| Glioma is the most common intracranial malignancy tumor with strong invasive ability.The characteristics of high morbidity,high recurrence rate,high mortality and low cure rate determine the bad prognosis of glioma.According to the recent clinical data,the median survival time after surgical operation of GBM patients is no longer than 14.6 months.At present,it has not been proved that new therapeutic methods or medicine could prolong the survival time of glioma significantly.Therefore,finding new therapeutic targets and strategies are required to improve the prognosis of glioma patients.Under the current situation,this study intends to explore new molecular mechanisms to regulate progression and malignant behavior of glioma cells through in vivo and in vitro experiments.The objective of this study is to lay a foundation for developing novel finding potential therapeutic targets for the treatment of glioma.This research was divided into four parts to elucidate the regulatory functions and possible mechanisms of long non-coding RNA GAS5(Lnc RNA-GAS5)in malignant behavior of glioma cells.Firstly,the human glioma tissues were collected to detect the expression levels of GAS5.And the Cox regression and survival analysis were carried out with clinical data to analyze the correlation of GAS5 expression and glioma prognostic.Secondly,different glioma cell lines were over-expressed with GAS5 and the proliferation,apoptosis and migration were determined though CCK-8 assay,apoptosis detection,ound healing and trans-well assay.Thirdly,the primary glioma stem cells were infected with GAS5-overexpression lenti-virus to evaluate the influence of GAS5 on GSC stemness,which is significant to glioma progression.The proliferation and self-renew ability were determined by clone spheres formation,stemness markers detection,CCK-8 and Edu staining.Lastly,RAP pull-down assay were performed to screen the GAS5-interacted RNAs and the candidates were further predicted by Starbase database and verified by using RT-PCR.The mechanisms of GAS5 regulating GSCs malignant phenotype was explored through RT-PCR,Western Blot,reporter assay and rescue experiments.The results showed that the expression of GAS5 in glioma tissue was negatively correlated with the malignant degree of glioma and positively correlated with the survival time of patients after operation;Overexpression of GAS5 repressed the proliferation and migration and promoted the apoptosis of glioma cells.GAS5 could also suppress the stemness and self-renew of GSCs and attenuate proliferatio of GSCs.The further mechanisms study indicated that GAS5 protected the co-host gene SPACA6 of let-7e/mir-125 a from degradation,and further inhibited the expression of IL-6R,which is the common target of let-7e and mir-125 a.Therefore,the proliferatio and migration of glioma cells were reduced through inhibiting the activation of IL-6R/STAT3 pathway.The inhibitor of let-7e or/and mi R-125 a could rescue the effect of GAS5.Based on the above results,the following conclusions can be drawn: Lower expression of GAS5 is related to the higher malignant degree of tumors and worse survival time after operation;GAS5 can negatively regulate the malignant biological behavior of glioma cells and GSCs;GAS5 upregulates the expression of let-7e and micro RNA-125 a by reducing SPACA6 degradation,and further represses the activation of downstream IL-6R/STAT3 pathway.The results of this study provide a theoretical basis for finding novel potential targets of molecular diagnosis and treatment of glioma.This study presents strong clinical transformation significance and application value. |
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