2+ signaling pathway, ABA signaling pathway and MAPK signaling cascade, were found to be differentially expressed. In addition, 158 transcription factors from 21 different families, such as MYB, WRKY and AP2/ERF, were differentially expressed during chilling and freezing treatments. Moreover, the measurement of physiological indicators and bioinformatics observations demonstrated the altered expression pattern of genes involved in reactive oxygen species balance and the sugar metabolism pathway during chilling and freezing stresses. This is the first report of the early responses of P. ussuriensis to cold stress, which lays the foundation for future studies on the regulatory mechanisms in cold-stress response. In addition the full-length reference transcriptome of P. ussuriensis deciphered could be used in future studies on P. ussuriensis.

PacBio and Illumina RNA Sequencing Identify Alternative Splicing Events in Response to Cold Stress in Two Poplar Species.

In eukaryotes, alternative splicing (AS) is a crucial regulatory mechanism that modulates mRNA diversity and stability. The contribution of AS to stress is known in many species related to stress, but the posttranscriptional mechanism in poplar under cold stress is still unclear. Recent studies have utilized the advantages of single molecular real-time (SMRT) sequencing technology from Pacific Bioscience (PacBio) to identify full-length transcripts. We, therefore, used a combination of single-molecule long-read sequencing and Illumina RNA sequencing (RNA-Seq) for a global analysis of AS in two poplar species (Populus trichocarpa and P. ussuriensis) under cold stress. We further identified 1,261 AS events in P. trichocarpa and 2,101 in P. ussuriensis among which intron retention, with a frequency of more than 30%, was the most prominent type under cold stress. RNA-Seq data analysis and annotation revealed the importance of calcium, abscisic acid, and reactive oxygen species signaling in cold stress response. Besides, the low temperature rapidly induced multiple splicing factors, transcription factors, and differentially expressed genes through AS. In P. ussuriensis, there was a rapid occurrence of AS events, which provided a new insight into the complexity and regulation of AS during cold stress response in different poplar species for the first time.

Anticancer roles of let-7f-1-3p in non-small cell lung cancer via direct targeting of integrin ß1.

Lung cancer is one of the most common types of cancer, with the highest mortality rate worldwide. MicroRNAs play notable roles in the chemotherapeutic effects of anticancer drugs. The present study used reverse transcription-quantitative PCR, western blotting and cell migration and invasion assays to reveal the role of let-7f-1-3p in non-small cell lung cancer (NSCLC) and explore the effect of let-7f-1-3p on doxorubicin (DOX) treatment. It was demonstrated that the levels of let-7f-1-3p in carcinoma tissues were lower compared with those in paracarcinoma tissues. Thus, let-7f-1-3p may act as a suppressor gene. The present study also explored the role of let-7f-1-3p in A549 and NCI-H1975 cells. Results revealed that let-7f-1-3p could inhibit the viability, migration and invasion of NSCLC cells and induce their apoptosis. Integrin ß1 acted as a target gene regulated by let-7f-1-3p. This suggested that let-7f-1-3p could enhance DOX-inhibited cell viability, migration and invasion in vitro. Overall, the present study demonstrated that let-7f-1-3p may act as a target for drug design and lung cancer therapy.

Sequence-Specific Detection of DNA Strands Using a Solid-State Nanopore Assisted by Microbeads.

Simple, rapid, and low-cost detection of DNA with specific sequence is crucial for molecular diagnosis and therapy applications. In this research, the target DNA molecules are bonded to the streptavidin-coated microbeads, after hybridizing with biotinylated probes. A nanopore with a diameter significantly smaller than the microbeads is used to detect DNA molecules through the ionic pulse signals. Because the DNA molecules attached on the microbead should dissociate from the beads before completely passing through the pore, the signal duration time for the target DNA is two orders of magnitude longer than free DNA. Moreover, the high local concentration of target DNA molecules on the surface of microbeads leads to multiple DNA molecules translocating through the pore simultaneously, which generates pulse signals with amplitude much larger than single free DNA translocation events. Therefore, the DNA molecules with specific sequence can be easily identified by a nanopore sensor assisted by microbeads according to the ionic pulse signals.

Resveratrol induces cervical cancer HeLa cell apoptosis through the activation and nuclear translocation promotion of FOXO3a.

Resveratrol (trans-3,4'V,5-trihydroxystilbene) presents antioxidant, anti-inflammatory, and cardioprotective functions in addition to its anticancer potential. In this study, we explored how resveratrol, as an anticancer agent, effectively influences cervical cancer HeLa cells. Our data showed that resveratrol could significantly inhibit HeLa cell proliferation and induce their apoptosis, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and flow cytometry. The immunofluorescence staining results in the present study suggested that resveratrol could facilitate FOXO3a nuclear translocation. We then focused on the mechanism of resveratrol in promoting HeLa cell apoptosis. The following experiments suggested that the possible initial mechanism involves the upregulation Forkhead box O (FOXO) 3a expression, which further increases the expression of Bcl-2 interacting mediator of cell death (BIM), the gene transcribed in apoptosis. Resveratrol could also inactivate the basal extracellular signal-regulated kinase (ERK) activity, causing FOXO3a activation and resulting in HeLa cell apoptosis. In summary, both mechanisms stimulated the accumulation of activated FOXO3a, promoted its nuclear translocation, and ultimately caused HeLa cell apoptosis. Thus, resveratrol may have a potential in the treatment of cervical cancer.

Delivery of MicroRNA-let-7c-5p by Biodegradable Silica Nanoparticles Suppresses Human Cervical Carcinoma Cell Proliferation and Migration.

Human cervical cancer is the most common gynecological malignancy. The continuous development of nanotechnology has allowed the wide use of nanomaterials in cancer treatment. Nanoparticles can be used as gene carriers because of their surface effect and small-size effect. MicroRNA-let-7c-5p (miR-let-7c-5p) belongs to the let-7 family. Although it has been reported to exert a tumor suppressive effect in a variety of cancers, the exact role and mechanism of miR-let-7c-5p in the progression of cervical cancer are unclear. In this study, we synthesized flower-shaped SiO2 -PEI nanoparticles with high pDNA/siRNA loading rates. This nanoparticle with miR-let-7c-5p-expressed plasmid could effectively transfer miR-let-7c-5p to human epithelial carcinoma (HeLa) cells. In addition, the combination of nanomaterials and gene therapy could inhibit the development of cancer under the conditions of extremely low cytotoxicity. These findings provided a new anticancer strategy based on F-SiO2 -polyethyleneimine/miR-let-7c-5p (FSP-let-7c-5p)nanoparticles and indicated that miR-let-7c-5p/IGF-1R/PI3K/AKT and -catenin/SLUG could be used as new potential targets for the treatment of cervical cancer.

Prevalence and risk factors of abnormal left ventricular geometrical patterns in untreated hypertensive patients.

BACKGROUND: The various prevalence of LVH and abnormal LV geometry have been reported in different populations. So far, only a few reports are available on the prevalence of LV geometric patterns in a large Chinese untreated hypertensive population. METHODS: A total of 9,286 subjects (5167 men and 4119 women) completed the survey and 1641 untreated hypertensive patients (1044 males and 597 females) enrolled in the present study. The LV geometry was classified into four patterns: normal; abnormal,defined as concentric remodeling;concentric or eccentric hypertrophy based on the values of left ventricular mass index (LVMI) and relative wall thickness (RWT). Logistic regression model was applied to determine the odds ratio (OR) and 95% confidence intervals (CI) of the risk factors of left ventricular hypertrophy. RESULTS: The prevalence of LVH was 20.2% in untreated hypertensive patients, much higher in women (30.8%) than in men (14.2%) (P < 0.01). The prevalence of LV geometrical patterns was 34.9%, 11.1%, 9.1% for concentric remodeling, concentric and eccentric hypertrophy,respectively. After adjustment by using Logistic regression model, the risk factors for LVH and abnormal LV geometry were age, female, systolic blood pressure, and body mass index. And low high density lipoprotein maybe a positive factor. CONCLUSIONS: The prevalence of LVH and abnormal LV geometric patterns was higher in women than in men and increased with age. It is crucial to improve the awareness rate of hypertension and control the risk factors of CV complications in untreated hypertensive population.

A pharmacogenetics-based warfarin maintenance dosing algorithm from Northern Chinese patients.

Inconsistent associations with warfarin dose were observed in genetic variants except VKORC1 haplotype and CYP2C9*3 in Chinese people, and few studies on warfarin dose algorithm was performed in a large Chinese Han population lived in Northern China. Of 787 consenting patients with heart-valve replacements who were receiving long-term warfarin maintenance therapy, 20 related Single nucleotide polymorphisms were genotyped. Only VKORC1 and CYP2C9 SNPs were observed to be significantly associated with warfarin dose. In the derivation cohort (n = 551), warfarin dose variability was influenced, in decreasing order, by VKORC1 rs7294 (27.3%), CYP2C9*3(7.0%), body surface area(4.2%), age(2.7%), target INR(1.4%), CYP4F2 rs2108622 (0.7%), amiodarone use(0.6%), diabetes mellitus(0.6%), and digoxin use(0.5%), which account for 45.1% of the warfarin dose variability. In the validation cohort (n = 236), the actual maintenance dose was significantly correlated with predicted dose (r = 0.609, P<0.001). Our algorithm could improve the personalized management of warfarin use in Northern Chinese patients.

Phenotype-genotype analysis in two Chinese families with Liddle syndrome.

The families with Liddle syndrome show marked phenotypic variation in blood pressure, serum potassium and other clinical manifestations. Here we analyzed the correlation of genotype-phenotype in two Chinese families with Liddle syndrome. The sequence of C-terminus of SCNN1B and SCNN1G were screened in the two families with likely Liddle syndrome. In addition to hypertension and hypokalemia, one of the two pedigrees had sudden death in their family members, so the exons of 428 reported genes-related to cardiovascular diseases were screened as well in the family. A heterozygous ßR566X nonsense mutation was found in the proband-1 in the first pedigree, and the proband's sister and father. They showed mild phenotype with hypertension under control. In contrast, two of the four previous studies report that the mutation causes severe phenotype. A heterozygous ßR597PfrX607 frameshift mutation was identified in the proband-2 in the second pedigree, showing malignant phenotype including resistant hypertension, hypokalemia, higher PRA and plasma angiotensin II levels. Both the proband-2 and the proband-2's father had sudden death in their twenties, but no meaningful mutations were found by screening of the exons in 428 cardiovascular disease-related genes. However, the same mutation has been related to moderate phenotype in previous studies. Our results confirmed that the phenotypes of Liddle syndrome are varied significantly even with the same mutation. The mechanisms why the same mutation causes very different phenotype need to be explored because intervention of these modifiers may change the disease course and prognosis accordingly.

Common genetic variants of MGP are associated with calcification on the arterial wall but not with calcification present in the atherosclerotic plaques.

BACKGROUND: Two endophenotypes of arterial calcification, calcification on arterial wall and calcification in atherosclerotic plaques, are associated with different types of cardiovascular events. Mgp-deficient mice showed matrix Gla protein (MGP) is strongly associated with calcification on arterial wall without atherosclerotic plaques, and MGP variants were not significantly associated with myocardial infarction. MGP may play different roles in the 2 endophenotypes. METHODS AND RESULTS: We analyzed the associations of MGP variants rs4236, rs1800801, and rs1800802 with the 2 endophenotypes determined by multidetector computed tomography angiography. A total of 585 with calcification on coronary artery wall, 675 with calcification in coronary atherosclerotic plaques, 454 with calcification on aortic wall, and 725 controls were enrolled. After Bonferroni correction, rs4236 and rs1800801 were still associated with calcification on arterial wall, the odds ratios were 0.708 (95% confidence interval, 0.540-0.928) for rs4236 and 0.652 (95% confidence interval, 0.479-0.888) for rs1800801 in coronary artery wall calcification, and 0.699 (95% confidence interval, 0.525-0.931) for rs4236 and 0.650 (95% confidence interval, 0.467-0.905) for rs1800801 in aortic wall calcification, respectively. The variants were correlated with calcification severity by ln(CAC Agatston score+1) in coronary artery wall calcification but not in atherosclerotic plaque calcification. In accordance with their associations with calcification on arterial wall, rs4236C and rs1800801A were associated with higher MGP plasma levels, whereas rs1800802C was associated with lower MGP levels in normal controls. Because of the role of calcification in plaque vulnerability, their associations with acute myocardial infarction were also determined in 771 controls and 752 patients, no association was found. CONCLUSIONS: MGP genetic variants showed association with calcification on arterial wall but not with calcification in atherosclerotic plaques.