ABSTRACT
Objective: To verify whether EFNB2 gene is targeted by miRNA-497 using molecular biology methods. Methods: Bioinformatic method predicted that EFNB2 gene is targeted by miRNA-497. PCR methods amplified the fragment in EFNB2 gene 3'-UTR including the putative miRNA-497 binding site. Then the sequences of wide type (WT) and mutant (MUT) were cloned into the pmirGLO luciferase vector, respectively. The DNA sequences of the amplified fragments were identified by restriction enzyme digestion and sequencing, and were consistent with the reference sequence from UCSC. This constructed vector was marked as pmirGLO-EFNB2 vector. Finally, the pmirGLO vector, the pmirGLO-EFNB2 vector, miRNA-497 mimics and negative control (NC) were divided into five groups and transfected into HEK393T cells, and the luciferase activity was tested after 24 h and 48 h by dual luciferase reporter gene assay. Results: The results of DNA sequencing demonstrated that the PCR fragment was successfully cloned into pmirGLO vector. The transfection results showed that the recombinant plasmid of WT and MUT was successfully transfected into HEK293T. The results of dual luciferase activity assay demonstrated that miRNA-497 significantly decreased the reporter gene activity compared with the NC. Conclusion: At the cellular level, the schizophrenia susceptibility gene EFNB2 was verified to be targeted by miRNA-497, which provides a new idea and new clue for subsequent studies on miRNA-497 function in the molecular mechanism of schizophrenia.
ABSTRACT
Objective To investigate the gene expression of Efnb2 and Ephb4 in the lung tissue of congenital esophageal atresia and tracheoesophageal fistula(EA-TEF) rat,and to explore the effect on lung development in EA rat.Methods Twenty-four female Wistar rats were randomly divided into experimental group(EATEF group)and control group(CON group)with 12 rats in each group.Adriamycin(ADR)-treated rats were established by intrapedtoneal injection of adriamycin at 1.75 mg / kg on day E7-9.The rats were sacrificed by caesarean on day 15 (E15),day 18 (El8) and day 21 (E21).The expression of Ephb4 and Efnb2 in fetal lung was detected by Q-PCR and immunohistochemistry.Results (1) It showed significantly higher mRNA expression levels of Efnb2 at the canalicular stage,which decreased and returned to pseudoglandular level at the sacular stage.The trend of Ephb4 mRNA and Efnb2 mRNA in the two groups was basically the same.The expression of Efnb2 mRNA in EA-TEF group(50.65 ± 12.65) was significantly higher than that in control group(23.63 ± 11.31) at the canalicular stage (P ＜ 0.05),but there was no statistical significance between the two groups at both pseudoglandular stage and sacular stage;The expression of Ephb4 mRNA in EA-TEF group (4.32 ± 2.88) was significantly higher than that in control group (1.01 ± 0.19) at the pseudoglandular stage (P ＜ 0.05),but there was no statistical significance between the two groups at both canalicular stage and sacular stage.(2)Efnb2 and Ephb4 proteins were expressed on the surface of vascular epithelium,alveolar and bronchial epithelium at different developmental stages,and there was no statistical significance in the distribution pattern.The expression of Efnb2 (162.70 ± 10.04) and Ephb4 (152.20 ± 12.32) in EA-TEF group was enhanced at the canalicular stage,while returned to normal level at the sacular stage.Conclusion The angiogenesis factor Efnb2 and its receptor Ephb4 can regulate the branching development of fetal lung tissue,which maybe a compensatory mechanism for pulmonary dysplasia in adriamycin-induced EA-TEF model.