Investigating The Roles Of ER Stress In Inducing NPC Cells Differentiation

BackgroungThe molecular machinery of endoplasmic reticulum(ER)integrates various intracellular and extracellular cues to maintain homeostasis in diverse physiological or pathological scenarios.Disruptions in Ca2+ homeostasis,inhibition of protein glycosylation,and accumulation of misfolded proteins can all challenge the function of the endoplasmic reticulum-Golgi network,resulting in ER stress.ER stress and the unfolded protein response(UPR)have been found to mediate molecular and biochemical mechanisms that affect cell proliferation,differentiation,and apoptosis.Although a number of reviews on the ER stress response have been published,comprehensive reviews that broadly summarize ER physiology in the context of pluripotency embryonic development and tissue homeostasis.Different ER stress transducers located on the ER membrane.Such as PERK,IRE land ATF6 can initiate distinct UPR signalling pathways.In the absence of stress,all ER stress transducers are sequestered by the ER HSP70 family protein Bip(GRP78).ER stress regulates complex functions involved in tumor development,growth and cellular differentiation of pluripotent stem cells in many cancers,including neuronal cells,hematopoietic progenitors and smooth muscle cells.ER stress plays an important role in maintaining the stability of the blood stem cell pool.ER Stress-Induced Differentiation Sensitizes Colon Cancer Stem Cells to Chemotherapy.Nasopharyngeal carcinoma(Nasopharyngeal carcinoma,NPC)is the low differentiation and high metastatic nasopharyngeal mucosa malignant tumor,which is common in Southern China and Southeast Asia.Most of NPC belong to low differentiated squamous cell carcinoma,which provides a unique model to study solid tumor differentiation therapy.If "differentiation" theory can be introduced to solid tumors(such as NPC)in the clinical treatment,it will bring the gospel to the patients.At present,the research of ER stress in NPC has not been reported.NPC cells as epithelial cells,whether it can be induced to differentiate by ER stress.Based on the above research background,we will Investigate the roles of ER stress in inducing NPC cells differentiation.As a transcription factor Hesl plays an important role in the development of some tumors.Hesl is closely related with adult stem cells and CSCs.Studies have shown that,Hesl is often used as a stem cell marker,which is essential in the adult stem cells of self-renewal and proliferation,maintaining stemness,survival and inhibition of stem cell differentiation.Study on the relationship between Hes1 and NPC CSCs has reported that Hesl co-expressed with Oct4 and Sox2 in NPC cells.Our previous study found that Sternness-related genes expression were down-regulated by RNA interference Hesl expression in NPC cells.Hesl is high expression in undifferentiated NPC than differentiated NPC.The expression trend of Hesl is opposite to NPC differentiation index CK8 and Involucrin,and same to CK13 in tissue samples.Based on the research background,we intend to investigate whether Hesl is involved in ER stress induced NPC cell differentiation process.Methods1.Activation of the ER stress in NPC cells with Tunicamycin and Thapsigargin1)To determine the optimal concentration and time of Tunicamycin and Tg.2)qRT-PCR and Western methods were used to detect the effect of ER stress in NPC cells.2.ER stress induces NPC cells differentiation1)The morphological changes of ER stress pre-treated NPC cells.2)Tumor sphere formation and ratio of side populations(SP)cells detected by flow cytometry to evaluate the "stemness" of ER stress pre-treated NPC cells3)qRT-PCR and Western blot were used to detect the expression level of NPC differentiation related markers in ER stress pre-treated NPC cells.3.To Investigate the effect of ER stress on the biological characteristics of the ER Stress-induced differentiation NPC cells1)CCK-8 assay and colony formation assay were used to detect the differ-entiated NPC cells induced by ER Stress growth and proliferation.2)The migration and invasion of NPC cells induced differentiation by ER stress were detected by scratch wound assay and transwell.3)SA-?-Gal staining was used to detect the effects of ER stress on aging of NPC cells.4)The effect of ER stress inducer on the apoptosis of NPC cells was detected by flow cytometry.5)qRT-PCR and Western blot were used to detect the expression of epithelial related marker genes in differentiated NPC cells.6)Subcutaneous tumor formation in nude mice to evaluate the effect of ER stress on the tumorigenicity of NPC cells.7)Histopathology of xenograft tumors:The tumor sections were under H&E staining and IHC staining using antibodies against ER stress related marks,BrdU,Ki-67 and P21.4.To detect the expression level of Hesl in NPC cells induced by ER stress qRT-PCR and Western blot was used to detect the expression of Hes1 gene in ER stress of NPC cells.Results1.Establishment of ER stress model of NPC cellsThe optimal concentration and time of ER stress inducer,Tm1.0?g/mL 24h;Tg 1.0 ?M 24h.qRT-PCR and Western blot showed that compared with the control group.The ER stress signaling pathway chaperone GRP78 was significantly higher in ER stress treatment group(P<0.05),PERK,ATF6 and IRE-1 pathway were activated in varying degrees.2.ER stress induces NPC cells differentiation NPC cells pretreated by ER stress:cell morphology transformed from low differentiation cobblestone to higher differentiation.CK8 and Involucrin in ER induced differentiation of stress NPC cells was significantly rised,and the CK13 on the contrary.SP side population cell proportion was significantly reduced(P<0.05).3.Biological characteristics of NPC cells induced by ER stressCCK-8 and clone formation assay showed that ER stress induced NPC cell proliferation significantly decreased(P<0.05).Wound healing assay and Transwell assay showed that ER stress induced NPC cell migration ability was significantly decreased in vitro(P<0.05).SA-?-Gal staining showed that NPC cells aging was significantly increased after ER stress(P<0.05).Flow cytometry was used to detect the effect of ER stress inducer on apoptosis of NPC cells(P>0.05).qRT-PCR and Western blot showed that the expression of E-ca was significantly up-regulated in ER stress treated NPC cells,and the expression of Vimentin was decreased(P<0.05).4.Expression of Hes1 in ER stress-induced NPC cells qRT-PCR and Western blot showed that the expression of Hes1 in NPC cells was significantly decreased after ER stress(P<0.05).Conclusions1.Activation of the ER stress in NPC cells with Tunicamycin and Thapsigargin successfully.2.ER stress promotes the differentiation of NPC cells3.ER stress inhibits NPC proliferation,migration and invasion.4.The expression of Hesl was significantly down regulated in NPC cells after ER stress.