A Preliminary Study Of MiR-375-3p Mediated Rasd1 Gene On Rat Circadian Disorder Caused By HIBD

Objective:To study the expressions of microRNA-375-3p(miR-375-3p),dexamethasone-induced Ras-related protein 1(Rasd1)and Cryptochrome1(Cry1)in pineal gland of neonatal rats sufferring hypoxic-ischemic brain damage(HIBD),to confirm the targeted regulation of Rasd1 by miR-375-3p via Dual-Luciferase reporter gene system,and to explore the possible role of mi R-375-3p in the pathogenesis of circadian rhythm disorders after HIBD via regulating Rasd1.Method: Seven-day-old Sprague-Dawley(SD)rats were randomly divided into two groups: HIBD and sham-operated.HIBD was induced according to the Rice-Vannucci method.RT-qPCR was performed to measure the expression profiles of miR-375-3p,Rasd1 and Cry1 in the pineal gland at 0 h,8 h,16 h,24 h,32 h,40 h and 48 h after HIBD.Luciferase reporter gene assay was used to confirmed the targeted regulation of Rasd1 expression by miR-375-3p.Results:1.Compared with the sham-operated group,the expression of miR-375-3p in the HIBD group was significantly up-regulated in the pineal gland at 8 h after HIBD(P<0.05),down-regulated in the pineal gland at 24 h,32 h,40 h and 48 h after HIBD(P<0.05),while there were no statistically significant differences at 0 h,16 h(P>0.05).The level of mi R-375-3p in HIBD peaked at 16 h,decreased at 48 h after HIBD and remained till 48 h.2.Compared with the sham-operated group,the expression of Rasd1 in the HIBD group was significantly down-regulated in the pineal gland at 8 h,16 h and 48 h after HIBD(P<0.05),while Rasd1 was significantly up-regulated in the pineal gland at 0 h,24 h,32 h and 40 h after HIBD(P<0.05).The level of Rasd1 in HIBD began to rise at 24 h,decreased at 32 h after HIBD and reached the lowest point at 48 h.3.The expression of miR-375-3p was up-regulated in the pineal gland at 8 h and 16 h after HIBD,while Rasd1 was down-regulated;The expression of Rasd1 was significantly up-regulated in the pineal gland at 24 h after HIBD,while mi R-375-3p was significantly down-regulated.4.Compared with the sham-operated group,the expression of Cry1 in the HIBD group was significantly up-regulated in the pineal gland at 16 h and 24 h after HIBD(P<0.05),down-regulated in the pineal gland at 32 h and 40 h after HIBD(P<0.05),while there were no statistically significant differences at 0 h,8 h and 48 h(P>0.05).The level of Cry1 in HIBD increased at 16 h,reached a peak level at 24 h,decreased at 32 h,and remained till 48 h.5.RT-qPCR showed that the expression of Cry1 and Rasd1 in the HIBD group displayed the tendency rising up at the begining and declining in late,it was observed with a peak on 24 h that then decreased at 32 h.6.The luciferase reporter gene assay confirmed the targeted regulation of Rasd1 by mi R-375-3p.Conclusion:1.The expression of miR-375-3p was up-regulated in the pineal gland at 8 h and 16 h after HIBD,while Rasd1 was down-regulated;The expression of Rasd1 was significantly up-regulated in the pineal gland at 24 h after HIBD,while mi R-375-3p was significantly down-regulated.2.The expression of Cry1 and Rasd1 in the HIBD group displayed the tendency rising up at the begining and declining in late,which suggested that Rasd1 may take part in the pathogenesis of circadian rhythm disturbance after HIBD.3.Rasd1 is one of the target genes of miR-375-3p.