The Effect And Mechanism Of MRTF-A Regulating MiR-1273g-3p On Autophagy In SH-SY5Y Cells Treated With A?_25-35

Objective:To investigate the effect on regulation of autophagy and molecular mechanism of MRTF-A interaction with miRNAs in SH-SY5 Y cells treated with A?_25-35.Methods:1.Build the SH-SY5 Y cell' model;Screen of MRTF-A-related target miRNAs via Microarray data:Establishment the model of A?_25-35-induced SH-SY5 Y Cells;and overexpression of MRTF-A.The cells were cultured and divided into control group,A?_25-35 group,MRTF-A group,MRTF-A with A?_25-35 group.The cells were transfected MRTF-A plasmid?2.5?g?for 48 h,then treated with 40?mol/L A?_25-35 for 24h.The protein level of MRTF-A was detected by western blot.Then,total RNA was extracted and tested by the Agilent RNA 6000 Nano/Pico Assay method.Using miRNA One Array chip technology to detect significant difference in each group of miRNAs.Then,differentially expressed miRNAs were compared with each other to obtain MRTF-A regulated miRNAs.Finally,miR-1273g-3p was chosen and detected by RT-q PCR assay.2.The effect and mechanism of MRTF-A on autophagy in the model:1)The effect of MRTF-A on autophagy: In the previous,we found that MRTF-A can promote autophagy in AD transgenic mice.In this study,the effect of MRTF-A on autophagy was verified in SH-SY5 Y cells.The cells were cultured and divided into control group,A?_25-35 group,MRTF-A group,MRTF-A with A?_25-35 group.The cells were transfected MRTF-A plasmid?2.5?g?for 48 h,then treated with40?mol/L A?_25-35 for 24 h.The protein was extracted and the expression of autophagy-related genes LC3 and Beclin1 were detected by western blot.2)The relationship of MRTF-A on autophagy with the miR-1273g-3p: The cells were cultured and divided into control group,A?_25-35 group,MRTF-A with A?_25-35 group,MRTF-A and miR-1273g-3p inhibitor control with A?_25-35 group,MRTF-A and miR-1273g-3p inhibitor with A?_25-35 group.The cells were transfected with MRTF-A alone or co-transfected with miR-1273g-3p inhibitor for 48 h,then treated with 40?mol/L A?_25-35 for 24 h.The protein was extracted and the expression of autophagy-related genes LC3 and Beclin1 were detected by western blot.3)The effect of miR-1273g-3p on autophagy.The cells were cultured and divided into miR-1273g-3p mimic control group,miR-1273g-3p mimic group,miR-1273g-3p mimic control with A?_25-35 group,miR-1273g-3p mimic with A?_25-35 group.After treated with miR-1273g-3p for 48 h,the cells were treated with 40?mol/L A?_25-35 for 24 h.The protein levels of LC3 and Beclin1 were detected by Western blot.4)The effect of miR-1273g-3p on target gene m TOR: miR-1273g-3p's target gene was predicted by mirwalk and Targetcan software and m TOR is one of its targets.The cells were cultured and divided into miR-1273g-3p mimic control group,miR-1273g-3p mimic group.RNA and protein were extracted after transfected with miR-1273g-3p mimic for 48 h,and the m RNA and protein level of LC3 and Beclin1 were detected respectively by Western blot.5)The effect of MRTF-A on autophagy is related to the inhibition of m TOR by miR-1273g-3p: the cells were cultured and divided into control group,A?_25-35 group,MRTF-A with A?_25-35 group,MRTF-A and miR-1273g-3p inhibitor control with A?_25-35 group,MRTF-A and miR-1273g-3p inhibitor with A?_25-35 group.After transfected with MRTF-A alone or co-transfected with miR-1273g-3p inhibitor for 48 h,the cells were treated with 40?mol/L A?_25-35 for 24 h.Protein expression level of m TOR were detected by Western blot.Results:1.8 potential significance of miRNAs related with MRTF-A were found by miRNA microarray analysis: Western blot assay showed that the protein level of MRTF-A was significantly decreased in the model treated with 40?mol/L A?_25-35;and transfection of MRTF-A plasmid could increase the expression of MRTF-A.295 miRNAs were with significant difference according to the conditions of absolute value of Fold change greater than or equal to 0.585 and P-value less than 0.05.The upregulated was covered miR-1273g-3p,and the downregulated were composed with hsa-miR-6736-3p,hsa-miR-6740-3p,hsa-miR-7106-3p,hsa-miR-6747-3p,hsa-miR-6776-3p,hsa-miR-3653-5p.RT-q PCR results showed that MRTF-A can increase the expression of miR-1273g-3p,the express level of miR-1273g-3p was consistent with result of miRNA microarray data.2.Both of MRTF-A and miR-1273g-3p can promote the autophagy in SH-SY5 Y cells treated with A?_25-35: Western blot assay showed that overexpression of MRTF-A increased the protein ratio of LC3II/LC3 I and the protein expression of Beclin1 in SH-SY5 Y cells treated with 40?mol/L A?_25-35,indicating that MRTF-A can promote the autophagy.And transfection of miR-1273g-3p mimic also increased the protein ratio of LC3II/LC3 I and the protein expression of Beclin1 in SH-SY5 Y cells treated with 40?mol/L A?_25-35,indicating that miR-1273g-3p can also promote the autophagy.MRTF-A co-transfection with miR-1273g-3p inhibitor decreased the protein ratio of LC3II/LC3 I and the protein expression of Beclin1 compared with single transfection of MRTF-A in SH-SY5 Y cells treated with 40?mol/L A?_25-35,indicating that miR-1273g-3p is relative to the effect of MRTF-A on promoting autophagy.3.The mechanism of MRTF-A promoting autophagy may be through mediating miR-1273g-3p inhibiting its target gene,m TOR: a total of 1090 target genes of miR-1273g-3p were predicted by mirwallk,Targetscan software.m TOR,on of its targets,is the negative regulation gene of autophagy.RT-q PCR and western blot showed that miR-1273g-3p significantly inhibited the m RNA and protein level of m TOR.On the other hand,MRTF-A inhibited m TOR protein level,which was disappeared after co-transfected with miR-1273g-3p inhibitor.It's suggest that the mechanism of MRTF-A promoting autophagy may be that regulating miR-1273g-3p to inhibit its target gene,m TOR.Conclusion:MRTF-A can promote the autophagy in SH-SY5 Y cells treated with A?_25-35,and miR-1273g-3p can also promote the autophagy by inhibiting expression of m TOR in SH-SY5 Y cells treated with A?_25-35.On the other hand,MRTF-A can upregulate expression of miR-1273g-3p.It's proposed that MRTF-A promotes the autophagy in SH-SY5 Y cells with A?_25-35,which may be related to the up-regulation of miR-1273g-3p,thus,to inhibit the expression of m TOR.