Identification and effective regulation of scarb1 gene involved in pigmentation change in autotetraploid Carassius auratus.

The autotetraploid Carassius auratus (4nRR, 4 n=200, RRRR) is derived from whole-genome duplication of Carassius auratus red var. (RCC, 2 n=100, RR). In the current study, we demonstrated that chromatophores and pigment changes directly caused the coloration and variation of 4nRR skin (red in RCC, brownish-yellow in 4nRR). To further explore the molecular mechanisms underlying coloration formation and variation in 4nRR, we performed transcriptome profiling and molecular functional verification in RCC and 4nRR. Results revealed that scarb1, associated with carotenoid metabolism, underwent significant down-regulation in 4nRR. Efficient editing of this candidate pigment gene provided clear evidence of its significant role in RCC coloration. Subsequently, we identified four divergent scarb1 homeologs in 4nRR: two original scarb1 homeologs from RCC and two duplicated ones. Notably, three of these homeologs possessed two highly conserved alleles, exhibiting biased and allele-specific expression in the skin. Remarkably, after precise editing of both the original and duplicated scarb1 homeologs and/or alleles, 4nRR individuals, whether singly or multiply mutated, displayed a transition from brownish-yellow skin to a cyan-gray phenotype. Concurrently, the proportional areas of the cyan-gray regions displayed a gene-dose correlation. These findings illustrate the subfunctionalization of duplicated scarb1, with all scarb1 genes synergistically and equally contributing to the pigmentation of 4nRR. This is the first report concerning the functional differentiation of duplicated homeologs in an autopolyploid fish, substantially enriching our understanding of coloration formation and change within this group of organisms.

Expression and localization of HPG axis-related genes in Carassius auratus with different ploidy.

Introduction: In the Dongting water system, the Carassius auratus (Crucian carp) complex is characterized by the coexistence of diploid forms (2n=100, 2nCC) and polyploidy forms. The diploid (2nCC) and triploid C.auratus (3n=150, 3nCC) had the same fertility levels, reaching sexual maturity at one year. Methods: The nucleotide sequence, gene expression, methylation, and immunofluorescence of the gonadotropin releasing hormone 2(Gnrh2), Gonadotropin hormone beta(Gthß), and Gonadotropin-releasing hormone receptor(Gthr) genes pivotal genes of the hypothalamic-pituitary-gonadal (HPG) axis were analyzed. Results: The analysis results indicated that Gnrh2, follicle-stimulating hormone receptor(Fshr), and Lethal hybrid rescue(Lhr) genes increased the copy number and distinct structural differentiation in 3nCC compared to that in 2nCC. The transcript levels of HPG axis genes in 3nCC were higher than 2nCC (P<0.05), which could promote the production and secretion of sex steroid hormones conducive to the gonadal development of 3nCC. Meanwhile, the DNA methylation levels in the promoter regions of the HPG axis genes were lower in 3nCC than in 2nCC. These results suggested that methylation of the promoter region had a potential regulatory effect on gene expression after triploidization. Immunofluorescence showed that the localization of the Fshß, Lhß, and Fshr genes between 3nCC and 2nCC remained unchanged, ensuring the normal expression of these genes at the corresponding sites after triploidization. Discussion: Relevant research results provide cell and molecular biology evidence for normal reproductive activities such as gonad development and gamete maturation in triploid C. auratus, and contribute to further understanding of the genetic basis for fertility restoration in triploid C. auratus.

The alternative transcription and expression characterization of Dmc1 in autotetraploid Carassius auratus.

Established autotetraploids often have a highly stable meiosis with high fertility compared with neo-autotetraploids. The autotetraploid Carassius auratus (4n = 200, RRRR) (4nRR), which stemmed from whole-genome duplication of Carassius auratus red var. (2n = 100, RR) (RCC), produces diploid gametes with an adopted diploid-like chromosome pairing in meiosis and maintains the formation of autotetraploid lineages. In this study, we focused on Dmc1, a meiosis-specific recombinase during the prophase of meiosis I, and elaborated on the genetic variation, alternative transcription, expression characterization, and epigenetic modification of Dmc1 in RCC and 4nRR. Two original Dmc1 from RCC were identified in 4nRR, and two duplicated Dmc1 differences in genetic composition were observed in 4nRR. Furthermore, we only noticed that one original and one duplicated Dmc1 were expressed in RCC and 4nRR, respectively. However, both possessed identical gene expression profiles, differential expression of sexual dimorphism, and hypomethylation levels. These results indicated that the specific expression of duplicated Dmc1 may be involve in the progression of meiosis of the diploid-like chromosome pairing in autotetraploid Carassius auratus. Herein, the findings significantly increase knowledge of meiosis of autopolyploid fish and provide meaningful insights into genetic breeding in polyploidy fish.

miR-767-3p suppresses melanoma progression by inhibiting ASF1B expression.

BACKGROUND: Melanoma, the type of skin cancer considered as most malignant, and known to be linked with a high incidence as well as high mortality rate. Although the dysregulation of ASF1B and miR-767-3p expression is involved in the progression of various cancers, their biological function in melanoma remains unclear. METHODS: Real-time qPCR was the primary source for determining the levels of ASF1B and miR-767-3p in melanoma. For the validation of association among miR-767-3p and ASF1B, luciferase activity assay was used. Quantification of cell apoptosis, proliferation, migration and viability in melanoma cells were carried out by flow cytometry, BrdU, transwell assays, and CCK-8, respectively. Further evaluation of tumor growth was achieved by xenograft in vivo. RESULTS: Results showed an increased expression of ASF1B while declined expression of miR-767-3p in melanoma. ASF1B knockdown repressed cell migration, viability, proliferation, and tumor growth whereas boosted apoptosis in A375 as well as in A875 melanoma cells. Moreover, miR-767-3p attenuated the migration and proliferation of melanoma cells and encouraged cell apoptosis by reducing ASF1B levels. CONCLUSION: In this study, miR-767-3p was shown to inhibit ASF1B which will attenuate melanoma tumorigenesis, and by this it can be a potential new effective biomarker for the treatment of melanoma.

MicroRNA-520d-3p suppresses melanoma cells proliferation by inhibiting the anti-silencing function 1B histone chaperone.

As the most common and aggressive malignant form of skin cancer, melanoma has a poor prognosis in its late stage. MicroRNA (miR)-520d-3p has been reported as a key modulator that regulates the development of different types of cancer, but its role in melanoma remains unclear. The purpose of this study was to investigate the role and mechanism of miR-520d-3p in melanoma. The expression of anti-silencing function 1B histone chaperone (ASF1B) and miR-520d-3p in melanoma tissues and cells was detected by reverse transcription-quantitative polymerase chain reaction. The interaction between ASF1B and miR-520d-3p was verified by luciferase activity detection. Cell counting kit-8, bromodeoxyuridine, fluorescein isothiocyanate, and cell adhesion assays were performed to detect cell viability, proliferation, apoptosis, and adhesion in melanoma cells. ASF1B expression was evidently increased, whereas miR-520d-3p level was downregulated in melanoma tissues and cells. Overexpression of ASF1B enhanced cell growth and adhesion and hampered cell apoptosis in melanoma cells. Furthermore, miR-520d-3p suppressed the tumorigenic effects of melanoma cells. Moreover, miR-520d-3p suppressed the expression of ASF1B to suppress melanoma tumorigenesis. In conclusion, we have found out that miR-520d-3p suppressed melanoma tumorigenesis by inhibiting ASF1B, which could be a promising target for melanoma therapy.

Organization and expression analysis of 5S and 45S ribosomal DNA clusters in autotetraploid Carassius auratus.

BACKGROUND: Autotetraploid Carassius auratus (4n = 200, RRRR) (abbreviated as 4nRR) is derived from whole genome duplication of Carassius auratus red var. (2n = 100, RR) (abbreviated as RCC). Ribosome DNA (rDNA) is often used to study molecular evolution of repeated sequences because it has high copy number and special conserved coding regions in genomes. In this study, we analysed the sequences (5S, ITS1-5.8S-ITS2 region), structure, methylation level (NTS and IGS), and expression level (5S and 18S) of 5S and 45S ribosomal RNA (rRNA) genes in 4nRR and RCC in order to elucidate the effects of autotetraploidization on rDNA in fish. RESULTS: Results showed that there was high sequence similarity of 5S, 5.8S and ITS1 region between 4nRR and RCC. This study also identified two different types of ITS2 region in 4nRR and predicted the secondary structure of ITS2. It turns out that both secondary structures are functional. Compared with RCC, there was no significant difference in NTS (5S rRNA) methylation level, but the expression level of 5S rRNA was lower in 4nRR, indicating that methylation had little effect on the expression level in 4nRR. IGS (45S rRNA) was hypermethylated in 4nRR compared to RCC, but the expression of 18S rRNA gene was no significantly different from that in RCC, indicating that methylation regulation affected gene expression in 4nRR. CONCLUSION: The above studies initially revealed the effects of autotetraploidization on the structure and function of 5S and 45S rRNA in Carassius auratus, and provided a theoretical support for the systematic study of the evolution pattern and characteristics of rDNA in vertebrates.

Formation of autotriploid Carassius auratus and its fertility-related genes analysis.

BACKGROUND: Formation of triploid organism is useful in genetics and breeding. In this study, autotriploid Carassius auratus (3nRR, 3n = 150) was generated from Carassius auratus red var. (RCC, 2n = 100) (♀) and autotetraploid Carassius auratus (4nRR, 4n = 200) (♂). The female 3nRR produced haploid, diploid and triploid eggs, whereas the male 3nRR was infertile. The aim of the present study was to explore fertility of potential candidate genes of 3nRR. RESULTS: Gonadal transcriptome profiling of four groups (3 females RCC (FRCC), 3 males 4nRR (M4nRR), 3 males 3nRR (M3nRR) and 3 females 3nRR (F3nRR)) was performed using RNA-SEq. A total of 78.90 Gb of clean short reads and 24,262 differentially expressed transcripts (DETs), including 20,155 in F3nRR vs. FRCC and 4,107 in M3nRR vs. M4nRR were identified. A total of 106 enriched pathways were identified through KEGG enrichment analysis. Out of the enriched pathways, 44 and 62 signalling pathways were identified in F3nRR vs. FRCC and M3nRR vs. M4nRR, respectively. A total of 80 and 25 potential candidate genes for fertility-related in F3nRR and M3nRR were identified, respectively, through GO, KEGG analyses and the published literature. Moreover, protein-protein interaction (PPI) network construction of these fertility-associated genes were performed. Analysis of the PPI networks showed that 6 hub genes (MYC, SOX2, BMP4, GATA4, PTEN and BMP2) were involved in female fertility of F3nRR, and 2 hub genes (TP53 and FGF2) were involved in male sterility of M3nRR. CONCLUSIONS: Establishment of autotriploid fish offers an ideal model to study reproductive traits of triploid fish. RNA-Seq data revealed 6 genes, namely, MYC, SOX2, BMP4, GATA4, PTEN and BMP2, involved in the female fertility of the F3nRR. Moreover, 2 genes, namely, TP53 and FGF2, were related to the male sterility of the M3nRR. These findings provide information on reproduction and breeding in triploid fish.