Effect Of UBE2L3 In Malignant Proliferation Of Hepatocellular Carcinoma And Its Mechanisms

Objective:1.To detect the expression level of UBE2L3 in HCC tissues.2.To study the role of UBE2L3 in the proliferation and apoptosis of HCC cells.3.To demonstrate the molecular mechanism of UBE2L3 in the proliferation and apoptosis of HCC cells.4.To illuminate the effect of UBE2L3 in vivo by using orthotopic xenograft model in nude mice.Methods:1.The microarray data was obtained from the NCBI GEO database(accession numbers GSE14520,GSE25097).The UBE2L3 expression in the tumor tissues and the adjacent non-tumoral tissues was illustrated by scatterplot.Kaplan-Meier analysis of the correlation between UBE2L3 expression level and overall survival or disease free survival in the TCGA cohort.The mRNA expression level of UBE2L3 in the tumor tissues and the adjacent non-tumoral tissues in 66 cases of HCC patients were detected by reverse transcription quantity polymerase chain reaction(RT-qPCR).The protein expression level of UBE2L3 in the tumor tissues and the adjacent non-tumoral tissues in 66 cases of HCC patients were detected by Western blot.2.After knock down or knock out the expression of UBE2L3,the proliferation of HCC cells was tested by typan blue exclusion assays.The ability of colony formation and anchorage-dependent growth was analyzed by colony formation assays and soft agar assays,respectively.3.After knock down or knock out the expression of UBE2L3,the apoptotic rate of HCC cells was assayed by Flow Cytometry.The protein expression of apoptosis-related factors were measured by Western blot.4.UBE2L3 knock out cell line was used to conduct the nude mice xenograft model,then the model was used to determine the role of UBE2L3 in HCC tumor growth.5.After overexpress UBE2L3,the proliferation of HCC cells was tested by typan blue exclusion assays.The ability of colony formation and anchorage-dependent growth was analyzed by colony formation assays and soft agar assays,respectively.And the apoptotic rate of HCC cells was assayed by Flow Cytometry.6.The mRNA expression level of p65 in the UBE2L3 knock down,knock out or overexpression cells were detected by RT-qPCR.The protein expression level and the phosphorylation of p65 in the UBE2L3 knock down,knock out or overexpression cells were detected by Western blot.Immunocytochemistry was used to analyze the protein expression level and subcellular location of p65 in the UBE2L3 knock down cells.The cytoplasmic and nuclear protein in UBE2L3 knock down cells were extracted and used for the detection of p65 protein expression level and subcellular location.7.The mRNA expression level of PUMA in the UBE2L3 knock down,knock out or overexpression cells were detected by RT-qPCR.The protein expression level of PUMA in the UBE2L3 knock down,knock out or overexpression cells were detected by Western blot.8.The effect of p65 inhibitor JSH-23 on the UBE2L3 KO induced changes in downstream signaling pathway were measured by Western blot.The effect of p65 inhibitor JSH-23 on the UBE2L3 KO induced cell proliferation decreasing was tested by typan blue exclusion assays.The effect of p65 inhibitor JSH-23 on the UBE2L3 KO induced cell apoptosis was assayed by Flow Cytometry.9.The influence of UBE2L3 knock down or GSK3? overexpression on transcriptional activity of NF-?B was tested by dual-luciferase reporter system.The UBE2L3 knock down or GSK3? overexpression plasmid was co-transfected with RL-TK and NF-?B luciferase reporter plasmid.The mRNA expression level of p65 in the GSK3? knock down or overexpression cells were detected by RT-qPCR.The protein expression level and the phosphorylation of p65 in the GSK3? knock down or overexpression cells were detected by Western blot.10 The mRNA expression level of GSK3? in the UBE2L3 knock down,knock out or overexpression cells were detected by RT-qPCR.The protein expression level and the phosphorylation of GSK3? in the UBE2L3 knock down,knock out or overexpression cells were detected by Western blot.The effect of UBE2L3 on the protein stability of GSK3? was determine by Western blot under the treatment of UBE2L3 overexpress and protein synthesis inhibitor CHX.Whether the UBE2L3-regulated GSK3? protein degradation was through the ubiquitin-proteasome degradation pathway,Western blot assay was employed under the treatment of UBE2L3 overexpress and proteasome inhibitor MG132.The association between UBE2L3 and GSK3? was measured by Co-IP.The ubiquitination level of GSK3? was analyzed by Co-IP in the UBE2L3 knock down or overexpression cells.11.The effect of GSK3? knock down or GSK3? inhibitor 1-Azakenpaullone on the UBE2L3 KO induced changes in downstream signaling pathway were measured by Western blot.The effect of GSK3? knock down or GSK3? inhibitor 1-Azakenpaullone on the UBE2L3 KO induced cell proliferation decreasing was tested by typan blue exclusion assays.The effect of GSK3? knock down or GSK3? inhibitor 1-Azakenpaullone on the UBE2L3 KO induced cell apoptosis was assayed by Flow Cytometry.12.UBE2L3 knock out cell line and GSK3? knock down celline were used to conduct the orthotopic xenograft model in nude mice.Then the mice were treated with p65 inhibitor JSH-23 to analyze the role of UBE2L3 and the effect of GSK3? knock down or or p65 inhibitor JSH-23 in HCC tumor growth.Results:1.The microarray data was obtained from the NCBI GEO database(accession numbers GSE14520;GSE25097).The UBE2L3 expression in the tumor tissues was significantly higher than that in the adjacent non-tumoral tissues.The analysis in the TCGA cohort shown that patients with high UBE2L3 expression level had significantly shorter overall survival and disease free survival than those with low UBE2L3 expression level.The mRNA expression level of UBE2L3 in the tumor tissues was significantly higher than that in the adjacent non-tumoral tissues.The protein expression level of UBE2L3 in the tumor tissues were significantly higher than that in the adjacent non-tumoral tissues in 50 cases(75.8%)of HCC patients.The statistical analysis shows that the protein expression level of UBE2L3 in the tumor tissues were significantly higher than that in the adjacent non-tumoral tissues.2.UBE2L3 knock down or knock out led to a significant decrease in proliferation of HCC cells.UBE2L3 knock down or knock out reduced the ability of colony formation and anchorage-dependent growth in HCC cells.3.UBE2L3 knock down or knock out led to a significant increase in the apoptotic rate of HCC cells and expression of apoptosis-related factors.4.Knock out UBE2L3 in HCC cells could inhibit tumor growth in nude mice xenograft model.5.UBE2L3 overexpression led to a significant increase in proliferation of HCC cells.UBE2L3 overexpression increased the ability of colony formation and anchorage-dependent growth in HCC cells.And had trend in inhibition of apoptotic rate of HCC cells.6.UBE2L3 knock down or knock out could increase the mRNA expression level,protein expression level and phosphorylation of p65.UBE2L3 overexpression could decrease the mRNA expression level,protein expression level and phosphorylation of p65.UBE2L3 knock down led to a significant increase in p65 expression level and its nuclear translocation.7.UBE2L3 knock down or knock out could increase the mRNA and protein expression level of PUMA.UBE2L3 overexpression could decrease the mRNA and protein expression level of PUMA.8.p65 inhibitor JSH-23 could reverse the UBE2L3 KO induced changes in downstream signaling pathway and could reverse the UBE2L3 KO induced cell proliferation decreasing,the UBE2L3 KO induced cell apoptosis.9.The transcriptional activity of NF-?B was activated by UBE2L3 knock down or GSK3? overexpression.GSK3? knock down could decrease the mRNA expression level,protein expression level and phosphorylation of p65.GSK3? overexpression could increase the mRNA expression level,protein expression level and phosphorylation of p65.10.UBE2L3 knock down,knock out or overexpression had no influence on GSK3? mRNA expression level.UBE2L3 knock down or knock out could increase the protein expression level of GSK3? and decrease the phosphorylation of GSK3?.UBE2L3 overexpression could decrease the protein expression level of GSK3? and increase the phosphorylation of GSK3?.UBE2L3 overexpression and the treatment of CHX could induced the degradation of GSK3?.And the treatment of UBE2L3 overexpress and MG132 shown that UBE2L3 degraded GSK3? protein was through the ubiquitin-proteasome degradation pathway.UBE2L3 and GSK3? associated with each other.The ubiquitination level of GSK3? was decreased in UBE2L3 knock down cells and increased in UBE2L3 overexpression cells.11.GSK3? knock down or GSK3? inhibitor 1-Azakenpaullone could reverse the UBE2L3 KO induced changes in downstream signaling pathway and could reverse the UBE2L3 KO induced cell proliferation decreasing,the UBE2L3 KO induced cell apoptosis.12.Knock out UBE2L3 in HCC cells could inhibit tumor growth in orthotopic xenograft model in nude mice.And the treatment of GSK3? knock down or p65 inhibitor JSH-23 could abolish the inhibitory effect of UBE2L3 knock out induced in HCC tumor growth.Conclusion:UBE2L3 expression is frequently upregulated in clinical HCC samples.UBE2L3 knock down or knock out could significantly inhibit the proliferation of HCC cells,increase the apoptotic rate of HCC cells and inhibit tumor growth in nude mice xenograft model.Its molecular mechanism is that silencing UBE2L3 could increase the protein stability of GSK3? and promote its activation,then the expression level,phosphorylation of p65 and p65 nuclear translocation is up-regulated,thus activate the expression of PUMA.In vivo,knock out UBE2L3 in HCC cells could inhibit tumor growth in orthotopic xenograft model in nude mice.The treatment of GSK3? knock down or p65 inhibitor JSH-23 could abolish those effects above.Overall,our data suggested that UBE2L3 is an important pro-tumorigenic factor in liver carcinogenesis.