| Part 1. Quantitative detection of the expression of MDR1 gene:establishment of real-time quantitative PCR methodObjective:To establish the real-time PCR method to detect the expression of MDR1 (multidrug resistance 1) gene mRNA.Methods:PCR primers were designed with Primer 5.0 software, Trizol were used to extract mRNA from MCF-7 cell line, RT-PCR (Reverse transcription polymerase chain reaction) were performed to amplify the target fragments, amplified target gene fragment was verified by T-A clone and sequencing. The expression of MDR1 gene andβ-actin was detected by the Bio-Rad icycler Fluorescence Quantitative PCR apparatus, the standard curve and amplification efficiency ofβ-actin and MDR1 were obtained, in order to determine the the specificity of amplification, the melting curve Analysis was performed. Results:(1) MDR1 target gene fragment was amplified successfully and confirmed by T-A cloned sequencing; (2) The melting curve peaks of MDR1 andβ-actin were single peak. No non-specific PCR amplification products were found; (3) The amplification efficiency ofβ-actin and MDR1 was in the same level.Conclusions:The real-time PCR method was successfully established to quantitatively detect the mRNA expression of MDR1 gene.Part 2. Associations of MDR1 C3435T polymorphism with MDR1 gene mRNA expressionObjective:To investigate the associations of MDR1 C3435T polymorphism with MDR1 gene mRNA expression in patients with cancer.Methods:The DNA was extracted from peripheral blood of 74 cases with cancer. The MDR1 C3435T polymorphism were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and confirmed by DNA sequencing. The real-time PCR method was used to detect the MDR1 gene mRNA expression of each case. Data were analyzed with the multiple independent samples non-parametric test (Kruskal-Wallis test) of SPSS 13.0 statistics analysis software system.Results:A statistically significant difference of MDR1 gene mRNA expression was observed in CC, CT and TT genotype at MDR1 gene C3435T locus(P=0.036); The relative expression level of MDR1 gene mRNA for the CC, CT and TT genotypes was 3.01,2.55 and 1.0 respectively.Conclusions:The MDR1 gene mRNA expression in the three genotypes of C3435T was significant difference. The expression level was CC> CT> TT.Part 3. The clinical significance of MDR1 C3435T polymorphismObjective:To investigate the associations of MDR1 C3435T polymorphism with clinical therapeutic effect and hematologic toxicities after chemotherapy.Methods:Clinical data of 74 cases with cancer were Collected and the associations of the MDR1 gene C3435T polymorphism with clinical therapeutic effect and hematologic toxicities after chemotherapy were analyzed. The X2 test programme of SPSS 13.0 statistics software was used to analyze the data.Results:(1) The associations of the MDR1 C3435T polymorphism with clinical therapeutic effect:①For non small-cell lung cancer patients, the response rate (CR+PR) in TT+CT genotype and CC genotype was 50%,21.4% respectively, the response rate of TT+CT genotype was higher than CC genotype, but no statistically significant difference was observed (P>0.05).②For gastrointestinal cancer patients, the response rate (CR+PR) in TT+CT genotype and CC genotype was 21.4%,16.7% respectively, the response rate of TT+CT genotype was higher than CC genotype, but no statistically significant difference was observed (P>0.05).③For non-Hodgkin lymphoma patients, the complete remission rate (CR) in TT+CT genotype and CC genotype was 25%,0% respectively, the CR rate of TT+CT genotype was higher than CC genotype; The response rate (CR+PR) in TT+CT genotype and CC genotype was 66.7%, 83.3% respectively. No statistically significant difference was observed (P>0.05). (2) The associations of MDR1 gene C3435T polymorphism with hematologic toxicities:①For non small-cell lung cancer patients, the severe leukopenia rate (III+IVdegree) in TT+CT genotype and CC genotype was 28.6%,14.3% respectively, the leukopenia rate of TT+CT genotype was higher than CC genotype, but no statistically significant difference was observed (P=0.648). The severe hemoglobin decrease rates (III+IVdegree) in TT+CT genotype and CC genotype was 7.1%,14.3% respectively, no statistically significant difference was observed (P= 1.000). The severe thrombocytopenia rate (III+IVdegree) in TT+CT genotype and CC genotype was 7.1%,21.4% respectively, no statistically significant difference was observed (P=0.596).②For gastrointestinal cancer patients, the severe leukopenia rate(III+IVdegree) in TT+CT genotype and CC genotype was 10.0%,33.3% respectively, no statistically significant difference was observed (P=0.518). The severe hemoglobin decrease rate (III+IVdegree) in TT+CT genotype and CC genotype was 10.0%,0% respectively, no statistically significant difference was observed (P=1.000). The severe thrombocytopenia rate (III+IVdegree) in TT+CT genotype and CC genotype were both 0%, no statistically significant difference was observed (P=1.000).③For non-Hodgkin lymphoma patients, the severe leukopenia rate (III+Ⅳdegree) in TT+CT genotype and CC genotype was 45.5%,40.0% respectively, no statistically significant difference was observed (P=1.000).The severe hemoglobin decrease rate (III+IVdegree) in TT+CT genotype and CC genotype were both 0%, no statistically significant difference was observed (P=1.000). The severe thrombocytopenia rate (III+Ⅳdegree) in TT+CT genotype and CC genotype was 9.1%,0% respectively, no statistically significant difference was observed (P= 1.000).Conclusions:(1)The chemotherapy response rate is higher in cancer patients with TT+CT genotype than that with CC genotype, the statistical difference between them had not been reached. (2) After chemotherapy, the severe hematologic toxicities of patients between TT+CT genotype and CC genotype, no statistical difference was obtained. (3) The samples of this study was small, the result should be verificated in a larger sample. |
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