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 H...

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Autores principales:	Yan-yan SHAO, Lin FU, Meng-ting ZHU, Ying NAN, Heng YANG, Zong-sheng ZHAO
Formato:	article
Lenguaje:	EN
Publicado:	University of Kafkas 2021
Materias:	3’-utr mirna target gene luteal cells sheep Veterinary medicine SF600-1100
Acceso en línea:	https://doaj.org/article/3f25c9ba259a44d9a94ea0c1bc070460
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spelling	oai:doaj.org-article:3f25c9ba259a44d9a94ea0c1bc0704602021-12-03T07:56:26ZMechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS10.9775/kvfd.2021.260801300-60451309-2251https://doaj.org/article/3f25c9ba259a44d9a94ea0c1bc0704602021-11-01T00:00:00Zhttp://vetdergikafkas.org/uploads/pdf/pdf_KVFD_2864.pdfhttps://doaj.org/toc/1300-6045https://doaj.org/toc/1309-2251Given 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. Yan-yan SHAOLin FUMeng-ting ZHUYing NANHeng YANGZong-sheng ZHAOUniversity of Kafkasarticle3’-utrmirnatarget geneluteal cellssheepVeterinary medicineSF600-1100ENKafkas Universitesi Veteriner Fakultesi Dergisi, Vol 27, Iss 6, Pp 681-690 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	3’-utr mirna target gene luteal cells sheep Veterinary medicine SF600-1100
spellingShingle	3’-utr mirna target gene luteal cells sheep Veterinary medicine SF600-1100 Yan-yan SHAO Lin FU Meng-ting ZHU Ying NAN Heng YANG Zong-sheng ZHAO Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS
description	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.
format	article
author	Yan-yan SHAO Lin FU Meng-ting ZHU Ying NAN Heng YANG Zong-sheng ZHAO
author_facet	Yan-yan SHAO Lin FU Meng-ting ZHU Ying NAN Heng YANG Zong-sheng ZHAO
author_sort	Yan-yan SHAO
title	Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS
title_short	Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS
title_full	Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS
title_fullStr	Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS
title_full_unstemmed	Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS
title_sort	mechanism of mir-665 regulating luteal function via targeting hpgds
publisher	University of Kafkas
publishDate	2021
url	https://doaj.org/article/3f25c9ba259a44d9a94ea0c1bc070460
work_keys_str_mv	AT yanyanshao mechanismofmir665regulatinglutealfunctionviatargetinghpgds AT linfu mechanismofmir665regulatinglutealfunctionviatargetinghpgds AT mengtingzhu mechanismofmir665regulatinglutealfunctionviatargetinghpgds AT yingnan mechanismofmir665regulatinglutealfunctionviatargetinghpgds AT hengyang mechanismofmir665regulatinglutealfunctionviatargetinghpgds AT zongshengzhao mechanismofmir665regulatinglutealfunctionviatargetinghpgds
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Mechanism of miR-665 Regulating Luteal Function Via Targeting HPGDS

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