The Experimental Research Of The Mechanism Of MiR-34a-5p In MLN8237-induced Senescence Of Neuroblastoma Cells

Research background and purpose: Neuroblastoma is the most common extracranial solid tumor in children.The prognosis of high-risk neuroblastoma is very poor.Increasing the intensity of chemotherapy can’t improve long-term survival rate,and it will increase more serious side effects at the same time,affecting children’s quality of life.Cell senescence is a state of the cell cycle arrest.The senescence of tumor cells can temporarily inhibit the development of tumors to a certain extent.Moreover,the dose of the drug that induces cell senescence is much lower than the dose required to induce cell apoptosis,which can also reduce drug-related side effects.Mi R-34a-5p is closely related to cell senescence,and it can cause cell senescence in a variety of tumors,and it is rarely reported in neuroblastoma.This study focused on the role and mechanism of MiR-34a-5p in the senescence of neuroblastoma cells induced by the small molecule AURKA inhibitor MLN8237.Methods: The neuroblastoma cell line(SK-N-SH)was used as the research object.The changes in cell morphology and volume after MLN8237 treatment and MiR-34a-5p transfection were observed under a microscope.Senescence associated-β-galactosidase(SA-β-gal)staining method was used to detect cell senescence,and flow cytometry was used to detect cell cycle.The changes in cell proliferation under various treatments were examined by CCK-8.The MiR-34a-5p mimic and MiR-34a-5p inhibitor were transfected into cells to change the expression level of MiR-34a-5p.Western Blot method was used to detect the expression of related proteins in cells after MLN8237 treatment or MiR-34a-5p transfection,and the expression of related genes was detected by RT-PCR.Results: 1.After the neuroblastoma SK-N-SH cells were treated by the 0.5μmol/L MLN8237 for 72 hours,the CCK-8 results indicated that the cell proliferation was significantly suppressed.The cell volume was observed to increase under the microscope,and the cells were stained blue significantly by SA-β-gal.The analysis of flow cytometry demonstrated that MLN8237 caused significant G2 / M phase arrest,indicating that MLN8237 could induce SK-N-SH cell senescence.2.Proteins were collected at different time points after MLN8237 treatment.The Western blot results showed that MLN82327 could inhibit the activity of AURKA by inhibiting its phosphorylation.In the cell senescence induced by MLN8237,the PTEN / P27 and RB / P16 pathway proteins were not significantly up-regulated,but it caused the up-regulation of P53,P21 proteins and mRNA expression,and activated the P53 / P21 senescence pathway.3.RT-PCR showed that MiR-34a-5p expression was increased during the cell senescence induced by MLN8237,and the expression of SIRT1,the target anti-senescence gene of MiR-34a-5p,was decreased.After MiR-34a-5p was overexpressed in SK-N-SH,β-galactosidase staining showed that the cell volume increased and the cells were stained blue significantly,and flow cytometry analysis demonstrated the overexpression of MiR-34a-5p increased the cells in G2 / M phase.These results indicated that MiR-34a-5p could induce SK-N-SH cell senescence.4.In SK-N-SH,the expression level of MiR-34a-5p was changed by cell transfection,which could regulate the expression of senescence-related target gene SIRT1 at the mRNA and protein levels.The overexpression of MiR-34a-5p up-regulated the expression of P53 and P21 and activated the P53 / P21 senescence pathway,while the PTEN / P27 and RB / P16 pathway proteins were not up-regulated.And the inhibition of MiR-34a-5p could down-regulate the expression of P53 and P21.5.The combination of MiR-34a-5p inhibitor and MLN8237,compared with MLN8237 alone,reduced the expression of P53,P21 and antagonized the activation of P53 / P21 pathway caused by MLN8237;β-galactosidase staining showed the combination attenuated the cell senescence caused by MLN8237.The CCK-8 results also indicated that the combination of both could alleviate the inhibitory effect on cell proliferation of MLN8237.RT-PCR demonstrated that the overexpression of MiR-34a-5p in combination with MLN8237 increased the expression of P53 and P21 more significantly than MLN8237 alone,and the CCK-8 results also showed a synergistic effect of inhibiting cell proliferation after the combination.This revealed that MLN8237 induced SK-N-SH cells senescence by up-regulating MiR-34a-5p.Conclusions: In neuroblastoma SK-N-SH cells,MLN8237 activated the P53 / P21 senescence pathway.The up-regulation of P53 increased the expression of MiR-34a-5p,which caused the down-regulation of the target anti-senescence gene SIRT1,and the decrease of SIRT1 could reduce the degradation of P53.So a feedback loop was formed to participate in cell senescence induced by MLN8237,and the combination of MiR-34a-5p and MLN8237 could enhance the inhibitory effect on neuroblastoma.