Genome sequences and population genomics reveal climatic adaptation and genomic divergence between two closely related sweetgum species.

Understanding the genetic basis of population divergence and adaptation is an important goal in population genetics and evolutionary biology. However, the relative roles of demographic history, gene flow, and/or selective regime in driving genomic divergence, climatic adaptation, and speciation in non-model tree species are not yet fully understood. To address this issue, we generated whole-genome resequencing data of Liquidambar formosana and L. acalycina, which are broadly sympatric but altitudinally segregated in the Tertiary relict forests of subtropical China. We integrated genomic and environmental data to investigate the demographic history, genomic divergence, and climatic adaptation of these two sister species. We inferred a scenario of allopatric species divergence during the late Miocene, followed by secondary contact during the Holocene. We identified multiple genomic islands of elevated divergence that mainly evolved through divergence hitchhiking and recombination rate variation, likely fostered by long-term refugial isolation and recent differential introgression in low-recombination genomic regions. We also found some candidate genes with divergent selection signatures potentially involved in climatic adaptation and reproductive isolation. Our results contribute to a better understanding of how late Tertiary/Quaternary climatic change influenced speciation, genomic divergence, climatic adaptation, and introgressive hybridization in East Asia's Tertiary relict flora. In addition, they should facilitate future evolutionary, conservation genomics, and molecular breeding studies in Liquidambar, a genus of important medicinal and ornamental values.

Hsa_circ_0040809 regulates colorectal cancer development by upregulating methyltransferase DNMT1 via targeting miR-515-5p.

BACKGROUND: Circular RNAs (circRNAs) are key regulators in the progression of various cancers. Abnormal DNA methylation patterns feature prominently in the regulation of the expression of tumor-related genes. This study is aimed at investigating the molecular mechanism of circ_0040809 affecting colorectal cancer (CRC) progression by regulating DNA methyltransferase 1 (DNMT1). METHODS: circ_0040809 was selected from the circRNA microarray datasets (GSE142837 and GSE138589). Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine the expression of circ_0040809, miR-515-5p, and DNMT1 mRNA in paired cancerous and paracancerous tissues of 40 CRC patients, as well as in cell lines. Western blotting was conducted for detecting DNMT1 protein expression in CRC cells. Cell proliferation, migration, and apoptosis were assessed through CCK-8, Transwell, and flow cytometry assays. Bioinformatics and dual-luciferase gene assay were conducted to predict and verify, respectively, the targeted relationships between circ_0040809 and miR-515-5p, as well as between miR-515-5p and DNMT1 mRNA. RESULTS: In CRC tissues and cells, circ_0040809 and DNMT1 expression are markedly increased, whereas miR-515-5p expression is decreased. Also, high circ_0040809 expression is significantly linked to shorter overall survival. Cell function compensation experiments reveal that circ_0040809 silencing inhibits CRC cell proliferation and migration and promotes apoptosis, while circ_0040809 overexpression has the opposite effects. Mechanistically, circ_0040809 competitively binds to miR-515-5p to elevate DNMT1 expression. Rescue assay reveals that overexpressed miR-515-5p partly counteracts the tumor-facilitating impact of circ_0040809. CONCLUSIONS: circ_0040809 facilitates CRC cell proliferation and migration, and inhibits apoptosis, through modulating miR-515-5p/DNMT1 axis. Our study implies that targeting circ_0040809 may be a therapy strategy for CRC treatment.

Genomic insights on the contribution of balancing selection and local adaptation to the long-term survival of a widespread living fossil tree, Cercidiphyllum japonicum.

'Living fossils' are testimonies of long-term sustained ecological success, but how demographic history and natural selection contributed to their survival, resilience, and persistence in the face of Quaternary climate fluctuations remains unclear. To better understand the interplay between demographic history and selection in shaping genomic diversity and evolution of such organisms, we assembled the whole genome of Cercidiphyllum japonicum, a widespread East Asian Tertiary relict tree, and resequenced 99 individuals of C. japonicum and its sister species, Cercidiphyllum magnificum (Central Japan). We dated this speciation event to the mid-Miocene, and the intraspecific lineage divergence of C. japonicum (China vs Japan) to the Early Pliocene. Throughout climatic upheavals of the late Tertiary/Quaternary, population bottlenecks greatly reduced the genetic diversity of C. japonicum. However, this polymorphism loss was likely counteracted by, first, long-term balancing selection at multiple chromosomal and heterozygous gene regions, potentially reflecting overdominance, and, second, selective sweeps at stress response and growth-related genes likely involved in local adaptation. Our findings contribute to a better understanding of how living fossils have survived climatic upheaval and maintained an extensive geographic range; that is, both types of selection could be major factors contributing to the species' survival, resilience, and persistence.

Phylogenomic and Comparative Analyses of Complete Plastomes of Croomia and Stemona (Stemonaceae).

The monocot genus Croomia (Stemonaceae) comprises three herbaceous perennial species that exhibit EA (Eastern Asian)⁻ENA (Eastern North American) disjunct distribution. However, due to the lack of effective genomic resources, its evolutionary history is still weakly resolved. In the present study, we conducted comparative analysis of the complete chloroplast (cp) genomes of three Croomia species and two Stemona species. These five cp genomes proved highly similar in overall size (154,407⁻155,261 bp), structure, gene order and content. All five cp genomes contained the same 114 unique genes consisting of 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Gene content, gene order, AT content and IR/SC boundary structures were almost the same among the five Stemonaceae cp genomes, except that the Stemona cp genome was found to contain an inversion in cemA and petA. The lengths of five genomes varied due to contraction/expansion of the IR/SC borders. A/T mononucleotides were the richest Simple Sequence Repeats (SSRs). A total of 46, 48, 47, 61 and 60 repeats were identified in C. japonica, C. heterosepala, C. pauciflora, S. japonica and S. mairei, respectively. A comparison of pairwise sequence divergence values across all introns and intergenic spacers revealed that the ndhF⁻rpl32, psbM⁻trnD and trnS⁻trnG regions are the fastest-evolving regions. These regions are therefore likely to be the best choices for molecular evolutionary and systematic studies at low taxonomic levels in Stemonaceae. Phylogenetic analyses of the complete cp genomes and 78 protein-coding genes strongly supported the monophyly of Croomia. Two Asian species were identified as sisters that likely diverged in the Early Pleistocene (1.62 Mya, 95% HPD: 1.125⁻2.251 Mya), whereas the divergence of C. pauciflora dated back to the Late Miocene (4.77 Mya, 95% HPD: 3.626⁻6.162 Mya). The availability of these cp genomes will provide valuable genetic resources for further population genetics and phylogeographic studies on Croomia.

Exploring the Diagnostic and Prognostic Predictive Values of Ferroptosis- Related Markers in Lung Adenocarcinoma.

BACKGROUND: Lung Adenocarcinoma (LUAD), a common and aggressive form of lung cancer, poses significant treatment challenges due to its low survival rates. AIM: To better understand the role of ferroptosis driver genes in LUAD, this study aimed to explore their diagnostic and prognostic significance, as well as their impact on treatment approaches and tumor immune function in LUAD. METHOD: To accomplish the defined goals, a comprehensive methodology incorporating both in silico and wet lab experiments was employed. A comprehensive analysis was conducted on a total of 233 ferroptosis driver genes obtained from the FerrDB database. Utilizing various TCGA databases and the RT-qPCR technique, the expression profiles of 233 genes were examined. Among them, TP53, KRAS, PTEN, and HRAS were identified as hub genes with significant differential expression. Notably, TP53, KRAS, and HRAS exhibited substantial up-regulation, while PTEN demonstrated significant down-regulation at both the mRNA and protein levels in LUAD samples. The dysregulation of hub genes was further associated with poor overall survival in LUAD patients. Additionally, targeted bisulfite-sequencing (bisulfite-seq) analysis revealed aberrant promoter methylation patterns linked to the dysregulation of hub genes. RESULT & DISCUSSION: Furthermore, hub genes were found to participate in diverse oncogenic pathways, highlighting their involvement in LUAD tumorigenesis. By leveraging the diagnostic and prognostic potential of ferroptosis driver hub genes (TP53, KRAS, PTEN, and HRAS), significant advancements can be made in the understanding and management of LUAD pathogenesis. CONCLUSION: Therapeutic targeting of these genes using specific drugs holds great promise for revolutionizing drug discovery and improving the overall survival of LUAD patients.

The Comprehensive Analysis of Hub Gene ARRB2 in Prostate Cancer.

Methods: The differential expressed genes (DEGs) were screened from the gene expression profile GSE30994 related to PRAD and then analyzed by protein-protein interaction (PPI) to screen the hub gene. Subsequently, the relation between hub gene and pan cancers, PRAD prognosis, and immunotherapy was analyzed. Besides, the effects of hub gene on the growth and metastasis of PRAD cell lines and inflammatory factors (IFs) were detected by functional experiments. Results: 276 upregulated and 1,861 downregulated DEGs were analyzed from GSE30994 gene expression profiles. Through enrichment analysis, it was found that upregulated DEGs were significantly enriched in nitric oxide-mediated signal transduction, insulin signaling pathway, etc. Through PPI networks, ARRB2 was determined as the hub gene that was highly expressed in pan cancers, including PRAD, and contributed to poor prognosis of PRAD patients. Immunoassay showed that ARRB2 was associated with B cells, NK cells, endothelial cells, etc. and also connected with tumor-infiltrating lymphocytes (TILs). Next, the signature model analysis revealed that ARRB2 had a clinical value in predicting PRAD prognosis. In functional experiments, ARRB2 was highly expressed in PRAD cell lines, promoted PRAD cell growth and metastasis, and positively associated with IFs. Conclusion: ARRB2 has a good prognostic ability in PRAD, and it could be a potential target of PRAD immunotherapy, which offers new directions for PRAD research.

Association of PD-1/PD-L1 expression and Epstein--Barr virus infection in patients with invasive breast cancer.

PURPOSE: Causative factors of breast cancer include infections, such as Epstein-Barr virus (EBV) infection. The aim of this study was to analyze the clinicopathological features of EBV-positive (IBC) and determine if EBV affects programmed cell death receptor 1 (PD-1)/PD ligand 1 (PD-L1) expression in IBC, similar to other EBV-infected tumors with PD-L1/PD-1 expression. METHODS: We collected 140 samples of IBC tissues and 25 samples of adjacent tissues. All patients were followed-up by telephone from the day of surgery to December 2020. Chromogenic in-situ hybridization was performed to evaluate EBV-encoded RNA (EBER). Immunohistochemistry was performed to evaluate PD-L1 and PD-1 expressions. The correlation between PD1/PDL1 expression and clinicopathological features was also analyzed. RESULTS: EBER was detected in 57 of 140 (40.7%) IBC tissues and not detected in any adjacent tissue (P < 0.05). Clinicopathologic features of patients were consistent with EBV-associated IBC. EBV infection was correlated with the mass size, menopausal status, axillary lymph node metastasis, vascular invasion, Ki-67 index, clinical stage, and estrogen receptor and progesterone receptor expressions (all P < 0.05), but not with the histological type, invasive ductal carcinoma histological grade, or human epidermal growth factor receptor 2 (HER2) expression (all P > 0.05). The positive rate of PD-1/PD-L1 expression was higher in the EBV-positive group than in the EBV-negative group (P < 0.05). The Kaplan-Meier univariate survival analysis showed that EBV was associated with poor disease-free survival and overall survival in patients with IBC. PD-L1/PD-1 expression could predict a poor prognosis. CONCLUSIONS: In this study, clinicopathologic characteristics of patients were consistent with EBV-infected IBC. Patients with EBV-positive breast cancer were more likely to have elevated PD-1/PDL-1 expression compared to those with EBV-negative breast cancer. This finding could serve as a basis to explore therapeutic targets, particularly immunotherapy, for patients with IBC.

Long non-coding RNA LINC00662 promotes proliferation and migration of breast cancer cells via regulating the miR-497-5p/EglN2 axis.

Previous reports indicated that long noncoding RNA 662 (LINC00662) plays a crucial role in several human cancers. Here, we studied the expression pattern of LINC00662 and explored its function in human breast cancer. The expression level of LINC00662 was determined in human breast cancer cell lines and tissues by real-time quantitative polymerase chain reaction (RT-qPCR). Cytoplasmic and nuclear RNA from MDA-MB-157 cells were extracted to analyze the subcellular location of LINC00662. Moreover, the MTT assay, wound-healing assay, colony-forming assay and transwell assay were employed in MDA-MB-157 cells to detect the effect of LINC00662 on cell apoptosis, invasion, migration and proliferation, respectively. LINC00662-specific miRNA and miRNA-gene axis were examined in a dual-luciferase reporter assay and Western blot. We found that LINC00662 was overexpressed in both breast cancer cell lines and tissue compared to normal breast cell lines and healthy breast tissue. Analysis of subcellular localization revealed that LINC00662 was mainly found in the cytoplasm. Furthermore, LINC00662 silencing reduced cell viability and inhibited the proliferation, migration and invasion of MDA-MB-157 cells. Bioinformatics analysis predicted that LNC00662 binds to miR-497-5p. A series of studies confirmed that LINC00662 directly interacted with miR-497-5p and downregulated its expression in MDA-MB-157 cells. MiR-497-5p knockdown significantly reversed the inhibitory effect of shLINC00662. Moreover, egl-9 family hypoxia inducible factor 2 (EglN2) was verified as a target of miR-497-5p. Overall, our results demonstrated that overexpression of LINC00662 accelerated the malignant growth of breast cancer cells via sponging miR-497-5p and upregulating EglN2 expression, and indicate that targeting LINC00662 may represent a novel strategy for breast cancer therapy.

Mining and characterization of novel EST-SSR markers of Parrotia subaequalis (Hamamelidaceae) from the first Illumina-based transcriptome datasets.

Parrotia subaequalis is an endangered Tertiary relict tree from eastern China. Despite its important ecological and horticultural value, no transcriptomic data and limited molecular markers are currently available in this species. In this study, we first performed high-throughput transcriptome sequencing of two individuals representing the northernmost (TX) and southernmost (SJD) population of P. subaequalis on the Illumina HiSeq 2500 platform. We gathered a total of 69,135 unigenes for P. subaequalis (TX) and 84,009 unigenes for P. subaequalis (SJD). From two unigenes datasets, 497 candidate polymorphic novel expressed sequence tag-simple sequence repeats (EST-SSRs) were identified using CandiSSR. Among these repeats, di-nucleotide repeats were the most abundant repeat type (62.78%) followed by tri-, tetra- and hexa-nucleotide repeats. We then randomly selected 54 primer pairs for polymorphism validation, of which 27 (50%) were successfully amplified and showed polymorphisms in 96 individuals from six natural populations of P. subaequalis. The average number of alleles per locus and the polymorphism information content values were 3.70 and 0.343; the average observed and expected heterozygosity were 0.378 and 0.394. A relatively high level of genetic diversity (HT = 0.393) and genetic differentiation level (FST = 0.171) were surveyed, indicating P. subaequalis maintained high levels of species diversity in the long-term evolutionary history. Additionally, a high level of cross-transferability (92.59%) was displayed in five congeneric Hamamelidaceae species. Therefore, these new transcriptomic data and novel polymorphic EST-SSR markers will pinpoint genetic resources and facilitate future studies on population genetics and molecular breeding of P. subaequalis and other Hamamelidaceae species.

Generation and classification of transcriptomes in two Croomia species and molecular evolution of CYC/TB1 genes in Stemonaceae.

The genus Croomia (Stemonaceae) is an excellent model for studying the evolution of the Eastern Asia (EA)-Eastern North America (ENA) floristic disjunction and the genetic mechanisms of floral zygomorphy formation. In addition to the presence of both actinomorphic and zygomorphic flowers within the genus, species are disjunctively distributed between EA and ENA. However, due to the limited availability of genomic resources, few studies of Croomia have examined these questions. In this study, we sequenced the floral and leaf transcriptomes of the zygomorphic flowered C roomia heterosepala and the actinomorphic flowered Croomia japonica, and used comparative genomic approaches to investigate the transcriptome evolution of the two closely related species. The sequencing and de novo assembly of transcriptomes from flowers of C. heterosepala (ChFlower), flowers of C. japonica (CjFlower), and leaves of C. japonica (CjLeaf) yielded 57,193, 62,131 and 64,448 unigenes, respectively. In addition, estimation of Ka/Ks ratios for 11,566 potential orthologous groups between ChFlower and CjFlower revealed that only six pairs had Ka/Ks ratios significantly greater than 1 and are likely under positive selection. A total of 429 single copy nuclear genes (SCNGs) and 21,460 expression sequence tags-simple sequence repeats (EST-SSRs) were identified in this study. Specifically, we identified seven CYC/TB1-like genes from Stemonaceae. Phylogenetic and molecular evolution analyses indicated that these CYC/TB1-like genes formed a monophyletic clade (SteTBL1) and were subject to strong purifying selection. The shifts of floral symmetry in Stemonaceae do not appear to be correlated with TBL copy number.

Complete chloroplast genome sequences of two endangered Phoebe (Lauraceae) species.

BACKGROUND: Phoebe (Lauraceae) comprises of evergreen trees or shrubs with approximately 100 species, distributed in tropical and subtropical Asia and Neotropical America. A total of 34 species and three varieties occur in China. Despite of economic and ecological value, only limited genomic resources are available for this genus. RESULTS: We sequenced the two complete chloroplast (cp) genomes of Phoebe chekiangensis and P. bournei using Illumina sequencing technology via a combined strategy of de novo and reference-guided assembly. We also performed comparative analyses with the cp genomes of P. sheareri and P. sheareri var. oineiensis previously reported. The chloroplast genomes of P. chekiangensis and P. bournei identically contain 112 genes consisting of 78 protein coding genes, 30 tRNA genes, and 4 rRNA genes, with the size of 152,849 and 152,853 bp, respectively. From the two chloroplast genomes, 131 SSRs were identified and 12 different SSRs located in five protein coding genes. The analysis showed the extremely conserved structure of chloroplast genomes with surprisingly little variations at the LSC/IR and SSC/IR boundaries. Moreover, the mean nucleotide diversity was found to be 0.162% for 77 regions, suggesting an extraordinarily low level of sequence divergence. Four highest divergent regions (trnH-psbA, rps14-trnT, petA-psbJ, ccsA-ndhD) with the percentage of nucleotide diversity higher than 0.50% were identified, which had potential use for species identification and phylogenetic studies. CONCLUSION: This study will facilitate our understanding of population genetics, phylogenetic relationship and plant evolution of Phoebe species.

Comparative Genomics and Phylogenomics of East Asian Tulips (Amana, Liliaceae).

The genus Amana Honda (Liliaceae), when it is treated as separate from Tulipa, comprises six perennial herbaceous species that are restricted to China, Japan and the Korean Peninsula. Although all six Amana species have important medicinal and horticultural uses, studies focused on species identification and molecular phylogenetics are few. Here we report the nucleotide sequences of six complete Amana chloroplast (cp) genomes. The cp genomes of Amana range from 150,613 bp to 151,136 bp in length, all including a pair of inverted repeats (25,629-25,859 bp) separated by the large single-copy (81,482-82,218 bp) and small single-copy (17,366-17,465 bp) regions. Each cp genome equivalently contains 112 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 78 protein coding genes. Gene content, gene order, AT content, and IR/SC boundary structure are nearly identical among all Amana cp genomes. However, the relative contraction and expansion of the IR/SC borders among the six Amana cp genomes results in length variation among them. Simple sequence repeat (SSR) analyses of these Amana cp genomes indicate that the richest SSRs are A/T mononucleotides. The number of repeats among the six Amana species varies from 54 (A. anhuiensis) to 69 (Amana kuocangshanica) with palindromic (28-35) and forward repeats (23-30) as the most common types. Phylogenomic analyses based on these complete cp genomes and 74 common protein-coding genes strongly support the monophyly of the genus, and a sister relationship between Amana and Erythronium, rather than a shared common ancestor with Tulipa. Nine DNA markers (rps15-ycf1, accD-psaI, petA-psbJ, rpl32-trnL, atpH-atpI, petD-rpoA, trnS-trnG, psbM-trnD, and ycf4-cemA) with number of variable sites greater than 0.9% were identified, and these may be useful for future population genetic and phylogeographic studies of Amana species.