Hypermethylation Of SEPP1 Promotes The Tumorigenesis Of Clear Cell Renal Cell Carcinoma

Objective:1. Using gene microarray technology to screen out genes that have significant difference in the degree of methylation and in gene expression level. And then verify this difference in the primitive culture ccRCC cell lines and tumor tissues.2. To construct a recombinant lenti viral expression vector of the target gene. To investigate the effects of its infection on cell proliferation of clear cell renal cell carcinoma cell lines, and to supplement the mechanism of the tumorigenesis in renal clear cell carcinoma.Methods:1. Use DNA sequencing technology to screen out 5 cases of VHL mutation-type ccRCC and 5 cases of VHL wild-type ccRCC, match one with another according to gender, age, tumor Furman stage, tumor diameter. Extract the total RNA, use Affymetrix HTA2.0 expression profile microarray to screen out genes that express differently in two groups. Extract the total DNA, use Illumina 450 k methylation microarray to screen out genes that have different methylation level.2. Extract the total RNA from 20 cases of VHL wild-type ccRCC primary cultured cell lines, then reverse into cDNA, use RT-PCR to validate expression microarray results.3. Extract the total DNA from 20 cases of VHL wild-type ccRCC primary cultured cell lines, after DNA sulfuration, use methylation-Specific PCR to validate methylation microarray results.4. Use Sequenom MassArray methylation detection methods to detect the CpG island methylation level of target genes.5. Western blot and immunohistochemical methods were used to further validate the expression difference of target gene SEPP1.6. Polymerase chain reaction (PCR) method was used to obtain the whole length of target SEPP1 sequence, using cDNA of 293T cells as template. The recombinant lentiviral vector and the packaging vector were cotransfected into 293T cells and virus was packaged. After the clear cell renal cell carcinoma cell line 769-P and 786-O were infected by the virus supernatant, RT-PCR and western blot were used to measure the expression of SEPP1.7. Western blot was used to measure the expression of SEPP1. Cells were divided into three groups:infected by recombinant lentiviral vector group; infected by empty vector group and black control group. The effects of SEPP1 on proliferation of 769-P cells were analyzed by the MTS test and Colony-forming assay and cell cycle test.Results:1. Eighty genes were selected by expression microarray with fold change greater than 1.5 and P value less than 0.05. We further choose 12 genes with fold change greater than 2 and use RT-PCR to validate the difference in ccRCC primary cultured cell lines. For 5 genes:SLC34A2, CXCL5, SULT1C4, AKAP12, SEPP1, the difference was verified.2. Combining the results of expression microarray,4 genes were selected from the methylation microarray:SULT1C4, SEPP1, SLC34A2 and AKAP12, the methylation level difference of SEPP1 was validated by Sequenom MassARRAY methylation detection method.3. The expression level difference of SEPP1 was validated by western-blot and immunohistochemistry using ccRCC primary cultured cell lines and ccRCC tissues.4. After enzyme digestion, polyacrylamide gel electrophoresis (PAGE) showed the stripes of the same size with the SEPP1 DNA and empty vector. Sequencing result completely consistent with the human SEPP1 sequence. The 786-O and 769P cell lines showed enhanced green fluorescent under the fluorescence microscope. Compared with the empty vector group and the blank control group, the experimental group expressed significantly increased SEPP1 protein. 5. MTS experiment results prompt that the proliferation ability of over-expession cells have been reduced.6. Tablet cloning test showed that the colony forming ability of SEPP1 over-expression cell lines have been evidently reduced.7. Cell cycle experiments showed that SEPP1 over-express give rise to G2/M period retardation.Conclusion:1. SEPP1 was selected as the target gene using microarray technology. It is differently expressed and methylated in mutation type VHL ccRCCs and wild type VHL ccRCCs.2.Recombinant lentiviral vector plv-EGFP(2A)Puro-SEPP1 were constructed successfully. Overexpression of SEPP1 can significantly reduce the proliferation of 786-0 and 769P cells, and can cause cell cycle G2/M arrest in 786-O cells.