MiR-140-5p Inhibits Glioma Cell Proliferation And Invasion By Regulating GLUL Gene

Background and Objective Gliomas are the most frequent primary malignant tumours in the central nervous system.According to the degree of malignancy,gliomas are categorized into four grades: ???????.WHO grade ?-? gliomas are considered low-grade glioma,whereas WHO grade ? and ? are high-grade glioma.The pathological features of glioma are mainly invasive growth,leading to no obvious boundary between the tumour tissue and normal brain tissue.Even treatment with surgery,radiotherapy and chemotherapy,it is still difficult to completely remove the tumour tissue.In particular,the median survival time of glioblastoma(WHO ?)is only 12-15 months,and the prognosis is very poor.Glutamine addiction in glioma describes that glioma cells are highly dependent on glutamine providing energy and biosynthesis needs.Glutamate-ammonia ligase(GLUL),also known as glutamine synthase,is an enzyme that converts glutamate and ammonia to glutamine.GLUL,as a key enzyme in the synthesis of glutamine,is crucial for the study of regulation mechanism of glutamine metabolism in malignant transformation of gliomas.The regulation of mi RNAs belongs to the epigenetic regulation mechanism.At the post-transcriptional level,mi RNAs hybridize to the 3'-untranslated regions(3'-UTR)of target gene m RNAs,resulting in translational inhibition or m RNA degradation,which play an important role in cell proliferation,growth,metabolism and differentiation.However,whether mi RNAs affect glioma proliferation and invasion by regulating GLUL has not been reported.We predicted that mi R-140-5p can target the 3'-UTR region of GLUL by querying mi RNAs bioinformatics software.Therefore,our study will explore the relationship between mi R-140-5p and GLUL and their effects on the biological function of gliomas.Materials and Methods In this study,q PCR was performed to examine the expression of GLUL m RNA and mi R-140-5p in glioma cells;The protein expression level of GLUL in glioma cells was detected by Western blot;To construct glioma cells that knock down GLUL and overexpress mi R-140-5p,Lipfectmine 2000 liposome was used to transfect GLUL-si RNA and mi R-140-5p mimics into LN18 and U251,respectively.A series of experiments including CCK-8,Transwell migration and invasion assays were performed to evaluate the ability of proliferation,invasion and migration of glioma cells.Dual luciferase assays verified whether mi R-140-5p directly targeted to GLUL3'-UTR.Results In the present study,we firstly detected the expression of GLUL in glioma cells by q PCR and Western blot.We found that expression of GLUL in high grade glioma cells(LN18 and U251)was apparently higher than low grade glioma cells.After knocking down GLUL expression by si RNA technology,the proliferation and invasion ability of glioma cells was significantly inhibited.The bioinformatics software such as Target Scan,mi Randa and PITA were used to predict that mi R-140-5p targeted the3'-UTR region of GLUL,and dual-luciferase reporter assays further confirmed the results.q PCR showed the expression of mi R-140-5p in high grade glioma cells(LN18and U251)was significantly lower than that in low grade glioma cells,and it was negatively correlated with the expression of GLUL.After overexpression of mi R-140-5p,the expression levels of GLUL m RNA and protein were significantly down-regulated,and the proliferation and invasion ability of glioma cells was inhibited.Conclusion Our results showed that mi R-140-5p significantly inhibited the proliferation,invasion and migration of glioma cells by down-regulating GLUL expression.In addition,our results revealed there was a regulation axis of mi R-140-5p-GLUL in glioma cells.These results helped us to explain the molecular mechanism of malignant progression of glioma at the metabolic level,and provided an important theoretical basis to further explore the regulation mechanism of Gln metabolic reprogramming in glioma.