RESUMO
In this study, a multiplex amplification system including 47 autosomal InDels, 2 Y-chromosome InDels, and the sex-determining marker (Amelogenin) was developed with six fluorescent dyes labeling. These InDels were selected from the previous study based on a series of criteria (0.3 < MAF < 0.5, HET > 0.4, etc). The system was designated the AGCU InDel 50 kit and was validated in a series of studies, including a degradation study; tests for sensitivity, species specificity, reproducibility, stability, applicability to case samples, balance of peak height, and PCR conditions; and a population study. The results showed that AGCU InDel 50 kit was quite sensitive, specific, stable in several PCR conditions or exposure to PCR inhibitors, especially against degradation. 74 case samples and 50 paternity cases with STR mutation events were tested using PowerPlex® 21 System, AGCU InDel 50 kit, and Investigator DIPplex kit, and the results showed that the ratio of loci detected with the developed kit were close to Investigator@ DIPplex kit, but considerably higher than PowerPlex® 21 System for case samples containing low amounts of degraded DNA. As for 50 paternity cases, no mutation was observed in any InDels locus, and the CPIs based on 47 autosomal InDels contained in the AGCU InDel 50 kit were all higher than those based on 30 InDels contained in Investigator® DIPplex kit, except 3 cases. In the population study, 203 unrelated individuals from the Guangdong Han population were detected using the AGCU InDel 50 kit, and the values of combined power of discrimination and combined power of exclusion were 0.999 999 999 999 999 and 0.9997, respectively. Thus, AGCU InDel 50 kit is suitable for individual identification and as a supplemental tool for paternity testing. It is reproducible, accurate and robust for forensic applications and human genetic studies.
Assuntos
Impressões Digitais de DNA , Mutação INDEL , Reação em Cadeia da Polimerase Multiplex/instrumentação , Alelos , Amelogenina/genética , Animais , Cromossomos Humanos Y , Degradação Necrótica do DNA , Corantes Fluorescentes , Genética Populacional , Humanos , Repetições de Microssatélites , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
MicroRNAs (miRNAs) play critical roles in breast cancer cell biological processes, including proliferation and apoptosis by inhibiting the expression of their target genes. Herein, we reported that miR-630 overexpression initiates apoptosis, blocks cell cycle progression and suppresses cell proliferation in breast cancer cells. Furthermore, BMI1, a member of polycomb group family, was identified as a direct target of miR-630, and there was a negative correlation between the expression levels of BMI1 and miR-630 in human breast cancer samples. With a series of biology approaches, subsequently, we proved that BMI1 was a functional downstream target of miR-630 and mediated the property of miR-630-dependent inhibition of breast cancer progression. Taken together, these findings provide further evidence on the tumor-suppression function of miR-630 in breast cancer, and clarify BMI1 as a novel functional target gene of miR-630.
Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Apoptose/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , HumanosRESUMO
MicroRNAs have been integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. The miR-630 was reported to be deregulated and involved in tumor progression of several human malignancies. However, its expression regulation shows diversity in different kinds of cancers and its potential roles remain greatly elusive. Herein, we demonstrate that miR-630 is significantly suppressed in human breast cancer specimens, as well as in various breast cancer cell lines. In aggressive MDA-MB-231-luc and BT549 breast cancer cells, ectopic expression of miR-630 strongly inhibits cell motility and invasive capacity in vitro. Moreover, lentivirus delivered miR-630 bestows MDA-MB-231-luc cells with the ability to suppress cell colony formation in vitro and pulmonary metastasis in vivo. Further studies identify metadherin (MTDH) as a direct target gene of miR-630. Functional studies shows that MTDH contributes to miR-630-endowed effects including cell migration and invasion as well as colony formation in vitro. Taken together, these findings highlight an important role for miR-630 in the regulation of metastatic potential of breast cancer and suggest a potential application of miR-630 in breast cancer treatment.