| Part one The expression of BMI-1 and RNF2 in esophageal squamous cellcarcinomas and the effect of them on clinicopathologiccharacteristics and prognosticObjective: Esophageal squamous cell carcinoma(ESCC)has been a common cancer with serious health threat.Radiotherapy(RT)is an important way for esophageal carcinoma,however,the prognosis of pateins is poor owing to radioresistance.B-cell-specific Moloney murine leukemia virus integration site-1 and Ring finger protein 2,as the core components in the polycomb transcriptional repressors,have been dysregulated in various malignancies.The purpose of this study is to detect the expression of B-cell-specific Moloney murine leukemia virus integration site-1 and Ring finger protein 2 in human esophageal squamous cell carcinoma(ESCC)and matched adjacent normal tissues,and to assess the clinicopathologic and prognostic significance of it in ESCC.Methods:1 The expression of BMI-1 and RNF2 at protein level in ESCC and normal mucosal tissues were assessed through immunohistochemistry.2 Western blotting was employed to detecte the expression of BMI-1 and RNF2 in ESCC and normal mucosal tissues.3 The relationship between the expression of BMI-1,RNF2 and clinicopathological parameters or prognosis was analyzed.Results:1 The expression of BMI-1 and RNF2 in esophageal tissues analyzed by immunohistochemistry stainingImmunohistochemical assay found that BMI-1 and RNF2 proteins localized mainly in cellular nuclei of ESCC.BMI-1 positive staining was present in 32 of 60 ESCC tissue sections(53.33%),but only 5 of 60 normal tissue sections(8.33%).RNF2 positive staining was present in 35 of 64 ESCC(54.69%),but only 7 of 64 normal tissue sections(10.94%).The results revealed that the expression of BMI-1 and RNF2 at protein levels was markedly higher in ESCC tissues than that in adjacent normal tissues(P<0.001).2 The expression of BMI-1 and RNF2 in esophageal tissues analyzed by Western blotting analysisThe results demonstrated that the expression of BMI-1 and RNF2 at protein levels was obviously higher than that of adjacent normal tissues by Western blotting analysis(0.317±0.098 vs 1.032±0.210,0.206±0.028 vs 1.070±0.153)(P<0.05),which was accordance with those of Immunohistochemical assay.3 The correlation between BMI-1,RNF2 and clinicopathologic and prognostic significance of them in ESCCsAdditionally,the expression of BMI-1 and RNF2 at protein levels was markedly correlated with tumor size(P=0.022,P=0.024),lymph node metastasis(P=0.011,P=0.015)and TNM stage(P=0.021,P=0.027).However,it was no significant differences between the expression of BMI-1,RNF2 and the other parameters(P>0.05).Kaplan-Meier survival analysis revealed that patients with positive expression of BMI-1 and RNF2 had a shorter obviously overall survival than negative patients(P<0.001).The overall 3-year survival rate for patients in BMI-1 positive and negative expression group was 18.80% and 58.30%,respectively.The median survival time was 14 months and 26 months,respectively.The overall 3-year survival rate for patients in RNF2 positive and negative expression group was 14.30% and 48.30%,respectively.The median survival time was 15 months and 24 months,respectively.Conclusions:1 The expression of BMI-1 and RNF2 was very high in ESCC.2 The expression of BMI-1 and RNF2 was obviously correlated with tumor malignant degree,lymph node metastasis and poor prognosis,which provides new ideas for molecular targeted therapy of ESCC.Part two The effect of BMI-1 and RNF2 depletion on radiosensitivity inESCC in vitroObjective: RNA interference was employed to study the effect of BMI-1 and RNF2 depletion on the cell proliferation,migration,cell cycle distribution,apoptosis and the correlation between BMI-1,RNF2 and DNA damage repair genes of ESCC after exposure to irradiation,and to expore the regulatory effect of them on the radiosensitivity of ESCC.Methods:1 The expression of BMI-1 and RNF2 at protein levels was detected and evaluated by Western blotting assay in different ESCC cell lines,including TE13,KYSE30,ECA109,and KYSE170.2 The BMI-1 and RNF2 eukaryotic expression vectors(pGLV3-H1-BMI-1 and pGLV3-H1-RNF2)were transfected into ECA109 and TE13 cells.The cells were observed with puromycin for two weeks and monoclonal cells were picked to amplificate.In the end,shRNA-mediated stable ECA109-shBMI-1(ECA109-sh RNF2),TE13-shBMI-1(TE13-shRNF2)cells,and their respective negative controls(ECA109-NC and TE13-NC cells)were obtained,respectively.qRT-PCR and Western blotting assays were employed to evaluate the transfection efficiency,respectively.3 MTS and colony formation assays were employed to detecte the effect of BMI-1 and RNF2 depletion on proliferation and radiosensitivity of tumor cells,respectively.4 Transwell migration assay was used to evaluate the effect of BMI-1 and RNF2 depletion on migratory ability of tumor cells.5 Western blotting and Immunofluorescence staining were used to explore the dose-and time-effect relationship in protein expression and nuclear foci of BMI-1,RNF2,and their downsteam targeted genes γH2AX,H2AK119 ub by in different ESCCs after irradiation(IR)for different doses at different times,respectively.Co-immunoprecipitation(Co-IP)assay was employed to assess the interaction of BMI-1,RNF2 with γH2AX and H2AK119 ub.6 Flow cytometry was employed to analyze cell cycle distribution and apoptosis of tumor cells in each group.7 Western blotting assay was used to detect the expression of cell cycle-related proteins(CDK4,Cycle D2 and P16)and cell apoptosis-related proteins(bcl-2 and bax)at protein levels.Results:1 The expression of BMI-1 and RNF2 in different ESCCsWestern blotting analysis found that the expression of BMI-1 and RNF2 at protein levels in different ESCCs,including TE13,KYSE30,ECA109,and KYSE170 were very high.Additionally,ECA109 and TE13 cells exhibited relatively higher expression of BMI-1 and RNF2 at protein levels(P<0.01).2 The silencing effect of BMI-1 shRNA and RNF2 shRNA on ESCCsThe expression BMI-1 and RNF2 in ECA109 and TE13 cells was depleted to explore their effect on biological characteristics of tumor cells.qRT-PCR and Western blotting analysis found that the expression of BMI-1 at mRNA and protein levels was markedly decreased in ECA109 sh BMI-1 and TE13 shBMI-1 cells than those in their corresponding blank control or negative control cells(P<0.01).The expression of RNF2 at mRNA and protein levels was consistent with those of BMI-1 in ECA109 and TE13 cells,which displayed that BMI-1 and RNF2 shRNA-mediated stable transfection can effectively inhibit the expression of BMI-1 and RNF2 in ECA109 and TE13 cell lines.3 The effect of BMI-1 and RNF2 depletion on cell viability and colony formationMTS assay showed that cell viability was markedly lower in ECA109-shBMI-1,ECA109-shRNF2,TE13-shBMI-1 and TE13-sh RNF2 cells than that in their corresponding blank control or negative control cells at 24,48 and 72 h before irradiation(P<0.05),and the tendence is more obvious at 48,72 h after IR(P<0.01).Colony formation assays revealed that the values of D0,Dq,and SF2 in BMI-1 shRNA and RNF2 shRNA groups were lower obviously than those in control group and NC group,while the values of N in BMI-1 shRNA and RNF2 shRNA groups were markedly higher than those in other groups(P<0.05),which showed higher radiosensitivity in BMI-1 shRNA and RNF2 shRNA groups(P<0.05).It was not significantly different between control and NC groups(P>0.05).4 The effect of BMI-1 and RNF2 depletion on cell migrationTranswell migration assay found that the numbers of migrated cells through the transwell hole in ECA109-BMI-1 sh RNA and ECA109-RNF2 shRNA groups were significantly lower than those of other groups(P<0.05).The results of TE13 cells were accordance with those of ECA109 cells(P<0.05).5 The correlation between BMI-1,RNF2 and DNA damage repair genesThe expression of BMI-1,RNF2,H2AK119 ub,and γH2AX at protein levels increased with the increasing doses,their levels were highest at 1~2h after accepted 6Gy irradiation,then gradually declined,and was close to the pre-irradiation level at 24 h after IR.Their change tendence is similar,and there is in a time-and dose-dependent.Ionizing radiation induced the levels of BMI-1,RNF2,γH2AX nuclear foci in a dose-dependent in cells,and the greater the radiation doses,the greater the numbers of foci.The BMI-1,RNF2,γH2AX foci achieved the peak at 1~2h,reverted to the pre-irradiation level at 24 h after IR.Co-immunoprecipitation(Co-IP)assay revealed that the interaction between BMI-1,RNF2 and H2AK119 ub,γH2AX was markedly increased after exposure to IR,while it was not dramatically correlation between BMI-1,RNF2 and H2AK119 ub,γH2AX before IR.6 The effect of BMI-1,RNF2 depletion on cell cycle distribution of esophageal squamous cellsIt was not obviously different in different period ratios of each group before IR(P>0.05).The percentage of G0/G1 phase in each group was dramatically lower after IR compared with corresponding unirradiated groups(P<0.05),while depletion of BMI-1,RNF2 expression upregulated the percentage of G0/G1 phase(P<0.01),and downregulated the percentage of G2/M phase(P<0.01).Additionally,the percentage of G2/M phase in each group was obviously higher after IR compared with corresponding unirradiated groups(P<0.05).It was not significantly different in the percentage of S phase in each group before and after IR(P>0.05).7 The effect of BMI-1,RNF2 knockdown on cell apoptosis of esophageal squamous cellsThe apoptosis rates in each group was obviously high after IR compared with corresponding unirradiated groups(P<0.05).It was not obviously different in the three groups before IR although the apoptosis rate after BMI-1 depletion was slightly higher(P>0.05),however,the apoptosis rate after BMI-1 silencing was higher than NC and control groups after IR(P<0.01).In addition,the apoptosis rate after RNF2 depletion was higher than NC and control groups before IR(P<0.05),and the tendence was more obviously after IR(P<0.01).8 The effect of BMI-1,RNF2 knockdown on cell cycle-related and apoptosis-related proteins of esophageal squamous cells1)The effect of BMI-1,RNF2 knockdown on cell cycle-related proteins of tumor cells such as Cyclin D2,CDK4 and P16.Western blotting assay revealed that the expression of Cyclin D2,CDK4 and P16 in tumor cells after IR significantly recreased compared with the corresponding unirradiated group(P<0.05).Silencing of BMI-1 and RNF2 markedly downregulated the expression of Cyclin D2 and CDK4,and upregulated P16 compared with control and NC groups before IR(P<0.05),and the tendence was more obvious after IR(P<0.01).2)The effect of BMI-1 and RNF2 knockdown on cell apoptosis-related proteins of tumor cells such as bcl-2 and bax.Western blotting assay showed that the expression of bcl-2 and bax in each group was obviously high after IR compared with corresponding unirradiated groups(P<0.05).Silencing of BMI-1 and RNF2 expression dramatically downregulated the expression of bcl-2,and upregulated the expression of bax before IR compared with control and NC groups(P<0.05).The tendence was more obvious after IR(P<0.01).Conclusions:1 Knockdown of BMI-1 and RNF2 inhibits cell proliferation and migration,and enhances the radiosensitivity of esophageal cancer through arresting cell cycle at G0/G1 phase and inducing cell apoptosis.2 The regulation effect of BMI-1 and RNF2 on cell cycle distribution and cell apoptosis is through interaction with ubiquitylation and phosphorylation of H2 AX to regulate the expression of cell cycle-related and cell apoptosis-related proteins in tumor cells.Part three The effect of BMI-1 and RNF2 knockdown on radiosensitizati-on in transplanted tumor of nude mice in vivoObjective: Transplantation tumor models in nude mice were constructed to study the tumorigenicity,analyze related molecular changes in the tumor tissues,and explore the mechanisms in the development of esophageal squamous cancer.Methods:1 TE13-control,TE13-pGLV3-H1,TE13-BMI-1 shRNA and TE13-RNF2 shRNA cells were inoculated to set up transplantation tumor models in BALB/c nude mice,and then to observe the tumor formation and the growth of subcutaneous.2 Nude mice were irradiated when the transplantation tumors were approximately 0.8 to 1.0 cm in diameter,and sacrificed at 24 h after IR.Western blotting assay was used to analyze the expression of BMI-1 and RNF2 at protein levels in transplantation tumor tissue.3 The apoptosis of tumor tissues was analyzed by TUNEL assay in vivo.4 The expression of BMI-1,RNF2 and apoptotic related-genes bcl-2 and bax in transplantation tumor of nude mice was observed by immunohistochemical assay.Results:1 The effect of BMI-1 and RNF2 depletion on tumor formation and inhibition rate of transplantation tumorThe effect of BMI-1 and RNF2 knockdown on tumor formation and tumor growth inhibition rates revealed that it was not dramatical effect of transfection on transplanted tumor growth,however,the tumor volume and weigh of nude mice in silencing BMI-1 and RNF2 groups were markedly small compared with other groups(P<0.05).Moreover,the tendence was more significantly after IR.The tumor growth was fast in control and NC groups,knockdown of BMI-1,RNF slowed the growth rate of xenografts(P<0.05).The growth rate slowed down after IR,and depletion of BMI-1 and RNF2 increases the tendence.Tumor growth inhibition rates were 8.22%,52.52%,29.71%,76.26%,33.42%,and 75.73% in different groups,respectively.The tumor volume was smaller obviously after knockdown of BMI-1 and RNF2 combined with IR,which displayed that depletion of BMI-1 and RNF2 promoted the radiosensitivity in vivo.2 The silencing effect of shRNA on BMI-1 and RNF2 in vivoWestern blotting aaasy revealed that the expression of BMI-1 and RNF2 at protein levels was markedly lower in knockdown of BMI-1 and RNF2 groups than other groups(P<0.01),and the silencing efficiency of BMI-1 and RNF2 were 66.67% and 68.21%,respectively.3 The effect of BMI-1 and RNF2 knockdown on apoptosis of transplantation tumor in nude miceTUNEL assay revealed that the apoptosis rates in each group were significantly high after IR compared with corresponding unirradiated groups(P<0.05).Additionally,the apoptosis rates in tumor tissues of BMI-1 shRNA and RNF2 shRNA groups was markedly higher after IR than those of control and NC groups(P<0.05),which indicated that depletion of BMI-1 and RNF2 combined with IR induced cell apoptosis,and thus enhanced the radiosensitivity of transplantation tumor.4 The expression of BMI-1 and RNF2 at protein levels in transplantation tumor of nude miceThe results found that BMI-1 and RNF2 proteins mainly located in the nucleus,and less in cytoplasm by Immunohistochemical assay.Compared with their corresponding unirradiated groups,the expression of BMI-1 and RNF2 protein in the tumor tissues of each group was slightly high after irradiation,but had no statistical difference(P>0.05).In addition,the expression of BMI-1 and RNF2 proteins was significantly lower in the depletion of BMI-1 and RNF2 groups than those of control and NC groups before IR(P<0.05),the tendence is more obvious after IR.5 The expression of apoptosis-related proteins in transplantation tumor of nude miceImmunohistochemical results revealed that the expression of bcl-2 protein in each group was significantly lower after IR than the corresponding unirradiated groups(P<0.05).Compared with control and NC groups,the expression of bcl-2 protein was obviously lower in depletion of BMI-1 and RNF2 groups before IR(P<0.05).In addition,compared with control or NC combined with irradiation group,the expression of the bcl-2 at protein levels significantly decreased in the depletion of BMI-1 or RNF2 combined with irradiation group(P<0.05).The expression of bax at protein level in each group was significantly higher after irradiation than the corresponding unirradiated groups(P<0.05).Compared with control and NC groups,the expression of bax protein was markedly higher in depletion of BMI-1 and RNF2 groups before IR(P<0.05).In addition,compared with control or NC combined with irradiation group,the expression levels of the bax at protein levels significantly increased in the depletion of BMI-1 or RNF2 combined with irradiation group(P<0.05).Conclusions: Depletion of BMI-1 and RNF2 induces apoptosis of transplantation tumor tissues in nude mice,inhibits the growth of tumor and improves the radiotherapy sensitization effect of esophageal cancer cells in vivo through downregulating the expression of bcl-2 and upregulating the expression of bax. |
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