Molecular analysis of MLH1 variants in Chinese sporadic colorectal cancer patients.

Single nucleotide polymorphisms (SNPs) in mismatch repair genes, especially in the MLH1 gene, are closely associated with susceptibility to hereditary nonpolyposis colorectal cancer. However, few relevant findings are available regarding the association between sporadic colorectal cancer (SCRC) and SNPs of MLH1 in Chinese patients. Therefore, the present study aimed to describe the pathogenic association between three important MLH1 polymorphisms and SCRC in the Chinese population. Peripheral blood samples from 156 SCRC patients and 311 healthy controls were collected. DNA was purified from peripheral blood, and the V384D, R217C, and I219V polymorphisms were evaluated using high-resolution melting analysis and direct sequencing. The association between the three important MLH1 polymorphisms and clinical pathological features of the SCRC patients was analyzed. In addition, PMS2-MLH1 protein interactions were determined by co-immunoprecipitation (Co-IP) to determine the protein functional alteration induced by these SNPs. Among the three polymorphisms, V384D was significantly associated with the risk of SCRC (OR = 31.36, P < 0.0001). The allele frequencies were 4.81 and 0.16% in the SCRC group. No association was found between SCRC and R217C, or between SCRC and I219V. Moreover, the allele frequency of R217C was significantly higher in the SCRC patients younger than 60 years than in those older than 60 years. Co-IP showed that the MLH1 R217C, V384D, and I219V variants had relative binding abilities with PMS2 of 0.59, 0.70, and 0.80, respectively, compared with the wild-type. These findings suggest that MLH1 V384D could be a promising genetic marker for susceptibility to SCRC.

Molecular cloning and expression analysis of MAT1 gene in black tiger shrimp (Penaeus monodon).

MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the CDK-dependent kinase (CAK), can regulate the cell cycle, transcription, and DNA repair. This study was intended to investigate the role of MAT1 in the reproductive maturation of black tiger shrimp (Penaeus monodon). In this study, the P. monodon MAT1 (PmMAT1) gene was identified and characterized. The full-length cDNA of PmMAT1 was 1490 bp in length with an open-reading frame of 993 bp corresponding to 330 amino acids. The temporal expression of PmMAT1 in various tissues was measured by quantitative real-time PCR with the highest expression observed in ovaries. In the ovaries, the PmMAT1 gene was continuously but differentially expressed during the maturation stages. Comparative analyses of MAT1, CDK7, and cyclin H in the CAK complex of P. monodon indicated that the expression of CDK7 and cyclin H coincided with that of MAT1 during the ovary maturation stages. Serotonin (5-HT) injection promoted the expression level of PmMAT1 in the ovaries of shrimp at 6-48 h post-injection. These results indicate that PmMat1 plays a prominent role in the process of ovarian maturation.

MicroRNA-26b inhibits osteosarcoma cell migration and invasion by down-regulating PFKFB3 expression.

MicroRNAs regulate target gene expression and are involved in cell proliferation, apoptosis, differentiation, tumor invasion, and cancer stem cell regulation, among other processes. MicroRNA-26b (miR-26b) is closely related to tumor occurrence and development. In this study, we analyzed miR-26b expression in osteosarcoma tissue, its effect on Saos-2 osteosarcoma cell proliferation and invasion, and its relationship with 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression. Osteosarcoma tissue was obtained from surgical patients and normal tissue adjacent to the tumor was used as a control. Real-time polymerase chain reaction was applied to detect miR-26b expression in cancer tissue and normal tissue. A vector expressing miR-26b was constructed and transfected into Saos-2. An MTT assay, cell invasion assay, and scratch experiment were used to analyze the effect of miR-26b on Saos-2 cell proliferation, invasion, and migration abilities. Western blotting analysis was performed to investigate the role of miR-26b on PFKFB3 expression. miR-26b expression in normal tissue was 7.55-fold higher than in osteosarcoma tissue (t = 10.20, P = 0.006). Compared with control tissue, miR-26b significantly inhibited osteosarcoma proliferation, migration, and invasion (P < 0.05). Western blotting results revealed that PFKFB3 protein expression decreased in Saos-2 cells after transfection with miR-26b. miR-26b was down-regulated in osteosarcoma tissue. miR- 26b may inhibit osteosarcoma cell proliferation, migration, and invasion by regulating PFKFB3 protein expression. miR-26b may have a tumor suppressor role in tumor occurrence and development.

Characterization and expression analysis of a cyclin B gene from black tiger shrimp (Penaeus monodon).

The open reading frame of black tiger shrimp (Penaeus monodon) cyclin B (Pmcyclin B) was identified, based on cDNA sequence registered in GenBank (accession No. EF015590). The target sequence was 1206 bp, corresponding to 401 amino acids. Two conserved signature sequences of the cyclin B gene family were found in the Pmcyclin B deduced aa sequence. Temporal expression of Pmcyclin B in different tissues, including ovary, lymphoid organ, brain, blood, muscle, heart, gill, hepatopancreas, and intestine, were quantified by quantitative real time PCR. Messenger RNA expression levels of Pmcyclin B were greatest in the ovary, compared to other tissues (P < 0.05). Temporal expression of Pmcyclin B in the ovary at six different developmental stages was investigated by real-time PCR; no significant difference was observed (P < 0.05). Recombinant Pmcyclin B protein and its polyclonal antibody were successfully produced. Western blot analysis revealed differential expression of Pmcyclin B in ovaries in developmental stages II to IV; a positive signal (45 kDa) was observed in all ovarian stages assessed, but was most intense at stage III. Pmcyclin B protein was assessed by immunohistochemistry and was localized to the cytoplasm of prophase oocytes at stage II and enriched in the nuclei of pro-metaphase oocytes at stages III and IV. Results from this study indicate that Pmcyclin B is constitutively expressed and plays an important role in ovarian maturation in P. monodon.