| Objective:PROX1 is a transcription factor homologous to Drosophila melanogaster Prospero. PROX1 is a new gene first cloned from the embryonic human brain cDNA library in 1996, and this contig was mapped to human chromosome 1q32.2-q32.3 which consists of 5 extrons and 4 introns. The available sequence of the human PROX1 cDNA is 2924 bp long which contains an open reading frame of 2208 nt. The amino acid sequence is 737 amino acids long with a calculated molecular mass of 83 kDa. Recent studies have shown that PROX1 controls cell differentiation and plays essential roles during embryonic development of the central nervous system, lens, retina, liver, pancreas, and lymphatic vasculature. At the same time, aberrant expression of PROX1 was detected in a variety of human cancers. On the one hand, PROX1 was highly expressed in glioma, colorectal carcinoma and hepatocellular carcinoma, and PROX1 also promoted tumor metastasis by enhancing the cell migration capability. On the other hand, the expression of PROX1 was down-regulated in virtue of DNA methylation in pancreatic carcinoma, breast cancer and hematopoietic tumors, further, the expression level of PROX1 was positively relative with the degree of differentiation of tumor cell and high PROX1 expression indicated favorable prognosis. During the development of embryo, PROX1 exerts its function in a context-dependent manner. Similarly, both oncogenic and tumor-suppressive functions have been ascribed to PROX1 in a variety of different human cancers, which suggests that PROX1 plays an important role during the development of cancer. The purpose of this study is to confirm the expression pattern of PROX1 in renal cell carcinoma and discuss the role of PROX1 in the pathogenesis of renal cell carcinoma, and further explore the potential molecular mechanism in order to provide the theoretical bases for the prevention and treatment of renal cell carcinoma.Methods:1. The real-time quantitative PCR, Western blot and immunohistochemistry are applied to determine the expression of PROX1 in renal cancer tissue, adjacent normal tissue, renal cell carcinoma cell lines and renal cell line. 2. Construct the recombinant lentivirus expression vectors of PROX1 and siRNA targeting PROX1 and perform the packaging of the recombinant lentivirus for the subsequent study of cell function.3. Infect the renal cell carcinoma cell ACHN which has been confirmed to be with high expression of PROX1 by Western blot with recombinant lentivirus of siRNA targeting PROX1 to knockdown the endogenous expression level of PROX1, simultaneously, infect the renal cell carcinoma cell 786-0 which has been confirmed to be with low expression of PROX1 by Western blot with recombinant lentivirus of PROX1 to increase the expression level of PROX1, and further investigate the cell growth by CCK-8 cell proliferation and cytotoxicity assay kit, detect the cell colony formation ability by colony-formation assay and analyze the cell migration capability by scratch-wound assay, finally use Western blot to determine the change of cell proliferation and migration related proteins.Results:1. We first detected the mRNA expression of PROX1 in 92 fresh renal cancer tissue specimens using real-time quantitative RCR. the data showed that PROX1 expression was significantly reduced in tumor tissues compared with matched adjacent renal tissue (P<0.001). Unexpectedly, after stratifying by stage and grade, expression of PROX1 mRNA trended higher in T3/4 and G3/4 RCC tissues compared with T1/2 and G1/2 tissues, respectively, although this difference did not reach statistical significance; to extend our observations, we tested PROX1 protein expression in 115 paraffin-embedded RCC sections by immunohistochemistry, the results displayed that renal tubules in adjacent normal tissue showed intense PROX1 expression in 52 (89.7%) RCC patients, however, RCC tissues showed variable PROX1 expression levels, that is a total of 39 RCC specimens (33.9%) showed high expression and 76 (66.1%) showed low expression. Similar to PROX1 mRNA expression, PROX1 protein expression was also higher in T3/4 and G3/4 RCC specimens than in T1/2 and G1/2 specimens, respectively. When specimens were stratified according to clinicopathological factors, PROX1 expression was found to be significantly related to tumor nuclear grade (P<0.001) and tumor N stage (P=0.012). Overall survival was significantly decreased in the high PROX1 expression group compared to the low PROX1 expression group (P=0.001). Cox proportional hazard model indicated that PROX1 may serve as an independent prognostic factor for renal cancer.2. Replication defective lentiviral vector pLKO.1 was used to construct the recombinant lentiviral packaging plasmid of siRNA targeting PROX1, lentiviral vector pWPI containing the enhanced green fluorescent protein (EGFP) sequence was used to construct the recombinant lentiviral packaging plasmid of PROX1, then the recombinant lentiviral packaging plasmid and the recombinant lentiviral packaging helper plasmid were co-transfected into the human embryonic kidney cell line 293 through the liposomal transfection to obtain recombinant lentiviral suspension.3. The results of Western blot confirmed that infection of recombinant lentivirus vLKO.1-si259 and vLKO.1-si1646 significantly decreased endogenous expression of PROX1 in renal cell carcinoma cell line ACHN. The subsequent results of CCK-8 cell proliferation and cytotoxicity assay showed that the OD values of CCK-8 at different detected time points in the ACHN cell group infected with vLKO.1-si259 and vLKO.1-si 1646 were all significantly lower than those corresponding values in the ACHN cell group infected with vLKO.1-siSCR expressing a scrambled control siRNA, and this discrepancy in growth behavior between PROX1-knockdown cells and scrambled control cells increased over time, which suggested that the expression of PROX1 enhanced the growth of the cell line ACHN. The further colony-formation assay showed that colony-formation rate of ACHN cells infected with vLKO. 1-si259 and vLKO.1-si1646 (27.3% and 21.3% respectively) was notably lowed that the ACHN cells infected with vLKO.1-siSCR. Besides, scratch-wound assay showed that 24 hour mean migrating distance (μm) of vLKO.1-si259 and vLKO.1-si1646 infected ACHN cells (537.6 and 533.9 respectively) was markedly shorter than the vLKO.1-siSCR infected ACHN cells (692.6). Furthermore, the infection of recombinant lentivirus vWPI.1-PROX1 significantly increased endogenous expression of PROX1 in renal cell carcinoma cell line 786-O. In contrast with PROX1 RNAi experimental results, the OD values at different detected time points and colony-formation rate (45%vs 32%) of vWPI.1-PROX1 infected 786-Ocells were both high than the wild type 786-Ocells, and the mean migrating distance (μm) (479.6 vs 326.9) of vWPI.1-PROX1 infected 786-O cells was also longer than the wild type 786-O cells. Finally, the result of Western blot indicated that overexpression of PROX1 correlated with decreased E-cadherin expression and increased Vimentin expression in 786-O cells, conversely, reduced PROX1 correlated with E-cadherin overexpression and reduced Vimentin expression in ACHN cell.Conclusion:PROX1 is of high expression level in adjacent renal tissues but of low expression level or without expression in tissues of renal cell carcinoma, PROX1 protein expression level was positively associated with the pathologic stage, Furhman nuclear grade and lymph node metastasis and negatively correlated with patient prognosis, PROX1 may serve as an independent prognostic factor for renal cancer. Additionally, the expression of PROX1 can down-regulate the expression of E-cadherin and up-regulation the expression of Vimentin, and promote renal cancer cells proliferation, colony formation, as well as accelerate renal cancer cells migration. The present preliminarily study explores the potential role the PROX1 gene plays in the development and progression of renal cell carcinoma at the gene level, and contributes to better understanding of the molecular pathogenesis of RCC, providing new ideas for the diagnosis and treatment of kidney cancer and important experimental evidence of PROX1 as a target for cancer gene therapy and anticancer drugs. |
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