CircRNA (circ_0008057) promotes uremic serum-mediated proliferation and migration of vascular smooth muscle cells via miR-370/PLk1 signaling pathway.

In order to explore the mechanism of gefitinib-acquired resistance in lung cancer, a new biomarker has been developed for early clinical diagnosis and intervention; human NSCLC (Non-Small Cell Lung Cancer) cell lines H292 (denoted as H292S) and PC9 (denoted as PC9S) were used to establish gefitinibresistant NSCLC cell lines H292 and PC9 models. CCK-8 (Cell Counting Kit-8) method was used to test the drug resistance of the cells. circRNAs (circular RNAs) that were differentially expressed before and after resistance were screened by RNA sequencing technology. The effects of circSETD3 overexpression and interference on the sensitivity of gefitinib was observed to analyze the nuclear localization of circSETD3 and verify the interaction between circSETD3-miR-520h-ABCG2. The results showed that the most significant change in differential expression of human NSCLC cell lines before and after drug resistance was hsa_circ_0000567, that is, circSETD3, which is mainly present in the cytoplasm. In H292S and PC9S, compared with the negative control group, the cell proliferation ability of the overexpression group was significantly increased, and the apoptosis ability was significantly decreased. In H292R and PC9R, compared with the negative control group, the proliferation ability of the interference group was significantly decreased, and the apoptosis ability was significantly increased. Overexpression of circSETD3 to H292S and PC9S, the expression of ABCG2 increased significantly. Also, the expression of ABCG2 decreased significantly after transfection with miR-520h mimics. H292R and PC9R interfered with circSETD3, the expression of ABCG2 decreased significantly. Moreover, the expression of ABCG2 increased significantly after transfection with miR-520h inhibitor. In conclusion, circSETD3 can be used as a novel biomarker for lung cancer. It relieves miR-520h degradation of the transporter ABCG2 by down-regulating the miR-520h expression, causing gefitinib to be pumped out of the cell.

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.

MicroRNA-221 promotes myocardial apoptosis caused by myocardial ischemia-reperfusion by down-regulating PTEN.

OBJECTIVE: The aim of this study was to investigate whether microRNA-221 could promote cardiomyocyte apoptosis by down-regulating the expression of PTEN (gene of phosphate and tension homology deleted on chromosome ten), thereby participating in the development of myocardial ischemia-reperfusion. MATERIALS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to analyze the expression levels of microRNA-221 and PTEN in human cardiomyocytes (HCM) cells treated with hypoxia/reoxygenation (H/R). The expressions of myocardial injury markers, including lactic dehydrogenase enzyme (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were determined by qRT-PCR as well. The binding relationship between microRNA-221 and PTEN was verified by the Dual-Luciferase reporter gene assay. Subsequently, microRNA-221 inhibitor and si-PTEN were transfected into cells. The proliferation and apoptosis of cells were analyzed using Cell Counting Kit-8 (CCK-8) and flow cytometry, respectively. In addition, the expression levels of apoptosis-related proteins were determined by Western blot. RESULTS: The qRT-PCR results confirmed that the expression level of microRNA-221 in H/R treated cells was significantly up-regulated when compared with the normoxic treated group, whereas PTEN expression was markedly down-regulated. After silencing microRNA-221, the expression levels of myocardial injury markers, including LDH, MDA, GSH-PX in H/R cells were significantly decreased. However, SOD levels were remarkably increased. At the same time, down-regulation of microRNA-221 markedly increased cell proliferation, whereas decreased apoptosis. However, microRNA-221 enhanced the expression of apoptosis-related genes, including Bax and cytochrome C. Meanwhile, the expression level of anti-apoptotic gene Bcl-2 was significantly inhibited. The Dual-Luciferase reporter gene assay showed that microRNA-221 could target bind to PTEN and inhibit its expression. Similarly, down-regulation of PTEN markedly decreased cell proliferation and increased cell apoptosis. Furthermore, PTEN down-regulation remarkably promoted protein expression of pro-apoptosis-related genes, whereas inhibited the protein expression of anti-apoptotic genes. CONCLUSIONS: MicroRNA-221 promoted myocardial apoptosis induced by myocardial ischemia-reperfusion by down-regulating PTEN. Therefore, microRNA-221 might be a potential therapeutic target for myocardial ischemia-reperfusion injury.