The Roles Of Hes1 In The Cell Differentiation Of Nasopharyngeal Cancer (NPC) And Underlying Mechanisms

Background and Objective:Nasopharyngeal carcinoma(NPC)is a type of head and neck tumor with high incidence in Southern China,especially in Guangdong and Guangxi provinces.The etiology study of NPC showed that genetic,EB virus infection and environmental factors are related to its occurrence.Because its occulted diseased parts and early lymph node metastasis,it is difficult to take surgical treatment radically.So many patients are seemly to be diagnosed in advanced stage.Radiotherapy,chemotherapy and combined therapy remain the main treatment for NPC,while normal cells could be killed at the same time and tumors could become drug resistance which would lead to relapse.Therefore,the efficacy is not so satisfied that new treatment therapies need to be developed.More than 95%of NPCs are pathologically diagnosed as low differentiated and undifferentiated carcinomas.This key feature of NPC presents as a unique model for intervention by differentiation therapy.The differentiation therapy could be a new clinical treatment for NPC.Tumor induced differentiation therapy is an approach which induces the malignant cells differentiation so that their morphology,and biological characteristics are similar to the mature cells during the process of maturation and differentiation under the treatment of differentiation inducing agents.After the differentiation of the immature cells,some of which are similar to normal cells,some restore part functions of the normal cells.Differentiation therapy aims to force the malignant cells to resume the process of maturation.The degree of tumor cells differentiation is often prompted its extent of malignancy.Generally,the malignant degree of high differentiation carcinoma is low and its progression is slow,while the low differentiatiated carcinoma and undifferentiated carcinoma are the opposite.Therefore,it colud be a truth by the use of induced differentiation therapy strategy to achieve the goal of reducing the degree of tumor malignancy or even cure cancer.Now the most crucial and successful drugs of differentiation inducing agent applied in clinical treatment is all-trans-retinoic acid(ATRA).This drug induces M3 leukemia cells to differentiate into mature cell.That makes acute promyelocytic leukemia patients achieved complete remission.After that the researchers have studied differentiation therapies of some Solid tumors,but the research is still in vitro experimental stage and the therapeutic effect is ineffective.Based on our former studies,we examined the high expression levels of Hesl both in NPC cells and tissues.It promoted the proliferation of NPC cells in vitro and tumorigenesis in vivo.Hesl gene(hairy and enhancer of split-1)is the mammalian homology of the hairy gene in Drosophila.Hesl is one of the seven members of the Hes gene family(Hesl-7).As a member of the family of the basic helix-loop-helix(bHLH)transcriptional repressors,it is not only a transcriptional repressor but also a classical downstream target of the Notch signaling pathway.Recent studies have shown that Hesl plays an important role in adult stem cells and inhibits the process of tumor cell differentiation in rhabdomyosarcoma and colon cancer.In our previous studies we analyzed the histological types of clinical NPC specimens from 103 patients.The results showed that Hesl was expressed highly in undifferentiated carcinoma compared with differentiated carcinoma.Although the P value had not yet reached 0.05,it is still suggested a certain relationship between Hes1 and NPC differentiation.Last year an article published in Nature Communications revealed the relationship between EZH2 and NPC differentiation.The author concluded that EZH2 can inhibit the differentiation of NPC cells by targeted IKKa gene.Through the means of bioinformatics,we predicted that EZH2 could be a target of Hes1,which was later be confirmed by CHIP assay and double luciferase reporter assay and could be positively regulated by Hes1 in our previous studies.Based on the background as well as our previous studies,we aim to solve two problems:1.Investigate the roles and mechanisms of Hes1 in the process of NPC cell differentiation;2.Identify whether Hes1 could be a potential target in induced differentiation therapy of NPC.This study will lay a foundation for the clinical induced differentiation therapy of NPC theoretically.Methods:1.Using clinical specimens to identify the correlation between Hes1 and different Histological types of NPCWe collected 32 non-cancerous nasopharyngeal epithelial tissues and 122 NPC tissues to analysis the expression of Hes1 in clinical specimens and the correlation between Hes1 and the clinicopathological features by using immunohistochemistry.2.Using clinical specimens to identify the associations between Hes1 and its target gene EZH2 and the biological markers for NPC differentiationWe distinguished 122 NPC tissues into 34 differentiated non-keratinizing c arcinoma and 88 undifferentiated non-keratinizing carcinoma based on the clini cal pathological parameters and histological types of WHO in 2005.By using immunohistochemistry,we clarified the relationship of Hes1 expression with its target gene EZH2 and NPC differentiated markers.3.Using the model of all-trans-retinoic acid(ATRA)induced tumor cell differ entiation,to investigate the changes of expression of Hes1 and its target gene EZH2 in the process of NPC cell differentiationNPC cell lines CNE2 and S18 were treated with different doses of ATRA.After 48h and 72h,we respectively collected total RNA and protein of the treated cells.Quantitative real-time PCR and Western bolt were used to identify the expression of NPC differentiated markers to clarify the success of foundation of the induced differentiation model by ATRA.Based on this model we investigated the changes of expression of Hes1 and its target gene EZH2 in the process of NPC cell differentiation.4.To investigate the impact of Hes1 in NPC cell differentiation in vivo and vitroWe purchased four shRNA-Hesl lentiviral vectors from ABM Company.After plasmid transformation,extraction and correct enzyme digestion,by using liposome-mediated transient transfection techniques,four shRNA-Hesl lentiviral vectors were transfected 293T cells.48 and 72 hours later,we collected total RNA and protein of the above cells.Quantitative real-time PCR and western blot were used to evaluate the effect of suppressing Hesl expression and then selected the best of interfering shRNA-Hesl-c producing lentivirus carrying shHes1.Viral supernatants were collected 72 hours later and then infected NPC cell lines CNE2 and S18.Puromycin was applied about 2 weeks to kill the Puro(一)cells and achieved the Puro(+)cells.Stable cell lines were expanded for experiments later.Based on the stably interfered shHesl cell lines above,we used colony fo rmation assay,CCK-8 assay and β-gal staining assay to identify cell proliferati on ability and senescence ability with Hes1 knokdown in vitro.Hes1,EZH2 a nd NPC differentiated markers were also tested by qRT-PCR and Western blot in order to determine the impact of Hes1 in the process of NPC cell differenti ation.In vivo we will conduct the tumorigenesis assay in nude mice.5.To investigate the mechanisms of Hesl in the process of NPC cell differentiationPackaging plasmids(psPAX2 and pMD2.G),along with pLV-R2P Blank L entiviral Vector and overexpression EZH2 Lentiviral Vector,which were purch ased from American Addgene Company,were transfected into 293T cells by li posome-mediated transient transfection techniques.Then stable NPC cell lines CNE2-shHes1 and S18-shHesl were transfected 72 hours later by the viral sup ernatant respectively.Stable cell lines were expanded for experiments later.Based on the above established stable cell lines,CCK-8 assay,colony for mation assay and β-gal staining assay were used to identify cell proliferation a bility and senescence ability in vitro.By using qRT-PCR and Western blot in order to determine EZH2 over-expression in Hes1 knockdown CNE2 and S18 cells could reverse the effect induced by Hes1 knockdown in CNE2 and S18 cells.In vivo we will conduct the tumorigenesis assay in nude mice.6.To investigate whether Hesl could be a potential target of NPC induced differentiation treatmentpLV-CDH-GFP-Puro Lentiviral Vector and overexpression Hesl Lentiviral Vector,along with packaging plasmids(psPAX2 and pMD2.G)were transfected with lipofectine 2000 to 293T cells.Then NPC cell lines CNE2 and S18 were transfected 72 hours later by the viral supernatants respectively.Puromycin was applied about 2 weeks to kill the Puro(一)cells and achieved the Puro(+)cells.Stable cell lines were expanded for experiments later.ATRA inducing differentiation assay was conducted on the stably established cell lines above,and then NPC differentiated markers were detected through qRT-PCR and Western blot to determine whether Hesl could suppress the differentiation of NPC cells induced by ATRA.Results:1.Using clinical specimens to identify the correlation between Hes1 and different Histological types of NPC1.1 High Hesl expression was found in NPC tissues comparing with non-cancerous epithelial tissuesBy using immunohistochemistry,we detected the expression of Hesl in 32 non-cancerous nasopharyngeal epithelial tissues and 122 NPC tissues.The results showed that High Hes1 expression was found in 44.3%(54/122)cases of NPC,which is higher than 12.5%(4/32)cases of non-cancerous nasopharyngeal epithelial tissue(x2=10.893,P=0.001).Compared with non-cancerous epithelial tissues,High Hesl expression was found in NPC tissues.1.2 Up-regulated Hesl expression was found in undifferentiated non-keratinizing carcinoma comparing with differentiated non-keratinizing carcinomaThe results showed that up-regulated Hesl expression was found in 50%(44/88)cases of undifferentiated carcinoma,which is higher than 29.4%(10/34)cases of differentiated carcinoma(χ2=4.214,P=0.045).Compared with differentiated non-keratinizing carcinoma,up-regulated Hesl expression was found in undifferentiated non-keratinizing carcinoma.1.3 Correlation between Hes1 and the clinicopathological featuresAnalyzed with Pearson χ2 test,Hesl expression was found with no significantly associations between gender and age.But we founded that the gene was positively correlated with histological type,T classification,N classification,distant metastasis and clinical stage.2.Using clinical specimens to identify the associations between Hes1 and its target gene EZH2 and the biological markers for NPC differentiation2.1 Expression of Hesl was correlated with a low level of CK8 as well as involucrin while a high level of CK13The results showed high CK8 expression was found in 67.6%(23/34)cases of differentiated non-keratinizing carcinoma,which is higher than 38.6%(34/88)cases of undifferentiated non-keratinizing carcinoma(χ2=8.292,P=0.005).High involucrin expression was found in 55.9%(19/34)cases of differentiated non-keratinizing carcinoma,which is higher than 32.9%(29/88)cases of undifferentiated non-keratinizing carcinoma(χ2=5.402,P=0.018).High CK13 expression was found in 45.4%(40/88)cases of undifferentiated non-keratinizing carcinoma,which is higher than 26.5%(9/34)cases of differentiated non-keratinizing carcinoma.The epithelial cell differentiation markers Involucrin and CK8 were highly expressed in differentiated non-keratinizing carcinoma while expression of Hes1 and CK13 were on the contrary.2.2 Expression of Hesl was positively correlated with its target gene EZH2Hesl target gene EZH2 were expressed in 56.8%(50/88)undifferentiated non-keratinizing carcinoma higher than 32.3%(11/34)differentiated non-keratinizing carcinoma,the difference was statistically significant(P<0.05).Compared with differentiated non-keratinizing carcinoma,high EZH2 expression was found in undifferentiated non-keratinizing carcinoma.We therefore examined the correlated relationship between Hesl and its target gene EZH2,which showed that Hes1 was positively correlated with EZH2.3.Using the model of all-trans-retinoic acid(ATRA)induced tumor cell differentiation,to investigate the changes of expression of Hesl and its target gene EZH2 in the process of NPC cell differentiation3.1 Successfully established the ATRA induced model of NPC differentiationNPC cell lines CNE2 and S18 were treated with different doses of ATRA(OμtM,10μM,15μM,20μM,25μM,30μM,40μM).48h and 72h later,we identified changes of morphologies and collected total RNA and protein of the treated cells respectively.We identified the expression of NPC differentiated markers by using qRT-PCR and western bolt,to clarify the successful foundation of the induced differentiation model by ATRA.The results revealed that the epithelial cell differentiation markers expression of CK8 and involucrin were increased with the increasing doses of ATRA,while CK13 was on the contrary.That showd we had successfully established the inducing model of NPC cells by ATRA.3.2 The expression of Hesl and EZH2 were suppressed in the process of NPC differentiation induced by ATRABased on the successfully established inducing model of NPC cells by ATRA,we tested the RNA and protein expression of Hesl and its target gene EZH2 through qRT-PCR and Western blot.And we found the reduced expression of Hesl and its target gene EZH2 in the process of NPC cell differentiation.4.To investigate the impact of Hesl in NPC cell differentiation in vivo and in vitro4.1 To select the best site of the four shRNA-Hes1 lentiviral vectorsBased on the liposome-mediated transient transfection techniques,four shR NA-Hesl lentiviral vectors were transfected 293T cells.We collected the total RNA and protein 48 and 72 hours later of the above cells.qRT-PCR and wes tern blot were used to evaluate the best effective vector suppressing Hesl expr ession.Then we selected the best interfering shRNA-Hesl-c producing lentiviru s carrying shHesl.The interfering efficiency was more than 50%.4.2 To establish stably NPC cell lines with Hesl knockdownThe shRNA-Hesl-c lentivirus vector and its shSCR lentivirus vector along with packaging plasmids(pMD2.G and psPAX2)were transfected into 293T cells.72 hours later,Virus supernatant was collected and then infected NPC cell lines CNE2 and S18 along with polybrene(2μg/ml).Puromycin was applied about 2 weeks to kill the Puro(-)cells and achieved the Puro(+)cells.We tested the RNA and Protein expression of Hesl through qRT-PCR and Western blot of the two stable cell lines.The results revealed that the levels of Hes1 in the two stable cell lines were lower than the shSCR vector cells,while the Western blot results was as same as qRT-PCR.4.3 To detect cell growth and proliferation ability by using CCK-8 assay and colony formation assayCCK-8 assay and colony formation assay were used to detect cell growth and proliferation ability of the stable cell lines.CCK-8 assay showed that CNE2 and S18 cells with Hesl knockdown grew slower than shSCR cells.Colony formation assay also indicated that the colonies numbers of NPC cells were decreased.In Conclusion,the ability of proliferation in CNE2 and S18 cells with Hesl knockdown were depressed.4.4 To detect cell senescence ability by using β-gal staining assayWe used β-gal staining assay to detect cell senescence ability of the stable cell lines.β-gal staining assay showed that the proportion of β-gal positive cells were increased in CNE2 and S18 cells with Hesl knockdown.4.5 To detect the expression of NPC differentiation markers by using western blotBy using western blot,we identified the expression of NPC differentiation markers of the stable cell lines with Hesl knockdown.The results indicated that CK8 and involucrin were increased while CK13 was on the contrary in CNE2 and S18 cells with Hesl knockdown.5.To investigate the mechanisms of Hes1 in NPC cell differentiation5.1 To establish stably NPC cell lines with EZH2 knockdownThe shRNA-EZH2 lentivirus vector and its shSCR lentivirus vector along with packaging plasmids(pMD2.G and psPAX2)were transfected into 293T cells.72 hours later,Virus supernatant was collected and then infected NPC cell lines CNE2 and S18 along with polybrene(2μg/ml).Puromycin was applied about 2 weeks to kill the Puro(-)cells and achieved the Puro(+)cells.We tested the RNA and Protein expression of EZH2 through qRT-PCR and Western blot of the two stable cell lines.The results revealed that the levels of EZH2 in the two stable cell lines were lower than that shSCR vector cells,while the Western blot results was as same as qRT-PCR.5.2 To detect cell growth and proliferation ability by using CCK-8 assay and colony formation assayCCK-8 assay and colony formation assay were used to detect cell growth and proliferation ability of the stable cell lines.CCK-8 assay showed that CNE2 and S18 cells with EZH2 knockdown grew slower than shSCR cells.Colony formation assay also indicated that the colonies numbers of NPC cells were decreased.In Conclusion,the ability of proliferation in CNE2 and S18 cells with EZH2 knockdown were depressed.5.3 To detect cell senescence ability by using β-gal staining assayWe used β-gal staining assay to detect cell senescence ability of the stable cell lines.β-gal staining assay showed that the proportion of β-gal positive cells were increased in CNE2 and S18 cells with EZH2 knockdown.5.4 To detect the expression of NPC differentiation markers by using western blotBy using western blot,we identified the expression of NPC differentiation markers of the stable cell lines with EZH2 knockdown.The results indicated that CK8 and involucrin were increased while CK13 was on the contrary in CNE2 and S18 cells with EZH2 knockdown.Conclusion:1.Compared with non-cancerous epithelial tissues,High Hes1 expression is found in NPC tissues.Up-regulated Hes1 expression is also found in undifferentiated non-keratinizing carcinoma and positively correlated with its target gene EZH2.2.Expression of Hes1 and EZH2 are suppressed in the process of NPC differentiation induced by all-trans-retinoic acid(ATRA).That prompts the two may play the roles of suppressing differentiation in the process of NPC differentiation.3.The ability of proliferation is depressed in CNE2 and S18 cells with Hesl knockdown,And the cell senescence and differentiation ability are promoted.