Insulin-like growth factor I (IGF-1) plays an important role in fish growth and development. As the promoter of IGF-I regulates the expression of IGF-I, polymorphisms in the promoter region could be functionally important to differences in IGF-I expression and function. In this study, the 5 0 flanking region (1,770 bp) of the largemouth bass IGF-I gene was cloned, sequenced and characterized. Several putative liver-enriched transcriptional factor binding sites, including HNF-1a, HNF-3b and C/EBP, were found to have multiple copies in the promoter sequence of the largemouth bass IGF-I gene. It did not contain consensus TATA, CCAAT-like or GAGA sequences. Sequencing of 5 0 flanking sequences from individual fish revealed the presence of a deletion (GTTT) and two single-nucleotide substitutions, causing two different haplotypes: ATTTTTGTTTTT(A) and ATAATT-TT(B). The haplotypes were tested for their association with growth traits in a population of 91 largemouth bass individuals using polymerase chain reaction. The association analysis was carried out using a general linear model. Results showed the haplotypes had significant effects on body weight and body width (P \ 0.05). The individuals with the AA genotype had significantly higher body weight and body width than those with the AB and BB genotypes (P \ 0.05). Moreover, the effect of the genotypes on IGF-I mRNA expression in the liver was evaluated through the use of a semiquantitative RT-PCR method. The results showed that the IGF-I mRNA expression levels were affected by the promoter polymorphisms (P \ 0.01), and that levels of IGF-I mRNA of the AA genotype in liver were greater than those of both the AB and the BB genotypes (P \ 0.05 and P \ 0.01). Our results implied that the IGF-I gene could be a potential quantitative trait nucleotide that affects growth traits in largemouth bass.

No abstract

Objectives: Immune thrombocytopenia (ITP) is an autoimmune disease. T helper cell 17 (Th17) cells are increased in peripheral blood of ITP patients. NOTCH signaling is involved in Th17 cell differentiation and function. Besides, lncRNA Plasmacytoma variant translocation 1 (PVT1) was decreased in experimental autoimmune encephalomyelitis, and overexpressing PVT1 inhibited Th17 cell differentiation. Here, we aimed to investigate the effect of lncRNA PVT1 on ITP and its related mechanism. Methods: The number of Th17 cells and Treg cells was carried out using flow cytometry. PVT1 levels were detected by quantitative real-time PCR. Interleukin-17 (IL-17) levels and transforming growth factor-β (TGF-β) levels were detected by enzyme-linked immunosorbent assay. Protein levels of retinoid acid-related orphan receptor γ t (RORγt), forkhead box P3 (Foxp3), and NOTCH1 were carried out by western blot. NOTCH1 ubiquitylation was detected by ubiquitination assay. Results: PVT1 was down-regulated and Th17 cells were up-regulated in ITP patients. Overexpression of PVT1 decreased the number of Th17 cells, and also decreased the levels of IL-17, RORγt, and NOTCH1. Besides, PVT1 could bind to NOTCH1 and mediated NOTCH1 degradation by increasing its ubiquitination. Additionally, excessive expression of PVT1 could increase the levels of PVT1, reduce the amount of Th17 cells, as well as the levels of IL-17, RORγt, and NOTCH1, while co-overexpressing NOTCH1 reversed the results. Conclusion: PVT1 was down-regulated in ITP patients. Overexpressing PVT1 might reduce Th17 cell differentiation by down-regulating NOTCH1, and further alleviated the development of ITP.

Effect of miRNA-200c (miR-200c) on the proliferation, invasion and apoptosis of prostate cancer cell line LNCaP was investigated. The difference in miR-200c expression was observed using RT-qPCR in the NC group (transfected empty plasmid), simulation group (simulation sequence) and inhibition group (transferred inhibition sequence), which were established by transfecting LNCaP cells with a kit. The proliferation, invasion and apoptosis of cells after transfection were detected using the cell counting kit-8 (CCK-8) method, Transwell chamber and flow cytometry. RT-qPCR detection showed that the relative expression of miR-200c in LNCaP cells significantly increased compared with RWPE-1 cells (P<0.05). The difference was statistically significant in the relative expression of miR-200c cells among NC group, simulation group and inhibition group after transfection (P<0.05) and they significantly decreased in NC group of cells compared with the simulation group (P<0.05). CCK-8 detection showed that there were differences at the 2nd, 3rd, 4th and 5th days of growth in the NC group, simulation group and inhibition group of cells (P<0.05) and there was a difference in the proliferation ability between NC group and simulation group (P<0.05). Transwell chamber detection showed that there was a difference in the invasion ability among NC group, simulation group and inhibition group of cells (P<0.05), among which the number of passed membrane cells in inhibition group was significantly smaller than that in NC group and simulation group (P<0.05), and the difference was not statistically significant between NC group and simulation group (P>0.05). Flow cytometry detection of the apoptosis ability of each group of cells showed that there was a difference in the apoptotic rate in the NC, simulation and inhibition groups (P<0.05). The low expression of miR-200c is beneficial to inhibit the proliferation and invasion of LNCaP cells in vitro and to promote apoptosis, which may be a potential target for prostate cancer biotherapy.

Growth hormone releasing hormone (GHRH) regulates the secretion of growth hormone (GH) in the pituitary gland. A 66-bp deletion (c.-923_-858del) was detected in the 5'-flanking sequence of the largemouth bass (Micropterus salmoides) GHRH gene. In two cultured random populations of adult individuals (A: n = 170 and B: n = 150), the genotype ratios of +/+:+/- were 2.5:1 and 2.8:1 respectively. Only one -/- fish was detected. A Largemouth bass family was constructed with two heterozygous individuals (+/-) as parents. The genotype ratio of +/+:+/-:-/- in the filial generation embryos was 1:1.6:0.1 at the neurula and 1:2:0 at hatched larvae stages. This indicated that the 66-bp deletion was a recessive lethal site and that homozygous individuals (-/-) died off in embryonic development. The growth traits (body weight, body length and body depth) were measured, and the GHRH mRNA expression levels in brain tissue were detected using real-time PCR. The effects of genotype (+/-) on growth traits and GHRH mRNA expression were not significant. Although the cause of death was not clear, the results hint that the 66-bp deletion site in GHRH 5'-flanking sequence significantly affects the livability in largemouth bass embryonic development.