Objective To explore the influence and the underlying mechanism of transmembrane protein 16A(TMEM16A)on glioma.Methods U87MG and LN229 glioma cell lines with TMEM16A knockdown were constructed by using shRNA or lentivirus,and negative control group(Scrb shRNA)and shRNA group were set up.TMEM16A overexpression was constructed by using the GV141-TMEM16A plasmid and empty vector control group(Vector)and TMEM16A overexpression group were set up.The mRNA levels of TMEM16A and IκBα were determined by quantitative real-time PCR(qRT-PCR).Western Blot and immunohistochemistry(IHC)were conducted to analyze protein expressions.In vitro,the Cell Counting Kit-8,EdU,flow cytometry,wound healing,and Transwell assays were employed to measure glioma cells'viability,proliferation,cycle,apoptosis,migration and invasion.An assay of dual-luciferase reporter was conducted to assess the influence of TMEM16A on NF-κB activation.NF-κB activation mechanism was ascertained by cycloheximide chase assay and coimmunoprecipitation(co-IP).An intracranial mouse model was used to evaluate the effects of TMEM16A on glioma tumorgenesis in vivo.Results Compared with adjacent non-tumor tissues or normal human astrocytes(NHA),TMEM16A were highly expressed in glioma tissues and cells.The expression level of TMEM16A increased with the grade of glioma.Compared to the Scrb shRNA group,knockdown of TMEM16A in U87MG and LN229 cells had a considerable inhibitory effect on viable,proliferative,migratory and invasive abilities and it can cause cell cycle arrest and apoptosis.Western Blot showed that compared with the Scrb shRNA group,the expression of p21,p27,Bax,cl-caspase-3,and E-cadherin was significantly increased and expression of cyclin D1,cyclin E1,CDK2,CDK4,Bcl-2,TWIST1,N-cadherin,Vimentin,Slug,MMP2,and MMP9 decreased.TMEM16A activated NF-κB signaling pathway by ubiquitination-mediated IκBα degradation in glioma cells.Compared to the Scrb shRNA group,knockdown of TMEM16A hindered glioma tumorgenesis in vivo.Conclusions The results provide strong evidence that TMEM16A/NF-κB axis drives glioma progression and provide the basis for the treatment of glioma.
维普
万方数据
