[Effect of REG3A on proliferation and invasion of glioma cells by regulating PI3K/Akt signaling pathway].

Objective: To investigate the effects of regenerating islet-derived protein 3A (REG3A) on the proliferation and invasion of glioma cells and its molecular mechanism. Methods: Five low-grade, five high-grade glioma tissues and ten adjacent tissues from glioma patients who underwent surgery at Linyi People's Hospital from October 17, 2017 to October 18, 2018 were collected. Human glioma cell lines (SF295, U251, TG905, A172, CRT) and a primary glioma cell line PT-1 were cultured in vitro. The protein and mRNA expressions of REG3A in these tissues and glioma cell lines were detected by Western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). SF295 cells were infected with lentivirus and labeled as REG3A plasmid transfection group, and the TG905 cells were transfected with si-REG3A by liposome transfection reagent and labeled as si-REG3A transfection group. At the same time, the empty transfection control and blank control groups were set up. Glioma cells were treated with REG3A recombinant protein alone or in combination with Akt1/2 inhibitors. Cell counting kit-8 (CCK-8) and cell scratch assay were used to detect cell proliferation and invasion, respectively. Western blot was used to detect the protein expression of N-cadherin, vimentin and phosphorylation of Akt (p-Akt) in REG3A overexpressed and knockdown glioma cells. Results: RT-qPCR results showed that the mRNA expression levels of REG3A in glioma cells in each group were U251 (2.129±0.13), TG905 (2.22±0.59), CRT (5.02±0.31), A172 (6.62±1.34) and PT-1 (9.18±0.61), respectively, higher than its expression in SF295 cells (1.00±0.18, P<0.001). The mRNA expression level of REG3A in high-grade glioma tissue samples (3.18±2.92) was higher than that in the control group (1.00±1.14, P=0.031) and low-grade glioma group (0.90±0.67, P=0.014). The results of western blot and immunohistochemical staining were consistent with that of RT-qPCR. The migration rate of cells in si-REG3A transfection group [(60.57±5.30)%] was lower than that of the empty transfection group [(84.18±13.63)% (P=0.038)] and blank control group [(79.65±12.09)% (P=0.076)]. The results of the scratch experiment showed that the migration rate of cells in REG3A plasmid transfected cells in the SF295 group was (96.05±6.41)%, which was significantly higher than that of empty transfected cells [(74.47±8.23)%, P=0.021)]. REG3A recombinant protein could up-regulate the expression of N-cadherin, vimentin and p-Akt in SF295 cells. Compared with the control group [(100.00±2.53)%], the proliferation rate in the REG3A recombinant protein group [(117.70±10.24)%] was significantly up-regulated, and the proliferation rate in the REG3A recombinant protein+ Akt inhibitor group [(98.31±3.64)%] was significantly lower than that of the REG3A recombinant protein group (P=0.017). The migration rate of the REG3A recombinant protein+ Akt inhibitor group was (63.35±4.06)%, which was significantly lower than (89.26±11.07)% of the REG3A recombinant protein group (P=0.019). Conclusion: REG3A can promote the proliferation and invasion of human glioma cells by activating the PI3K/Akt signaling pathway.

Transcriptome analysis of the grass carp (Ctenopharyngodon idella) using 454 pyrosequencing methodology for gene and marker discovery.

Total RNA isolated from the brain, muscle, liver, gonad, and intestinal tissues of grass carp was pooled to construct cDNA libraries. Using 454 pyrosequencing, a total of 738,604 high-quality reads were generated from the normalized cDNAs of the pooled individuals. Clustering and assembly of these reads produced a set of 37,086 all-unigene sequences after BLAST. Of these, 24,010 (64.74%) were annotated in the National Center for Biotechnology Information database, and 3715 simple sequence repeats and 2008 single nucleotide polymorphisms were identified in this EST dataset as potential molecular markers. This study provides new data for functional genomic and biological research on grass carp. The markers identified in this study will enrich the currently used molecular markers and facilitate marker-assisted selection in grass carp-breeding programs. These results also demonstrate that transcriptomic analysis based on 454 sequencing is a powerful tool for gene discovery and molecular marker development in non-model species.

A 66-bp deletion in growth hormone releasing hormone gene 5'-flanking region with largemouth bass recessive embryonic lethal.

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.