The Mechanism Of MicroRNA-17-5p In Attenuating The Neuroprotective Effect Of Act A/Smads Signal Loop Through SARA In Cerebral Ischemic Injury

Cerebral ischemia is the leading cause of death and disability,threatening the human health worldwide.Activin A?Act A?,as an endogenous neuroprotective factor,has been reported to ameliorate cerebral ischemic injury through Act A/Smads signal pathway.Our previous study has demonstrated that Act A exists a self-amplification mechanism,and the Act A/Smads signal loop can be rapidly activated to exert neuroprotective effect.However,it was self-limited because of the temporary activation and spontaneous attenuation of Smad anchor for receptor activation?SARA?.microRNA-17-5p?miR-17-5p?is an important member of miR-17 family.It has been reported that miR-17-5p inhibited the expression of SARA in esophageal squamous cell carcinoma.Therefore,we hypothesized that downregulation of miR-17-5p could amplify the neuroprotective effect of Act A/Smad signal loop by regulating the level of SARA.To test our hypothesis,well-differentiated PC12 cells were successfully cultured,and oxygen glucose deprivation?OGD?model was established to simulate the process of cerebral ischemia in vitro.The dynamic changes of miR-17-5p and Act A/Smads signal loop at different times of OGD were detected.Then,well-differentiated PC12 cells were transfected with miR-17-5p mimic or inhibitor,and the activation of Act A was evluated by the level of phosphorylated Smad3?p-Smad3?.The results showed that miR-17-5p activated Act A/Smads signal loop through increasing the level of p-Smad3 and the accumulation of Act A receptors.The luciferase reporter activity verified that SARA is the target gene of miR-17-5p,which may shed light on the endogenous neuroprotective effect of Act A signal pathway.To investigate whether miR-17-5p could serve as a target for promoting Act A/Smads activation,we detected the exprssion of other members of miR-17 family in different time points of OGD,and detected whether SARA was their target gene using the dual luciferase assay system.1.In vitro model of cerebral ischemia?1?ObjectiveTo simulate the process of cerebral ischemia,well-differentiated PC12 cells were successfully cultured,and OGD model was established.?2?MethodsFirstly,the neuronal characteristics of well-differentiated PC12 cells were identified by MAP2 fluorescent staining.Secondly,an OGD model of highly differentiated PC12 cells was constructed by treating with DMEM containing Na₂S₂O₄ and no glucose to establish OGD model.Subsequently,blood gas analysis was used to detect the indicators in the medium at 0 h,1.5 h,3 h,6 h and 12 h of OGD.Finally,CCK-8 and Hoechst 33342 fluorescence staining were used to detect the cell viability and apoptotic cell morphology of OGD at 0 h,1.5 h,3 h,6 h and12 h.?3?ResultsThe immunofluorescence staining showed that PC12 cells are MAP2-positve.Blood gas analysis showed that pO₂ was always 0 mmHg,and there was no difference in pCO₂,Glu,pH,Na⁺,K⁺in the medium with sodium dithionite concentration of 1mM during OGD 0 h to OGD 12 h.After OGD treatment,CCK-8 assay showed the cell viability was decreased,and Hoechst 33342 staining showed the number of apoptotic cells was increased.?4?ConclusionsThe neuronal characteristics of well-differentiated PC12 cells were successfully verified,and in vitro model of cerebral ischemia was successfully established.2.miR-17-5p was involved in the regulation of Act A/Smads signal pathway?1?ObjectiveTo elucidate the dynamic changes of miR-17-5p and Act A/Smads signal loop in ischemic brain injury,miR-17-5p reduces the activation time course and intensity of Act A/Smads signal loop by inhibiting the expression of SARA gene and the regulation of miR-17-5p on the level of apoptosis.?2?MethodsFirst,real-time quantitative PCR was used to detect the dynamic changes of miR-17-5p in OGD injury,and Western blot was used to determine the changes of SARA and the key molecules of Act A/Smads signal loop during OGD injury.Subsequently,well-differentiated PC12 cells were transfected with miR-17-5p mimic,inhibitor and corresponding NC,followed by OGD treatment at 0 h,3 h and 6 h,and the protein levels of SARA and key molecules in Act A/Smads pathway were detected by Western blot.Finally,CCK-8 was used to detect the cell survival rate,and Hoechst33342 fluorescence staining was used to observe the nuclear morphology of apoptosis.?3?ResultsReal-time quantitative PCR showed that the expression of miR-17-5p increased gradually during OGD 0 h to 3 h;peaked at OGD 3 h;decreased after OGD 6 h and12 h,basically no difference between OGD 0 h.Western blot results showed that the expression of SARA protein increased during OGD 0 h to 3 h;peaked at OGD 3 h;OGD decreased at 6 h and 12 h,and there was no difference between OGD 0 h.The protein expression of Act A,p-Smad3 and Smad3 also showed a change in the first rise and then decrease.After transfection of miR-17-5p mimic or inhibitor,Western blot results showed that the protein expression of SARA was significantly down-regulated in well-differentiated PC12 cells transfected with 50 nM miR-17-5p mimic for 24-72 h.The expression gradually recovered with the time extanding.After OGD treatment,the expression of SARA in the mimic group was not different from that in the control group.100 nM miR-17-5p mimic significantly inhibited the expression of SARA,Act A,p-Smad3 and Smad3 at OGD 0 h,3 h,6 h,while 200 nM miR-17-5p inhibitor could make them significantly increased.CCK-8 results showed that there was no statistical difference among mimic,mimic NC and control groups at OGD 0 h,and there was no statistical difference among inhibitor,inhibitor NC and control groups;at OGD 3 h and 6 h,miR-17-5p mimic significantly reduced the survival rate of well-differentiated PC12 cells,and the use of miR-17-5p inhibitor significantly improved the survival rate.Hoechst 33342 fluorescence staining showed that there was no significant difference between the transfected group and the untransfected group at OGD 0 h.After OGD treatment,the number of apoptotic cells in the miR-17-5p mimic group was significantly increased,The number of apoptotic cells in the miR-17-5p inhibitor group was significantly reduced.?4?ConclusionsmiR-17-5p was short-term highly expressed after OGD injury;Act A/Smads signal loop showed short-term activation and self-limiting after OGD injury.miR-17-5p negatively regulated Act A/Smads signal loop activity by inhibiting SARA protein expression.miR-17-5p inhibitor could improve the survival rate of well-differentiated PC12 cells after OGD injury and negatively regulated the level of apoptosis.3.The prediction,identification and validation of miR-17-5p targeting on SARA?1?ObjectiveTo explore the effect of miR-17-5p on SARA and its underlying mechanisms.?2?MethodsTargetScan and miRDB target gene predictive bioinformatics softwares were utilized to predict the interaction between miR-17-5p and SARA.To clarify whether SARA was the targeted gene of miR-17-5p,wild-type and mutant 3'UTR of SARA was inserted into plasmid and then transfected into 293T cells and measured its activity by dual luciferase reporter assay.?3?ResultsThe miRDB and TargetScan software showed that SARA is a target gene of miR-17-5p.Subsequently,sequencing confirmed that the construction of the wild type vectors WT1 and WT2 was in line with expectations.The dual luciferase reporter activity assay showed that miR-17-5p can targetly inhibited SARA expression by binding to the site of?57-64?,?403-409?and?1592-1598?,and synergistic effect was observed by binding to the site of?57-64?and site?403-409?.?4?ConclusionsSARA is a gene-specific target of miR-17-5p.4.The temporal expression of miR-17 family during cerebral ischemic injury in vitro and its targeted regulation of SARA?1?ObjectiveTo explore the temporal expression of miR-17 family during cerebral ischemic injury in vitro and the effect on SARA expression,as well as its underlying mechanisms.?2?MethodsFirstly,Real-time quantitative PCR was utilized to detect the time course expression of miR-17 family in well-differentiated PC12 cells treated with OGD.Secondly,miRDB and TargetScan target gene prediction softwares were used to predict the interaction between miR-17 family and SARA.To clarify whether SARA was the targeted gene of miR-17 family,wild-type and mutant 3'UTR of SARA was inserted into plasmid,and then transfected into 293T cells and measured its activity by dual luciferase reporter assay.?3?ResultsReal-time quantitative PCR showed that the expression of miR-17 family increased immediately after OGD,peaked at 3 h after OGD,and started to decline at 3h after OGD.The results of miRDB and TargetScan indicated that SARA is a direct target of miR-17 family,including miR-20a,miR-20b,miR-93 and miR-106b.The wild type vectors?WT1 and WT2?were constructed successfully.The dual luciferase reporter activity assay showed that both miR-20a-5p and miR-20b-5p were able to inhibit SARA expression alone through the site?57-64?,site?403-409?and site?1592-1598?,and synergistic effect was observed by binding to site?57-64?and?403-409?.miR-93-5p inhibited the expression of SARA through site?403-409?or site?1592-1598?.miR-106b-5p inhibited the expression of SARA only through binding to site?57-64?.?4?ConclusionsmiR-17 family exhibits transient high expression during cerebral ischemic injury in vitro.Members of the miR-17 family are capable of targeted inhibition of SARA.