Role And Mechanism Study Of Long Non-coding RNAs In Kidneys Injured By Calcium Oxalate Crystal

?Background?The formation of kidney stones is a common urological disorder,and the number of patients presenting with this disorder is on the rise.Calcium oxalate(CaOx)is the major constituent of most kidney stones and accounts for 80% of them.In addition,acute oxalosis(for example,in glyoxylate poisoning)can induce acute kidney damage as a result of renal tubular blockage caused by the deposition of calcium oxalate crystals.Kidney stones associated with a significant loss of kidney function are frequently accompanied by interstitial fibrosis,which is a common result of many kidney diseases.Several previous studies have suggested that kidney fibrosis causes renal epithelial cells to transition to mesenchymal cells,a so-called epithelial-to-mesenchymal transition(EMT).This phenomenon is related to cell proliferation.In cultured mouse kidney epithelial cells,TGF-?1 stimulates G2/M arrest and induces the production of profibrotic cytokines that can stimulate the proliferation and transition of pericytes to myofibroblasts.In mouse models of experimentally induced renal fibrosis,the functional consequence of EMT during fibrotic injury is an arrest in the G2 phase of the cell cycle.In our previous study,EMT also occured in mouse model injected with 100mg/kg glyoxylate.In recent years,an increasing number of reseachers are concentrating on long non-coding RNAs(lncRNAs),which are a subgroup of non-coding RNAs greater than 200 nucleotides without obvious protein coding functions.Many studies have indicated that lncRNAs play key roles in a variety of biological processes,such as proliferation and apoptosis,through complex mechanisms(Mercer et al.,2009).However,there are few studies on the role of lncRNAs in kidney stone diseases,and the overall pathophysiological contributions of lncRNAs to kidney stones remain largely unknown.?Aims?The aim of our study is to investigate human lncRNAs by using a lncRNA microarray and BLAST algorithm to mouse lncRNAs.We got a human lncRNA,termed CHCHD4P4,to determine its biological functions and molecular mechanisms in HK-2 cells exposed to COM.Understanding the precise molecular mechanisms by which the lncRNAs function will be critical for exploring these potential new strategies for the early diagnosis and treatment of kidney stone disease.?Methods?1.The mRNA and lncRNA expression profiles of three experimental mice and three normal mice were examined and analyzed by using a Agilent 8×60K Microarray.Differential gene based on the functional analysis of genes,were categorized as significantly up-regulated GO-Term and significantly down regulated GO-Term.Differential gene based on the KEGG pathway analyses,were classified into significantly up-regulated pathway and significantly down regulated pathway.2.To analyze the human lncRNAs that are homologous to mouse lncRNAs,we applied the BLAST algorithm to mouse lncRNAs and human lncRNAs.CHCHD4P4 was found to be upregulated in vitro in human proximal tubular epithelial cells exposed to COM as compared to its expression in the control group of cells by Real-time PCR.3.We observed the function of CHCHD4P4 on biological behavior of HK-2 cells in vitro.HK-2 cell with high expression of CHCHD4P4 was selected for study.HK-2 cell was transfected with siRNAs to down-regulate CHCHD4P4 expression and transfected with CHCHD4P4-pcDNA3.1 to up-regulate CHCHD4P4 expression.Real-time PCR,immunofluorescence,western-blot were used for evaluating Epithelial mesenchymal markers.CCK8 assays,Edu assays,Flow cytometry were employed for investigating the cell proliferation and cell apoptosis.?Results?1.We identified 2165 differentially expressed genes;among these,1421 genes were upregulated and 744 genes were downregulated in the glyoxylate-exposed mice.Totally 376 different lncRNAs were found expressed in crystal group,with 154 of lncRNAs expression were up-regulated and 222 of lncRNAs expression were down-regulated.2.15 pairs of lnc RNAs homologous between mice and humans were found.To validate these findings,we analyzed the expression of these pairs using real-time PCR,and found that the expression of the mouse lncRNA AU015836 and human lncRNA CHCHD4P4.3.The expression of the CHCHD4P4 was higher in the CaOx-exposed HK-2 cells than the control group.In our model,overexpression of CHCHD4P4 increased the transcript levels of ZEB1,Vimentin,and Snail and decrease the expression of E-cadherin.CHCHD4P4 possibly limits HK-2 cell proliferation by promoting apoptosis in vitro,and not by altering the cell cycle.?Conclusions?1.In the vitro,EMT occured in the HK-2 cells exposed to COM.The result was consistent with our previous annimal model.2.The expression of the CHCHD4P4 was higher in the CaOx-exposed HK-2 cells than the control group.The knockdown of CHCHD4P4 reduced the process of EMT.In contrast,the exogenous expression of CHCHD4P4 promoted EMT.3.CHCHD4P4 possibly limits HK-2 cell proliferation by promoting apoptosis in vitro,and not by altering the cell cycle.4.CHCHD4P4 may inhibit cell proliferation in kidneys with calcium oxalate crystals by promoting EMT.CHCHD4P4 may play an important role in the pathogenesis and development of kidney stone.