| ObjectiveTo investigate the transcription expression and promoter methylalion status of P73and SIX2gene in tissue and blood among children with nephroblastoma and to discuss their clinicopathological correlations,so as to explore its pathogenesis from epigenetics angle,provide a theoretical basis for its future gene targeted therapy.MethodsThe methylation-specific PCR (MSP) and real-time quantitative PCR (qRT-PCR) were used to detect the mRNA expression level and methylation status of P73and SIX2gene in tissue and blood,then analysed their clinicopathological correlations and how gene methylation affected its transcription in wilms’tumor.Results1. Identification of RNA extracted from the tissue and peripheral blood Electropherogram of RNA extracted from tissue and peripheral blood display strip of both18S and28S, brightness of28S was about twice18S,meant that RNA integrity is better; Values of measured A260/A280was greater than1.90, indicating high purity of RNA.2. Relative Quantity(RQ) of P73and SIX2in tissue and blood from the mRNA expression level Values of measured RQ was denominated in a selected peripheral blood of normal child as control. Relative Quantity(RQ) of P73〠SIX2mRNA in tumor and peritumoral tissue was both higher than that of the normal tissue (p<0.05), but difference in RQ value among tumor tissue and peritumoral tissue was not statistically significant(P>0.05); Average Relative Quantity(RQ) of SIX2mRNA in three fetal kidneys was330.55±18.97. Relative Quantity(RQ) of P73〠SIX2mRNA in case group was both higher than that of the control group (p<0.05).3. Promoter methylalion status of P73and SIX2gene in tissue and blood P73〠SIX2gene methylation-positive rate in cancer and peritumoral tissue was both tower than that of the normal tissue (p<0.05), but difference in methylation-positive rate among tumor tissue and peritumoral tissue was not statistically significant(P>0.05);P73〠SIX2gene methylation-positive rate in case group was both tower than that of the control group(p<0.05);3cases of fetal kidney were all unmethylated.4. Relationship between the clinical data and both Relative Quantity(RQ)〠promoter methylalion status of P73and SIX2gene in tumor tissue and case group RQ values of P73in the tumor tissue was associated with tumor size, clinical stage, pathological type, lymph node metastasis (p<0.05),but not with gender, age, vascular invasion, treatment and outcome (p>0.05);RQ values of SIX2in the tumor tissue was associated with tumor size, clinical stage, pathological type, lymph node metastasis and vascular invasion (p<0.05),but not with gender, age and treatment and outcome (p>0.05); RQ values of P73〠SIX2in case group was associated with tumor size, clinical stage, pathological type, lymph node metastas and treatment and outcome (p <0.05), but not with gender, age and vascular invasion (p>0.05); P73〠SIX2gene methylation-positive rate in tumor tissue and case group was both independent of the clinical data (p>0.05). 5. Relationship between the promoter methylalion status and the mRNA Relative Quantity(RQ) of P73and SIX2gene in tissue and blood RQ values of P73SIX2in tumor tissue was both higher than that of the normal renal tissue among methylated group (p<0.05), but difference in RQ values among unmethylated tumor, adjacent tumor and normal renal tissue was not statistically significant (p>0.05);RQ values of P73〠SIX2in case group was both greater than that of the control group in methylated group (p<0.05); There was no statistical significance in RQ values between the case group and control group in unmethylated group (p>0.05)6. Relationship between survival of children and the mRNA Relative Quantity(RQ) of P73and SIX2gene in tissue and blood Sort out children’s survival data from38cases of tumor tissue and45cases of preoperative blood in case group,choose the average of the mRNA Relative Quantity(RQ) of P73and SIX2as the boundary, the median survival time of whose RQ values was greater than or equal to the average boundary was significantly less than that of less than of the average boundary (P <0.05).7. Correlation among the mRNA Relative Quantity(RQ) and methylation status about P73and SIX2gene in tissue and blood Pearson and Spearman rank correlation analysis indicated that RQ values of P73gene had negative correlation with that of SIX2gene in tumor tissue(γs=0.252,P=0.128),but it was positive correlation in case group(γ=0.785,P=0.000); Meanwhile, p73and SIX2gene promoter methylation status had positive correlation among both tumor tissue and case group(Y=0.558,P=0.000;γ=0.539,P=0.000).Conclusion1. P73and SIX2gene is highly expressed in tissue and blood of Wilms’Tumor, and it is positively correlated with the degree of malignancy;2. There is a negative correlation tendency between the overexpression in transcriptional level and its promoter methylation status about p73and SIX2gene;3.There is certain close relationship between the high expression of P73and SIX2 gene and low survival rate in children with nephroblastoma. |
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