A Study On The Role Of TMEM216 In ER Stress-mediated Apoptosis In Human Lung Cancer Cells

Currently,lung cancer possesses the world's highest mortality of cancer.Chemotherapy is still the primary means of cancer treatment,but the side effects is so serious that it is important to find a more specific target for cancer therapy.During the recent years,apoptosis of tumor cell induced by chemotherapeutic drugs through ER stress has been widely reported.It is of great significance to investigate the regulatory mechanism of ER stress for apoptosis mechanism researchment and searching for new therapeutic targets.EIF2AK3,one of the ER stress sensor proteins,whose regulatory mechanism is mainly focus on its protein activity and mRNA stability regulation,is little reported about its deregulation regulation.TMEM216 belongs to the TMEM family.It is a tetraspan transmembrane protein,and is of vital important in maintaining normal cellular physiological state.It is reported that TMEM216 may interact with EIF2AK3.Therefore,we hope to take TMEM216 as a breakthrough to study the regulatory mechanism of EIF2AK3,and then to further explore the molecular mechanism of TMEM216 regulation of tumor cell apoptosis and to find a new target for cancer therapy.First of all,we detected the protein level of TMEM216 and EIF2AK3 in defferent NSCLC cell lines by western blot analysis,we found that there maybe correlation between the levels of the two proteins;the TMEM216 was knockdown and we found that the protein levels of EIF2AK3 and its downstream proteins were down-regulated significantly,but had no effect on ERN1;we also found that the protein level of EIF2AK3 was increased after TMEM216 was overexpressed.These researches indicated that TMEM216 up-regulates the level of EIF2AK3 in NSCLC cells.Secondly,we the apoptosis was decreased significantly after the expression of TMEM216 was inhibited.We over-expressed TMEM216,western blot analysis and flow cytometry showed that TMEM216 promotes apoptosis.These results suggested that TMEM216 promotes apoptosis of NSCLC cells.Thirdly,the TMEM216 plasmid and EIF2AK3 siRNA were co-transfected in H1299 cell line,we found that inhibition the expression of EIF2AK3 weakens apoptosis induced by TMEM216.Therefore,TMEM216 promotes apoptosis via up-regulation of EIF2AK3.Next,the interaction between TMEM216 and EIF2AK3 was detected by immunoprecipitation analysis;and TMEM216 regulates EIF2AK3 degrading through ubiquitin-proteasome pathway.Then,RNA interference,western blot analysis and immunoprecipitation assay were conducted to screen the EIF2AK3's E3 ubiquitin ligase and we found SYVN1 maybe one of the E3 ligases of EIF2AK3.We also found that knockdown the expression of TMEM216 enhances EIF2AK3-SYVN1 interaction and promoted EIF2AK3's polyubiquitination level;meanwhile,over-expression of TMEM216 weakened EIF2AK3-SYVN1 interaction and downregulated EIF2AK3's polyubiquitination level.So we drew a conclusion that TMEM216 could stabilize EIF2AK3.Finally,our data suggested that ER stress may regulate the expression of TMEM216 by positive feedback through up-regulation of DDIT3.In summary,we used human NSCLC cell lines as models and definited that TMEM216 could up-regulate EIF2AK3 and promote apoptosis;demonstrated that EIF2AK3 degradates through the ubiquitin-proteasome pathway,and screened the SYVN1 as the E3 ubiquitin ligase of EIF2AK3.We also explored the molecular mechanism of EIF2AK3 deregulation:TMEM216 could weaken EIF2AK3-SYVN1 interaction and stabilize EIF2AK3.Finally we verified that ER stress could regulate TMEM216 in a positive feedback maner.