Up-regulation Of Beta-Adrenergic Receptors-Mediated MALAT1 Promotes The Proliferation Of Glioma Cells And Its Molecular Mechanisms

Background and Aims:Glioma is one of the most common malignant tumors in the brain,accounting for more than 50%of the intracranial tumors.With strong invasiveness,rapid postoperative recurrence and high mortality,it has become one of the most intractable early death solid tumors in neurosurgery.Molecular targeted therapy is an investigating hotspot in recent years due to its advantages such as clear target,high safety and mild side effects.G protein coupled receptor（GPCR）is considered as the most successful drug target developed,and more than 50%of clinical drugs and drug targets under development are related to GPCR.Some studies showed thatβ-adrenergic receptor（β-AR）,a member of GPCR superfamily,is widely expressed in many tumor tissues and closely related to the occurrence of many tumors.Some studies have suggested thatβ-AR is related to the occurrence of astroglioma,but the mechanism ofβ-AR regulating the occurrence of glioma needs further study.Therefore,this project aims to investigate the roles and molecular mechanisms ofβ-AR regulating the proliferation in glioma,so as to provide effective molecular targets and certain theoretical basis for clinical treatment of glioma.Methods:1.Detection of expression levels ofβ₁-AR andβ₂-AR genes in glioma cells（U251-MG,U373-MG）:The total RNA was extracted from glioma cells,and the mRNA expression levels ofβ₁-AR andβ₂-AR were detected by qPCR or RT-PCR.2.Effects of isoproterenol（ISO）on proliferation of glioma cells（U251-MG,U373-MG）:After 24 h of ISO stimulation with various concentrations of 0,0.1,1,5,10,30,50μM,the cells were stimulated with 10μM ISO for 24 h,48 h and 72 h,respectively.The proliferation of cells was detected by MTT assay.Finally,the proliferation of cells was detected by cell immunofluorescence and colony formation assay with 10μM ISO after 48 h of cell stimulation.3.Gene microarray screens for genes with altered transcriptional expression levels afterβ-AR activation:The U251-MG cells were stimulated with 10μM ISO for30 min and 60 min respectively,and the alternations of the expression levels in genes were detected by gene microarray.4.Validation of the interference efficiency of MALAT1 and detection ofβ-AR-induced cell proliferation in the condition of the up-regulation of MALAT1:The interference of MALAT1 gene was performed in U251-MG cells to extract cellular RNA,and the interference efficiency was detected by qPCR.After knockdown of MALAT1 by siRNA,the effects of ISO stimulation on cell proliferation were detected by MTT assay.5.Molecular mechanism analysis ofβ-AR promoting glioma cell proliferation:Firstly,effects ofβ-AR on the phosphorylation of ERK1/2 and CREB proteins in U251-MG and U373-MG cells were stimulated by ISO for 0,1,2,5,10,20 and 30 min,respectively,and then extracted the proteins to detect the phosphorylation of ERK1/2and CREB proteins in the two cell lines by Western Blot.Secondly,activated beta-AR regulated cell proliferation through ERK1/2/CREB pathway in U251-MG cells were treated with U0126 at different times,and an appropriate time point was selected to inhibit the phosphorylation of ERK1/2.Then the cells were pretreated on this time point,followed by U0126 and ISO co-stimulation,and the collection protein was used to detect the phosphorylation degree of ERK1/2 and CREB protein.Cell proliferation was detected by Ki67 immunofluorescence assay and MTT.Thirdly,β-AR regulated ERK1/2/CREB pathway through up-regulation of MALAT1 to promote glioma cell proliferation were performed in U251-MG and U373-MG cells.The expression levels of MALAT1 gene were reduced by siRNA,and then cells were stimulated for 5 min by ISO to collect protein,and the phosphorylation levels of ERK1/2 and CREB protein in the two cell lines were detected by Western Blot.Result:1.RT-PCR results showed that bothβ₁-AR andβ₂-AR were significantly expressed in glioma cells U251-MG and U373-MG,and the mRNA expression level ofβ₂-AR was significantly higher than that ofβ₁-AR.2.ISO,a specific agonist of beta-AR,significantly promoted the proliferation of glioma cells in a concentration-dependent and time-dependent manner.3.β-AR promotes the proliferation of glioma cells by up-regulating MALAT1.4.Mechanism analysis:The ISO significantly increased the phosphorylation of ERK1/2 and CREB in glioma cells.Secondly after activation of beta-AR,glioma cell proliferation is regulated by ERK1/2/CREB signaling pathway.Thirdly interference with the expression of MALAT1 can significantly inhibit the activation of ERK1/2/CREB signaling pathway byβ-AR.Conclusion:1.Bothβ₁-AR andβ₂-AR were expressed in glioma cells（U251-MG and U373-MG）,and the expression ofβ₂-AR was higher.2.β-AR regulates ERK1/2/CREB signaling pathway through up-regulating lncRNA MALAT1 to promote glioma cell proliferation.