Human glioblastoma multiforme (GBM) accounts for the majority of human brain gliomas. Several TMEM proteins, such as TMEM 45A, TMEM 97, and TMEM 140, are implicated in human brain gliomas. However, the roles of TMEM168 in human GBM remain poorly understood. Herein we found that mRNA levels of TMEM168 were overexpressed in GBM patients (n = 85) when compared with healthy people (n = 10), which was also supported by data from The Cancer Genome Atlas (TCGA). Kaplan‐Meier analysis of Gene Expression Omnibus dataset GSE16011 suggested that enhanced TMEM168 expression was associated with shorter survival time. To investigate whether and how TMEM168 functioned in the tumorigenesis of human GBM cells, two human GBM cell lines (U87 and U373) were used for study. Lithium chloride (LiCl), an activator for Wnt/β-catenin pathway, was used for the treatment. Our data suggested that siRNA-TMEM168 (siTMEM168) prevented viability of U87 and U373 cells, induced cell cycle arrest (G₀/G₁ phase) and promoted apoptosis, and the mechanisms involved in blocking Wnt/β-catenin pathway, as evidenced by reducing expression of β-catenin, C-myc, cyclin D1, and survivin. Furthermore, the inhibited effect of siTMEM168 on human GBM cell growth was significantly alleviated with additional LiCl treatment, substantiating the involvement of the Wnt/β-catenin pathway in this process. In summary, our data demonstrated that TMEM168 may represent a therapeutic target for the treatment of human GBM.

Background. Paeoniflorin is an active component of a widely used traditional Chinese medicine with antitumor activity through ferroptosis induction. It has been reported recently that ferroptosis is emerging in certain types of cancer; however, its relevance in glioma is still not well studied. Methods. CCK8 assay was performed for cell proliferation. Expression of mRNA and protein was tested by qPCR and western blot, respectively. Clinical section samples were detected by IHC. The relationship between NEDD4L and STAT3 was validated by a coimmunoprecipitation assay. Apoptosis was identified by TUNEL assay. A xenograft mouse model was utilized to validate the potential of paeoniflorin toward glioma cancer cells. Results. The data suggested that paeoniflorin could increase NEDD4L expression in glioma cells. The NEDD4L expression level was lower in glioma cancer tissues compared to adjacent normal tissues, and it correlates with poor prognosis. Meanwhile, NEDD4L mediates the ubiquitination of STAT3. Furthermore, increased NEDD4L significantly inhibited cell viability and induced accumulation of intracellular ROS levels, accompanied by decreased expression of key ferroptosis factors Nrl2 and GPX4, while NEDD4L knockdown had a reverse effect, suggesting that ferroptosis could be involved. NEDD4L-induced ferroptosis could be rescued by forced expression of STAT3. A xenograft nude mouse model showed that paeoniflorin inhibits tumor growth and further sensitizes glioma cells to RSL3, another well-known ferroptosis inducer. Conclusions. In summary, this study demonstrated that paeoniflorin might function as an effective drug for glioma by inducing ferroptosis via upregulation of NEDD4L and repression of Nrl2, GPX4, and STAT3.