The Mechanism Of MicroRNA-29a-3p In Promoting SH-SY5Y Cell Proliferation And Neurite Outgrowth

Background and aimsNeurogenic bladder(NB)is caused by damage to the central or peripheral nervous system,resulting in bladder storage and voiding dysfunction,bladder detrusor dysfunction or urethral sphincter dysfunction,secondary to a variety of congenital diseases or Acquired injury.nerve" anastomosis surgery can restore the innervation of the bladder and fundamentally treat NB.However,due to the slow rate of nerve regeneration,the amount of nerve regeneration after the nerve anastomosis and the recovery of bladder function were not ideal.MicroRNA(miRNA)was a type of endogenous single-stranded non-coding RNA with regulatory functions found in eukaryotes.The main research on miRNA focused on tumors,but in recent years,studies have found that miRNAs were closely related to the normal function of the nervous system and the occurrence of diseases.In our previous experiments,we used bioinformatics to detect the expression level of miRNA in the regenerated nerve tissue after neural anastomosis,and showed that the expression level of miRNA-29a in the regenerated nerve tissue was the highest,however,its effect on SH-SY5Y human neuroblastoma and its mechanism of action have not been reported yet.miRNA-29a was divided into two types:miRNA-29a-3p and miRNA-29a-5p according to the different processing methods of its precursors,and it mainly existed in the form of miRNA-29a-3p in cells.PTEN gene and PI3K/Akt/mTOR signaling pathway played key biological activities in cell proliferation and regeneration,and studies have reported that miRNA-29a can down-regulate PTEN expression in PC 12 cells.Therefore,miRNA-29a may exert its neurotrophic activity through this signaling pathway.This article aimed to study the effects of miRNA-29a-3p on SH-SY5Y cell proliferation and neurite growth and explore its mechanism of action,so as to provide new targets and molecular mechanisms for the use of miRNA to treat NB.Methods1.SH-SY5Y cells were cultured in vitro and transfected with mimic miRNA-29a-3p and inhibitor miRNA-29a-3p,respectively.The expression levels of miRNA-29a-3p in each group of cells were detected by qPCR to confirm the transfection results.2.QPCR was used to detect the mRNA expression levels of PTEN,Akt and mTOR in each group of cells,and the effect of miRNA-29a-3p on the PTEN gene and PI3K/Akt/mTOR signaling pathway was analyzed at the RNA level.3.Western blotting was used to detect the total protein levels of PTEN,Akt,and mTOR in each group of cells,and the phosphorylation levels of Akt and mTOR proteins were detected respectively,and then p-Akt/Akt and p-mTOR/mTOR were calculated respectively.The effect of miRNA-29a-3p on the total protein level and activation level PTEN and key proteins in the PI3K/Akt/mTOR signaling pathways was analyzed from the protein level.4.The MTT method was used to detect the proliferation activity of each group of cells,and the images of each group of cells were taken by photomicrography and the length of cell neurites was measured using Image-Pro Plus 6.0 software.The results of molecular biology experiments were verified.Results1.The qPCR results showed that the miRNA-29a-3p in the mimic miRNA-29a-3p group was higher than the control group,the inhibitor miRNA-29a-3p group was lower than the control group,and the mimic NC group and inhibitor NC group were similar to the control group,confirming that the transfection was successful.2.The qPCR results showed that miRNA-29a-3p can significantly reduce the expression level of PTEN mRNA in SH-SY5Y cells,while the level of PTEN mRNA increased significantly after miRNA-29a-3p were knocked down;There was no significant difference in the mRNA expression levels of Akt and mTOR in each group of cells.3.Western blotting results showed that the trend of PTEN protein was consistent with that of mRNA,and miRNA-29a-3p could inhibit the expression of PTEN protein;The expression levels of Akt and mTOR proteins were not significantly different among the groups,but the phosphorylation levels of the two proteins were significantly increased in the mimic miRNA-29a-3p group,and significantly decreased in the inhibitor miRNA-29a-3p group;The results of p-Akt/Akt and p-mTOR/mTOR are consistent with the phosphorylation level results.4.The results of MTT experiment showed that the cell proliferation activity of mimic miRNA-29a-3p group was significantly increased,while that of inhibitor miRNA-29a-3p group was significantly decreased;The increased expression of miRNA-29a-3p can promote the growth of neurites in SH-SY5Y cells。Conclusion1.miRNA-29a-3p can enhance the proliferation activity of SH-SY5Y cells and promote the growth of their neurites.2.miRNA-29a-3p can inhibit the mRNA expression of PTEN but does not affect the mRNA expression levels of Akt and mTOR.3.miRNA-29a-3p exerts its neurotrophic effect by inhibiting the expression levels of PTEN gene and protein,and promoting the phosphorylation of Akt and mTOR proteins in the PI3K/Akt/mTOR signaling pathway.