| Glioma is an intracranial primary tumor with a high incidence of more than 50%of all intracranial tumors.As the glioma is invasive and aggressive,so it is difficult to completely remove the tumor through the operation.Treatment effect and prognosis is usually poor.Glioma is divided into four grades by WHO and glioma with the higher grade is more malignant.Glioblastoma multiforme(GBM),which is WHO grade IV,has a high incidence,accounting for about 15%of all intracranial tumors.After the patients with glioblastoma get surgery supplemented by standard radiotherapy and chemotherapy,the median survival of patients is still not more than 15 months.Recent studies have shown that glioma,particularly the most malignant GBM,contain a subset of cells that have the characteristics of some normal stem cells,which are called glioblastoma stem-like cells(GSCs).GSCs play a strong role in promoting the proliferation of glioblastoma.Meanwhile,GSCs play a key role in promoting the infiltration and diffusion of tumor into the brain.Currently surgery combined with radiotherapy and chemotherapy can not kill the GSCs effectively,so the tumor often relapses fast,predicting poor prognosis.Thus,it is necessary to explore effective treatment of GSCs.Melatonin is a hormone secreted by the pineal gland.Melatonin has long been considered as a hormone that regulates biological rhythms.In recent years,more and more studies have shown that melatonin has a variety of tumors have an inhibitory effect on a variety of tumors including glioma.However,the effect of melatonin on GSCs is rarely studied,and its mechanism is not clear.EZH2 has a histone methyltransferase function,which is one of the components of the polycomb repressive complex 2(PRC2).EZH2 abnormal increase has been found in a variety of tumors including glioma,breast cancer,stomach cancer,colon cancer and so on.And the higher the expression level of EZH2 is,the higher the degree of malignancy is,predicting worse prognosis.At the same time,there is definite evidence that EZH2 is one of the indicators of GSCs.Overexpression of NOTCH receptors is also present in a variety of malignancies including glioma.The EZH2-NOTCH 1 signaling axis has been verified to exist in pancreatic cancer.Whether the EZH2-NOTCHl signal axis also exists in GSCs,and whether the EZH2-NOTCH1 signal axis is involed in the effect of melatonin on GSCs have not been reported.In this study,we studied the effect of melatonin on GSCs and the specific mechanism beneath it.OBJECTIVE:1.To study the effect of melatonin on the proliferation,self-renewal and stem cell properties of GSCs.2.To study the specific role of EZH2-NOTCH1 signaling axis in the effectual procedure of melatonin.3.To explore the specific mechanism of EZH2 in the regulation of NOTCH1.METHODS:PART 1:EFFECTS OF MELATONIN ON PROLIFERATION,SELF-RENEWAL AND STEM CELL PROPERTIES OF GSCS.1.Culture and verification of GSCs from U251 and T98 cell lines1.1 U251 and T98G cell lines were cultured in stem cell enrichment medium(serum-free,B27,bFGF and EGF factors)for at least three weeks to establish GSCU251 ? GSCT98G cell models.1.2 Neurological scoring and TTC staining were used to evaluate the damage to central nervous system.The expression of CD 133 and SOX2 in GSCs were detected by qRT-PCR to verify whether the GSCU251 and GSCT98G cell models were established successfully.2.To investigate the effects of different concentrations of melatonin on the survival and proliferation of GSCs2.1 Medium containing different concentrations of melatonin(0.1-1000?M)were prepared to culture GSCU251 and GSCT98G.2.2 CCK-8 assay was used to detect the effect of melatonin on the proliferation of GSCU251 and GSCT98G.3.To investigate the effect of melatonin on GSCs self-renewal abilityThe effects of different concentrations of melatonin on the self-renewal ability of GSCU251 and GSCT98G were detected by sphere formation assay.4.To investigate the effect of melatonin on the stem cell properties of GSCsTo detect the effect of melatonin on the expression of CD 133 and SOX2 in GSCU251 and GSCT98G by qRT-PCR and Western Blot.PART 2:THE ROLE OF EZH2 IN THE INHIBITION OF GSCS BY MELATONIN1.To investigate the effect of melatonin on the expression of EZH2 in GSCsEffect of melatonin on expression of EZH2 in GSCs was explored by qRT-PCR and Western Blot.2.Establishment of EZH2 knockdown GSCU251 model.2.1 The EZH2 knockdown GSCU251 model was established by transfection of GSCU251 with EZH2 shRNA2.2 To verify whether EZH2 knockdown GSCU251 model was established successfully by Western Blot.3.To study the effect of EZH2 knockdown on the proliferation,self-renewal and stem cell properties of GSCU251After knocking down EZH2 in GSCU251 CCK-8 assay was used to detect the growth and proliferation ability of GSCU251;Sphere formation assay was used to detect the self-renewal ability of GSCU251;qRT-PCR was used to detect the expression ofCD133 in GSCU251.4.Establishment of EZH2 overexpression GSCU251 model4.1 EZH2 overexpression GSCU251 model was established by transfection of GV230-EZH2 plasmid into GSCU251.4.2 Western Blot was used to detect whether the EZH2 overexpression GSCU251 model was established successfully.5.To study the proliferation,self-renewal and stem cell properties of GSCU251 after EZH2 was overexpressedAfter EZH2 was overexpressed in GSCU251,CCK-8 assay was used to detect growth and proliferation of GSCU251;Sphere formation assay was used to detect the self-renewal ability of GSCU251;qRT-PCR was used to detect the expression of CD133 in GSCU251.6.To investigate the proliferation ability,self-renewal ability and stem cell properties of control group and EZH2 overexpression group GSCU251 after melatonin was added6.1 After melatonin was added into the control group GSCU251,CCK-8 assay and sphere formation assay were used to detect GSCU251 proliferation and self-renewal ability;The expression of CD133 in GSCU251 was detected by qRT-PCR.6.2 After melatonin was added into the EZH2 overexpression group GSCU251,CCK-8 assay and sphere formation assay were used to detect GSCU251 proliferation and self-renewal ability;The expression of CD 133 in GSCU251 was detected by qRT-PCR.PART 3:THE FUNCTION AND MECHANISM OF NOTCH1 IN THE EFFECTUAL PROCEDURE OF EZH21.To investigate the effect of melatonin on the expression of NOTCH1 in GSCs1.1 qRT-PCR was used to detect the mRNA expression of NOTCH1,CCND1,CCNE2 and HES1 in GSCU251 and GSCT98G after melatonin was added.1.2 Western Blot was used to detect the expression of NICD1(intracellular functional region of NOTCH1)and HES1 in GSCU251 and GSC-T98G after melatonin was added.2.To investigate the effect of EZH2 on the regulation of NOTCH12.1 The changes of NOTCH 1 transcription activity in EZH2 knockdown and overexpression GSCU251 were detected by dual-luciferase reporter assay.2.2 Western Blot was used to detect the protein expression of NICD1 and HES1 in EZH2 knockdown and overexpression GSCU251.2.3 Chromosome immunoprecipitation assay(ChIP)was used to detect the aggregation of EZH2,H3K27me3 and SUZ12 in the promoter positions of NOTCH1 in GSCU251.PART 4:A PRELIMINARY STUDY ON THE EZH2-NOTCH1 SIGNALING AXIS IN CLINICAL SAMPLES1.To explore the correlation between expression of EZH2 and NICD1 in glioblastoma samples and normal brain tissue samples1.1 67 cases of glioblastoma samples and 12 cases of normal brain tissue samples were taken for EZH2 and NICD1 immunohistochemical staining.1.2 Statistical analysis of the expression of EZH2 and NICD1 in glioblastoma samples.RESULTS:PART 1:EFFECTS OF MELATONIN ON PROLIFERATION,SELF-RENEWAL AND STEM CELL PROPERTIES OF GSCS1.Culture and verification of GSCs from U251 and T98 cell linesAfter U251 and T98G cell lines were cultured in stem cell enrichment medium for at least three weeks,GSCU251 ? GSCT98G cell models were established.By qRT-PCR,we found that the levels of stem cell markers CD 133 and SOX2 in the GSCU251 and GSCT98G cell models were significantly higher than those in the conventional U251 and T98G cell lines.2.To investigate the effects of different concentrations of melatonin on the survival and proliferation of GSCsAfter treatment of GSCU251 and GSCT98G with different concentrations of melatonin,the results of CCK-8 assay showed that 100?M and ImM melatonin inhibited the growth of GSCs.3.To investigate the effect of melatonin on GSCs self-renewal abilityAfter treatment of GSCU251 and GSCT98G with different concentrations of melatonin,the results of sphere formation assay showed that 100?M and ImM melatonin inhibited the self-renewal of GSCs.4.To investigate the effect of melatonin on the stem cell properties of GSCsThe results of qRT-PCR and Western Blot showed that 1 mM melatonin significantly inhibited the expression of CD133 and SOX2 in GSCU251 and GSCT98G,PART 2:THE ROLE OF EZH2 IN THE INHIBITION OF GSCS BYMELATONIN1.To investigate the effect of melatonin on the expression of EZH2 in GSCsThe results of qRT-PCR and Western Blot showed that the intervention of melatonin significantly reduced the expression of EZH2 in GSCU251 and GSC-T98G2.Establishment of EZH2 knockdown GSCU251 model.Western blot analysis showed that the expression of EZH2 protein in EZH2 knockout GSCU251 model was significantly decreased.3.To study the effect of EZH2 knockdown on the proliferation,self-renewal and stem cell properties of GSCU251After knocking down EZH2 in GSCU251,CCK-8 assay showed that the growth and proliferation ability of GSCU251 were reduced significantly;Sphere formation assay showed that the self-renewal ability of GSCU251 was inhibited;The expression of CD133 in GSCU251 was significantly decreased by qRT-PCR.4.Establishment of EZH2 overexpression GSCU251 modelWestern blot analysis showed that the expression of EZH2 protein in EZH2 overexpression GSCU251 model was significantly increased.5.To study the proliferation,self-renewal and stem cell properties of GSCU251 after EZH2 was overexpressedAfter EZH2 was overexpressed in GSCU251,CCK-8 showed that the growth and proliferation ability of GSCU251 were significantly enhanced;Sphere formation assay showed that the self-renewal ability of GSCU251 was enhanced;The expression of CD133 in GSCU251 was significantly increased by qRT-PCR.6.To investigate the proliferation ability,self-renewal ability and stem cell properties of control group and EZH2 overexpression group GSCU251 after melatonin was added6.1 After melatonin was added into the control group GSCU251,CCK-8 assay and sphere formation assay showed that GSCU251 proliferation and self-renewal ability of GSCU251 were significantly reduced;The expression of CD 133 in GSCU251 was significantly decreased by qRT-PCR.6.2 After melatonin was added into the EZH2 overexpression group GSCU251,CCK-8 assay and sphere formation assay showed that GSCU251 proliferation and self-renewal ability of GSCU251 were significantly reduced;qRT-PCR showed that the expression of CD133 in GSCU251 was significantly decreased.PART 3:THE FUNCTION AND MECHANISM OF NOTCH1 IN THE EFFECTUAL PROCEDURE OF EZH21.To investigate the effect of melatonin on the expression of NOTCH1 in GSCs1.1 qRT-PCR was used to detect the mRNA expression of NOTCH1,CCND1,CCNE2 and HES1 in GSCU251 and GSC-T98G after melatonin was added.1.2 Western Blot was used to detect the expression of NICD1(intracellular functional region of NOTCH1)and HES1 in GSCU251 and GSCT98G after melatonin was added.1.1 The results of qRT-PCR showed the mRNA expression of NOTCH1,CCND1,CCNE2 and HES1 in GSCU251 and GSCT98G was significantly decreased after melatonin intervention.1.2 Western Blot results also showed that the expression of NICD1 and HES1 protein in GSCU251 and GSCT98G was decreased significantly after melatonin intervention.2.To investigate the effect of EZH2 on the regulation of NOTCH12.1 By dual-luciferase reporter assay,we found that,after EZH2 knockdown and overexpression of GSCU251,the transcription activity of NOTCH1 was attenuated and enhanced respectively.2.2 Western Blot results showed that the protein expression of NICD1 and HES1 increased and decreased respectively after EZH2 knockdown and overexpression in GSCU251.2.3 By ChIP assay,aggregation of EZH2 could be detected in the promoter position of NOTCH1 in GSCU251,while H3K27me3 and SUZ12 could not be detected in the same position.At the same time,the intervention of melatonin significantly reduced the aggregation of EZH2 in the promoter region of NOTCH1.PART 4:A PRELIMINARY STUDY ON THE EZH2-NOTCH1 SIGNALING AXIS IN CLINICAL SAMPLES1.To explore the correlation between expression of EZH2 and NICD1 in glioblastoma samples and normal brain tissue samples1.1 Immunohistochemical staining analysis showed that 73%of EZH2 overexpression glioblastoma samples also had high expression of NICD1.1.2 Statistical analysis revealed a high positive correlation between the expression of EZH2 and NICD1 in glioblastoma samples.CONCLUSION:1.Specific concentration of melatonin could significantly inhibit the proliferation and self-renewal ability of GSCs,and could significantly reduce the stem cell potency of GSCs.2.Through the mechanism experiments,we found that melatonin could play a role in inhibiting GSCs by inhibiting the EZH2-NOTCH1 signaling axis.3.During the EZH2-NOTCH1 signal axis,EZH2 controlled the expression of NOTCH1 by directly combining the promoter region of NOTCH1. |
PDF Full Text Request