| Glioma is a neuroectodermal tumor derived from glial cells and/or has the neurotissue characterized by gial cell differentiation.As a most common primary intracranial malignant tumors,glioma accounts for about 50%of brain malignant tumors.It has the biological characteristics of fast proliferation,high invasiveness and diffuse diffusion to the adjacent tissues.However,due to lack of surgical clearance,drug resistance,and brain blood barrier,the clinic treatment of glioma is poor and recurrence rate is high with less than 1%five-year survival rate.Therefore,a new and more targeted therapy is urgent need for glioma.At present,the pathological grade of patients is mainly evaluated according to the degree of tumor necrosis,microvascular proliferation,cell heterotypic degree and nuclear division,but this method has certain limitations.Therefore,it is urgent to find new markers for pathological grading of glioma in order to improve the objectivity and accuracy of evaluation.As known as potassium channel(K+channel)is a kind of hydrophilic micro-channel located in cell membrane,and participate in maintaining resting potential and the repolarization of action potential.In addition,K+channel is involved in many physiological processes such as volume stabilization,proliferation,differentiation,migration and apoptosis.K+ channel is known as the most species,the most complex ion channels,which are encoded by more than 90 genes.According to its physical,chemical property,and structural characteristic,K+channe can be divided into four families:voltage-gated dependent K+channel(Kv channel),Calciumactivated K+channel(KCa channel),inward-rectifier K+channel(Kir channel)and two-poredomain K+channel(K2P channel).Kv1.5 channel is a member of Kv channel subfamily,encoded by KCNA5 gene.Under depolarized membrane potential,the Kv1.5 current has characteristics of outward delayed,rapid activation,slow inactivation.It is widely distributed in heart,brain,kidney,gland,muscle and plays an important role in repolarization of excitable cells and signal transduction of non-excitable cells.In recent years,more and more studies found that Kv1.5 channel is important to tumor genesis and development.Our previously studies found that Kv1.5 channel protein was correlated with Cavolin-1 protein expression in different breast epithelial cells,blocking K+channel could inhibit the proliferation of breast epithelial cells,and Caveolin-1 could promote the expression of Kv1.5 channel protein through AKT signaling pathway.Recently studies have found that Kv1.5 channel was closely related to glioma,and can participate in the process of glioma cell proliferation and apoptosis through various channels such as cell cycle,cell volume change,calcium signal and cytoplasmic pH value.The first part of this paper was to explore the biological characteristics of Kv1.5 channel and its relationship with histopathological grading of human glioma tissues.Whole-cell patch clamp was used to record a transmembrane,voltage-dependent,outward rectifying K+current which could be inhibited by 4-AP.RT-PCR,Western Blot and immunofluorescence were used to detect the protein expression of Kv1.5 channel.It was found that the protein was distinguishing positively expressed in different cells and distributed on the cell membrane in a punctiform manner.And then we use human brain glioma biopsy specimens to detect the expression level of Kv1.5 channel protein.HE staining and immunohistochemical results suggested the epithelial tissue source was malignant glioma tissue specimens,and found that the expression level of Kv1.5 channel was negative related with glioma pathology classification.The second part was to investigate the effect of Kv1.5 channel on the survival rate and its relationship with autophagy in glioma cells.MTT assay results suggested different inhibitors of K+channels(TEA,4-AP and DPO-1)on cell survival rates were decreased in a dose and time dependent way for 24 h,48 h and 72 h.At the same time,short-time inhibition of survival rate was first significantly increased(P<0.05)with DPO-1 for 1 h,a specific inhibitor of Kv1.5 channel,and then decreased.It was similar to the bidirectional regulation of autophagy.Western Blot showed that it promoted the autophagy realted proteins LC3-? and LC3-?,inhibited the phosphorylation of mTOR signaling protein.And acridine orange staining found that acidic hydrolysis enzyme activity was increased,suggested that specific inhibition of Kv1.5 channel induced autophagy in U251 cells.The third part of this paper was to explore the mechanism of Kv1.5 channel involved in the autophagy of glioma cells.We constructed siRNA and overexpression vector of Kv1.5 channel protein,and then transient trasfect in glioma cells.Western Blot showed that downregulating Kv1.5 channel protein promoted cell autophagy and activited ERK signaling pathway.While up-regulating Kv1.5 channel protein inhibited cell autophagy and the phosphorylation level of ERK was decreased.It indicated that Kv1.5 channel was involved in autophagy of glioma cells,and its mechanism was related with ERK signaling pathway.Then Rh123 staining and flow cytometry showed that the down-regulating Kv1.5 channel protein lead to early apoptosis,and MMP was decreased when autophagy inhibitors CQ were added.While up-regulating Kv1.5 channel protein decreased the number of apoptotic cells,and MMP was increased when autophagy inducer Rapa was added.It indicated that Kv1.5 channel was involved in the early apoptosis of glioma cells,which was related to autophagy.The fourth part of this paper is to analyze the differentially expressed genes of distinguishing expression of Kv1.5 channel in glioma cell.Transfection was used to change the expression level of Kv1.5 channel protein,and then RNA-Seq analysis was preformed to select differentially expression genes.It found that after down-regulating Kv1.5 channel protein,411 differentially expression genes was selected.GO,KEGG,Reactome and protein network interaction analysis showed that ERK was up-regulated,endocytosis pathway and chaperonmediated autophagy were enrichment,and Cav-3 protein was down-regulated.Taken together,this study revealed Kv1.5 channel negatively correlated with the pathological grade of glioma:the expression of Kv1.5 channel was more in low grade glioma tissue than in high grade glioma tissume;and proved that regulating Kv1.5 channel induced autophagy at the transcriptome and protein levels in U251 cells which was related to ERK signaling pathway.What's more,the involvement of Kv1.5 channel in early apoptosis of glioma cells was related autophagy,too.This paper provided a new theoretical basis for understanding the Kv 1.5 channel biological function and developing new ideas for clinical treatment of glioma. |
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