Multi gene mutation signatures in colorectal cancer patients: predict for the diagnosis, pathological classification, staging and prognosis.

BACKGROUND: Identifying gene mutation signatures will enable a better understanding for the occurrence and development of colorectal cancer (CRC), and provide some potential biomarkers for clinical practice. Currently, however, there is still few effective biomarkers for early diagnosis and prognostic judgment in CRC patients. The purpose was to identify novel mutation signatures for the diagnosis and prognosis of CRC. METHODS: Clinical information of 531 CRC patients and their sequencing data were downloaded from TCGA database (training group), and 53 clinical patients were collected and sequenced with targeted next generation sequencing (NGS) technology (validation group). The relationship between the mutation genes and the diagnosis, pathological type, stage and prognosis of CRC were compared to construct signatures for CRC, and then analyzed their relationship with RNA expression, immunocyte infiltration and tumor microenvironment (TME). RESULTS: Mutations of TP53, APC, KRAS, BRAF and ATM covered 97.55% of TCGA population and 83.02% validation patients. Moreover, 57.14% validation samples and 22.06% TCGA samples indicated that patients with mucinous adenocarcinoma tended to have BRAF mutation, but no TP53 mutation. Mutations of TP53, PIK3CA, FAT4, FMN2 and TRRAP had a remarkable difference between I-II and III-IV stage patients (P < 0.0001). Besides, the combination of PIK3CA, LRP1B, FAT4 and ROS1 formed signatures for the prognosis and survival of CRC patients. The mutations of TP53, APC, KRAS, BRAF, ATM, PIK3CA, FAT4, FMN2, TRRAP, LRP1B, and ROS1 formed the signatures for predicting diagnosis and prognosis of CRC. Among them, mutation of TP53, APC, KRAS, BRAF, ATM, PIK3CA, FAT4 and TRRAP significantly reduced their RNA expression level. Stromal score, immune score and ESTIMATE score were lower in patients with TP53, APC, KRAS, PIK3CA mutation compared non-mutation patients. All the 11 gene mutations affected the distributions of immune cells. CONCLUSION: This study constructed gene mutation signatures for the diagnosis, treatment and prognosis in CRC, and proved that their mutations affected RNA expression levels, TME and immunocyte infiltration. Our results put forward further insights into the genotype of CRC.

Cloning and Functional Analysis of MADS-box Genes, TaAG-A and TaAG-B, from a Wheat K-type Cytoplasmic Male Sterile Line.

Wheat (Triticum aestivum L.) is a major crop worldwide. The utilization of heterosis is a promising approach to improve the yield and quality of wheat. Although there have been many studies on wheat cytoplasmic male sterility, its mechanism remains unclear. In this study, we identified two MADS-box genes from a wheat K-type cytoplasmic male sterile (CMS) line using homology-based cloning. These genes were localized on wheat chromosomes 3A and 3B and named TaAG-A and TaAG-B, respectively. Analysis of TaAG-A and TaAG-B expression patterns in leaves, spikes, roots, and stems of Chinese Spring wheat determined using quantitative RT-PCR revealed different expression levels in different tissues. TaAG-A had relatively high expression levels in leaves and spikes, but low levels in roots, while TaAG-B had relatively high expression levels in spikes and lower expression in roots, stems, and leaves. Both genes showed downregulation during the mononucleate to trinucleate stages of pollen development in the maintainer line. In contrast, upregulation of TaAG-B was observed in the CMS line. The transcript levels of the two genes were clearly higher in the CMS line compared to the maintainer line at the trinucleate stage. Overexpression of TaAG-A and TaAG-B in Arabidopsis resulted in phenotypes with earlier reproductive development, premature mortality, and abnormal buds, stamens, and stigmas. Overexpression of TaAG-A and TaAG-B gives rise to mutants with many deformities. Silencing TaAG-A and TaAG-B in a fertile wheat line using the virus-induced gene silencing (VIGS) method resulted in plants with green and yellow striped leaves, emaciated spikes, and decreased selfing seed set rates. These results demonstrate that TaAG-A and TaAG-B may play a role in male sterility in the wheat CMS line.

Genistein attenuates monocrotaline-induced pulmonary arterial hypertension in rats by activating PI3K/Akt/eNOS signaling.

INTRODUCTION: Phytoestrogen genistein may be useful to treat pulmonary arterial hypertension (PAH). However, its mechanism is still not clear. The aim of the present study was to confirm the therapeutic effects of phytoestrogen genistein on PAH in monocrotaline-induced rat model and to explore its mechanism. MATERIALS AND METHODS: Sprague-Dawley male rats were randomly divided into 4 groups: control group (n=8), PAH group (n=8), genistein treament group with three different doses (n=8 in each dose group) and group of PI3K inhibitor LY294002. The rat model of PAH was induced by monocrotaline (MCT). The situation of survival of rats was observed. Pathological studies of lung and heart tissues were performed. Western-blot detection of P-Akt and P-eNOS expression levels in lung tissue was carried out. Nitrate reductase analysis was used to measure nitric oxide (NO) in lung tissue. RESULTS: Genistein treatment resulted in significant improvement in the speed of tricuspid regurgitation, diameter of pulmonary artery, mean pulmonary artery pressure and right ventricular hypertrophy index. Genistein treatment also resulted in significant improvement in the stenosis of pulmonary artery, proliferation of smooth muscle, right ventricular hypertrophy and myocardial hypertrophy. These therapeutic effects were more obvious with increasing dose of genistein. After genistein treatment, amelioration in survival rates of PAH rats was observed. PI3K inhibitor LY294002 could block these therapeutic effects. In rat lung tissue, P-Akt, P-eNOS and NO expressions were increased significantly in genistein treatment group when compared with PAH group (p<0.05, respectively). The increase in expression level of P-Akt, P-eNOS and NO was correlated with genistein dose. P-Akt, P-eNOS and NO expressions in lung tissue increased slightly in the PI3K inhibitor LY294002 group when compared with PAH group, but the difference was not statistically significant (p>0.05). CONCLUSIONS: We confirmed that genistein could relax pulmonary vascular resistance, reduce pulmonary artery pressure, improve right heart function and ameliorate survival rate in the rat model of PAH. Our study suggested that its mechanism was related with PI3K/Akt/eNOS signal pathway. Phytoestrogen genistein may become a new and effective drug for patients with PAH.

Transcription factor gene TuGTγ-3 is involved in the stripe rust resistance in Triticum urartu.

Wheat stripe rust, caused by Puccinia striiformis West. f. sp. tritici Eriks. ＆Henn. (Pst), is a serious fungal disease. Identification of new genes associate with stripe rust resistance is important for developing disease resistant wheat cultivars and studying the mechanism of disease resistance. Trihelix is a plant specific transcription factor family, which is involved in regulation of growth and development, morphogenesis, and response to stresses. So far, no study reports on the relationship between the Trihelix family and wheat stripe rust. In this study, a gene in the GTγ subfamily of Trihelix family, designated TuGTγ-3, was cloned from Triticum urartu Tum. (2n=2x=14, AA). The results of sequencing demonstrated that TuGTγ-3 gene consisted of a complete open reading frame (ORF), and its coding sequence was 1329 bp in length, which encoded a protein with 442 amino acids. The predicted molecular weight of this protein was 50.31 kDa and the theoretical isoelectric point was 6.12. Bioinformatic analysis revealed that TuGTγ-3 protein had a monopartite nuclear localization signal (GLPMQKKMRYT), and had neither transmembrane domain nor signal peptide. The conserved trihelix domain, the fourth α-helix and the CC domain were located in the regions of Q115?R187, F234?Y241 and K362?K436, respectively. Dissection of secondary structure showed that TuGTγ-3 protein comprised of 43.89% α-helix, 9.51% extended strand, 9.95% ß-turn and 36.65% random coil structures. Based on the BLAST search against the genome database of common wheat from IWGSC, TuGTγ-3 was located on the long arm of chromosome 5A. Transient expression experiment using onion inner epidermal cell showed that the fusion protein TuGTγ-3-GFP distributed mainly in nuclear and slightly in cytoplasm. Expression profiles in different organs indicated that expression level of TuGTγ-3 was much higher in leaves than that in roots or leaf sheaths, and the expression in leaves was extremely up-regulated by infection of the Pst race CYR32. Furthermore, the BSMV-VIGS experiment demonstrated that the transcription factor TuGTγ-3 positively regulated resistance to stripe rust in T. urartu.

Molecular Characterization of Three GIBBERELLIN-INSENSITIVE DWARF2 Homologous Genes in Common Wheat.

F-box protein is a core component of the ubiquitin E3 ligase SCF complex and is involved in the gibberellin (GA) signaling pathway. To elucidate the molecular mechanism of GA signaling in wheat, three homologous GIBBERELLIN-INSENSITIVE DWARF2 genes, TaGID2s, were isolated from the Chinese Spring wheat variety. A subcellular localization assay in onion epidermal cells and Arabidopsis mesophyll protoplasts showed that TaGID2s are localized in the nuclei. The expression profiles using quantitative real-time polymerase chain reaction showed that TaGID2s were downregulated by GA3. The interaction between TaGID2s and TSK1 (homologous to ASK1) in yeast indicated that TaGID2s might function as a component of an E3 ubiquitin-ligase SCF complex. Yeast two-hybrid assays showed that a GA-independent interaction occurred between three TaGID2s and RHT-A1a, RHT-B1a, and RHT-D1a. Furthermore, TaGID2s interact with most RHT-1s, such as RHT-B1h, RHT-B1i, RHT-D1e, RHT-D1f, etc., but cannot interact with RHT-B1b or RHT-B1e, which have a stop codon in the DELLA motif, resulting in a lack of a GRAS domain. In addition, RHT-B1k has a frame-shift mutation in the VHIID motif leading to loss of the LHRII motif in the GRAS domain and RHT-D1h has a missense mutation in the LHRII motif. These results indicate that TaGID2s, novel positive regulators of the GA response, recognize RHT-1s in the LHRII motif resulting in poly-ubiquitination and degradation of the DELLA protein.

The 160 bp Insertion in the Promoter of Rht-B1i Plays a Vital Role in Increasing Wheat Height.

The extensive use of two alleles (Rht-B1b and Rht-D1b) at the Rht-1 locus in wheat allowed dramatic increases in yields, triggering the so-called "Green Revolution." Here, we found that a new natural allelic variation (Rht-B1i) containing a single missense SNP (A614G) in the coding region significantly increased plant height against the genetic background of both Rht-D1a (11.68%) and Rht-D1b (7.89%). To elucidate the molecular mechanism of Rht-B1i, we investigated the promoter region. Sequence analysis showed that the Rht-B1i promoter could be divided into two classes depending on the presence or absence of a specific 160 bp insertion: Rht-B1i-1 (with the 160 bp insertion) and Rht-B1i-2 (without the 160 bp insertion). The promoter of Rht-B1i-1 contained 32 more possible cis-acting elements than Rht-B1a, including a unique auxin response element AUXREPSIAA4. Quantitative RT-PCR analysis indicated that the 160 bp insertion is likely to promote the transcription of the Rht-B1i-1 gene. The coleoptile lengths of wheat varieties treated with IAA, GA3, and IAA/GA3, combined with the histochemical staining of transgenic Arabidopsis containing the Rht-B1i-1 promoter, showed that the height-increasing effect of Rht-B1i-1 may be due to the synergistic action of IAA and GA3. These results augment our understanding of the regulatory mechanisms of Rht-1 in wheat and provide new genetic resources for wheat improvement.