Application Of Bioinformatics To Screen Novel Drug NS398 For ADPKD And Verification Of Its Effects

Objective:ADPKD is the most common hereditary kidney disorder.The crime gene mostly is mutated PKD1 or PKD2 gene.The pathogenesis of ADPKD is characterized by the increased cell proliferation and unstricted expansion of renal cysts,which is tumor-like.ccRCC is one of the most common malignant t?mors in kidney.Bioinformatics analysis of the RNA sequencing profiles of kidney tissues from ADPKD and ccRCC patients revealed the similarities and differences in genes of the two diseases,which can be further utilized to explore pathways,functions and predict survival.Connectivity Map was utilized to screen potential molecules which can inhibit covariant genes.The most promising molecule was applied in experiemnts in vivo and in vitro to confirm its actual effect and further illustrate the mechanism.Methods:(1)The Renal Gene Expression Database(RGED)is a plaform providing kidney tissue microarray data of kidney diseases.Datasets of RGED7869 and RGED53757 were selected respectively for searches centering ADPKD and ccRCC.(2)The complete data were obtained from NCBI Gene Expression Omnibus(NCB0-GEO).Covariant DEGs of the two diseases were identified.Then enriched pathways and functions were unravelled.Survival analysis was conducted to explore functions of non-covariant genes.A small molecule compound NS398 was also obtained to inhibit the covariant genes.(3)Cultured Pkd1-/-cells and human renal clear carcinoma cells in vitro,the effect of NS398 on cell proliferation was detected,and the mechanism for anti-proliferation was explored.(4)Utilizing early induced polycystic kidney disease mouse model,the effect of NS398 on cyst growth was detected.Results:(1)Bioinformatics analysis on GSE7869 and GSE53757 revealed 264 and 1914 DEGs in ADPKD and RCC patients' renal tissues respectively.The common parts extracted from the DEGs were found enriched in the arachidonic acid metabolism and modulating cell cycle.The PPI analysis of DEGs revealed the main functions as cell cycle and energy metabolism.NS398,a small molecule compound was identified via Connectivity Map to inhibit the covariant genes.(2)In vitro experiments,NS398 suppressed the proliferation of Pkd1-/-cells and human RCC cells 786-0.Flow cytometry revealed that NS398 prohibited cell cycle at G1 phase with no interference to apoptosis.The cellular expressions of Cyclin D1 decreased while p21 increased with P-Akt and P-ERK expressions both declining after NS398 treatment.Thus,NS398 may affect cell cycle via PI3K/Akt and ERK signaling pathways.(3)In vivo experiment,NS398 retarded the growth of renal cysts in the early-induced Pkdl mice and protected renal function.Conclusions:(1)The results of bioinformatics analysis of human kidney tissue RNA profiles of ADPKD and ccRCC disclosed novel research targets.The covariant DEGs mainly participate in cell cycle,and NS398 can inhibit the covariant DEGs.(2)In vitro experiments,NS398 suppressed the proliferation of Pkd1-/-cells and human RCC cells 786-0.NS398 also regulated the expression of cyclins such as Cyclin D1 and p21.P-Akt and P-ERK expressions were reduced after NS398 treatment in both cells,indicating NS398 may inhibit cell proliferation through PI3K/Akt and ERK pathways.(3)In vivo experiments,NS398 retarded the growth of renal cysts and protected renal function in the early-induced Pkdl mice.