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Osteosarcoma is frequently metastasized at the time of diagnosis in patients. However, the underlying mechanism of osteosarcoma metastasis remains poorly understood. In this study, we evaluated DNA methylation profiles combined with gene expression profiles of 21 patients with metastatic osteosarcoma and 64 patients with non-metastatic osteosarcoma from TARGET database and identified PKIB and AIM2 as hub genes related to the metastasis of osteosarcoma. To verify the effects of PKIB on migration and invasion of osteosarcoma, we performed wound-healing assay and transwell assay. The results showed that PKIB significantly inhibited the migration and invasion of osteosarcoma cells, and the Western blot experiments showed that the protein level of E-cad was upregulated and of VIM was downregulated in 143-B cell recombinant expression PKIB. These results indicate that PKIB inhibit the metastasis of osteosarcoma. CCK-8 assay results showed that PKIB promote the proliferation of osteosarcoma. In addition, the Western blot results showed that the phosphorylation level of Akt was upregulated in 143-B cells overexpressing PKIB, indicating that PKIB promotes the proliferation of osteosarcoma probably through signaling pathway that Akt involved in. These results give us clues that PKIB was a potential target for osteosarcoma therapy. Furthermore, combined clinical profiles analysis showed that the expression of AIM2- and PKIB- related risk scores was significantly related to the overall survival of patients with osteosarcoma. Thus, we constructed a nomogram based on AIM2 and PKIB expression–related risk scores for osteosarcoma prognostic assessment to predict the 1-, 2-, 3-, and 5-year overall survival rate of patients with metastatic osteosarcoma, assisting clinicians in the diagnosis and treatment of metastatic osteosarcoma.

The caleosin (CLO) protein family displays calcium-binding properties and plays an important role in the abiotic stress response. Here, a total of 107 CLO genes were identified in 15 plant species, while no CLO genes were detected in two green algal species. Evolutionary analysis revealed that the CLO gene family may have evolved mainly in terrestrial plants and that biological functional differentiation between species and functional expansion within species have occurred. Of these, 56 CLO genes were identified in four cotton species. Collinearity analysis showed that CLO gene family expansion mainly occurred through segmental duplication and whole-genome duplication in cotton. Sequence alignment and phylogenetic analysis showed that the CLO proteins of the four cotton species were mainly divided into two types: H-caleosins (class I) and L-caleosins (class II). Cis-acting element analysis and quantitative RT–PCR (qRT–PCR) suggested that GhCLOs might be regulated by abscisic acid (ABA) and methyl jasmonate (MeJA). Moreover, transcriptome data and qRT–PCR results revealed that GhCLO genes responded to salt and drought stresses. Under salt stress, gene-silenced plants (TRV: GhCLO06) showed obvious yellowing and wilting, higher malondialdehyde (MDA) content accumulation, and significantly lower activities of superoxide dismutase (SOD) and peroxidase (POD), indicating that GhCLO06 plays a positive regulatory role in cotton salt tolerance. In gene-silenced plants (TRV: GhCLO06), ABA-related genes (GhABF2, GhABI5, and GhNAC4) were significantly upregulated after salt stress, suggesting that the regulation of salt tolerance may be related to the ABA signaling pathway. This research provides an important reference for further understanding and analyzing the molecular regulatory mechanism of CLOs for salt tolerance.

The early flowering 4 (ELF4) family members play multiple roles in the physiological development of plants. ELF4s participated in the plant biological clock’s regulation process, photoperiod, hypocotyl elongation, and flowering time. However, the function in the ELF4s gene is barely known. In this study, 11, 12, 21, and 22 ELF4 genes were identified from the genomes of Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. There ELF4s genes were classified into four subfamilies, and members from the same subfamily show relatively conservative gene structures. The results of gene chromosome location and gene duplication revealed that segmental duplication promotes gene expansion, and the Ka/Ks indicated that the ELF4 gene family has undergone purification selection during long-term evolution. Spatio-temporal expression patterns and qRT-PCR showed that GhELF4 genes were mainly related to flower, leaf, and fiber development. Cis-acting elements analysis and qRT-PCR showed that GhELF4 genes might be involved in the regulation of abscisic acid (ABA) or light pathways. Silencing of GhELF4-1 and GhEFL3-6 significantly affected the height of cotton seedlings and reduced the resistance of cotton. The identification and functional analysis of ELF4 genes in upland cotton provide more candidate genes for genetic modification.

Glycerol-3-phosphate acyltransferases (GPATs), critical for multiple biological processes like male fertility, have been extensively characterized. However, their precise functions and underlying regulatory mechanism in cotton anther development are unclear. This research demonstrated the importance of GhGPAT12/25 (a paralogs pair on A12/D12 sub-chromosome of cotton) to regulate the degradation of tapetum, anther cuticle formation, and pollen exine development. GhGPAT12 and GhGPAT25 exhibited specifically detected transcripts in tapetum and pollen exine during the early anther developmental stages. GhGPAT12/25 are sn-2 glycerol-3-phosphate acyltransferases and can transfer the acyl group of palmitoyl-CoA to glycerol-3-phosphate (G3P). CRISPR/Cas9-mediated knockout identified the functional redundancy of GhGPAT12 and GhGPAT25. Knockout of both genes caused completely male sterility associated with abnormal anther cuticle, swollen tapetum, and inviable microspores with defective exine and irregular unrestricted shape. RNA-seq analysis showed that the loss of function of GhGPAT12/25 affects the processes of wax metabolic, glycerol monomer biosynthesis, and transport. Consistently, cuticular waxes were dramatically reduced in mutant anthers. Yeast one-hybrid system (Y1H), virus-induced gene silencing (VIGS), and dual-luciferase (LUC) assays illustrated that GhMYB80s are likely to directly activate the expression of GhGPAT12/25. This study provides important insights for revealing the regulatory mechanism underlying anther development in cotton.

The potassium transporter family HAK/KUP/KT is a large group of proteins that are important in plant potassium transport and plays a crucial role in plant growth and development, especially in economic crops. Although HAK/KUP/KT genes have been identified in many species, research on these genes in cotton is still quite rare. In this study, in total, 21, 24, 45, and 44 HAK/KUP/KT genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. Phylogenetic analysis showed that these genes were divided into four clusters. The G. hirsutum gene promoters contained diverse cis-regulatory elements, such as drought-responsive elements, low temperature-responsive elements, and other elements. The RNA-seq data and qRT-PCR results showed that HAK/KUP/KT genes had different expression patterns in fiber development. The qRT-PCR results of drought and NaCl treatment indicated that HAK/KUP/KT genes might play important roles in abiotic stress responses. These results will provide molecular insights into potassium transporter research in cotton.

The sodium/hydrogen antiporter (NHX) gene family with the Na+/H+ exchange protein domain is a transporter of sodium and hydrogen ions and plays an important role in the response of plants to salt stress. Studying the response of cotton to salt stress through comprehensive identification and analysis of NHX genes in several species and their roles in salt tolerance mechanisms is of great significance. In this study, 23, 24, 12, and 12 NHX genes were identified from Gossypium hirsutum (Gh), G. barbadense, G. arboreum and G. raimondii, respectively. Phylogenetic analysis showed that these genes were mainly divided into three clades with significant subcellular localization, namely, endosome (Endo-class), plasma membrane (PM-class) and vacuole (Vac-class). By analyzing the structure of NHX genes and proteins, each branch of the NHX gene family was found to be structurally conserved, and collinearity analysis showed that NHX genes were mainly expressed through whole genome and segmental duplication. The non-synonymous (Ka)/synonymous (Ks) values showed that the NHX gene family experienced strong purifying selection during long-term evolution. Cis-acting element analysis showed that the NHX gene family may be related to the regulation of abscisic acid (ABA) and methyl jasmonate (MeJA) hormones. Additionally, transcriptomic data analysis and qRT-PCR showed that GhNHXs exhibited different expression patterns in each tissue and under different salinities. These results provide an important reference for us to further understand and analyze the molecular regulation mechanism of cotton NHX genes.