MicroRNA-145 Induce Apoptosis Of Glioma Cells By Targeting BNIP3 And Notch Signaling

Human glioma is the most common intracranial malignant tumor in clinical neurosurgery with highly mortality.In China,glioma is the second incidence of intracranial tumor.In recent years,the incidence is mainly in young adults.Pathological types including oligodendrocytes glioma tumor,medulloblastoma,ependymoma,polymorphous gliomas and astrocyte tumors,etc.The pathogenesis is located in the cerebral hemisphere.According to the histopathological characteristics,the primary glioma was classified as 4 grades,and the level of higher was the increase of malignancy.Low grade glioma includes grade astrocytoma and hair cell type cell tumor,while high grade glioma includes glioblastoma and mesenchymal astrocytoma.Because the pathogenesis of glioma is not clear,the current treatment method is difficult to cure.The main therapy is glioma resection,but they always recur and are ultimately fatal.Therefore,it is one of the focal points of glioma research to find the factors that cause the development of glioma and induce improvement.With the development of molecular biology,the targeted therapy of specific molecules has become a hot spot in the treatment of glioma and achieved some results.MicroRNA(miRNA)play a regulatory role in the process of tumor disease in various tissues and organs.miRNA can regulate various tumor progression related factors,and in turn,the occurrence and development of tumors can affect the expression changes of multiple miRNAs.Recent studies have also found that miR-145 is closely related to the development of glioma,but the role of miR-145 in the apoptosis of glioma cells remains unknown.This study mainly explore the expression of miR-145 in the process of glioma.The main study contents as follows:(1)The expression of BNIP3 and miR-145 in gliomaFirst,we collected glioma samples and normal brain tissue samples,and detected the expression of miR-145 by qRT-PCR.Then,we used stereotaxy intracranial injection of rat C6 cells.Hematoxylin and Eosin(H&E)staining and Magnetic Resonance Imaging(MRI)verified glioma model was successfully established.qRT-PCR showed that miR-145 was reduced in glioma tissue and U87/U251 glioma cell lines.The immunohistochemistry,qRT-PCR and Western blot showed that the expression of BNIP3 was increased.It is showed that miR-145 was decreased while BNIP3 was increased in glioma.(2)The effect of miR-145 in U87/U251 cells apoptosisIn U87/U251 cells,the cell model of miR-145 overexpression was established by mir-145 mimics.The result of Hoechst 33342 staining,Tunel staining,flow cytometry and Western blot showed that overexpression of miR-145 could increase the apoptosis of cells in glioma cells.(3)The effect of BNIP3 in U87/U251 cells apoptosisIn U87/U251 cells,BNIP3 siRNA was used to knockdown the BNIP3 expression.The result of Hoechst 33342 staining,Tunel staining,flow cytometry,and Western blot indicated that deregulation of BNIP3 could result in apoptosis of cells.The results indicated that silencing BNIP3 could increase the apoptosis of glioma cells.(4)The target effect between miR-145 and BNIP3It found that the 3'-UTR of BNIP3 contains the putative binding sites for miR-145.In U87/U251 cells,BNIP3 level was decreased after transfected with miR-145 mimics and the BNIP3 level was increased by miR-145 inhibitor.Then,the 3'-UTR of BNIP3 was cloned into a luciferase expression vector to evaluate its response to miR-145.Co-transfected the luciferase reporter with the miR-145 mimic into U87/U251 cells,and the result showed the 3'-UTR conveyed decreased expression.Moreover,it was found that miR-145 mimics mainly increased the expression of BNIP3 in the nucleus.(5)The effect of miR-145 and BNIP3 in Notch pathwayIn U87/U251 cells,transfected with miR-145 mimics,inhibitor,BNIP3-siRNA,or BNIP3-vetor,western blot detected the expression of Notch1 and downstream target protein Hes1 and P21 in Notch signaling pathway.The results showed that overexpression of miR-145 and siBNIP3 can inhibit the Notch signaling pathway,and suppress miR-145 and overexpression of BNIP3 can promote the Notch signaling pathway.In conclusion,all the dates suggested that miR-145 can regulate Notch signaling pathway by targeting BNIP3 and thus affect the apoptosis of U87/U251 cells.