Identification Of Key Gene And Cell Signal Pathway And Study On Molecular Mechanisms Of Kidney Stone Based On The Network Analysis

Nephrolithiasis(kidney stone)is a joint disease of the urinary system.It is due to mineral crystals deposited in the kidney or moved to the ureter.Calcium-containing kidney stones are the main type in a variety of kidney stone,its accounting for 75%.It is known that kidney stones are familial and significantly hereditary,and is affected by diet and many other environmental factors.The interaction between crystals and renal tubular epithelial cells is an important stage affecting the formation of kidney stones.However,the mechanism of it is not certain yet.In recent years,with the development of computational biology,network analysis technology has been one of the fastest developing directions in the field of bioinformatics research.It can macroscopically investigate the overall relationship between genes/proteins in diseases,discover molecular interactions and find the key targets and key signal pathways in the biological process more objectively.Here,we use bioinformatics methods and cell molecular biology methods to analyze the formation and development of kidney stones,aiming to find the key genes and key cell signaling pathways that affecting the occurrence and development of kidney stones.The potential targets and key signal pathways of kidney stones are found through gene/protein interaction network analysis.And these results are further researched on the molecular pathological mechanisms of kidney stone diseases.Finally,the key genes and cell signaling pathways that affect the formation and development of kidney stones are discovered.This research has a profound significance for elucidating the molecular mechanism of calcium-containing kidney stones,as well as the early prevention,treatment and prevention of recurrence of the disease.Firstly,a total of 839 kidney stone-related proteins were collected in the Poly Search2 database and literature which-related with kidney stone.Kidney stone related PPI network and the direct interaction network were obtained by using In Web database.The network contained 340 proteins and 740 interactions.The Gene Net Toolbox was utilized to verify the authenticity of the network,and the P value was0.0009.Kidney stone core protein was obtained by using the network analyzer function of Cytoscape 3.6.1 software according to the Degree and Btweeness of the direct interaction network.The core protein was disposed by using GO analysis.The analysis result shows that the core protein is significantly enriched in the endoplasmic reticulum stress(ERS)and the endoplasmic reticulum-related protein post-translational modification,such as"protein folding","protein stabilization","ATF6-mediated unfolded protein response"and"response to stress",it implies ERS is an important biological process in the pathogenesis of kidney stones.Next,in order to verify the results of proteomics data analysis,we conducted further research using cell and molecular biology experiments.Western Blot results showed that the expression of BIP and CHOP proteins in the cells was increased after COM stimulated HK2 cells for 12 hours.Phospho-ERK(PERK)protein was activated,and ATF6 protein expression increased.Semi-quantitative PCR showed that spliced XBP-1 m RNA was up-regulated from 3hours to 12 hours.The above experimental results showed that COM crystals through three main pathways(including PERK,IRE1 and ATF6)induce ER stress.TUNEL cell apoptosis showed that the number of apoptosis cells in the COM treatment group was significantly more than that of the control group.Western Blot analysis showed that the activated Caspase12(ERS-specific caspase)of HK2 cells was increased after 3 hours of COM stimulation.The expression of activated caspase3 increased at 3 hours and 12hours.PJNK,PP38 expression is markedly up-regulated.The above experimental results indicate that ERS is linked to the apoptosis induced by COM crystals.Atomic absorption experiments showed that the adhesion of Ca²⁺on the cell surface of the ERS inducer-tunicamycin(TM)treatment group were significantly increased,and that of the ERS inhibitor-Salburinal(SAL)treatment group were significantly reduced,which proved that ERS in HK2 cells affects crystallization-cell adhesion.The occurrence of ERS is the fundamental biological event of crystallization-cell adhesion in HK2.The MTT experiment showed that the cell viability of the tunicamycin treatment group decreased,and the cell viability of the SAL treatment group increased.It indicated that ERS affects the viability of HK2 cells.Western blot detected the OPN protein expression is increased and the MGP protein expression is decreased under the ERS conditions.It indicated that ERS affects the expression of proteins associated with stone formation.The above results found that whether in vivo or in vitro,the formation of kidney stones can trigger ERS.ERS through several aspects promotes the formation of kidney stones,cell apoptosis,affecting the expression of stone formation-related proteins,reduced cell viability,and aggravate crystal adhesion.Our results suggest that ERS may be a possibility target for the prevention and treatment of kidney stones.Secondly,this study collected and analyzed data on the pathogenicity of kidney stone at the genome and transcriptome levels.At the genomic level,256 single nucleotide polymorphism(SNP)related to the occurrence of kidney stones were obtained by using four internationally influential clinical GWAS studies.Through the statistical analysis of the genetic location of tag SNP and LD SNP in the genome by using the genetic variant enrichment analysis(Variant Set Enrichment,VSE)method revealed that these sites are not mainly enriched in the DNase I hypersensitive region(DNase Hypersensitive Site,DHS)and other functional segments related to histone modifications,suggesting that kidney stone risk genes are involved in the molecular mechanism of kidney stone disease has little correlation with transcription level regulation.Next,in the transcriptome data analysis of clinical samples of kidney stones,we selected a DNA microarray data containing 62 clinical tissue samples of kidney stones,and analyzed differentially expressed genes of the patients with kidney stones(with or without stones in the renal papillary)and normal people.Related differential genes obtained at the genomic level and the transcriptome level were processed respectively to construct a kidney stone protein direct interaction network and identify the core protein of the network.Through the KEGG enrichment analysis of the proteins in the direct interaction network,two main enrichment pathways for kidney stone-related proteins were obtained.Finally,the results of integrating the genome level and the transcriptome level revealed the key core protein at these two levels is caveolin-1(CAV1).At the same time,the analysis at the genomic level also showed that EGFR has a direct interaction with CAV1,and EGFR is the protein with the most direct interactions with other proteins in this network(edge9).KEGG enrichment analysis found that the differential genes at the two levels were significantly enriched in the focal adhesion kinase signaling pathway.The strategic core proteins CAV1 and EGFR are both important signaling molecules in the focal adhesion kinase signaling pathway.The above bioinformatics research results indicate that the occurrence of kidney stones is likely to be related to focal adhesion signaling pathway through CAV1 and EGFR proteins.This signaling pathway is tightly related to cell survival and apoptosis.Finally,we used cell molecular biology experiments to further study this prediction result.First,after HK2 cells were stimulated by COM crystal for 48 hours,the TUNEL test results showed that the number of apoptosis increased dramatically.RT-PCR experiments showed that the expression of CAV1 and EGFR m RNA at the cellular level remained unchanged.Western Blot analysis confirmed that the protein expression was reduced of intracellular CAV1,EGFR,AKT.The immunofluorescence experiment proved that the cell caveolae structure was drastically reduced.After CAV1 was overexpressed,the COM-induced apoptosis and the increase in Ca²⁺residues on the cell surface were improved.It proves that CAV1 protein plays an essential role in apoptosis and cell adhesion induced by calcium oxalate crystals.We have shown that crystallization can induce ERS in HK2 cells and studies have shown that ERS can induce ubiquitin-dependent proteasomal degradation of some proteins.Therefore,we speculate that the mechanism of crystallization may be linked to the activation of the ubiquitin-proteasome degradation pathway by ERS induced by calcium oxalate stimulation.It is for this reason that calcium oxalate stimulation is not affect the m RNA levels of CAV1 and EGFR but down-regulates the protein levels of CAV1 and EGFR.The experimental results confirmed that after the cells were treated with the proteasome inhibitor MG132.The decrease in CAV1 and EGFR protein expression induced by COM stimulation was reversed.The results of the rat kidney stone model test complied with this.The rats in the experimental group showed obvious stone after 8weeks.The RT-PCR experiment showed that the expression of CAV1 and EGFR m RNA at the tissue level was unchanged.Western Blot showed that the expression of CAV1.EGFR,and AKT was reduced in kidney tissue.Immunohistochemical detection showed expression of CAV1 and EGFR in rat kidney cortex and medulla reduced with the increase of modeling time.At the same time,the pathological results of the kidney tissue of experimental rats showed that inflammation occurred 8 weeks after the model was created.The pathological consequences of the renal tissue of experimental rats showed that the renal cortex and medulla inflammation occurred in the kidney stone model rats with renal tubular cell damage,and the time of its severity was basically consistent with the downward trend of CAV1 expression.In summary,this study combined bioinformatics and cell molecular biology methods to explore the molecular mechanism of kidney stone disease.Our results prove that crystallization induces endoplasmic reticulum stress in cells.On the one hand,it directly leads to apoptosis and aggravates cell-crystal interaction.On the other hand,it degrades key proteins such as CAV1 and EGFR by activating the ubiquitination-proteasome pathway and damage CAV1-EGFR-AKT and other cell adhesion signaling pathways,aggravate cell apoptosis and induce inflammation,further promote cell-crystal interaction.Our results provide a new explanation for the basic mechanism of crystal cell interaction in the kidney,and provide a reference for the development of novel targets for the prevention and treatment of kidney stones.