Expression And Functional Role Of Reprogramming Related Long Non-coding RNA(lincRNA-ROR) In Glioma

Objective: To investigate the expression and function of reprogramming related longnon-coding RNA (lincRNA-ROR) in glioma and GSCs (Glioma Stem Cells).Methods: Real-time quantitative PCR, we analyzed lincRNA-ROR expression levels in26glioma patients, and the expression correlation of lincRNA-ROR with SOX11, KLF4.To explore its functional role, gain-and loss-of-function studies were performed toassess the effect of lincRNA-ROR on cell proliferation,expression rate of GSCs markerCD133, and glioma stem spheres forming ability in vitro.Results:The lincRNA-RoR expression level was assessed in a group of26gliomapatients. We analyzed paired specimens obtained from them. From eachpatient, lncRNA was isolated from cancerous tissue and adjacentnoncancerous glioma tissue. The lincRNA-RoR expression in23glioma specimenswas declined compared with matched noncancerous tissue. Furthermore we also foundthe expression of lincRNA-ROR was positively correlated SOX11mRNA expression,and negatively correlated with KLF4mRNA expression.LincRNA-RoR shRNA plasmids were constructed and stably transfectedinto U87cells. A markedly decreased expression of LincRNA-RoR was detected withqPCR. Quantification analysis showed that lincRNA-RoR expressionlevel was significantly knocked down in lincRNA-RoR shRNA groups(P<0.05). Meanwhile, the expression of KLF4mRNA was up-regulated bylincRNA-RoR shRNA1inversely (P<0.05).We then performed the knockdown functional assays with U87cellsby suppressing lincRNA-RoR expression (lincRNA-RoR shRNAs) in comparisonwith U87cells transfected with negative control shRNA. The results showed that theU87cells in lincRNA-RoR shRNA transfected groups grew significantly faster compared with the cells in the NC group (P<0.05).To determine the effects oflincRNA-RoR knockdown on GSCs in U87cells in vitro, we assessed theCD133+expression rate by FCM and glioma stem spheres forming ability by spheroidformation assay. The results revealed that after lincRNA-RoRshRNA treatment, theCD133+GSCs number and glioma stem spheres number were both significantlyincreased in U87cells in vitro (P<0.05).PcDNA-lincRNA-RoR was constructed and stably transfected into U87cells. Amarkedly increased expression of lincRNA-RoR was detected with qPCR.Quantification analysis showed that lincRNA-RoR expression level was evidentlyelevated in pcDNA-lincRNA-RoR group. Meanwhile, the expression of KLF4mRNAwas inversely modulated by pcDNA-lincRNA-RoR and significantly decreased.To further identify the role of lincRNA-RoR in U87cells, we performedfunctional assays with U87cells by lincRNA-RoR overexpression(pcDNA-lincRNA-RoR) in comparison with U87cells transfectedwith pcDNA-control. The result showed that the U87cells in pcDNA-lincRNA-RoRtransfected groups grew significantly slower than the cells in the pcDNA-controlgroup.To determine the effects of lincRNA-RoR overexpression on GSCs in U87cellsin vitro, we assessed the CD133+expression rate by FCM and glioma stem spheresforming ability by spheroid formation assay. The results revealed that afterpcDNA-lincRNA-RoR treatment, the CD133+GSCs number and glioma stem spheresnumber were both significantly decreased in U87cells in vitro.Conclusions: Our results suggest that the reprogramming related lincRNA-ROR mayserve as a novel tumor suppressor gene in glioma, which can inhibit the proliferation ofcancer cell and self-renewal of GSCs, partly by inhibiting the KLF4expression. Furtherresearch about lincRNA-ROR may provide a novel biomarker and therapeutic target ofglioma for cancer clinic in future.