MiR-137 Inhibits Cell Growth By Repression Of SPHK2 In Glioma

Objective:To investigate the role of mi R-137 targeting SPHK2 in the proliferation of glioma cellsMaterials and methods:1.Insituhybridization(ISH)was used to detect the expression of micro RNA-137 in 60 glioma tissues of WHO grade II-IV and 10 non-tumor control brain tissues;immunohistochemistry(IHC)was used to detect the expression of SPHK2,Ki-67 in the above tissues;SPHK2,micro RNA-137 were analyzed.The relationship between SPHK2 and Ki-67 expression level,and the Relationship between the expression level of the above indicators and tumor benign and malignant grade were analyzed to clarify the significance of SPHK2 in auxiliary pathological diagnosis of glioma.2.Western blot was used to detect and compare the expression of SPHK2 in common glioblastoma(GBM)cell lines and human astrocyte lines(HA),and immunofluorescence was used to detect the localization and expression of SPHK2 in U87 and U251 cells.3.Bioinformatics software was used to predict the target genes of micro RNAs-137 and the predicted results were verified by luciferase assay.The effects of micro RNAs-137 on the expression of SPHK2 m RNA and protein were observed in GBM cells transfected with micro RNAs to confirm the regulatory effect and mechanism of micro RNAs on target gene expression.4.The effects of micro RNAs-137 mimics transfection on the proliferation of GBM cells were detected by clone formation assay and Ed U cell proliferation assay,and the effects of micro RNAs on the proliferation of GBM cells were observed by SPHK2 eukaryotic expression plasmidtransfection.Results:1.ISH showed that the positive marker index of micro RNA-137(LI%)in gliomas was significantly lower than that in non-tumorous control brain tissues.IHC showed that SPHK2 and Ki-67 positive marker index(LI%)in gliomas were significantly higher than those in non-tumour control brain tissues.Only Ki-67 increased with the increase of tumour grade,while other indicators showed no significant difference among gliomas of different grades.The correlation analysis showed that there was a significant negative correlation between micro RNA-137 LI% and Ki-67 LI%,while SPHK2 LI% and Ki-67 LI% were positively correlated.ROC curve analysis showed that both mi R-137 and SPHK2 could be used as diagnostic markers for glioma,and mi R-137 was also useful in the diagnosis of high and low grade glioma.2.Only A172 and U118 GBM cell lines showed slightly lower expression of SPHK2 than HA cells,while the expression of SPHK2 in other glioma cell lines was significantly higher than that in HA.Immunofluorescence assay also showed that SPHK2 was highly expressed in U87 and U251 cell lines,and mainly located in the nucleus.3.Bioinformatics analysis and luciferase assay confirmed that SPHK2 gene was the target gene of microRNAs-137 in glioma cells.The results of qRT-PCR and Western blot showed that micro RNAs transfection could decrease the protein level of SPHK2 in GBM cell line,while the m RNA level of SPHK2 remained unchanged,which confirmed that micro RNAs-137 could inhibit the translation of SPHK2.4.It inhibits the expression of target gene encoded protein after transcription.Plate cloning assay and Ed U assay showed that the proliferation of GBM cells was inhibited by microarray-137,and the above effects could be partly reversed by supplementing exogenous SPHK2Conclusion:1.The expression of micro RNA-137 was abnormally decreased and SPK2 was abnormally increased in human glioma cells and the expression levels of mi R-137,SPHK2 are closely related to glioma,which can be used as an important reference index for the diagnosis of glioma,and there is correlation between the above indicators.2.In glioma cells,micro RNAs-137 is an important negative regulator of SPHK2 expression.Abnormal decrease of micro RNAs-137 expression is an important reason for the abnormal over expression of SPHK2 in glioma cells and plays an important role in the regulation of the occurrence,development and proliferation of glioma.