The Role And Mechanism Of FOXO3a Down Regulation In The Metastasis Of Clear Cell Renal Cell Carcinoma

Background:Renal cell carcinoma (RCC) represents approximately2-3%of all human malignancies and the most common histological subtype is clear cell renal cell carcinoma (ccRCC), accounting for80-90%of RCC. Localised and metastatic ccRCC differs considerably in prognosis and therapy. The5-year cancer-specific survival is below27.1%in metastatic ccRCC and exceeds70%in localised ccRCC. However, up till now, the only method to identify a metastatic ccRCC is imaging evidence, lacking reliable molecular markers which could be used to determine the metastatic liability of ccRCC early. So, there is a high probability that when the primary tumor was excised, the tumor cells had invaded a distant organ and formed a micrometastasis which was undetectable by current image technique. We believe that it is the root of tumor recurrence and postoperative metastasis.Aimed to explore the underlying mechanisms of ccRCC metastasis, the genome-wide expression profiling of primary metastasis (PM) and primary non-metastasis (PN) ccRCC tissues were studied. And the bioinformatics analysis, real-time PCR validation, clinical follow-up, and biological function assays in vitro and in vivo et al were performed in this study.Methods:1. Sample collection:135tissue specimens, including21primary metastasis and114primary non-metastasis tissue specimens were obtained from the patients with ccRCC undergoing partial or radical nephrectomy from the period of2009to2012in Chinese People’s Liberation Army (PLA) General Hospital.2. Follow up:The114PN ccRCC pa’tients were followed up for a median of28.2months (rang from14.1to41.5months) and were observed for the postoperative metastasis or recurrence.3. Expression profiling:5PM and5PN ccRCC samples were analyzed by expression profiling after RNA extraction, purification, quality control, reverse transcription, fragmentation, labeling and hybridization by using Affymetrix GeneChip Human Genome U133Plus2.0Array.4. Bioinformatics analysis:Gene Ontology, pathway analysis and signal-net analysis were employed to analyze the biological process of ccRCC metastasis and to filter the core genes potentially promoting ccRCC metastasis.5. RNA extraction and real-time PCR:Total RNA was extracted by Trizol Reageant. Real-time reverse transcription PCR were performed by using SYBR Green and the expression of mRNA was compared by the difference of threshold cycle (ACT).6. Immunohistochemistry staining:10PM and10PN ccRCC tissues were selected randomly to perform immunohistochemistry staining against FOXO3a protein.7. Cell culture:The RCC cell lines786-0,769-P, A-498, Caki-2, Caki-1and ACHN were maintained in this study.8. Plasmids, small interfering RNA transfection:pCR3.1cloned with the coding domain sequence of FOXO3a and siRNA targeting FOXO3a were used for transient transfection of tumor cells. pIRES2-EGFP cloned with the CDS of FOXO3a and small hairpin RNA vector pGFP-V-RS against FOXO3a were used for stable transfection of tumor cells.9. Protein extraction and western-blot assays:Cell proteins were extracted by RIPA. Western-blot assays were performed according to standard procedures.10. Flow cytometry assays for cellular apoptosis:Annexin V-FITC/PI double staining was used to evaluate cellular apoptosis.11. Migration and invasion assays:Boyden Chambers in transwell plates were used for migration assays. Chambers coated with matrigel were used for invasion assays. Scratch wound assays were also performed for evaluating cellular migration.12. Nude mice orthotopic xenograft model and metastasis lesion detection: Stable transfected tumor cells were injected into the right kidney of nude mice. Seven weeks after injection, the mice were sacrificed and kidney and lungs were harvest. HE staining was performed to identify metastasis lesions. The signal intensity of the GFP fluorescence from the lung tissues represented the amount of lung metastatic lesions, which were able to be detected and analyzed by molecular imaging system.Results:1. A total of1257differently expressed genes (DEGs) were identified between PN and PM ccRCC groups by expression profiling. GO and pathway analysis of these DEGs indicated a more mesenchymal trait and suggested a probable epithelial-mesenchymal transition (EMT) occurred in the PM ccRCC group. Signal-net analysis and PubMed data mining suggested that FOXO3a was a potential key player promoting ccRCC metastasis.2. FOXO3a mRNA level in21PM and42PN ccRCC tissues was compared by real-time PCR assays. Results from the assays indicated that FOXO3a was down regulated in PM ccRCC tissues, which was in line with the microarray results.3. Immunohistochemistry staining for FOXO3a protein indicated that FOXO3a protein was down regulated in PM ccRCC samples. Western-blot assay for FOXO3a level in a panel of RCC cell lines suggested that protein level of FOXO3a was highest in786-0cell line and lowest in SN12-PM6cell line.4. Kaplan-Meier analysis demonstrated that the patients with low FOXO3a level had lower disease free survival rates than the high level group. And Cox regression analysis indicated that the mRNA level of FOXO3a was an independent prognostic factor for ccRCC metastasis.5. Flow cytometry assay indicated that cellular apoptosis was increased in FOXO3a forced expression SN12-PM6cell line and decreased in FOXO3a knock-down786-0cell line.6. Transwell assays indicated that cellular migration and invasion were increased in FOXO3a knock-down786-0cell line and increased in FOXO3a forced expression SN12-PM6cell line. Results from the scratch wound assays were in line with the transwell assays.7. In orthotopic xenograft mice model demonstrated that FOXO3a suppressed the metastatic behavior of SN12-PM6cell line in vivo.8. Knock-down FOXO3a increased the mRNA level of SNAIL1in786-0cell line, which induced the EMT of tumor cells thereby promoting ccRCC metastasis.Conclusions: FOXO3a was decreased in PM ccRCC samples and low level of FOXO3a was a independent prognostic factor for ccRCC metastasis. Loss of FOXO3a induced EMT of tumor cells by up regulating SNAIL1, which promoting tumor cells metastasis in vitro and in vivo.