Mechanism Of UORF Regulating The Malignant Behaviors Of Glioma Cells Via UCA1-miR-627-5p-NR2C2 Feedback Loop

Objective: Glioblastoma is one of the most common primary malignant intracranial tumors in adults,accounting for up to 10%-15% of all intracranial tumors.At present,surgical treatment combined with radiotherapy and chemotherapy is the main method for the treatment of glioma,but because of the recurrence of malignant glioma,the median survival of patients is 12-15 months.The aim of this study was to investigate the role of NR2C2 5' UTR-47aa-ORF,UCA1,miR-627-5p and NR2C2 in regulating the biological behavior of glioma cells,and to explore its molecular mechanism.We further aimed to investigate how UCA1 targeted miR-627-5p to regulate the transcription factor NR2C2 and affect the expression of the downstream protein SPOCK1 to regulate the malignant behaviors of glioma cells;and how NR2C2-47aa-uORF directly regulates the expression of NR2C2 to inhibit the biological effects of this feedback loop.Methods: We conducted Real-time PCR assay to evaluate the RNA expression levels of lnc-UCA1,miRNA-627-5p and NR2C2 in glioma tissues and cells.And then we used Western blot assay to explore the expression levels of NR2C2 ?SPOCK1?47aa-uORF in glioma tissues and cells.We next performed Cell Counting Kit-8,transwell assays,and flow cytometry to investigate the viability,abilitiy to migration and invasion and apoptosis of glioma cells.Lnc-UCA1 inhibition,over-expression or inhibition of NR2C2?over-expression or inhibition of miR-627-5p plasmids and over-expression uORF plasmids were transfected into the cells.The mRNA expression of lnc-UCA1,NR2C2andmiR-627-5p was measured by Real-time PCR.The expression of NR2C2,SPOCK1 ?47aa-uORF were detected by Western blot.Interactions between miR-627-5p and lnc-UCA1 or interactions between miR-627-5p and NR2C2 were evaluated using the luciferase reporter assay.We conducted Chromatin Immunoprecitation(ChIP)assays to identify interactions between NR2C2 and lnc-UCA1 or NR2C2 and SPOCK1.We finally measured the effects of lnc-UCA1,NR2C2 and 47aa-uORF on the growth of transplanted tumors and the survival time of nude mice bearing tumors.Results: We found that lnc-UCA1 is highly expressed in glioma tissues and cells.Knocking down of lnc-UCA1 inhibits the proliferation,migration and invasion of U87 and U251 cells but leads to a promotion in apoptosis.Mi R-627-5p expresses at a lowlevel in glioma,indicating it acts as an tumor suppressor.Over-expression of miR-627-5p causes a significantly deduction in proliferation,migration and invasion of glioma cells but a promotion in apoptosis;silencing miR-627-5p can significantly promote the proliferation,migration and invasion of glioma cells,and inhibit cell apoptosis.MiR-627-5p is a component target of UCA1.Silencing UCA1 or overexpression of miR-627-5p reduces NR2C2 expression.NR2C2 is highly expressed in glioma tissues and cells.Inhibition of NR2C2 also leads to a significantly decrease in proliferation,migration and invasion of U87 and U251 cells but an increase in apoptosis;while over-expression of NR2C2 promotes the proliferation,migration and invasion of U87 and U251 cells and inhibit apoptosis.SPOCK1 is highly expressed in glioma tissues and cells.MiR-627-5p binds to the 3 ? UTR region of NR2C2 mRNA.Silencing UCA1 inhibits the malignant behaviors of glioma cells by increasing the miR-627-5p level in order to reduce the expression of NR2C2.NR2C2 increases SPOCK1 promoter activity by binding to its promoter region.Over-expression of NR2C2 promotes the expression of SPOCK1,and silencing NR2C2 has an opposite result.Over-expression of miR-627-5p reduces the expression of SPOCK1 by negatively regulating NR2C2,and silencing miR-627-5p promotes the expression of SPOCK1 by up-regulating NR2C2,thereby regulating the malignant behaviors of glioma cells.NR2C2 can also bind to the promoter region of UCA1.Over-expression of NR2C2 significantly increases the expression of UCA1,forming a feedback loop to regulate the malignant behaviors of glioma cells.47aa-uORF is underexpressed in glioma tissues and glioma cells.Overexpression of47aa-uORF inhibits NR2C2 expression and inhibits the biological functions produced by the feedback loop to regulate the malignancies of glioma cells.Silencing UCA1 combined with over-expression of 47aa-uORF and silencing NR2C2 inhibited the growth of xenografted tumors and prolonged the survival of nude mice bearing tumors.Conclusions:1.Lnc-UCA1 is highly expressed in glioma tissues and cells,and miR-627-5p is low.Silencing lnc-UCA1 or overexpressing miR-627-5p inhibits the proliferation,migration and invasion ability of glioma cells and promotes apoptosis.2.Lnc-UCA1 specifically binds to miR-627-5p.NR2C2 is a target gene of miR-627-5p.NR2C2 directly binds to the promoter of SPOCK1 and enhances its transcriptionalactivity.Silencing lnc-UCA1 increases the negative regulation effect of miR-627-5p on NR2C2 by reducing its binding to miR-627-5p,inhibits the expression of NR2C2,inhibits the transcription effect of NR2C2 on SPOCK1,and down-regulates the expression of SPOCK1,thereby inhibit the proliferation,migration and invasion ability of glioma cells and promotes apoptosis.3.NR2C2 combines with the promoter region of lnc-UCA1 to promote its transcription and form a positive feedback loop.4.47aa-uORF reduces the expression of NR2C2 mRNA and inhibits the malignant biological behaviors of gliomas.5.47aa-uORF inhibits the malignant biological behaviors of glioma cells by inhibiting the Lnc-UCA1-mi R-627-5p-NR2C2 feedback loop.