MicroRNA-489-3p plays a significant role in congenital hypothyroidism through regulating neuronal cell apoptosis via targeting translationally controlled tumor protein 1

Abstract

Accumulating reports have indicated that congenital hypothyroidism (CH) is an endocrine disorder caused by underdeveloped thyroid gland or thyroid dyshormonogenesis. It has been also reported that certain microRNAs (miRNAs) may exert protective effects against the development of CH. However, whether miR-489-3p regulates CH progression remains unclear. The aim of the present study was to investigate the effects of miR-489-3p on CH and elucidate the underlying mechanisms. Therefore, Sprague Dawley rats were injected with propylthiouracil (50 mg/day) to establish a CH model. Reverse transcription-quantitative PCR (RT-qPCR) assay demonstrated that miR-489-3p was upregulated in the hippocampal tissues of CH rats. Furthermore, the TargetScan software was employed to predict the target gene of miR-489-3p, and a dual luciferase reporter assay revealed that translationally controlled tumor protein 1 (TPT1) was directly targeted by miR-489-3p. Additionally, RT-qPCR and western blot assays suggested that TPT1 was markedly downregulated in the hippocampal tissues of CH rats compared with control rats. In addition, inhibitor control, miR-489-3p inhibitor, control-shRNA or TPT1-shRNA were injected into CH rats. The results of the open-field and forced swimming tests revealed that miR-489-3p inhibitor notably improved the behavior of CH rats. Flow cytometry was applied to explore the effects of miR-489-3p inhibitor on neuronal cell apoptosis, and the findings indicated that miR-489-3p inhibitor attenuated CH-induced neuronal cell apoptosis, whereas these effects were reversed by treatment with miR-489-3p inhibitor and TPT1-shRNA. Finally, the function of miR-489-3p in neuronal cells was investigated in vitro. Neuronal cell viability, apoptosis and the expression of apoptosis-related proteins were determined using MTT assay, flow cytometry and western blot analysis, respectively. The results demonstrated that miR-489-3p inhibitor enhanced cell viability, suppressed apoptosis and upregulated Pim-3, phosphorylated (p)-Bad (Ser112) and Bcl-xL expression. Rescue experiments indicated that these effects were reversed following silencing of TPT1. Taken together, the findings of the present study demonstrated that miR-489-3p inhibitor could relieve CH-induced neurological damage through regulating TPT1 expression.

Keywords: congenital hypothyroidism; microRNA-489-3p; neuronal cells; translationally controlled tumor protein 1.

Figure 1

miR-489-3p is overexpressed in CH rats and acts via targeting TPT1. Pregnant rats were injected with propylthiouracil to induce CH in rat pups. (A) RT-qPCR analysis was performed to examine the expression levels of miR-489-3p in the hippocampal tissues of rats with or without CH. (B) Bioinformatics analysis predicted a miR-489-3p-binding site on the TPT1 3'-UTR. (C) RT-qPCR analysis was performed to examine the expression levels of miR-489-3p in 293T cells transfected with mimic control or miR-489-3p mimic for 24 h. (D) Dual luciferase reporter assay was performed to verify the potential association between miR-489-3p and TPT1. (E) RT-qPCR analysis was applied to measure TPT1 mRNA expression levels. (F) TPT1 protein expression levels in the hippocampal tissues of rats with or without CH were detected using western blot assay. ^**P<0.01 vs. the control group; ^##P<0.01 vs. the mimic control group. miR, microRNA; CH, congenital hypothyroidism; TPT1, translationally controlled tumor protein 1; 3'-UTR, 3'-untranslated region; RT-qPCR, reverse transcription-quantitative PCR; WT, wild-type; MUT, mutant.

Figure 2

Effect of miR-489-3p inhibitor and TPT1-shRNA on CH rats. Inhibitor control, miR-489-3p inhibitor, miR-489-3p inhibitor + control-shRNA or miR-489-3p inhibitor + TPT1-shRNA were injected into CH rats. (A) The miR-489-3p expression levels were determined by RT-qPCR analysis. (B) RT-qPCR analysis of TPT1 expression in the six groups. The effects of miR-489-3p inhibitor on (C) depressive- and (D and E) anxiety-like behaviors were determined using forced swimming and open-field tests, respectively. (F) Rat neuronal cell apoptosis was determined by flow cytometry in different groups. (G) Quantified cell apoptosis rate. ^**P<0.01 vs. the control group; ^##P<0.01 vs. the CH + inhibitor control group; ^&&P<0.01 vs. the CH + miR-489-3p inhibitor + control-shRNA group. miR, microRNA; TPT1, translationally controlled tumor protein 1; CH, congenital hypothyroidism; RT-qPCR, reverse transcription-quantitative PCR.

Figure 3

Effect of miR-489-3p inhibitor and TPT1-shRNA on TPT1 expression in cultured neuronal cells. miR-489-3p inhibitor, inhibitor control, miR-489-3p inhibitor + control-shRNA or miR-489-3p inhibitor + TPT1-shRNA were transfected into neuronal cells. (A) The levels of miR-489-3p were measured using RT-qPCR analysis in the inhibitor control and miR-489-3p inhibitor groups. (B) RT-qPCR and (C) western blot analysis of TPT1 expression in neuronal cells following treatment with control-shRNA and TPT1-shRNA. (D) RT-qPCR and (E) western blot assays were conducted to assess TPT1 expression in neuronal cells following transfection with miR-489-3p inhibitor + control-shRNA or miR-489-3p inhibitor + TPT1-shRNA. ^**P<0.01 vs. the inhibitor control group; ^##P<0.01 vs. the control-shRNA group; ^&&P<0.01 vs. the miR-489-3p inhibitor + control-shRNA group. miR, microRNA; TPT1, translationally controlled tumor protein 1; RT-qPCR, reverse transcription-quantitative PCR.

Figure 4

TPT1-shRNA reversed the effects of miR-489-3p inhibitor on neuronal cell viability and apoptosis via the TPT1/Pim-3 pathway. miR-489-3p inhibitor, inhibitor control, miR-489-3p inhibitor + control-shRNA or miR-489-3p inhibitor + TPT1-shRNA were transfected into neuronal cells. (A) Neuronal cell viability was assessed using an MTT assay. (B) Neuronal cell apoptosis was measured using flow cytometry. (C) The Pim-3, Bcl-xL, p-Bad (Ser112) and Bad protein expression levels in the six different groups were determined using western blot assay. (D-F) The Pim-3/GAPDH, Bcl-xL/GAPDH and p-Bad (Ser112)/Bad ratios were calculated. ^**P<0.01 vs. the inhibitor control group; ^##P<0.01 vs. the control-shRNA group. miR, microRNA; TPT1, translationally controlled tumor protein 1.