Mechanism of mir-665 regulating luteal function via targeting hpgds

Abstract

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<0.05). Whereas in luteal cells, compared to the blank/negative control group, RT-PCR and western blot assays showed lower HPGDS expression levels when miR-665 overexpression in miR-665-mimic group. Meanwhile, our result also showed that P4 concentration significantly increased using ELISA test in above group. In brief, our data verified that 3"-UTR wild-type dual luciferase reporter gene vector of HPGDS was successfully constructed, and miR-665 could target the gene by acting on the 3"-UTR region of HPGDS to regulate the ovine luteal cell function, providing a strong support to study the function and molecular mechanism of miR-665 in targeting HPGDS, especially in the ovarian-luteal tissue of sheep.

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