A Study On MicroRNA-34B-5P Regulating Apoptosis Of Hippocampal Astrocytes After Developing Seizures And Its Mechanism

PART ONE Analysis of differential expression profiles of hippocampus miRNA in developing brain injury induced by seizuresObjective To explore the microRNA(miRNA) differential expression profile in hippocampus of developing brain injury induced by seizures so as to provide the evidence that miRNAs are involved in the molecular pathogensis of developing brain injury induced by seizures.Methods16healthy and clean postnatal Sprague-Dawley rats of20-day old were randomly divided into a normal control group and a seizure group, each group contained8rats. Seizure model of rats were induced by inhalant flurothyl daily in6consecutive days, and the control group was treated in the same as seizure group except flurothyl inhalation. Rats were decapitated at1day in each group after recurrent seizures, and total RNA was extracted from hippocampus to do reverse transcription. TaqMan(?)MicroRNA Array was used to select the differential expressed hippocampus miRNAs of rats between the seizure group and the control group. Some differential expressed miRNAs were verified using Real-time RT-PCR.Results By using TaqMan(?) MicroRNA Array,30miRNAs were found to be differentially expressed between the seizure group and the control group, with9miRNAs up-regulated, while21miRNAs down-regulated respectively, whose difference was more than2folds. MiRNAs which had significant increase included miR-34b-5p, miR-204, etc, miR-582-3p, miR-672and so on expressed significiant reduction. Differential expression levels of miR-34b-5p, miR-204, miR-582-3p, miR-672found with miRNA microarray are validated by using real time RT-PCR, indicating the microarray results were reliable. MiR-34b-5p, miR-204and other miRNAs were involved in the processes of developing brain injury induced by seizures.Conclusion The expression of MiR-34b-5p, miR-204and other miRNAs had significant differences in the hippocampus between the seizure group and the control group, suggesting that they may be involved in the molecular mechanisms of developing brain injury induced by seizures. Part Two Dynamic expression and significance of microRNA-34b-5p and apoptosis-related proteins Bcl-2, Bax, caspase-3in the hippocampus after seizuresObjective To study the dynamic expressions of miR-34b-5p and apoptosis-related proteins Bcl-2, Bax, caspase-3in the hippocampus of developing rats with recurrent seizures, and to explore the role of miR-34b-5p on the apoptosis of hippocampal nerve cells in developing brain injury after suffering from seizures.Methods96healthy20-day-old Sprague-Dawley (SD) rats were randomly divided into two groups:the control group and the seizure group. Seizure model of rats were induced by inhalant flurothyl daily in6consecutive days. Real time RT-PCR was used to detect the expression of miR-34b-5p mRNA in the hippocampus of rats at2hrs,6hrs,1d,3ds and7ds after recurrent seizures, Western Blot detected the expression of Bcl-2, Bax and Caspase-3proteins of hippocampus.Results Real time RT-PCR showed the expression of miR-34b-5p in the hippocampus of seizure group was significantly higher than that in the control group at2hrs,6hrs and1d (P<0.01), and gradually returned to normal at3ds and7ds, it was no significant difference between the control group and the seizure group (P>0.05).(2) Western blot showed Bcl-2protein level in the hippocampus of seizure group had no significant change (P>0.05) at2hrs, but was significantly lower than that in the control group at6hrs,1d,3ds and7ds (P<0.01), and decreased the lowest at7ds. While the expression of Bax and Caspase-3(19kD/17kD) proteins showed the opposite trend after recurrent seizures, they were significantly higher than those in the control group at2hrs6hrs, Id,3ds and7ds (P<0.01), and up to peak at post-seizure1day. The ratio changes of Bax/Bcl-2in seizure group were significantly higher than those in control group at different time points (P<0.01).Conclusion The expression of miR-34b-5p in rat hippocampus was enhanced during early period after recurrent seizures, accompanied by increased levels of Bax protein and Bax/Bcl-2, activated Caspase-3and decreased levels of Bcl-2protein, suggesting that miR-34b-5p may play an important role in the apoptosis of hippocampal nerve cells in the developing brain injury induced by seizures. Part Three Prediction and validation of the target gene of microRNA-34b-5pObjective To predict and validate the target gene of miR-34b-5p, to know more about the biological function of miR-34b-5p and its mechanisms in developing brain injury induced by seizures.Methods Predict the target gene of miR-34b-5p by using multiple miRNA target prediction software tools.3’-UTR luciferase reporter gene vectors of the wild-type target gene and its mutant were constructed, and these two reporter vectors were cotransfected to hippocampus astrocytes together with miR-34b-5p mimics or miR-34b-5p negative control (miR-34b-5p m NC) respectively, the activity of luciferase was detected by Dual Lueiferase Reporter Assay so as to verify whether miR-34b-5p targets to this gene or not.Results (1) Bcl-2is the target gene of miR-34b-5p predicted by miRNA target prediction software tools.(2) Compared with the vehicle control group and the miR-34b-5p m NC, the luciferase activity of hippocampal astrocytes, which were cotransfected with miR-34b-5p mimics and wild-type Bcl-23’-UTR, decreased significantly, while the luciferase activity of hippocampal astrocytes, which was transfected with mutant Bcl-23’-UTR, remained, and confirmed Bcl-2gene is the direct target gene of miR-34b-5p.Conclusion3’-UTR luciferase reporter gene vectors of the wild-type target gene and its mutant were constructed successfully. The fact that Bcl-2is the target gene of miR-34b-5p has been predicted by miRNA target prediction software tools and identified by luciferase reporter gene assay. Part Four The effect of microRNA-34b-5p on apoptosis of hippocampal astrocytes after seizures and its mechanismsObjective To study the effect of miR-34b-5p on apoptosis of hippocampal astrocytes and the possible mechanisms of miR-34b-5p regulating apoptosis of hippocampal astrocytes after developing seizure.Methods (1) Hippocampal astrocytes of rat were cultured primarily, Kainic acid was used to induce cell excitotoxicity, Similar to seizure-induced cell injury. TUNEL staining was used to detect the apoptosis of hippocampal astrocytes induced by the different concentrations of kainic acid so as to determine the concentrations of kainic acid in the following experiments. Real time RT-PCR detected the expression of miR-34b-5p in hippocampal astrocytes, and Western Blot detected the expression of Bcl-2, Bax and Caspase-3proteins.(2) Liposome was used to transiently transfect miR-34b-5p mimics, miR-34b-5p inhibitors and the corresponding negative control (miR-34b-5p m NC, miR-34b-5p i NC) into hippocampal astrocytes, the expression of miR-34b-5p was compared between the transfected and the untransfected cells by qRT-PCR. All kinds of methods were used to detect the changes of apoptosis-related indictors in hippocampal astrocytes after kainic acid-induced seizure. Bcl-2mRNA was detected by qRT-PCR, TUNEL staining detected the apoptosis of hippocampal astrocytes, and Western Blot was used to detect the expression of Bcl-2, Bax and Caspase-3proteins.(3) Liposome was used to transfect control siRNA, Bcl-2siRNA, miR-34b-5p inhibitors or Bcl-2siRNA and miR-34b-5p inhibitors into hippocampal astrocytes, Western Blot detected the expression of Caspase-3protein.Results (1) The apoptosis of hippocampal astrocytes significantly increased after kainic acid-induced, as well as the expression of miR-34b-5p, the ratio of Bax/Bcl-2, Bax and Caspase-3(19kD/17kD) proteins, but the expression of Bcl-2protein was inhibited obviously.(2) Compared with the vehicle control group and the miR-34b-5p m NC group, the expression of miR-34b-5p, the ratio of Bax/Bcl-2, Bax and caspase-3(19kD/17kD) proteins significantly increased in hippocampal astrocytes which were transfected with miR-34b-5p mimics, as well as the hippocampal astrocytes apoptosis, whereas the expression of Bcl-2protein decreased obviously. The expression of miR-34b-5p, the ratio of Bax/Bcl-2, Bax and caspase-3(19kD/17kD) protein decreased significantly in hippocampal astrocytes which were transfected with miR-34b-5pinhibitor, as well as the hippocampal astrocytes apoptosis, whereas the expression of Bcl-2protein increased obviously.(3) MiR-34b-5p negatively regulated the expression of Bcl-2protein, but it had no effect on the expression of Bcl-2mRNA.(4) MiR-34b-5p inhibitors significantly decreased the expression of Caspase-3(19kD/17kD) protein, but the expression of Caspase-3 (19kD/17kD) protein in cotransfection with Bcl-2siRNA and miR-34b-5p inhibitors group was significantly higher than that in the miR-34b-5p inhibitors group, showed that Bcl-2siRNA can block the reduction of the expression of Caspase-3(19kD/17kD) protein induced by miR-34b-5p inhibitors.Conclusion (1) In the process of kainic acid-induced apoptosis in hippocampal astrocytes, the expression of miR-34b-5p significantly increased.(2) MiR-34b-5p over-expression and low expression of cell models were successfully constructed. Over-expression of miR-34b-5p can promote kainic acid-induced apoptosis of hippocampal astrocytes, whereas inhibition of miR-34b-5p could impede the process.(3) MiR-34b-5p may regulate the apoptosis of hippocampal astrocytes after developing seizures by suppressing the expression of Bcl-2at the post transcriptional level, then activating Caspase-3and the Caspase signal pathway.