Blood FOLR3 methylation dysregulations and heterogeneity in non-small lung cancer highlight its strong associations with lung squamous carcinoma.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers. Early detection is crucial to reduce lung cancer-related mortality. Aberrant DNA methylation occurs early during carcinogenesis and can be detected in blood. It is essential to investigate the dysregulated blood methylation markers for early diagnosis of NSCLC. METHODS: NSCLC-associated methylation gene folate receptor gamma (FOLR3) was selected from an Illumina 850K array analysis of peripheral blood samples. Mass spectrometry was used for validation in two independent case-control studies (validation I: n = 2548; validation II: n = 3866). Patients with lung squamous carcinoma (LUSC) or lung adenocarcinoma (LUAD), normal controls (NCs) and benign pulmonary nodule (BPN) cases were included. FOLR3 methylations were compared among different populations. Their associations with NSCLC clinical features were investigated. Receiver operating characteristic analyses, Kruskal-Wallis test, Wilcoxon test, logistics regression analysis and nomogram analysis were performed. RESULTS: Two CpG sites (CpG_1 and CpG_2) of FOLR3 was significantly lower methylated in NSCLC patients than NCs in the discovery round. In the two validations, both LUSC and LUAD patients presented significant FOLR3 hypomethylations. LUSC patients were highlighted to have significantly lower methylation levels of CpG_1 and CpG_2 than BPN cases and LUAD patients. Both in the two validations, CpG_1 methylation and CpG_2 methylation could discriminate LUSC from NCs well, with areas under the curve (AUCs) of 0.818 and 0.832 in validation I, and 0.789 and 0.780 in validation II. They could also differentiate LUAD from NCs, but with lower efficiency. CpG_1 and CpG_2 methylations could also discriminate LUSC from BPNs well individually in the two validations. With the combined dataset of two validations, the independent associations of age, gender, and FOLR3 methylation with LUSC and LUAD risk were shown and the age-gender-CpG_1 signature could discriminate LUSC and LUAD from NCs and BPNs, with higher efficiency for LUSC. CONCLUSIONS: Blood-based FOLR3 hypomethylation was shown in LUSC and LUAD. FOLR3 methylation heterogeneity between LUSC and LUAD highlighted its stronger associations with LUSC. FOLR3 methylation and the age-gender-CpG_1 signature might be novel diagnostic markers for the early detection of NSCLC, especially for LUSC.

Hypomethylation of DYRK4 in peripheral blood is associated with increased lung cancer risk.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. It is urgent to identify new biomarkers for the early detection of LC. DNA methylation in peripheral blood has been reported to be associated with cancers. We conducted two independent case-control studies and a nested case-control study (168 LC cases and 167 controls in study Ⅰ, 677 LC cases and 833 controls in study Ⅱ, 147 precancers and 21 controls in the nested case-control study). The methylation levels of DYRK4 CpG sites were measured using mass spectrometry and their correlations with LC were analyzed by logistic regression and nonparametric tests. Bonferroni correction was used for the multiple comparisons. LC-related decreased DYRK4 methylation was discovered in Study I and validated in Study II (the odds ratios [ORs] for the lowest vs. highest quartile of all three DYRK4 CpG sites ranged from 1.64 to 2.09, all p < 0.001). Combining the two studies, hypomethylation of DYRK4 was observed in stage I cases (ORs per -10% methylation ranged from 1.16 to 1.38, all p < 5.9E-04), and could be enhanced by male gender (ORs ranged from 1.77 to 4.17 via interquartile analyses, all p < 0.017). Hypomethylation of DYRK4_A_CpG_2 was significantly correlated with tumor size, length, and stage (p = 0.034, 0.002, and 0.002, respectively) in LC cases. Our study disclosed the association between DYRK4 hypomethylation in peripheral blood and LC, suggesting the feasibility of blood-based DNA methylation as new biomarker for LC detection.

Identification of FUT7 hypomethylation as the blood biomarker in the prediction of early-stage lung cancer.

Early detection of lung cancer (LC) is vital for reducing LC-related mortality. However, noninvasive diagnostic tools remain a great challenge. We aim to identify blood-based biomarkers for the early detection of LC. Here, LC-associated hypomethylation in alpha-1,3-fucosyltransferase VII (FUT7) is identified via the Illumina 850K array in a discovery study and validated by mass spectrometry in two independent case-control studies with blood samples from 1720 LC patients (86.8% LC at stage I, blood is collected before surgery and treatment) and 3143 healthy controls. Compared to the controls, blood-based FUT7 hypomethylation is identified in LC patients at stage I, and even in LC patients with malignant nodules ≤ 1 cm and in patients with adenocarcinoma in situ. Gender plays a role in the LC-associated FUT7 hypomethylation in blood, which is more significant in males than in females. We also reveal that FUT7 hypomethylation in LC could be enhanced by the advanced stage of cancer, involvement of lymph nodes, and larger tumor size. Based on a large sample size and semi-quantitative methods, our study reveals a strong association between blood-based FUT7 hypomethylation and LC, suggesting that methylation signatures in blood may be a group of potential biomarkers for detection of early-stage LC.

Hypomethylation of RPTOR in peripheral blood is associated with very early-stage lung cancer.

PURPOSE: Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Novel biomarkers for LC detection are urgently needed. Here we aimed to investigate the association between RPTOR methylation in peripheral blood and LC. METHODS: The methylation levels were measured by mass spectrometry in two independent case-control studies (159 LC cases vs. 188 controls in Study I, 413 LC cases vs. 687 controls in Study II). Logistic regression and Bonferroni correction were conducted to analyze the association. RESULTS: RPTOR hypomethylation was discovered in Study I and validated in Study II. Combining the two studies, RPTOR_CpG_2 and RPTOR_CpG_8 showed significantly lower methylation levels in stage I cases (ORs per -10% methylation = 1.22 and 1.27, respectively, both P-values < 0.005). The significance kept between RPTOR_CpG_8 and LC cases with tumor length ≤ 1 cm (OR per -10% methylation = 1.39, P = 0.001). Moreover, methylation levels of all CpG sites were lower in cases at stage II & III than in those at stage I (all P-values less than 0.017). CONCLUSION: Our study disclosed the association between RPTOR hypomethylation in peripheral blood and LC even in very early stage, suggesting the feasibility of blood-based DNA methylation for LC early detection.

Novel blood-based hypomethylation of SH3BP5 is associated with very early-stage lung adenocarcinoma.

BACKGROUND: Early detection is essential to improve the survival of lung cancer (LC). The quantitative measurement of specific DNA methylation changes in the peripheral blood could provide an efficient strategy for the detection of early cancer. OBJECTIVE: We applied a candidate approach and assess the association between blood-based SH3BP5 methylation and the risk of lung adenocarcinoma (LUAD) in a case-control cohort. METHODS: The methylation level of four CpG sites in the promoter of SH3BP5 gene was quantitatively determined by mass spectrometry in 171 very early-stage LUAD patients (93.6% LUAD at stage I) and 190 age and gender-matched controls. The logistic regression and non-parametric tests were used for the statistical analyses. RESULTS: We observed a significant association between decreased methylation of SH3BP5_CpG_4 in the peripheral blood and increased risk of LUAD (odds ratio (OR) per-10% methylation = 1.51, P = 0.006, FDR = 0.024), and even for the LUAD at stage I (OR per-10% methylation = 1.53, P = 0.006, FDR = 0.024). Moreover, the lower quartile of SH3BP5_CpG_4 methylation was correlated with increased risk for LUAD with a P trend of 0.011. Further investigation disclosed that the hypomethylation of SH3BP5_CpG_4 was mostly associated with LUAD in younger subjects (OR per-10% methylation = 2.02, P = 0.010, age < 55 years old) and probably could be enhanced by advance stage. CONCLUSION: Our study revealed an association between blood-based SH3BP5 hypomethylation and very early-stage LUAD, which provides a novel support for the blood-based methylation signatures as a potential marker for the evaluation of cancer risk.

The Association Between RAPSN Methylation in Peripheral Blood and Early Stage Lung Cancer Detected in Case-Control Cohort.

BACKGROUND: Early detection is essential to improve the survival and life quality of lung cancer (LC) patients. Changes of peripheral blood DNA methylation could be associated with malignancy but were mostly studied in Caucasians. METHODS: Here, in a Chinese population, we performed mass spectrometry assays to investigate the association between very early stage LC and methylation levels of RAPSN in the peripheral blood by a case-control cohort using of 221 LC patients (93.2% LC at stage I) and 285 unrelated cancer free control individuals. RESULTS: The odds ratios (ORs) of all CpG sites were evaluated for their risk to LC using inter-quartile analyses by logistic regression. In general, we observed an association between very early LC and decreased methylation of RAPSN_CpG_1.15 and RAPSN_CpG_3.4 (referring to Q4, OR range from 1.64 to 1.81, p<0.05). Stratified by gender, while hypomethylation of RAPSN_CpG_1.15, RAPSN_CpG_3.4 and RAPSN_CpG_7.14 were associated with LC in males (referring to Q4, ORs range from 1.94 to 2.31, p<0.05), RAPSN_CpG_2 and RAPSN_CpG_5 showed significantly lower methylation in female LC patients comparing to controls (referring to Q4, ORs range from 2.49 to 3.60, p<0.05). The risk of RAPSN hypomethylation to LC was enhanced by aging, and typically for people older than 55 years (referring to Q4, ORs range from 2.17 to 3.61 in six out of all 10 analyzed CpG groups, p<0.05). CONCLUSION: Our study reveals an association between RAPSN hypomethylation in peripheral blood and LC and suggests the occurrence of altered blood-based methylation at the early stage of cancer.

Genetic Mapping Combined with a Transcriptome Analysis to Screen for Candidate Genes Responsive to Abscisic Acid Treatment in Brassica napus Embryos During Seed Germination.

Brassica napus embryos contain precursor tissues for the leaves, stem, and root, as well as the cotyledons, and these precursor tissues play key roles in seed germination, seedling survival, and subsequent seedling growth. Abscisic acid (ABA) plays a prominent role in the inhibition of seed germination. The underlying molecular mechanisms of the embryo responses to ABA stress followed by inhibited seed germination have not been reported in B. napus to date. In this study, we conducted quantitative trait locus (QTL) analysis of B. napus seed in response to ABA stress using 170 recombinant inbred lines. Furthermore, we performed transcriptome sequencing (RNA-seq) analyses by using B. napus ZS11 embryos under sterile deionized water (control) and 10 mg/L (10A), 20 mg/L (20A), and 30 mg/L (30A) ABA treatment conditions. In total, 10 QTLs were screened for explaining 2.70-6.73% of the phenotypic variation under ABA stress. In addition, 1495, 3332, and 3868 differentially expressed genes (DEGs) were identified in the "control vs 10A," "control vs 20A," and "control vs 30A" comparisons, respectively. Gene Ontology (GO) enrichment analysis indicated that DEG functions are mainly related to response to stimuli, response to oxygen-containing compounds, response to lipids, and the transport and seed dormancy processes. These DEGs mainly participated in the response to plant hormone signal transduction, starch and sucrose metabolism, cutin, suberine, and wax biosynthesis, and phenylpropanoid biosynthesis processes pathways. Our results provide a foundation for further explorations of the molecular regulatory mechanisms of B. napus embryos in response to abiotic stress during the seed germination stage.

Genome-Wide Identification of the LAC Gene Family and Its Expression Analysis Under Stress in Brassica napus.

Lignin is an important biological polymer in plants that is necessary for plant secondary cell wall ontogenesis. The laccase (LAC) gene family catalyzes lignification and has been suggested to play a vital role in the plant kingdom. In this study, we identified 45 LAC genes from the Brassica napus genome (BnLACs), 25 LAC genes from the Brassica rapa genome (BrLACs) and 8 LAC genes from the Brassica oleracea genome (BoLACs). These LAC genes could be divided into five groups in a cladogram and members in same group had similar structures and conserved motifs. All BnLACs contained hormone- and stress- related elements determined by cis-element analysis. The expression of BnLACs was relatively higher in the root, seed coat and stem than in other tissues. Furthermore, BnLAC4 and its predicted downstream genes showed earlier expression in the silique pericarps of short silique lines than long silique lines. Three miRNAs (miR397a, miR397b and miR6034) target 11 BnLACs were also predicted. The expression changes of BnLACs under series of stresses were further investigated by RNA sequencing (RNA-seq) and quantitative real-time polymerase chain reaction (qRT-PCR). The study will give a deeper understanding of the LAC gene family evolution and functions in B. napus.

Screening of candidate gene responses to cadmium stress by RNA sequencing in oilseed rape (Brassica napus L.).

Cadmium (Cd) stress is one of the most serious threats to agriculture in the world. Oilseed rape (Brassica napus L.) is an important oil crop; however, Cd can easily accumulate in rapeseed and thus harm human health through the food chain. In the first experiment, our purpose was to measure the Cd accumulation in mature B. napus plants and its influences on fatty acid composition. The results showed that most Cd was accumulated in the root, and the seed fatty acid content was considerably different at different Cd toxicity levels. In the second experiment, 7-day-old B. napus seedlings stressed by Cd (1 mM) for 0 h (CK-0h), 24 h (T-24h), or 72 h (T-72h) were submitted to physiological and biological analyses, RNA-Seq and qRT-PCR. In total, 5469 and 6769 differentially expressed genes (DEGs) were identified in the comparisons of "CK-0h vs T-24h" and "CK-0h vs T-72h", respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the photosynthetic and glutathione (GSH) pathways were significantly enriched in response to Cd stress. Key factors in the response to Cd stress included BnPCS1, BnGSTU12, BnGSTU5, and BnHMAs. The transcription factors BnWRKY11 (BnaA03g51590D), BnWRKY28 (BnaA03g43640D), BnWRKY33 (BnaA03g17820D), and BnWRKY75 (BnaA03g04160D) were upregulated after Cd exposure. The present study revealed that upregulation of the genes encoding GST and PCS under Cd stress promoted the formation of low-molecular weight complexes (PC-Cd), and upregulation of heavy metal ATPase genes induced PC-Cd transfer to vacuoles. These findings may provide the basis for the molecular mechanism of the response of B. napus to Cd.

Combined QTL mapping, physiological and transcriptomic analyses to identify candidate genes involved in Brassica napus seed aging.

Seed aging is an inevitable problem in the germplasm conservation of oil crops. Thus, clarifying the genetic mechanism of seed aging is important for rapeseed breeding. In this study, Brassica napus seeds were exposed to an artificial aging environment (40 °C and 90% relative humidity). Using a population of 172 recombinant inbred lines, 13 QTLs were detected on 8 chromosomes, which explained ~ 9.05% of the total phenotypic variation. The QTLs q2015AGIA-C08 and q2016AGI-C08-2 identified in the two environments were considered the same QTL. After artificial aging, lower germination index, increased relative electrical conductivity, malondialdehyde and proline content, and reduced soluble sugar, protein content and antioxidant enzyme activities were detected. Furthermore, seeds of extreme lines that were either left untreated (R0 and S0) or subjected to 15 days of artificial aging (R15 and S15) were used for transcriptome sequencing. In total, 2843, 1084, 429 and 1055 differentially expressed genes were identified in R15 vs. R0, S15 vs. S0, R0 vs. S0 and R15 vs. S15, respectively. Through integrated QTL mapping and RNA-sequencing analyses, seven genes, such as BnaA03g37460D, encoding heat shock transcription factor C1, and BnaA03g40360D, encoding phosphofructokinase 4, were screened as candidate genes involved in seed aging. Further researches on these candidate genes could broaden our understanding of the regulatory mechanisms of seed aging.

Genome-Wide Analysis of the PYL Gene Family and Identification of PYL Genes That Respond to Abiotic Stress in Brassica napus.

Abscisic acid (ABA) is an endogenous phytohormone that plays important roles in the regulation of plant growth, development, and stress responses. The pyrabactin resistance 1-like (PYR/PYL) protein is a core regulatory component of ABA signaling networks in plants. However, no details regarding this family in Brassica napus are available. Here, 46 PYLs were identified in the B. napus genome. Based on phylogenetic analysis, BnPYR1 and BnPYL1-3 belong to subfamily I, BnPYL7-10 belong to subfamily II, and BnPYL4-6 and BnPYL11-13 belong to subfamily III. Analysis of BnPYL conserved motifs showed that every subfamily contained four common motifs. By predicting cis-elements in the promoters, we found that all BnPYL members contained hormone- and stress-related elements and that expression levels of most BnPYLs were relatively higher in seeds at the germination stage than those in other organs or at other developmental stages. Gene Ontology (GO) enrichment showed that BnPYL genes mainly participate in responses to stimuli. To identify crucial PYLs mediating the response to abiotic stress in B. napus, expression changes in 14 BnPYL genes were determined by quantitative real-time RT-PCR after drought, heat, and salinity treatments, and identified BnPYR1-3, BnPYL1-2, and BnPYL7-2 in respond to abiotic stresses. The findings of this study lay a foundation for further investigations of PYL genes in B. napus.