MicroRNA-448/EPHA7 axis regulates cell proliferation, invasion and migration via regulation of PI3K/AKT signaling pathway and epithelial-to-mesenchymal transition in non-small cell lung cancer.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is a primary subtype of lung cancers which has a high morbidity and poor prognosis. Emerging evidence has demonstrated that aberrantly expressed microRNAs (miRNAs) were implicated in the regulatory functions of multiple processes during tumorigenesis. In the current study, we explored the functional roles and underlying mechanisms of miR-448 in NSCLC. PATIENTS AND METHODS: Quantitative real-time polymerase chain reaction assays were conducted to measure miR-448 expressions in 51 pairs of NSCLC tissues and corresponding normal tissues. Moreover, the relationship between miR-448 expressions and clinicopathological characteristics of NSCLC patients was also determined. We then performed transwell assays to explore the functions of miR-448 in NSCLC cell invasion and migration. As we had identified EPHA7 as a functional target of miR-448 in NSCLC cells, the clinical significance of EPHA7 in NSCLC patients was further investigated. Finally, we detected the influence of miR-448 on tumor growth rate and tumor size of NSCLC using tumor xenografts. RESULTS: Underexpressed miR-448 was identified in NSCLC, and low miR-448 expression was confirmed to be associated with the poor prognosis and adverse clinicopathologic features of NSCLC patients. Moreover, functional assays demonstrated that miR-448 overexpression suppressed NSCLC cell proliferation, invasion and migration. EPHA7 was identified as a direct target of miR-448. Additionally, miR-448 restoration suppressed in vivo NSCLC cell growth. Finally, our studies also indicated that miR-448 exerted anti-NSCLC functions via regulating PI3K/AKT signaling pathway and EMT. CONCLUSIONS: These results showed that miR-448/EPHA7 axis maybe one of the useful diagnostic and prognostic biomarkers for NSCLC patients.

Position effect variegation and epigenetic modification of a transgene in a pig model.

Sequences proximal to transgene integration sites are able to regulate transgene expression, resulting in complex position effect variegation. Position effect variegation can cause differences in epigenetic modifications, such as DNA methylation and histone acetylation. However, it is not known which factor, position effect or epigenetic modification, plays a more important role in the regulation of transgene expression. We analyzed transgene expression patterns and epigenetic modifications of transgenic pigs expressing green fluorescent protein, driven by the cytomegalovirus (CMV) promoter. DNA hypermethylation and loss of acetylation of specific histone H3 and H4 lysines, except H4K16 acetylation in the CMV promoter, were consistent with a low level of transgene expression. Moreover, the degree of DNA methylation and histone H3/H4 acetylation in the promoter region depended on the integration site; consequently, position effect variegation caused variations in epigenetic modifications. The transgenic pig fibroblast cell lines were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. Transgene expression was promoted by reversing the DNA hypermethylation and histone hypoacetylation status. The differences in DNA methylation and histone acetylation in the CMV promoter region in these cell lines were not significant; however, significant differences in transgene expression were detected, demonstrating that variegation of transgene expression is affected by the integration site. We conclude that in this pig model, position effect variegation affects transgene expression.

Transgene insertion affects transcription and epigenetic modification of flanking host sequence in transgenic pigs.

Transgenic technology has been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, the efficiency of producing transgenic animal lines that retain normal function is extremely low. The low efficiency can be mainly attributed to the integrated transgene. A further understanding of the effects of transgene integration on transcription and epigenetic modification of the host genome would improve the transgenic efficiency. Therefore, we utilized three transgenic pigs produced by SCNT expressing GFP, to identify alterations of transcription, DNA methylation and histone acetylation resulting from integration of the GFP gene. Multiple copies of the transgene integrated into a single site of the three transgenic pigs were verified by TAIL—PCR and the integration sites were different in each pig. We observed that the integrated transgene frequently resulted in significantly low transcription of flanking sequences in various tissues of transgenic pigs in comparison with wild—type pigs. Corresponding with the low transcription, DNA hypermethylation and loss of acetylation of histone H3 and H4 were detected. Our results demonstrate that the abnormal transcription and epigenetic modification of sequences flanking the transgene were not correlated with the expression of the transgene. However, the disturbance caused by the insertion of the transgene, was dependent upon the integration site. This suggests that some sequences in the host genome could permit integration and expression of transgene without causing defects in the host.

[Antiarrhythmic effects of crebanine].

Crebanine (Cre) iv 5 mg/kg could convert BaCl2-induced arrhythmia into sinus rhythm in rats, and could significantly increase the tolerant dose of aconitine to produce ventricular fibrillation (VF) and cardiac arrest (CA) in rats. The drug could also decrease the incidence of VF and CA by CaCl2 in rats and by chloroform in mice, but had no protective effects on ouabain-induced arrhythmias in guinea pigs.