Biological Networks And Metabolic Pathway Analyses Of Renal Cell Carcinoma With And Without Metastasis

Renal cell carcinoma(RCC)is one of common malignant tumors of urinary system in China,which accounts for nearly 3% of all human malignancies,and is only secondary to bladder cancer.With the changes of life environment and improvement of diagnostic technologoes,an increased incidence of RCC was identified both in China and abroad,indicating that RCC might be a severe threaten to human health.Clear-cell RCC is an important component of RCC and accounts for70%-80% of RCC.Till now,surgery,radiotherapy,and chemotherapy are the main methods for RCC treatment in clinic.However,Due to the versatile biological behavior of renal clear cell carcinoma and limited diagnostic methods,most of the patients were diagnosed until the middle late stage and usually correlated with certain metastasis,which might lead to high frequent metastasis and recurrence after surgery,to result in poor prognosis for patients.Immunotherapy is the adjuvant therapy of end-stage RCC,but due to its toxicity and lower responses,the clinic therapeutic effect of immunotherapy is still unsatisfactory in clinic.Therefore,it is important to further explore the mechanism of RCC and metastasis of RCC provide some new insights in improving the therapy of RCC and exploring new target drugs.In this study,the microarray data provided by the public databases was used to screen the differentially expressed genes(DEGs)of non-metastatic and metastatic clear-cell RCC,as well as the underlying biofunctions of these DEGs in clear-cell RCC.Meanwhile,the protein interaction relationships among DEG-encoded proteins were analyzed to construct protein-protein interaction(PPI)network,and potential targeted small molecular drugs were also investigated,so that could provide some new insights for the clear-cell RCC treatment.Firstly,the DNA microarray datasets numbered GSE47352 and GSE22541 weredownloaded from Gene Expression Omnibus(GEO)database,including 4primary metastatic and 5 non-metastatic clear-cell RCC samples in GSE475352,and44 primary metastatic and 24 non-metastatic clear-cell RCC samples in GSE22541.In the current study,a total of 359 DEGs were identified in GSE475352,including196 upregulated and 163 downregulated genes.Meanwhile,a total of 2623 DEGs were identified in GSE22541,including 847 upregulated and 1776 downregulated genes.Then,DEGs were clustered according to the expression levels,and the biofunction and protein-protein interaction netwwork of DEGs were further revealed,as well as the disease associated small molecule drugs via Camp.Finally,DEGs were validated using the real-time PCR in metastasis and non-metastatistic renal cancer tissues and renal cancer cell lines.In GSE47352 dataset,a total of 5 pathways were significantly enriched by the DEGs,among which,renal cancer pathway was the most significant one,involving EPAS1,ETS1,JUN,SOS2,TGFB2,and PTPN11.Moreover,the PPI network analysis results showed that JUN(degree = 6)was the hub protein of PPI network.Small molecule drug analysis showed that thapsigargin might play a crucial role in anti-cancer therapy.In GSE22541,functional and pathway enrichment analyses presented that the upregulated genes were significantly enriched in ECM-receptor signaling pathway,cancer pathway and focal adhesion pathway,and the downregulated genes were significantly enriched in neurotrophin signaling pathway,insulin signaling pathway and lysosome.The PPI network analysis showed that ITGA2,CDK1,COL1A1,MMP2 and DCN were the hub proteins in PPI network of upregulated proteins,and TP53,AKT1,EP300,HSP90AB1 and RELA were the hub proteins in PPI network of downregulated proteins.To further explore some classical DEGs in metastasis of clear-cell RCC,shared DEGs of two datasets were screened,including 7 upregulated and 12 downregulated genes.Functional enrichment analysis showed that these genes were significantenriched in muscular organ development,but no significant pathway was enriched.Finally,an experimental verification was conducted for these shared DEGs.The expression levels of GPRC5 A,RAB25,SGCD and KRT7 were significantly upregulated in metastatic clear-cell RCC,while the expression levels of SLC6A19,SULT1C4,NFKBIL1 and INF were significantly downregulated in metastatic clearcell RCC compared with non-metastatic clear-cell RCC,which were consistent with bio-informatics analysis.Moreover,the expression of SGCD and KRT7 were significantly higher in CAKI cells than that in normal renal cells,and the expression of SLC6A19 was significantly lower than that in normal renal cells.Knockdown of KRT7 could significantly inhibited the migration of CKAI cells.In conclusion,in this study,a total of 19 shared DEGs were identified between two comparisons of non-metastatic and metastatic clear-cell RCC tissue samples.Meanwhile,the experimental verification showed that the GPRC5 A,RAB25,SGCD,KRT7,SLC6A19,SULT1C4,NFKBIL1 and INF might play important role in the metastasis of renal caner.Moreover,further investigation had verified that knocked down KRT7 coudl significantly inhibiting the migration of CKAI cells,indicating KRT7 might promote the metastasis of clear-cell RCC via increasing the migration of clear-cell RCC cells.Therefore,it might be important to further reveal the molecular mechanism of these genes =in metastasis of clear-cell RCC,so that it could provide us some useful information in improving the treatment and diagnosis efficiency of metastatistic clear-cell RCC.