The 1optimal 1protocol 1of 1miR-21 Transfected Into Neurons And The Mechanism Of Neuroprotective Effect In The Experimental Traumatic Brain Injury In Vitro

Aims: The study firstly aimed to establish a protocol to transfect miR-21 Agomir into neurons under different conditions in vitro, including different Serum-free medium,pretreatments of transfection and reagent concentrations, by evaluating the expression level of miR-21 and neurons injury, and was then designed to investigate whether miR-21 had the function of anti-apoptosis in experimental TBI model in vitro and to explore the possible regulatory mechanism of miR-21 on neuronal apoptosis,which could promote the further study and provide insights into developing a therapeutic approach gene therapy on TBI.Methods: Neurons were obtained from neonatal Wistar rats(<24h) and were cultured for 7 days. Labeling of the miR-21 agomir with a 5’ FAM oligonucleotide(FAM-miR-21) used to confirm whether miR-21 could transfected into neurons. Neurons randomly divided into 5 groups: starvation + DMEM(S-DMEM), unstavation + DMEM(N-DMEM), starvation + Neurobasal-A(S-Neurobasal), unstavation + Neurobasal-A(N-Neurobasal) and sham group(Sham). miRNA oligomers(20 mM, 2.5 μl) were combined with RNAiMAX(0.5/1/1.5/2/2.5 μl), and overall transfection solution reached to 500 μl(N-DMEM/Neurobasal-A) and 250 μl(S-DMEM/Neurobasal-A). Before transfection, neurons were treated with 250 μl DMEM/Neurobasal-A for 1 h in S-DMEM/Neurobasal-A. Added transfection solution to 6-well plates and maintained up to 6 h. Neurons were further cultured for 48 h prior to analysis. The expressions level of miR-21 were analyzed by RT-PCR and the function of miR-21 was detected the protein level of PTEN and PDCD4.The neurons injury was detected by TUNEL and MTT assay(To eliminate the biological effect of miR-21, miR-21 negative control(miR-21NC) was transfected into neurons).According to the trasfection protocol above, FAM-miR-21 used to tested the transfection efficiency. Then neurons were randomly assigned into 5 groups: Untreated Cell(UC), TBI treated(TBI-T), TBI+miR-21 agomir(TBI+A), TBI+miR-21 antagomir(TBI+AN) and TBI+miR-21 negative control(TBI+NC). miR-21 agomir, antagomir and negative control were transfected into the cortical neurons and After 24 h translation, neurons were injured. The expression levels of miR-21 and PTEN were analyzed by qRT-PCR after 48 h scratch injury. And neuronal apoptosis was detected by double immunofluorescence staining for MAP-2 and TUENL was performed after 48 h scratch injury. Western Blot detected the protein levels of PTEN, Total Akt, p-Akt, caspase-9, cleavaged caspase-3, Bcl-2 and Bax.Results: FAM-miR-21 could be transfected into neurons. The expression levels of miR-21 were higher in neurons transfected by Neurobasal-A than DMEM and the expression levels of mi-21 already attached to highest at ratios RNAiMAX: miR-21=3:5, but the increasing of RNAiMAX’s concentration had not caused the further up-regulation of expression level of miR-21. Neurons injury was condition-dependent and dose-dependent after transfection. Compared to S-Neurobasasl groups, Neurons have a smaller injury in N-Neurobasasl groups and compared to ratios RNAiMAX: miR-21=4/5:5, neurons injury was smaller at ratios of RNAiMAX: miR-21=1/2/3:5. The following study showed that the scratch cell injury was performed to mimic TBI-induced apoptosis in neurons, and miR-21 agomir/antagomir was transfected to up-/down-regulate the miR-21 level. Our data suggested that miR-21 can reduce the number of TUNEL-positive neurons. Meanwhile, miR-21 decreased the expression level of PTEN, and increased the phosphorylation of Akt significantly. In neurons transfected with miR-21 agomir, the expression of Bcl-2 was promoted while the caspase-3, caspase-9 and bax level were down-regulated, which are crucially the downstream apoptosis-related proteins of PTEN-Akt signaling pathway.Conclusion: The protocol of miR-21 transfected into neurons was RNAiMAX/miR-21 agomir complexes diluting in Neurobasal-A at ratio of RNAiMAX: miR-21=3:5 without pretreatment of starvation and miR-21 can exert the function of reducing neuronal apoptosis through activating the PTEN-Akt signaling pathway and regulating the expression levels of caspase-9、cleavaged caspase-3、Bcl-2 and Bax in vitro.