| Background and purpose:This study conducted in-depth research at the clinical,in vitro cell,and animal model experimental levels to explore the role and molecular mechanism of RBPMS-AS1 and its m6 A modification in GBM radiotherapy sensitivity,providing new research ideas and strategies for the treatment of GBM patients.Methods:1.The expression levels of RBPMS-AS1 and CAMTA1 were detected in GBM and normal brain tissues and cells.Plasmids overexpressing RBPMS-AS1 or CAMTA1 were constructed and transfected into GBM cells,which were then treated with different doses of X-ray irradiation.The vitality,proliferation ability and apoptosis of each group of cells were detected by MTT assay,clone formation assay and flow cytometry.2.Mutant and wild-type sequences of miR-301a-3p,RBPMS-AS1 and CAMTA1 were designed,as well as miR-301a-3p mimics,which were transfected separately or co-transfected into GBM cells to construct cell models.The ce RNA mechanism between miR-301a-3p and RBPMS-AS1 was verified by luciferase reporter gene assay and RNA pull-down assay.q RT-PCR and Western Blot were used to detect the expression of each group to verify the interaction between miR-301a-3p,RBPMS-AS1 and CAMTA1.MTT assay,clone formation experiment and flow cytometry were used to detect the effect of the RBPMS-AS1/miR-301a-3p/CAMTA1 axis on the radiosensitivity of GBM cells and its effect on downstream gene NRGN.3.A nude mouse intracranial GBM transplantation model was constructed to verify the effect of RBPMS-AS1 on GBM radiosensitivity in vivo using live imaging technology and immunohistochemistry.4.The expression levels of m6 A in RBPMS-AS1 in GBM and brain tissues were detected by Me-RIP method.The expression level of METTL3 was detected in GBM and normal brain tissues and cells.METTL3 knockdown vector sh-METTL3 was constructed and transfected into GBM cells.RNA immunoprecipitation(RIP),Me-RIP and radiolabeled streptomycin D experiments were used to verify the m6 A modification effect of METTL3 and YTHDF2 on RBPMS-AS1.MTT assay,clone formation experiment and flow cytometry were used to evaluate the effect of METTL3 regulating m6 A modification of RBPMS-AS1 on the biological characteristics of GBM and radiosensitivity.Results:1.Compared with normal brain tissues and cells,the expression levels of RBPMS-AS1 and CAMTA1 in GBM tissues and cells were significantly down-regulated(p<0.01).The overexpression of RBPMS-AS1 or CAMTA1 resulted in a significant decrease in cell vitality and proliferation ability(p<0.01),a significant increase in apoptosis rate(p<0.01),and a significant enhancement of radiosensitivity(p<0.01).2.The luciferase reporter gene assay showed that the fluorescence activity of RBPMS-AS1-WT(or CAMTA1-WT)+ miR-301a-3p-mimic group was significantly lower than that of mimic-NC group(p<0.01).There was no significant difference in fluorescence activity between RBPMS-AS1-Mut(or CAMTA1-Mut)group and mimic-NC group(p>0.05).RNA pull-down assay showed that the enrichment of RBPMS-AS1 and CAMTA1 in miR-301a-3p-WT group was significantly increased compared with that in miR-301a-3p-MUT group(p<0.001).The expression of CAMTA1 in miR-301a-3p mimic group was significantly lower than that in mimic-NC group(p<0.01),while there was no significant difference in the expression of CAMTA1 between miR-301a-3p-mimic+ pc DNA3.1-RBPMS-AS1 group and mimic-NC group(p>0.05).Compared with mimic NC group,the radiosensitivity of cells in miR-301a-3p mimic group was significantly reduced(p<0.05).Compared with miR-301a-3p mimic group,the radiosensitivity of GBM cells was significantly enhanced in miR-301a-3p mimic+ pc DNA3.1-RBPMS-AS1 group and miR-301a-3p mimic+ pc DNA3.1-CAMTA1group(p<0.05).Compared with pc DNA3.1 group,the expression level of NRGN in pc DNA3.1-RBPMS-AS1 group was significantly increased(p<0.01).Compared with pc DNA3.1-RBPMS-AS1 group,the expression of NRGN in pc DNA3.1-RBPMS-AS1+miR-301a-3p mimic group was significantly decreased(p<0.01).3.The in vivo imaging results showed that the fluorescence intensity of Vector-non-IR group was significantly higher than that of RBPMS-AS1-non-IR group(p<0.01),while the fluorescence intensity of Vector-IR group was significantly higher than that of RBPMS-AS1-IR group(p<0.01).Immunohistochemical results showed that the positive expression of Ki67 in Vector-IR group was significantly stronger than that in RBPMS-AS1-IR group(p<0.01),while the positive expression of Ki67 in Vector-non-IR group was significantly stronger than that in Vector-IR group(p<0.01).4.Compared with normal brain tissues,the m6 A modification level of RBPMS-AS1 in GBM was significantly up-regulated(p<0.05).METTL3 was significantly up-regulated in GBM tissues and cells compared with normal brain tissues and cells(p<0.001).RIP assay showed that the enrichment level of RBPMS-AS1 in YTHDF2 group was significantly increased compared with Ig G group and YTHDF1 group(p<0.05).Me-RIP assay showed that the m6 A modification level of RBPMS-AS1 in sh-METTL3 group cells was significantly decreased compared with sh-NC group cells(p<0.05).Radiosensitivity D assay showed that the stability of RBPMS-AS1 in sh-METTL3 group was significantly increased compared with sh-NC group(p<0.05).Compared with sh-NC group,the cell vitality and proliferation ability of sh-METTL3 group were significantly decreased(p<0.05),apoptosis rate was significantly increased(p<0.05),and radiosensitivity was significantly enhanced(p<0.05).Compared with sh-METTL3+sh-NC group,the cell vitality and proliferation ability of sh-METTL3+sh-RBPMS-AS1 group were significantly increased(p<0.05),apoptosis rate was significantly decreased(p<0.05),and radiosensitivity was significantly reduced(p<0.05).Conclusions:1.RBPMS-AS1 inhibits the proliferation of GBM cells and enhances radiotherapy sensitivity through the miR-301a-3p/CAMTA1/NRGN axis.2.In nude mice,RBPMS-AS1 significantly suppresses the proliferation and tumor growth of GBM cells while enhancing radiotherapy sensitivity.3.METTL3 suppresses the expression of RBPMS-AS1 by regulating its m6 A modification,thereby promoting GBM cell proliferation and radiotherapy resistance.YTHDF2,as a reader protein,can recognize and participate in the METTL3-mediated m6 A modification of RBPMS-AS1. |
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