Expression Of TFAP2? In Clear Cell Renal Cell Carcinoma And Its Influence On Cell Proliferation And Cell Cycle

Background:Clear cell renal cell carcinoma (ccRCC) is clinically common with malignant degree, especially in metastatic patients with advanced stages. Traditional surgery, radiotherapy and chemotherapy are not so effective, and the molecular target treatment shows greater clinical advantages. Various genes could be related to the early diagnosis and treatment of ccRCC. Transcription factor acting protein-2 alpha (TFAP2a) is a transcription factor abnormally expressed in many tumor tissues. It is involved in cell proliferation, differentiation, invasion and metastasis, and play an essential role on tumorigenesis and metastasis. But there is still rare report about TFAP2a in ccRCC.Objective:This study intends to clarify the expression of TFAP2a in ccRCC tissue and cell lines and its potential correlation with clinical pathology. The expression of TFAP2a is up and down regulated to study the influence on cell proliferation and cell cycle, investigating the role of TFAP2a on tumorigenesis in ccRCC.Methods:1. RT-PCR and western blot was respectively used to detect the expression of TFAP2a mRNA level and protein level in human kidney tubule epithelial cell lines (HKC, HK2) and ccRCC cell lines (A498?786-0?769-P?Caki-2?Caki-1?ACHN? SN12-PM6) by.49 pairs of ccRCC surgical specimens and their corresponding adjacent normal tissue also measured by RT-PCR and immunohistochemical staining. The correlation between TFAP2a mRNA level and clinical parameters was explored by statistical analysis,2. RNA interference method was used to transfect siRNA sequence targeting TFAP2a and negative control sequence as control into 786-0 cell to down regulate the expression of TFAP2a. The effects on cell proliferation and cell cycles were detected by MTS assay, cell clone formation assay and flow cytometric analyses.3. A recombinant plasmid expressing TFAP2a (pLV-EGFP(2A)-TFAP2a) was constructed and transfected into 786-0 and Caki-2 cells. The effects on cell proliferation and cell cycles were also detected by cell clone formation assay, MTS assay and flow cytometric analyses.Results:1.TFAP2? expression levels were significantly decreased in ccRCC cell lines compared with human kidney tubule epithelial cell lines (P<0.05). TFAP2? mRNA expression levels were significantly decreased in ccRCC tissue compared with normal tissue (P<0.05). Immunohistochemical staining showed TFAP2a both expressed in nucleus and cytoplasm, mainly in nucleus; the staining intensity of the tumor tissue was much higher than adjacent normal renal tissues. TFAP2? mRNA expression levels was negatively correlated with renal clear cell carcinoma Fuhrman grading (P<0.05).2. siRNA sequence successfully downregulated the expression of TFAP2a. MTS assay and cell clone formation assay showed the TFAP2a-siRNA group had a significantly higher growth rate(P<0.05). Flow cytometric analyses showed that TFAP2a-siRNA reduced the G0/G1 phases and increased the S phase.3. The plasmid pLV-EGFP(2A)-TFAP2a was successfully constructed and upregulated the expression of TFAP2a in 786-0 and Caki-2 cells. MTS assay and cell clone formation assay showed the recombinant plasmid group had a significantly lower growth rate(P< 0.05). Flow cytometric analyses showed reduction in the S phase of the recombinant plasmid group accompanied by decline in the Go/G1phases.Conclusions:Reduced expression of TFAP2a is associated with tumor development and malignant degree; TFAP2a influence tumor cell proliferation and cell cycle, and may be an tumor suppressor gene in ccRCC.