Kafkas Üniversitesi Veteriner Fakültesi Dergisi 2021 , Vol 27 , Issue 6
Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS
Yan-yan SHAO1, Lin FU2, Meng-ting ZHU1, Ying NAN1, Heng YANG3, Zong-sheng ZHAO1
1College of Animal Science and Technology, Shihezi University, Shihezi 832000, Xinjiang, CHINA
2Research institute of herbivorous livestock, Chongqing academy of animal sciences, Chongqing, CHINA
3College of Veterinary Medicine, Southwest University, Chongqing, CHINA
4Immunology Research Center, Medical Research Institute, Southwest University, Chongqing, CHINA
DOI : 10.9775/kvfd.2021.26080 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. Keywords : 3