[Single nucleotide polymorphism of rs28416520 in Piwil1 gene promoter region is associated with an increased risk of gastric cancer].

OBJECTIVE: To analyze the correlation between the single nucleotide polymorphisms (SNPs) in the promoter of Piwil1 gene and gastric cancer. METHODS: The expression of Piwil1 mRNA in the tumor tissues of 3 patients with gastric cancer was detected by RT-qPCR, and RNA-Sequencing data from the Cancer RNA-Seq Nexus were analyzed for Piwil1 mRNA expression in gastric patients. Blood samples were collected from 24 gastric cancer patients and 29 healthy control subjects for PCR amplification of Piwil1 gene promoter region. The SNP loci in the promoter region of Piwil1 gene were determined by direct sequencing, and the results were analyzed by SnapGene software. RESULTS: Analysis of the data from Cancer RNA-Seq Nexus and the results of RT-qPCR in 3 gastric cancer patients all showed significantly increased Piwil1 expression in gastric cancer tissues compared with the adjacent tissues. Seven SNP loci in two CpG regions of the Piwil1 gene promoter were genotyped, and only one SNP locus was found to be related to gastric cancer. The frequencies of GG, GA, and AA genotypes at the rs28416520 locus in CpG 67 region were 79.2%, 16.7%, and 4.1% in the gastric cancer group, and were 37.9%, 55.2%, and 6.9% in the control group, respectively, showing a significantly higher frequency of the GG genotype in gastric cancer group (OR=0.144, 95%CI: 0.045-0.564, χ2=9.071, P < 0.01). The frequency of allele G of the rs28416520 locus was significantly higher in gastric cancer group than in the control group (87.5% vs 65.5%; OR=0.271, 95%CI: 0.099-0.766, χ2=6.856, P < 0.01). The genotype or allele frequencies of the other 6 SNPs locus did not differ significantly between gastric cancer group and control group. CONCLUSIONS: The expression of Piwil1 is increased in gastric cancer tissues as compared with the adjacent tissues. The GG genotype and G allele of rs28416520 within CpG 67 region are associated with an increased risk of gastric cancer.

Cloning and expression profile of Bmlin-41 and its regulation by the silkworm microRNA let-7 / 生物工程学报

The heterochronic genes regulate cell proliferation and switch development stage transitions. Heterochronic genes might also play important roles in regulating the development of silkworm, but very few of their expression profiles, functions and their relationship with microRNAs are available so far. Firstly, in this work, the primers for cloning Bmlin-41 were designed based on the homologous sequence of known Drosophila melanogaster lin-41, which was used as the query to blast against SilkDB. The obtained full CDS (2 166 bp) of Bmlin-41 encodes 721 amino acids and contains B-box and NHL domains. Then, the spatiotemporal expression patterns of Bmlin-41 were characterized by RT-PCR, quantitative real time PCR as well as our lab's previous silkworm genome microarray data. Bmlin-41 was increasingly expressed from embryonic to adult stage. In diverse tissues of day-3 fifth instar, Bmlin-41 showed the highest accumulation in ovary, secondly in testis and midgut, but very low expression was observed in other tissues. Finally, 3'UTR of Bmlin-41 1 434 bp was cloned by rapid-amplification of cDNA ends (3'RACE) and was predicted to bare two binding sites of bmo-let-7 by using online RNAhybrid. To verify the binding effect, 3'UTR was cloned into psi-CHECK-2 vector and submitted to dual luciferase assay in the S2 cells in vitro. The dual luciferase assay demonstrated that Bmlin-41 was down-regulated by bmo-let-7 mimics and upregulated by bmo-let-7 antagomir, thus confirming the Bmlin-41 is negatively regulated by bmo-let-7. Our work might help further study on the roles of Bmlin-41 and bmo-let-7 and their regulation relationship involved in controlling metamorphosis of silkworm.

Cloning and expression profile of Bmyan in the silkworm (Bombyx mori) and experimental validation as one target of microRNA 7 / 生物工程学报

microRNAs (miRNAs) are an extensive class of -22-nucleotide (nt) endogenous noncoding RNAs regulating life activities ofmetazoans through binding to 3'-untranslated regions (3'-UTRs) of their target genes. This work aimed to identify yan gene in the silkworm, reveal its expression profile and confirm if it is one target of bmo-miR-7 and, as such, have potential for contributing to better understanding of the molecular mechanisms involved in the metamorphosis of silkworm. Based on homolog searching and PCR amplification, we cloned the coding sequence (CDS) of Bmyan, which encodes 476 amino acid residues and contains SAM-PNT and ETs domains. Quantitative PCR (q-PCR), RT-PCR and microarray data revealed high expression of Bmyan in the head, body wall and ovary of day-3 fifth instar larval silkworm, low or no expression in other tissues. It was lowly expressed in the early larval stages, but highly expressed from late spinning to day 4 pupa. The 3'-UTR of Bmyan was obtained by rapid-amplification of cDNA ends (3'RACE) and predicted to contain two potential recognition sites of bmo-miR-7. The luciferase reporter vector containing the 3'-UTR of Bmyan was constructed and co-transfected into BmE cell line with the mimic of bmo-miR-7 and the decreased relative activity of luciferase showed that Bmyan is one target of bmo-miR-7. This work helps further functional analysis of bmo-miR-7 and Bmyan in the silkworm.