| PART ⅠEstablishment of clear cell renal carcinoma cell tissue and paratumor tissue alignmentObjective:To establish the alignment of clear cell renal carcinoma cell tissue and paratumor tissue, and to definitude the pathological and immunohistological diagnosis of the tissueMethods:We chose9patients aged from47to64, diagnosed as clear cell renal cell carcinoma by imaging findings and clinical manifestation before operation, which are willing to donate their post-operation specimen to our research. All of these9patients chose to take the radical nephrectomy, including pre-operation staging T1aN0M04cases (44.4%) and T1bN0M05cases (55.6%), in which there are6male and3female cases, age55.2±7.1. For each post-operation specimen, we took two examples from the tumor(0.5cm in diameter), and two examples from the tissue2cm away from the tumor(0.5cm in diameter). We use hematoxylin-eosin staining, Cytokeratin and Vimentin staining on the paraffin section of one example from the tumor and one from the tissue. And we extracted RNA from rapid frozen examples of the other two with RNA TRIzol box.Results:All these9patients, aging from47to64, had the operation of renal nephrectomy.1) General Pathological diagnosis:The post-operation TNM staging are parallel to the pre-operation staging, with T1aN0M04cases (44.4%) and T1bN0M05cases (55.6%).2) Hematoxylin-eosin staining:From the cell-morphological analysis, case No.6was diagnosed as the chromophobe cell subtype of renal cell carcinoma instead the clear cell subtype, and the other eight cases were clear cell subtype. All these nine cases showed no signs of local metastasis.3) Immunohistochemistry:Vimentin staining showed that the signal of No.6tumor is negative, while the other eight cases are positive. All the signal of tumor showed positive results in Cytokeratin, pan staining. Both Vimentin and Cytokeratin staining showed negative results in the tissue away from the tumor in all these nine cases.4) The result of the RNA exaction is good, while the exaction dilution could meet the requirement for the next part.Conclusion:The existence of chromophobe cell tumor, which is uncommon in the clinical, was beyond our control. But the postoperative pathological TNM staging of all the renal cell cancer cases was entirely consistent with the preoperative staging. And it would also give us a relatively stable diagnostic result so that we could make better use of our tissue examples in the RNA testing. The rapid freezing could provide us a good example of renal tissue, with in which there is adaptive concentration and low rate of degradation. PART ⅡDetection of the expression change in clear cell renal cell carcinoma with RNA-seqObjective:To validate the known gene sets, signature and pathway and to find the novel gene sets or pathway related to the clear cell renal cell carcinoma, we applied a gene enrichment analysis method based on the whole transcriptome sequencing, with the technology of RNA-seq.Methods:RNAseq from9paired ccRCC tumors and normal kidney samples were produced as described in PART I. After the first strand cDNA synthesis, adding sequencing joints, and PCR program for40cycles, all the samples were applied to the sequencing of Illumina, HiSeq2000. Transcriptome expression levels obtained in the sequencing data, combined with the UCSC the Genome Browser and MsigDB database, was normalized. With the use of Blat(Blast-like alignment tool, Blat Bowtie、 RUM(RNA-Seq unified mapper, RUM)、 DEseq、 GSEA (Gene Set Enrichment Analysis, GSEA), we could get different levels analysis of the data, including chromosome, gene sets, signature and pathway.Results:From the normalized RNA-seq data, according to test results of nine cases of RNA extraction samples of transcriptome level, combined with a variety of genetic analysis software, it is obtained the following results:1) Through the application of RNA-seq technology, genome-wide transcriptomic analysis verified the known abnormal gene expression of renal clear cell carcinoma, such as EGLN3, CA9, and VEGFA, while sample six is unique in the tumor samples of gene expression, but consistent with the results of HE staining and immunohistochemistric results.2) It is found in our research, the loss of gene expression in3p and gain of gene expression in5q, both of which have been shown as popular genetic changes in ccRCC. Interestingly, tumor sample6showed a loss of chromosome1,2and17, gain of chromosome3and7, which is a typical characteristic of chromophobe subtype, but further pathological confirmation reveals that tumor sample6was diagnosed as a mixture of clear cell and chromophobe subtype. 3) In this study, through a collection of abnormal gene expression of RNA-seq analysis, we found35noteworthy gene sets, verified the differential expression level of the gene sets related to the VHL/HIF pathway and the expression loss of the gene sets for renal function cancer tissues, and also got the same results parellel to the recent PBRM1exon research in chromatin modification related genes.4) Combined with NCBI chip database GSE17895and GSE11024, it is estimated that RUNX1-RUNX1T1pathway has a specific expression rise in renal clear cell carcinoma.Conclusion:With the application of RNA-seq technology, nine renal cell carcinoma and adjacent tissues carried out the transcriptomic level of the whole genome-wide sequencing, which shows us the technological advantages of of RNA-seq technology like all-digital signals and so on. The known genes, gene sets, pathway or chromosome related to renal clear cell carcinoma shows specific expression level in our research, which is parellel to our findings before. And we even find some novel pathways, which may be related to the clear cell renal cell carcinoma, such as RUNX1-RUNX1T1pathway. PART ⅢPreliminary validation of RUNX1-RUNX1T1pathwayObjective:To verify the differential expression level of RUNX1-RUNX1T1pathway in clear cell renal cell carcinoma and adjacent tissuesMethods:We chose4patients aged from49to58, diagnosed as clear cell renal cell carcinoma by imaging findings and clinical manifestation before operation, which are willing to donate their post-operation specimen to our research. All of these4patients chose to take the radical nephrectomy, including pre-operation staging T1aN0M03cases (75%) and T1bN0M01cases (25%), in which there are3male and1female cases, age55.2+7.1. For each post-operation specimen, we took two examples from the tumor(0.5cm in diameter), and two examples from the tissue2cm away from the tumor(0.5cm in diameter). We use hematoxylin-eosin staining, Cytokeratin and Vimentin staining on the paraffin section of one example from the tumor and one from the tissue. And we extracted RNA from rapid frozen examples of the other two with RNATRIzol box. As representative of RUNX1-RUNX1T1pathway, we chose to do qRT-PCR test on the genes including NET02, GBP2and VCAN, of which the expression level is the top three in the whole pathway.Results:All these4patients, aging from49to58, had the operation of renal nephrectomy.1) General Pathological diagnosis:The post-operation TNM staging are parallel to the pre-operation staging, with T1aN0M03cases (75%) and T1bN0M01cases (25%).2) Hematoxylin-eosin staining:From the cell-morphological analysis, all the4cases were clear cell subtype. All these4cases showed no signs of local metastasis.3) Immunohistochemistry:All the signal of tumor showed positive results in Cytokeratin and Vimentin staining. Both Vimentin and Cytokeratin staining showed negative results in the tissue away from the tumor in all these4cases.4) The result of the RNA exaction is good, while the exaction dilution could meet the requirement for the next part.5) In the PCR test, the expression level of NETO2, GBP2and VCAN is higher in tumor tissue than in adjacent tissue. Conclusion:From the persuasive results of PCR, it is estimated that the RUNX1-RUNXlTl pathway is related to the clear cell renal cell carcinoma. |
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