The Effects Of MiRNA-338-3p On Malignant Glioma Cell Behavior Via Regulation Of MYT1L Expression

Background and Objective: Gliomas are the primary central nervous system tumors with the highest degree of malignancy and worst prognoses.Uncontrolled proliferation,biological heterogeneity,and invasiveness are the main drivers of poor prognosis in high-grade glioma patients.Sequencing techniques which can classify the degree of malignancy of gliomas at the molecular level have been rapidly developed and provide novel opportunities for advancing treatment.micro RNAs(miRNAs)are abnormally expressed in gliomas and play a key regulatory role in cell proliferation,invasion,metastasis,apoptosis,and other malignant biological processes—making them strong potential biomarkers for glioma grading and prognosis.Here,we first screened for differentially expressed genes and miRNAs in glioblastoma and paratumoral tissue with highthroughput second-generation sequencing.After bioinformatic analysis,we found that miRNA-338-3p,as well as its downstream target MYT1 L,may play important regulatory roles in the malignant biological behavior of gliomas.Thus,the overall purpose of our study was to explore the role of miRNA-338-3p in glioma cells,and to determine whether it could regulate MYT1 L to mediate malignant biological behavior,providing theoretical support in our search for potential molecular biomarkers and therapeutic targets in glioma patients.Content and methods:1.Identification of differentially expressed miRNAs and differentially expressed genes.(1)miRNA expression profile dataset(GSE90603)and transcriptome profile dataset(GSE90598)were downloaded from combined miRNAmRNA microarray chips in the Gene Expression Omnibus(GEO)database.(2)The expression of miRNA and mRNA were detected using the platform GPL21582 and GPL17692;Differentially expressed miRNAs and differentially expressed genes were identified using the R limma software package.(3)Fun Rich was used to perform functional enrichment analysis;miRNAs and their target genes regulatory network was constructed and visualized by Cytoscape 3.6.1 software.2.The regulatory relationship between miRNA-338-3p and MYT1 L in glioma cells.(1)Dual-luciferase reporter assays were used to verify the targeting of miRNA-338-3p to MYT1 L 3’-UTR.(2)RNA extraction and quantitative real time-PCR(qRT-PCR)and Western blot assays(Wb)were used to evaluate changes in MYT1 L mRNA and MYT1 L protein expression after overexpression or knockdown of miRNA-338-3p in glioma cells.3.Effect of miRNA-338-3p targeting MYT1L on biological behavior of glioma cells.(1)Edu experiments were used to verify the effects of miRNA-338-3p and MYT1 L on the proliferation of glioma cells,and rescue experiments were used to verify that miRNA-338-3p targeting of MYT1 L mediated glioma cells proliferation.(2)Flow cytometry and Hoechst/PI staining were used to verify the effects of miRNA-338-3p and MYT1 L on glioma cells apoptosis,and rescue experiments were used to verify miRNA-338-3p targeting in MYT1L-mediated glioma cells apoptosis.(3)The effects of miRNA-338-3p and MYT1L on glioma cells invasion and migration were verified with Transwell chamber invasion assays and scratch assays,and the effects of miRNA-338-3p targeting of MYT1 L on glioma cells regulation were verified with rescue assays.Results:1.miRNA-338-3p and its downstream MYT1 L may play an important regulatory role in glioma cells.59 differentially expressed miRNAs and 419 differentially expressed genes were identified using the R limma software package.All 59 DEMs were selected for the potential target genes(PTG)prediction.We then took the overlapping gene intersection of PTGs and DEGs,and analyzed the correlation of downregulated miRNAs with upregulated genes,upregulated miRNAs,and downregulated genes.There were 9 core DEMs(miRNA-338-3p,miRNA-137,miRNA-128-3p,miRNA-218-5p,miRNA-7-5p,miRNA-139-5p,miRNA-124-3p,miRNA-383-5p,miRNA-138-5p)and 129 overlapping genes with a targeted relationship.Further analysis revealed that miRNA-338-3p and MYT1 L not only have significant differential expression in glioma tissues and peritumor brain tissue,but also have a targeted regulatory relationship with MYT1 L.2.miRNA-338-3p is an upstream regulator of MYT1 L,and can directly target and negatively regulate MYT1 L expression.U251 and U87 cell lines were transfected with miRNA-338-3p mimic or inhibitor respectively to overexpress or knockdown miRNA-338-3p.RT-PCR verified the efficacy of transfection.We found that the relative expression of miRNA-338-3p in U251 and U87 cells was significantly increased after transfection with the miRNA338-3p mimic(P<0.05).Conversely,miRNA-338–3p inhibitor significantly downregulated miRNA-338-3p expression compared with the control group(P<0.05).The results of dual-luciferase reporter assay showed that upregulation of miRNA-338-3p could significantly reduce the luciferase of MYT1 L 3′UTR-Wild-type(WT)group(P<0.05),while there was no significant change in the luciferase activity of the mutant(MUT)(P>0.05),indicating that miRNA-338-3p could specifically bind to the 3′UTR region of the target gene MYT1 L and negatively regulate the expression of MYT1 L.RT-PCR and western blot analyses were used to confirm the regulation of MYT1 L by miRNA-338-3p.Results suggested that MYT1 L mRNA level or protein level were significantly decreased or increased after transfection with the miRNA-338-3p mimic or miRNA-338–3p inhibitor compared with the control and mimic NC group(P<0.05).All these findings suggest that miRNA-338-3p negatively regulates the expression of MYT1 L.3.miRNA-338-3p can also down-regulate the expression of MYT1 L and inhibit the malignant biological behavior of glioma cells.We used MYT1 L siRNA to downregulate MYT1L in U251 and U87cells and then chose the best interference efficiency siRNA-002 for further research(P<0.05).Edu assay showed that miRNA-338-3p inhibited the proliferation of glioma cells(P<0.05),MYT1 L overexpression promoted the proliferation of glioma cells(P<0.05);Rescue experiments showed that miRNA-338-3p targeted MYT1 L and inhibited glioma cell proliferation.The results of flow cytometry analysis and Hoechst/PI staining showed that the upregulation of miRNA-338-3p promoted the apoptosis of glioma cells(P<0.05),while MYT1L inhibited glioma cell apoptosis(P<0.05);Rescue experiments showed that the apoptosis of glioma cells was promoted by down-regulating the downstream gene MYT1 L.The results of Transwell assay and the wound-healing assay showed that miRNA-338-3p could inhibit the invasion and migration of glioma cells(P<0.05),while MYT1L overexpression promoted cell invasion and migration(P<0.05);Rescue experiments showed that miRNA-338-3p targeted MYT1 L to inhibit glioma cell invasion and migration.Conclusions:miRNA-338-3p affected the malignant biological behavior of glioma cells by negatively regulating MYT1 L expression levels,and are promising molecular targets for determining glioma prognosis and treatment.