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The Expression and Significance of Nrf2and NQO1in Clear Cell Renal Cell Carcinoma

Author: GaoQiang
Tutor: LiZuo
School: Hebei Medical University
Course: Surgery
Keywords: CcRCC Nrf2 NQO1 immunohistochemistry RT-PCR
CLC: R737.11
Type: Master's thesis
Year: 2014
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Abstract


Objectives: The morbidity and mortality of renal cell carcinoma, acommon urinary renal cell malignancy, is on rise year by year, and clear cellrenal cell carcinoma (ccRCC) accounts for60percent to85percent of it.ccRCC is highly malignant and likely to develope distant recurrence, and it isinsensitive to chemotherapies and radiotherapies. Therefore, it is important fordiagnosis, treatment and subsequent prognosis to elucidate the mechanism ofdevelopment and involution of ccRCC.Nuclear factor E2-related factor2(Nrf2) is a significant transcription factor that serves as a primary cellulardefense against the cytotoxic effects of oxidative stress. This is the mostimportant mechanism protecting cells from oxidative stress. NQO1, NADPHquinine oxidoreductase, is a protein expressed downstream in the Nrf2/AREanti-oxidative stress system.Abundant studies indicates that Nrf2/NQO1signal pathway playes acritical role in the oxidative stress reaction in diverse systems, and theexpression and activation level of Nrf2amd NQO1are closely associated withthe involution and develop of cancer. But no report has ever studied theexpression of Nrf2amd NQO1in the ccRCC. This study examines theexpression of mRNA and protein of Nrf2amd NQO1in ccRCC tissue and thecorresponding juxtacancerous tissue, and discuss the role they play in theinvolution and develop in ccRCC.Methods: Between December,2012and June,2013, we aquired ccRCCtissue samples from52patients admitted by the department of UrologicalSurgery, Second Hospital of Hebei Medical University, other21samples wereaquired from tissue located more than4cm away from the carcinoma entities(pathologically confirmed). The patients were between37to82years old,averaged56(56.13±13.98). No radiotherapy, chemotherapy or any other conservative treatment was given to them. All the cases have already beendiagnosed as ccRCC pathologically. The samples were assigned into2groups,one group was fixed by10%formalin solution, then embedded and slicedroutinely, the other group was stored in liquid nitrogen immediately afterbeing cut down and kept in the﹣80℃refrigerator. Pathological grading wasprocessed according to the Fuhrman grade established in1997by WHO,32cases were labeled as low grade group while20cases were labeled as highgrade group; divide the samples into clinical stage Ⅰ15cases, Ⅱ16cases,Ⅲ14cases and Ⅳ7cases according to the TNM standards established by theInternational Union against Cancer:1Immunohistochemical analysis: detect the expression of Nrf2andNQO1protein in all the tissue samples. Analyze data with SPSS17.0: calculateimmunohistochemical staining positive values, compare the expression ofNrf2and NQO1protein in ccRCC tissue and juxtacancerous tissue, andestimate their interdependence with clinical stage and pathological grade by χ2test or Fisher’s exact probability test; the correlativity of Nrf2and NQO1wasmeasured by spearman rank correlation analysis; P <0.05was consideredstatistically significant.2RT-PCR assay: detect the mRNA expression of Nrf2and NQO1in36ccRCC tissue samples and14juxtacancerous tissue samples. Extract mRNAfrom the frozen samples according to the Trizol reagent supplies user manual,examine the concentration and purity of the mRNA, reverse transcription andamplification were conducted with PE cycler, the RT-PCR products could thenbe analyzed with agarose gel electrophoresis. Regard DNA Marker(DL2000)as standard segment, observe the electrophoretic bands and take photos,analyze those bands with Quantity One gel image analysis software, referringto the appropriate internal reference electrophoretic bands, show the results asthe ratio of electrophoretic bands,the results were mean±standard deviation(x±s), P <0.05was considered statistically significant..Results:1Immunohistochemical staining results showed that protein ofNrf2and NQO1expressed in both ccRCC tissue and juxtacancerous tissue, with the respective positive rate76.9%and28.6%(χ2=15.005,P<0.01),which were statistically significant. NQO1protein expressed more in ccRCCtissue than in juxtacancerous tissue, the positive rate were73.1%and23.8%respectively(χ2=14.999,P<0.01),also statistically significant. The expressionof Nrf2and NQO1protein was positively associated with the clinical stageand pathological grade P<0.05. And the expression of NQO1was positivelyrelated with the expression of Nrf2in ccRCC tissue(r=0.594,P<0.05).2RT-PCR results showed that550bp was the target band of Nrf2,488bp was the target band of NQO1,162bp was he target band of internalreference. Half quantitive analyze showed that relative expression of themRNA of Nrf2and NQO1in ccRCC tissue were (0.767±0.125)and(1.025±0.148) respectively, that is, the relative expression of the mRNA ofNrf2and NQO1in ccRCC tissue was significantly higher than that in ctissue(p<0.05).Conclusions:1The expression of the protein and mRNA in ccRCC tissue are higerthan that in juxtacancerous tissue.2The expression of Nrf2and NQO1protein is positively realted with theclinical stage and pathological grade.3The expression of Nrf2and NQO1protein was positively associated.4It might be helpful to jointly monitor the expression of both Nrf2andNQO1for estimating the develop of the carcinoma; and Nrf2/NQO1maybecome a novel therapeutic target for ccRCC. Thus, we may get a fresh viewof the early detection and treatment for ccRCC.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Kidney,renal pelvis tumor
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