Genetic polymorphisms and preliminary association analysis with production traits of the porcine <Emphasis Type="Italic">SLC27A4</Emphasis> gene

Solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4) is a fatty acyl-CoA synthetase producing very long chain fatty acid-CoA for lipid metabolic pathways, suggesting that the SLC27A4 gene is a potential candidate gene for traits related to fat deposition in animals. This study was conducted to sequence the genomic region from exon 6 to 12 of porcine SLC27A4 and detect polymorphisms by comparative sequencing. In silico mapping assigned SLC27A4 gene between gene COQ4 (coenzyme Q4 homolog) and URM1 (ubiquitin related modifier 1 homolog) on pig chromosome 1q24-q2.12 where significant QTL affecting backfat depth had previously been identified. Thirty six putative sites of variation were detected, of which 31 polymorphisms including 28 SNPs and 3 indels were located in the intronic region, and 5 in the exonic regions. The g.1777G>A (EU703769) in intron 8 was confirmed by PCR-RFLP using HpaII restriction enzyme and further genotyped in four Chinese native pig breeds (Meishan, Erhualian, Tongcheng and Qingping) and three western meat-type pig breeds (Duroc, Large White and Landrace). Allele G was exclusively present in Tongcheng and Qingping pigs and predominant in the other pig populations analyzed. Significant differences of backfat at rump, body weight at birth and average daily gain on weaning between the AG and GG genotype were observed in Landrace pig population (P < 0.05).     

Rabbit SLC15A1, SLC7A1 and SLC1A1 genes are affected by site of digestion,stage of development and dietary protein content

Peptide transporter 1 (SLC15A1, PepT1), excitatory amino acid transporter 3 (SLC1A1, EAAT3) and cationic amino acid transporter 1 (SLC7A1, CAT1) were identified as genes responsible for the transport of small peptides and amino acids. The tissue expression pattern of rabbit (SLC15A1, SLC7A1 and SLC1A1) across the digestive tract remains unclear. The present study investigated SLC15A1, SLC7A1 and SLC1A1 gene expression patterns across the digestive tract at different stages of development and in response to dietary protein levels. Real time-PCR results indicated that SLC15A1, SLC7A1 and SLC1A1 genes throughout the rabbits’ entire development and were expressed in all tested rabbit digestive sites, including the stomach, duodenum, jejunum, ileum, colon and cecum. Furthermore, SLC7A1 and SLC1A1 mRNA expression occurred in a tissue-specific and time-associated manner, suggesting the distinct transport ability of amino acids in different tissues and at different developmental stages. The most highly expressed levels of all three genes were in the duodenum, ileum and jejunum in all developmental stages. All increased after lactation. With increased dietary protein levels, SLC7A1 mRNA levels in small intestine and SLC1A1 mRNA levels in duodenum and ileum exhibited a significant decreasing trend. Moreover, rabbits fed a normal level of protein had the highest levels of SLC15A1 mRNA in the duodenum and jejunum (P<0.05). In conclusion, gene mRNA differed across sites and with development suggesting time and sites related differences in peptide and amino acid absorption in rabbits. The effects of dietary protein on expression of the three genes were also site specific.     

Age-related expression profile of the <Emphasis Type="Italic">SLC27A1</Emphasis> gene in chicken tissues

The solute carrier family 27 (SLC27, also known as fatty acid transport proteins [FATPs]) plays important biological roles in cells. However, there is no report about the expression profile of SLC27 member in chicken. In this study, we quantified the expression of SLC27A1 (FATP1) mRNA in a mountainous black-boned chicken breed (MB) and a commercial meat type chicken breed (S01), to discern the tissue and age-related specific expression pattern and their potential involvement in fat deposition and muscle fatty acid metabolism. Real-time quantitative PCR assays were developed for accurate measurement of SLC27A1 mRNA levels in different tissues from chicken with different ages (0–12 weeks). Expression of SLC27A1 mRNA was detected in all tissues examined. There was a significantly age-related change of the SLC27A1 mRNAs in heart, breast muscle (BMW), leg muscle (LMW), liver, and abdominal fat (AF) tissues (P < 0.05). The breast muscle and leg muscle tissues had the highest expression of SLC27A1 mRNA than the other tissues from the same individual at 0, 2 and 4 weeks. The overall SLC27A1 mRNA level exhibited a “rise-decline” developmental change in all tissues except for breast muscle, subcutaneous fat, and brain. The S01 chicken had a higher expression of the SLC27A1 mRNA in breast muscle, subcutaneous fat, and heart tissues than the MB chicken. Our results showed that the expression of SLC27A1 mRNA in chicken tissues exhibits specific developmental changes and age-related patterns.     

The effects and mechanisms of SLC34A2 in tumorigenesis and progression of human non-small cell lung cancer

Background

SLC34A2 with highest expressions in lung, small intestine and kidney encoded a type 2b sodium-dependent phosphate transporter (NaPi-IIb). In lung, SLC34A2 only expressed in the apical membrane of type II alveolar epithelium cells (ATII cells) and played a pivotal role during the fetal lung development and embryonic development. ATII cells acting as multifunctional stem cells might transform into NSCLC after undergoing exogenous or endogenous factors. Increasing evidences showed that the genes performing critical roles during embryogenesis were also expressed during the development of cancer. In addition, recent research found the expression of SLC34A2 had a significant difference between the surgical samples of NSCLC and normal tissues, and SLC34A2 was down-regulated in lung adenocarcinoma cell line A549 and up-regulation expression of SLC34A2 could significantly inhibit cell viability and invasion of A549 in vitro. These results suggested SLC34A2 might play an important role in the development of NSCLC. However, the role of SLC34A2 in tumorigenesis and progression of NSCLC remains unknown.

Results

Our study found that SLC34A2 was also significantly down-regulated in 14/15 of examined NSCLC tissues. Moreover, we found that expressions of SLC34A2 were reduced in six NSCLC cell lines for the first time. Our result also revealed a dramatic inhibitory effects of SLC34A2 on cell growth, migration and invasion of several NSCLC cell lines. SLC34A2 also strongly inhibited tumor growth and metastasis ability in A549 subcutaneous tumor model and lung metastasis model, respectively. Further studies found that the suppressive effects of SLC34A2 on tumorigenesis and progression might be associated with the down-regulation of related protein in PI3K/Akt and Ras/Raf/MEK signal pathway.

Conclusions

For the first time, our data indicated that SLC34A2 could exert significantly suppressive effects on tumorigenesis and progression of NSCLC. SLC34A2 might provide new insights for further understanding the early pathogenesis of human NSCLC.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Prognostic significance and gene expression profiles of p53 mutations in microsatellite-stable stage III colorectal adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.     

Molecular characterization and expression patterns of the actinin-associated LIM protein (ALP) subfamily genes in porcine skeletal muscle

The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development.     

A survey of quantitative real-time polymerase chain reaction internal reference genes for expression studies in Brassica napus

Eight reference genes of Brassica napus were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) data, focusing on vegetative tissues and developing embryos. Analyses of expression stability indicated that UP1, UBC9, UBC21, and TIP41 were the top four choices as stably expressed reference genes for vegetative tissues, whereas ACT7, UBC21, TIP41, and PP2A were the top four choices for maturing embryos. In addition, radiolabeling of overall messenger RNA (mRNA) of maturing embryos indicated that the expression patterns of the top four ranked reference genes reflected the overall mRNA content changes in maturing embryos.     

The Mutant p53-Targeting Compound APR-246 Induces ROS-Modulating Genes in Breast Cancer Cells

TP53 is the most frequently mutated gene in human cancer and thus an attractive target for novel cancer therapy. Several compounds that can reactive mutant p53 protein have been identified. APR-246 is currently being tested in a phase II clinical trial in high-grade serous ovarian cancer. We have used RNA-seq analysis to study the effects of APR-246 on gene expression in human breast cancer cell lines. Although the effect of APR-246 on gene expression was largely cell line dependent, six genes were upregulated across all three cell lines studied, i.e., TRIM16, SLC7A11, TXNRD1, SRXN1, LOC344887, and SLC7A11-AS1. We did not detect upregulation of canonical p53 target genes such as CDKN1A (p21), 14-3-3σ, BBC3 (PUMA), and PMAIP1 (NOXA) by RNA-seq, but these genes were induced according to analysis by qPCR. Gene ontology analysis showed that APR-246 induced changes in pathways such as response to oxidative stress, gene expression, cell proliferation, response to nitrosative stress, and the glutathione biosynthesis process. Our results are consistent with the dual action of APR-246, i.e., reactivation of mutant p53 and modulation of redox activity. SLC7A11, TRIM16, TXNRD1, and SRXN1 are potential new pharmacodynamic biomarkers for assessing the response to APR-246 in both preclinical and clinical studies.     

PHGDH Inhibits Ferroptosis and Promotes Malignant Progression by Upregulating SLC7A11 in Bladder Cancer

Background: Bladder cancer (BCa) is a prevalent urologic malignancy that shows a poor prognosis. Abnormal metabolism and its key genes play a critical role in BCa progression. In this study, the role played by PhosphoGlycerol Dehydrogenase (PHGDH), an important molecule of serine metabolism, was investigated with regard to the regulation of ferroptosis in BCa.Methods: The BCa tissues of 90 patients were analyzed by RNA-sequencing for differential pathways and genes. Western blot, qPCR, and IHC were used to determine PHGDH expression in the cell lines (in vitro) and patient tissues (in vivo). R software was used to analyze PHGDH expression, prognosis, and PHGDH+SLC7A11 score. The biological functions of PHGDH were examined through organoids, and in vitro and in vivo experiments. C11 probes, electron microscopy, and ferroptosis inhibitors/ inducers were used to detect cellular ferroptosis levels. Protein profiling, co-IP, and RIP assays were used to screen proteins that might bind to PHGDH. PHGDH-targeted inhibitor NCT-502 was used to evaluate its effect on BCa cells.Results: PHGDH was highly expressed in patients with BCa. Knock-down of PHGDH promoted ferroptosis, while the decreased proliferation of BCa cells. Additionally, PHGDH knock-down downregulated the expression of SLC7A11. Co-IP and mass spectrometry experiments indicate that PHGDH binds to PCBP2, an RNA-binding protein, and inhibits its ubiquitination degradation. PCBP2 in turn stabilizes SLC7A11 mRNA and increases its expression. NCT-502, a PHGDH inhibitor, promotes ferroptosis and inhibits tumor progression in BCa. The PHGDH+ SLC7A11 score was significantly correlated with patient prognosis.Conclusions: To conclude, the PHGDH, via interaction with PCBP2, upregulates SLC7A11 expression. This inhibits ferroptosis and promotes the malignant progression of BCA. The results of this study indicated that NCT-502 could serve as a therapeutic strategy for BCa.     

Molecular and functional characterization of CpACS27A gene reveals its involvement in monoecy instability and other associated traits in squash (Cucurbita pepo L.)

A number of Cucurbita pepo genotypes showing instable monoecy or partial andromonoecy, i.e. an incomplete conversion of female into bisexual flowers, have been detected. Given that in melon and cucumber andromonoecy is the result of reduction of ethylene production in female floral buds, caused by mutations in the ethylene biosynthesis genes CmACS7 and CsACS2; we have cloned and characterized two related C. pepo genes, CpACS27A and CpACS27B. The molecular structure of CpACS27A and its specific expression in the carpels of female flowers during earlier stages of flower development suggests that this gene is the Cucurbita ortholog of CmACS7 and CsACS2. CpACS27B is likely to be a paralogous pseudogene since it has not been found to be expressed in any of the analyzed tissues. CpACS27A was sequenced in Bolognese (Bog) and Vegetable Spaghetti (Veg), two monoecious inbred lines whose F2 was segregating for partial andromonoecy. The Bog allele of CpACS27A carried a missense mutation that resulted in a substitution of the conserved serine residue in position 176 by an alanine. Segregation analysis indicated that this mutant variant is necessary but not sufficient to confer the andromonoecious phenotype in squash. In concordance with its involvement in stamen arrest, a reduction in CpACS27A expression has been found in bisexual flower buds at earlier stages of development. This reduction in CpACS27A expression was concomitant with a downregulation of other ethylene biosynthesis and signaling genes during earlier and later stages of ovary development. The role of CpACS27A is discussed regarding the regulation of ethylene biosynthesis and signaling genes in the control of andromonoecy-associated traits, such as the delayed maturation of corolla and stigma as well as the parthenocarpic development of the fruit.