Molecular Mechanisms Of Hsa-circ-001193 Regulating Glioma Cells Biological Behaviors By Targeting MiR-496

Introduction: Glioma is the most common primary malignant tumor in adult central nervous system.It has the characteristics of high recurrence rate,high lethality and poor prognosis.Despite the current domestic and international treatment of glioma with postoperative adjuvant chemotherapy and radiotherapy comprehensive treatment program,but the vast majority of glioma,especially glioblastoma prognosis has not been significantly improved.Therefore,in-depth study of the molecular mechanism of the occurrence and development of glioma is very important for the prevention and treatment of glioma and the survival of patients.Herein,we investigated whether hsa-circ-001193 affected the malignant biological behaviors of glioma cells,and the possible mechanisms.We further aimed to explore whether hsa-circ-001193 could promote RFX3 and DEF6 expression by negatively regulating mi R-496;whether hsa-circ-001193 could promote RFX3 by negatively regulating mi R-496.Methods: Human glioma cell line U87,U251,human astrocytes,and human embryonic kidney HEK293 T were cultured.Real-time PCR were used to detect the expression of hsa-circ-001193,mi R-496.Western blot was used to investigate the expression of RFX3 and DEF6.Cell proliferation was detected by Cell Counting Kit-8.Cell migration and invasion ability was measured using Transwell assay.Cell apoptosis was detected by flow cytometry.Over-expression or inhibition of hsa-circ-001193,mi R-496,RFX3 and DEF6 plasmids were transfected into the cells.The m RNA expression of hsa-circ-001193、mi R-496、RFX3 and DEF6 was measured by Real-time PCR.The expression of RFX3、DEF6、p-PI3 K,PI3K,p-AKT,AKT was determined by Western blot.Regulation of mi R-496 targeting RFX3 were evaluated by luciferase reporter assay.Regulation of mi R-496 targeting hsa-circ-001193 were evaluated by luciferase reporter assay.Co-Immunoprecipitation was conducted to investigate whether RFX3 bound to DEF6 promoter.Results: hsa_circ₀01193 was up-regulated in glioma tissues and cells.Knockdown of hsa_circ₀01193 attenuated malignant biological behaviors of glioma cells.Mi R-496 was down-regulated glioma tissues and cells.Over-expression of mi R-496 significantly impaired malignant biological behaviors of glioma cells.Mi R-496 targeted hsa-circ-001193 in a sequence-dependent manner.Knockdown of hsa-circ-001193 impaired malignant biological behaviors of glioma cells by decreasing RFX3 expression,which was negatively mediated by mi R-496.RFX3 was up-regulated in glioma tissues and cells.Inhibition of RFX3 inhibited glioma cells malignant biological behaviors,and decreased DEF6 expression by inactivated its promoter.DEF6 was up-regulated in glioma tissues and cells.Knockdown of DEF6 significantly inhibited glioma cells malignant biological behaviors.Over-expression of mi R-496 impaired malignant biological behaviors of glioma cells by decreasing RFX3 expression,which regulated PI3K/AKT pathways activity mediated by DEF6.Furthermore,nude mice carrying knockdown of hsa-circ-001193 combined with over-expression of mi R-496 had the lowest tumor volume and the longest survival period.Conclusion: 1.Hsa-circ-001193,RFX3 and DEF6 were up-regulated while mi R-496 was down-regulated in glioma tissues and cells.Knockdown of hsa-circ-001193,RFX3,DEF6 and over-expression of mi R-496 inhibited the malignant biological behavior of glioma cells.2.Hsa-circ-001193 targeted mi R-496;mi R-496 negatively regulated the expression of RFX3;RFX3 regulated biological behaviors of glioma cells by bounding to the promoter of DEF6 and promoting the transcription of DEF6.3.Knockdown of hsa-circ-001193 combined with overexpression of mi R-496 inhibited tumor growth and led to higher survival in nude mice.