Study Of Rat Circadian Rhythm Disturbance After Hypoxic-ischemic Brain Damage Caused By MiR-182 Mediated Clock Gene Regulation

Objective: To study the expressions of miR-182 and Clock gene in pineal gland of neonatal rats with hypoxic-ischemic brain damage(HIBD), and to test the effects on Clock gene when miR-182 is over-expressed or silenced in pineal cells in vitro, then we analyze the relationship between mi R-182 and Clock gene, and the possible role of mi R-182 in the pathogenesis of circadian rhythm disturbance after HIBD.Methods: In order to analyze the distribution, the expression of mi R-182 in different tissues(lung, intestine, stomach, kidney, cerebral cortex, pineal) was detected by quantitative real-time PCR(RT-PCR). 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-PCR analysis was performed to measure the expression profiles of miR-182 and the targeted gene Clock mRNA, and Western blot was used to measure the Clock protein in the pineal gland at 0, 24, 48 and 72 h after HIBD. The pineal glands of neonatal rats were removed by sterile surgical. The pinealocytes were isolated by trypsin digestion followed by different speed adhesion. Then the cells were cultured and passaged. The proliferation of cells was measured by CCK-8 at different passages and their morphologic features were observed by the inverted phase contrast microscope. The special 5-HT and MAP-2 immunofluorescence were used to detect the pinealocytes. Pinealocytes were transfected respectively by recombinant lentivirus LV-miR-182 and by recombinant lentivirus LV-miR-182-inhibition in vitro. Cells were harvested after successful transfection. RT-PCR analysis was performed to measure the expression profile of Clock m RNA and Western blot was used to measure the Clock protein in cells.Results: 1、RT-PCR showed that miR-182 was highly expressed in the pineal gland.2、Compared with the sham-operated group, the expression of miR-182 in the HIBDgroup was significantly up-regulated in the pineal gland at 24 h and 48 h after HIBD(P<0.05), while there were no statistically significant differences at 0 h, 72 h(P>0.05).The level of mi R-182 in HIBD began to rise at 24 h and remained till 48 h. Compared with the sham-operated group, Clock mRNA expression in the HIBD group increased at 0 h after HIBD, decreased at 48 h after HIBD and increased at 72 h after HIBD(P<0.05),while there was no statistically significant differences at 24 h(P>0.05).3、The level of Clock protein in HIBD group decreased that of the sham-operated group at 0 h and surpassed that of the sham-operated group at 72 h(P<0.05), while there were no statistically significant differences at 24 h, 48 h(P>0.05).4 、 We obtained high-purity pinealocytes using the combination of trypsin, the different speed adhesion process and DMEM/F12 culture medium effectively and reliablely.5、Compared with the control group: Clock mRNA expressions in the experimental group were no statistically significant differences when miR-182 was over-expressed or silenced in pineal cells(P > 0.05). Compared with the control group: the level of Clock protein decreased in the experimental group when miR-182 was over-expressed(P<0.05),and the level of Clock protein increased in the experimental group when miR-182 was silenced(P<0.05).Conclusion: miR-182 is a post-transcriptional regulator that regulate Clock gene in the pineal. The results indicate that mi R-182 may take part in the pathogenesis of circadian rhythm disturbance after HIBD by binding to Clock.