Study On The Expression And Effection Of Keap1 In Renal Cell Carcinoma

The renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. Although its incidence is lower than bladder cancer, the prevalence is about 2 to 3% in all the adult malignancies. According to the 2009 national cancer registration statistics annual report, the morbidity and mortality of RCC are 4.5/100000 and 1.46/100000, the incidence of male patients is about 2 times higher than female patients, the incidence of urban population is 4 times higher than the rural areas. About half of RCC patients havemetastasis when they come to hospital, andabout 30% of thelocalized RCC patients who have surgerywith RCC recurrence. These are all threatening the health of people.The Kelch-like ECH-associated protein 1(Keap1), a 69kDa tumor suppressor protein, was reported in recent year. The research shows that Keaplhas different expression in several tumors. Several Keapl mutants have been identified in lung, ovarian and liver cancer. The high mortality of ovarian has relationship with the low expression of Keap1. The expression of Keapl has significant difference between normal tissue and lung tumor tissue. So the protein may be used in clinical test research.The mechanism of Keap1 in the development of tumor is still unclear. According to the researches, there are three different ways to influence the occurrence and progress of tumor.1. Keap1 functions as a substrate adaptor protein for Nrf2, a transcription factor that regulates the cellular redox status. Under basal conditions, Keap1 functions as an adaptor protein in the Cul3- based E3 ligase complex resulting in rapid ubiquitination and subsequent degradation of Nrf2 which stimulates Nrf2-ARE pathway and regulates antioxidants or phase Ⅱ genes. That affects the proliferation and apoptosis, and also affects the sensitivity to chemotherapy and radiotherapy.2. Keapl down-regulates the activation of NF-κB signaling pathway by way of functioning as an IKKβ E3 ligase, which links cancer genomic alternations and the aberrant activation of oncogenic signaling pathways.3. Keapl functions as an adaptor for B-cell lymphoma-2(Bcl-2), inhibited the expression of Bcl-2 and enhanced apoptosis in cells.The expression of Keap1 between paracarcinoma normal tissues and renal cell carcinoma tissues has not been considered. The relationship between Keap1 and renal cell lines has been rarely reported. Here, we discussed the expression of Keapl in paracarcinoma normal tissues and renal cell carcinoma tissues, analyzed the differences in different tissues. We also used in-vitro assays to analyze the influence of Keap1 on the proliferation and colony forming ability in renal cell line ACHN. Then we explored the possible role of Keapl about the occurrence and development in renal cell carcinoma.ObjectiveWe detected the expression of Keap1 and Nrf2 in the clinical samples and renal carcinoma lines, and to analyze the correlation between their expression and clinical pathological features. We also used in-vitro assays to detect the influence of Keapl on the proliferation and colony forming ability in renal carcinoma cell line ACHN and to identify the role of Keapl in renal carcinomas.Methods1.32 renal carcinoma samples were analyzed by immunohistochemistry and Q-PCR in order to study the expression of Keap1 and Nrf2 differences in carcinoma and paracarcinoma normal tissues. All subjects are taken from the patients who accepted operation treatment in the Second Affiliated Hospital of Shandong University from May 2014 to April 2015.2. The expression of Keapl and Nrf2 was detected in renal carcinoma cell line ACHN and renal immortalized cell line.3. To determine the effect of Keapl on the expression of Nrf2 and Bcl-2, ACHN cells were transfected with GV-230-Keap1.4. CCK-8 cell proliferation and clone formation assay were adopted to observe the changes of proliferation and colony forming ability of ACHN after overexpressed Keapl.Result1. On the level of mRNA and protein, the expression of Keap1 in carcinoma tissues was lower than in corresponding paracarcinoma normal tissues. However, for Nrf2 this was revered, with a significant statistical difference (P<0.05). Further analysis based on relevant clinic pathologic data demonstrated that the expression of Keapl and Nrf2 were related to pathological type, but not to sex and age.2. Q-PCR and Western blot results showed that Keapl and Nrf2 were all expressed in both cell lines, the expression of Keapl in HK-2 was higher than ACHN, the expression of Nrf2 was revered.3. The ubiquitination level of Nrf2 was increased after transfecting the GV-230-Keapl. Meanwhile, the expression of Bcl-2 was inhibited; the expression of Bax was increased.4. The proliferation ability and clone formation capability of ACHN were evidently declined.ConclusionKeapl and Nrf2 were expressed in renal cell carcinoma tissues and paracarcinoma normal tissues. The expression of Keap1 in paracarcinoma normal tissues was higher, and the expression of Nrf2 in tumor tissues was more obviously. There was a significantly negative correlation between Keapl and Nrf2 in tumor tissues. Our study used paired clinical samples, that enhanced the balance between different groups, which confirmed different expression of Keapl in renal cell carcinoma tissues and paracarcinoma normal tissues. We first reported overexpression of Keapl can inhibit Nrf2 and Bcl-2 in ACHNcell lines, and inhibit the proliferation and colony formation ability. Keapl plays an important role in RCC and also provides a new way for gene therapy, a new target for clinical diagnosis, treatment and prognosis evaluation.