Combination With TMZ And MiR-505 Inhibits The Development Of Glioblastoma By Regulating The WNT7B/Wnt/?-Catenin Signaling Pathway

[Objective] As well konwn,high grade gliomas are the most malignant and common primary brain tumors for adults.Among them,grade IV glioma,also known as glioblastoma(GBM),presents with devastating clinical progression,short survival,and poor quality of life.Temozolomide(TMZ)is the most successful chemotherapeutic drug for glioblastoma,but many GBM patients do not respond to TMZ-based chemotherapy.Mi RNAs are involved in a variety of biological processes through the regulation of target genes,including cell proliferation,cell differentiation,cell migration,invasion,autophagy and so on.Dysregulation in miRNA have been observed in many tumors,including glioblastoma.It has been reported that some specific miRNAs are dysregulated in GBM and are involved in the regulation of chemotherapy or radiotherapy.However,the role of miR-505 in GBM tumorigenesis and chemosensitivity has not been reported.Therefore,this study aims to explore the molecular mechanism that miR-505 plays an extremely important role in the occurrence and progression of GBM,find a new miRNA that can promote the sensitivity of GBM patients to TMZ and develop an effective treatment strategy for GBM.[Methods] Firstly,RT-q PCR assay was used to detect the expression of miR-505 in different grade tissues of GBM patients and normal brain tissues,the expression level of miR-505 in different GBM cell lines and the effect of TMZ treatment on the expression of miR-505.Survival rates of GBM patients with different miR-505 expression levels were analyzed by Kaplan-Meier survival curves.Second,we examined the effects of miR-505 on the proliferation and migration of GBM cells by CKK-8 assay,colony formation assay,Ed U incorporation assay,cell cycle assay,apoptosis assay,Western blot and Transwell migration assay.Then,we predicted the candidate target genes of miR-505 by bioinformatics software and verified the targeting effect of miR-505 on WNT7 B by EGFP fluorescent reporter vector,RT-q PCR and Western blot.The expression of WNT7 B in different grade of GBM patients and normal brain tissues was detected by RT-q PCR.Pearson correlation analysis was used to further verify the correlation between tissue levels of miR-505 and WNT7 B.Next,we examined the expression of WNT7 B in different GBM cell lines by RT-q PCR,Western blot,CKK-8 assay,colony formation assay,cell cycle assay,apoptosis assay and Transwell migration assay and the effect of WNT7 B on GBM cell proliferation and migration,and through functional rescue experiments to verify whether the biological function of miR-505 through its regulation of WNT7 B.Finally,we examined the effects of miR-505 on Wnt /?-catenin signaling pathway activity and its downstream molecules by immunofluorescence cell staining,Western blot and Top-Flash / Fop-Flash dual luciferase reporter assay.The effects of miR-505 and WNT7 B on the growth of GBM tumor were studied by tumor xenograft model,Western blot and immunohistochemical staining.[Results] The expression level of miR-505 is negatively correlated with the malignancy of GBM,and TMZ can promote the expression of miR-505 in GBM.miR-505 inhibits the viability and proliferation of GBM cells by inhibiting the transformation of cells from the G0/G1 phase to the S phase and promoting apoptosis.miR-505 inhibits the migration of GBM cells by inhibiting EMT transformation.The combination of TMZ and pri-miR-505 enhanced the tumor suppressor effect of miR-505.miR-505 targets WNT7 B directly and down-regulates WNT7 B expression at the m RNA and protein levels.In addition,miR-505 was negatively correlated with WNT7 B expression at the tissue level.WNT7 B can accelerate the viability and proliferation of GBM cells by accelerating cell cycle progression and inhibiting apoptosis.WNT7 B promotes the migration of GBM cells by promoting the EMT pathway.Meanwhile,functional rescue experiments also confirmed that WNT7 B is a functional target gene of miR-505,and the inhibitory effect of miR-505 on the malignant phenotype of GBM cells is mediated by WNT7 B.miR-505 can inactivate Wnt/?-catenin signaling pathway by downregulating the expression of Wnt7 B and inhibiting the entry of ?-catenin into the nucleus,thereby inhibiting the expression of downstream effector molecules of Wnt signaling pathway.miR-505 inhibits the growth and proliferation of GBM tumor cells in vivo by suppressing the expression of Wnt7 B and ?-catenin,thereby inhibiting the Wnt /?-catenin pathway and its downstream.[Conclusion] miR-505 inhibits tumorigenesis as a tumor suppressor in glioblastoma.In addition,TMZ can increase the expression level of miR-505 and the combination with miR-505 promoting miR-505-mediated inhibition in GBM cells.Moreover,miR-505 directly targets and inhibits WNT7 B,inactivating the Wnt/ ?-catenin pathway.In conclusion,induction of miR-505 in combination with TMZ may be a useful therapeutic strategy to inhibit GBM.