A Study On The Role Of TMEM231 In Chemotherapeutic Agent-induced Apoptosis In Non-small Cell Lung Cancer Cells

Apoptosis is a kind of programmed cell death,which enables multicellular organisms to maintain homeostasis and eliminate single cells that threaten their survival.It plays a crucial role in the health and survival of multicellular organisms.Apoptosis is also the most common cell death mode induced by anticancer drugs in tumor cells.TMEM231 belongs to two transmembrane proteins of the TMEM protein family,which are located in the ciliary transition zone in ciliated cells,and are mainly located in the plasma membrane,mitochondria,nucleus and endoplasmic reticulum in ciliate free cells.In addition,TMEM231 and other Meckel syndrome(MKS)complex proteins jointly establish the transition zone,which can prevent proteins from entering cells.Currently,studies on the TMEM family mainly focus on ciliary development and cilia-related diseases,and a few studies are related to cancer.We reviewed relevant databases and literatures and found that TMEM231 may interact with TNFRSF10B,so we speculated that TMEM231 may be related to apoptosis.First,we knocked down or exogenously expressed TMEM231 in non-small cell lung cancer cell lines calu-1,H460 and H1299.Western Blot results showed that TMEM231 could reduce the protein level of TNFRSF10B in NSCLC cells and inhibit the apoptosis induced by TNFRSF10B.Flow cytometry was used to detect the effect of overexpression or knockdown of TMEM231 on apoptosis,and the results were consistent with the above results.Then,two variants of TNFRSF10B were overexpressed in H1792.The pull down experiment detected an interaction between the short variant of TNFRSF10B and the endogenous TMEM231.TMEM231-FLAG was overexpressed in calu-1 Immunofluorescence showed that they had co-localization.The interaction between endogenous TMEM231 and endogenous TNFRSF10B was also detected by immunoprecipitation.Secondly,we detected the localization of TMEM231 on the endoplasmic reticulum by immunofluorescence under the confocal laser scanning fluorescence microscope.We also detected that TMEM231 inhibited TNFRSF10B distribution on the plasma membrane by flow cytometry.In A549,knockdown of TMEM231 was treated with the apoptosis-inducing drug PEM(pemetrexed)for different times.After immunofluorescence staining,the co-localization of-TNFRSF10B and early intracellular marker EEA1 was observed by laser scanning confocal fluorescence microscopy.The results showed that knockdown of TMEM231 reduced the co-localization of EEA1 and TNFRSF10B,while the localization of TNFRSF10B on the plasma membrane increased.Therefore,TMEM231 may increase its localization on the plasma membrane by decreasing the endocytosis of TNFRSF10B.Finally,TMEM231 was overexpressed in calu-1 for immunoprecipitation,proving the interaction between TMEM231 and RAB9A(mainly located in late intracellular body).This indicates that TMEM231 may affect the membrane vesicle transport of TNFRSF10B by affecting RAB9A.Therefore,we used non-small cell lung cancer cells as the model to conclude that TMEM231 down-regulates TNFRSF10B and may affect the membrane vesicle transport of TNFRSF10B through RAB9A,thereby down-regulating apoptosis.