Ying NAN, Meng-ting ZHU, Heng YANG, Zong-sheng ZHAO, Yan-yan SHAO, Lin FU

MiR-665’nin HPGDS Hedefli Luteal Fonksiyon DüzenlemeMekanizması

MiRNA-665/hedef gen çiftinin etkileşimi hakkında az sayıdaki rapor göz önüne alındığında, hematopoietik prostaglandin D sentaz (HPGDS) geninin 3’-kodlanmayan bölgesi (3’-UTR) için bir çift lusiferaz raportör gen vektörünün oluşturulması ve HPGDS’nin miR-665 regülasyonunun moleküler mekanizmasının aydınlatılması amaçlandı. HPGDS geninin 3’-UTR bölgesini hedefleyen miR-665’nin saptanması için biyoinformatik yazılım kullanıldı. Sentetik psiCHECK-HPGDS-w/m-3’-UTR’nin güvenilirliği, dual lusiferaz sindirim yöntemi ile belirlendi. Daha sonra miR-665 mimik/negatif kontrolün, koyun luteal hücreleri ile ayrı ayrı ko-transfeksiyonu yapıldı ve ardından lusiferaz aktivitesi ve HPGDS ekspresyonu tespit edildi. Sonuçlar, HPGDS için 3’-UTR yabanıl tip (psiCHECK2-HPGDS-w-3’UTR) ve mutant (psiCHECK2-HPGDS-m-3’UTR) ekspresyon vektörlerinin başarıyla oluşturulduğunu ve dual lusiferaz raportör gen analizi, relatif lusiferaz aktivite ekspresyonunun, blank/negatif kontrol grubuna kıyasla %52’lik bir düşüşle w-3’-UTR grubunda inhibe edildiğini ve farkın istatistiksel olarak anlamlı olduğunu gösterdi (P

Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS

Given few reports on the interaction of miRNA-665/target gene pair, we aimed to construct a dual luciferase reporter gene vector for 3’-untranslated region (3’-UTR) of the haematopoietic prostaglandin D synthase (HPGDS) gene and elucidate the underlying molecular mechanism of miR-665 regulation of HPGDS. Bioinformatics software was used to predict miR-665 targeting of 3’-UTR region of HPGDS gene. The reliability of the synthetic psiCHECK-HPGDS-w/m-3’-UTR was determined using double luciferase digestion method. Then, miR-665 mimic/negative control was separately co-transfected with sheep luteal cells, and then, luciferase activity and HPGDS expression were detected. Results showed that 3’-UTR wild-type (psiCHECK2-HPGDS-w-3’UTR) and mutant (psiCHECK2-HPGDS-m-3’UTR) expression vectors for HPGDS were successfully constructed, and dual luciferase reporter gene assay showed that the expression of relative luciferase activity was inhibited in the w-3’-UTR group, with 52% decrease compared to the blank/negative control group, and the difference was statistically significant (P

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