The Expression Pattern Of MRP Family Members (MRPs) In Pediatric Acute Lymphoblastic Leukemia And Their Clinical Significance

The thesis includes three parts: Part I: Optimalizing the conditions of fluorescencequantitative RT-PCR to identify specific primers for MRP1-6genes; Part II: MRP1expression in childhood acute lymphoblastic leukemia and its clinical significance; Part III:MRP2-6expression pattern in childhood acute lymphoblastic leukemia and their clinicalsignificance.Part I: Optimalizing the conditions of fluorescence quantitative RT-PCRto identify specific primers for MRP1-6genesObjective: In order to identify the primers’ specificity for amplifying MRP1-6genes,QRT-PCR conditions were optimalized.Methods: Leukemia cell lines were selected as temple for amplifying MRPs geneswith the primers and dissolution curve for each gene was nanlyzed to make sure whetherthe primers are specific for genes’ amplification.Result: Dissolution curve were satisfied for MRP1-6genes amplification andgenes’expression was detected within leukemia cell lines.Conclusion: We get the specific primers and conditions of MRP1-6for QRT-PCRwhich lain the foundation for clinical analysis. Part II: MRP1expression in pediatric childhood acute lymphoblasticleukemia and its clinical significanceObjective: Multidrug resistance-associated protein1(MRP1) has been reported aclose correlation with tumor multi-drug resistance. MRP1gene expression was detectedusing real-time quantitative PCR(QRT-PCR） in pediatric acute lymphoblasticleukemia(ALL) and its clinical significance was analyzed.Methods:146patients at different stages of ALL were enrolled in2013, including67cases at initial stage,70cases at complete remission, and9cases at relapse,10idiopathicthrombocytopenic purpura as control as well. MRP1gene expression was detected usingreal-time quantitative PCR（QRT-PCR）and its clinical significance was analyzed by the.SPSS18software. P value below0.05was regarded as statistic significance.Results：（1）The median expression of MRP1was5.82and8.49for initial and relapsegroup，respectively, which was statistic higher than that at complete remission, the latterwas1.99.（2）MRP1expression level had close correlation with ALL risk, the median ofMRP1expression was4.28,5.62and7.56for standard-risk group (SR), intermediate-riskgroup (IR) and high-risk group (HR), respectively.（3）Initial ALL children were dividedinto two groups including high expression group and low expression group by the medianexpression, the rate of leukemia cells’ sensitivity to prednisone on7th day was70.6%inhigh expression group (n=34), which was statistic lower than that in low expression group(n=33,90.9%, P=0.035). The rate of complete remission on33th day in high expressiongroup was64.7%, and87.9%in low expression group, which showed a significantdifference between them (P=0.026). The rate of complete remission on15th day in highexpression group was68.8%, and69.7%in low expression group, which showed nosignificance between them (P=0.664)（4）The transcription level of MRP1in initial groupof T-ALL（median=7.71）was signifcantly higher than that in B-ALL(median=5.18)(P=0.007).（5）Single factor analysis showed that MRP1mRNA has no relationship with sex、age、WBC count、 HB、 PLT、the blast cells percentage in bone narrow section and bloodsmear at diagnose. Conclusions: In children ALL, the expression of MRP1is closely related withimmunophenotyping, treatment response, hazard level and disease relapse which was apoor biomarker for ALL prognosis. Part III: The expression pattern of MRP2-6genes in childhood acutelymphoblastic leukemia and their clinical significanceObjective: Multidrug resistance-associated proteins2to6have been reportedinvolved in a large number of tumors and have a close correlation with tumor multi-drugresistance. In this work, we detect the MRP2-6genes expression in childhood acutelymphoblastic leukemia (ALL) by Q-RT-PCR and explore their clinical significance.Methods:133patients at different stage of ALL were enrolled in2013, including56cases at initial stage,68cases at remission, and9cases at relapse,10idiopathicthrombocytopenic purpura patients as control. Real-time quantitative PCR （QRT-PCR）was employed to detect the MRP2-6genes expression. The data was analyzed by SPSS18software and P value below0.05was regarded as statistic significance.Results:（1）The median expression of MRP2at initial stage is highest,higher thanthat at relapse and complete remission, but didn’t reach statistc significance. However, themedian expression of MRP3at initial stage was highest and staticticly higher thancomplete remission. MRP4and MRP5showed a similar patten in their expression, namely,high expression for relapse, intermediate expression for initial stage and low expression atcomplete remission which reached statisticly significance. The median expression ofMRP6for relapse was2.0, which was higher than initial and complete remission group,but the differences among them were not significant.(2) The median of MRP2、MRP5expression for intermediate-risk group (IR)（MedianMRP2=4.62、Median MRP5=1.71）was higher than standard-risk group (SR)（Median MRP2=3.27、Median MRP5=1.27）and high risk group (HR)（Median MRP2=2.14、Median MRP5=1.67）.However,there wasno statistical significance among them; The median of MRP4expression was increasecontinually with the risk of ALL, but didn’t reach statistical significance among them; the median of MRP6expression for high-risk group (HR) was higher than standard-risk group(SR，0.88vs0.62）and intermediate-risk group (IR，0.88vs0.40）, but the differencesamong them were not significant either.(3) Initial ALL children were divided into twogroup including high expression group and low expression group by median expression,and evaluated their prediaction on the treatment response on7th,15th and33th day.Thesults revealed no significant difference between them, neither between B-ALL andT-ALL.(5) Single factor analysis showed that MRP2has relationship with PLT count atdiagnosis and MRP4has relationship with sex.Conclusions: In childhood ALL, MRP2-6has a different expression pattern. MRP4and MRP6mRNA expression showed a close relation with relapse.