MiR-181b-5p Modulates Chemosensitivity Of Glioma Cells To Temozolomide By Targeting Bcl-2

Background: Glioma,is the most common primary central nervous system tumor,accounting for about half of all intracranial primary tumors.It is one of the most lethal types of malignant and aggressive human cancers according to World Health Organization(WHO)classification.However,it has poor prognosis and extremely high mortality due to invasive growth and recurrence of tumor,the median survival of patients(WHO grade IV)is only 14.6-17 months.Current therapies mainly include surgical resection,radiotherapy and chemotherapy.Chemotherapy is the primary postoperative and adjuvant therapy for glioma.Temozolomide(TMZ)is a clinical first-line chemotherapy drug that significantly inhibits glioma cell proliferation and induces apoptosis.Although TMZ is commonly used in glioma patients,the resistance of glioma cells obtained by congenital or acquired glioma cells to TMZ drugs has a poor prognosis.MicroRNAs(miRNAs),small non-coding RNAs that are about 22 nucleotides in length,could causes degradation of target gene mRNA or inhibits its translation by targeting the bases of mRNA 3′-untranslated region(3′-UTR)specificly.It has been documented that some miRNAs are dysregulated in several kinds of cancers and regulate the expression of oncogenes and tumor suppressor genes.The miR-181 family was discovered to play an important role in regulating biological functions in glioma,and miR-181 b is less expressed in human gliomas as a tumor-suppressive miRNA.Objectives: To explore the molecular mechanism of miR-181b-5p and its target geneBcl-2 on modulating TMZ chemosensitivity in glioma cells.Method: 1.Effect of miR-181b-5p on TMZ chemosensitivity of U87 MG and U251 glioma cells was detected by MTT.2.Immunofluorescence was used to analyze the effect of miR-181b-5p,TMZ and the combation of miR-181b-5p and TMZ on Ki67 expression.3.TargetScan was used to predict the downstream target gene of miR-181b-5p,and the target gene was identified by dual-luciferase reporter system.The effect of miR-181b-5p on Bcl-2 expression was analyzed by Q-PCR and Western blotting.4.Effect of miR-181b-5p,TMZ and miR-181b-5p+TMZ on the expression of apoptosis-related protein Bax and cycle-related protein CyclinD1 and CDK4 was analyzed by Western blotting.5.Flow cytometry was used to analyze the effect of miR-181b-5p,TMZ and miR-181b-5p+TMZ on apoptosis and cell cycle.6.Transwell was used to analyze the effect of the combination of miR-181b-5p and TMZ treatment on cell migration and invasion.7.Subcutaneous inoculation method was used to establish human glioma xenograft model in nude mice to detect the effects of miR-181b-5p、TMZ and miR-181b-5p+TMZ on tumor growth.Result: 1.miR-181b-5p can ehhanc the sensitivity of U87 MG and U251 cells to TMZ.2.The signal intensity in Ki67-positive stained cells was significantly lower after treatment with TMZ and miR-181b-5p,and most notably the combination of the two.3.miRNA target prediction showed that Bcl-2 is a potential target gene of miR-181b-5p.Dual-luciferase reporter assay,Real-time PCR and Western blotting proved that miR-181b-5p can target Bcl-2 directly and reduce its expression.4.miR-181b-5p+TMZ has a significant effect on increasing the expression of Bax and promoting the apoptosis of U87 MG cells compared with miR-181b-5p and TMZ alone.5.miR-181b-5p and TMZ can inhibit the migration and invasion of U87 MG cells,and the miR-181b-5p+TMZ group showed the most significant effect.6.miR-181b-5p and TMZ can both downregulate the expression of CyclinD1 and CDK4 and cell cycle was blocked in S phase.miR-181b-5p and TMZ combination treatment has a significant effect.Conclusion: 1.Overexpression of miR-181b-5p can enhance the sensitivity of U87 MGand U251 cells to TMZ.2.Bcl-2 is a direct target gene of miR-181b-5p,and miR-181b-5p can target Bcl-2 to inhibit cell proliferation,migration and invasion,promote apoptosis in U87 MG cells and inhibit tumor growth in vivo.3.The miR-181b-5p+TMZ combination plays a stronger role in the above aspects.