High-glucose Cultured Human Proximal Tubule Epithelial Cells Long Non-coding RNA Analysis And Validation

Background Diabetic nephropathy is a diabetic microvascular complication and the leading cause of end-stage renal disease?ESRD?.Diabetic nephropathy is characterized by a series of ultrastructural changes in almost all renal,including glomerular,tubular hypertrophy,thickening of the basement membrane,mesangial expansion,glomerulosclerosis,and tubulointerstitial fibrosis.Previous studies have focused on glomerular lesions,and renal tubular injury has been overlooked.As the research progresses,it is increasingly recognized that renal tubules play an important role in the pathogenesis of diabetic nephropathy.Tubular injury leads to oxidative stress,hypoxia,chronic inflammation,and fibrosis,which in turn affects glomerular filtration function.Therefore,both damages promote each other,worsen the deterioration of renal function,and jointly promote the development of DN.Therefore,it is of great significance to study the function changes of proximal renal tubular epithelial cells under high glucose and its mechanism of action on renal injury.Long non-coding RNA?lnc RNA?is a RNA with a length of more than 200 nt and no protein coding capacity.It is closely related to the development of many diseases.Initially,lnc RNAs were thought to be transcriptional “junks” that did not have biological functions.However,recent studies have shown that the expression of lnc RNAs is tissue-and cell-specific,spatially specific,capable of transcription from epigenetic levels,transcription Levels,post-transcriptional levels regulate life activities,affect individuals' growth and development,metabolism,and cell proliferation,apoptosis,and differentiation.More and more studies have found that lncRNA is closely related to the development of diabetes.Liu et al.found that the expression of lnc RNA-MALAT1 in the retina of diabetic animals was elevated.Through intervention of MALAT1,the retinal function was significantly improved,and microvascular damage,vascular leakage,and inflammation were alleviated.Wang et al.by knocking out the lnc RNA-PVT1 gene of mesangial cells to analyze the expression levels of extracellular matrix?ECM?proteins and their regulatory factors,and found that knocking out lnc RNA-PVT1 can reduce the expression level of extracellular matrix proteins.Thus,it was concluded that lnc RNA-PVT1 can promote the development of diabetic nephropathy.However,there are few studies on lnc RNA and diabetic kidney disease,especially in the renal tubules,and the specific regulatory mechanisms are still unclear.Therefore,this study intends to use lnc RNA microarray technology to screen abnormally expressed lnc RNAs and m RNAs in high-glucose cultured human proximal tubular epithelial cells,and conduct bioinformatics analysis and preliminary exploration of mechanisms to lay a foundation for further exploration of the pathogenesis of diabetic nephropathy.Objective In this study,a long-chain non-coding RNA and m RNA expression profile of human renal proximal tubular epithelial cells?HK-2 cells?cultured with high glucose and normal glucose was constructed using microarray analysis technology.Further bioinformatics analysis was used to find potential targets.Molecular,conductive pathways,biological effects,etc.,and preliminary verification based on the information provided by the chip data,provide potential diagnostic markers and targets for the clinical treatment of diabetic nephropathy,and preliminary explore its mechanism.Method Human proximal tubular epithelial cells?HK-2 cells?were treated with serum-free normal sugar concentration medium for 24 hours and switched to normal sugar?5.6 mmol/L?containing 10% fetal bovine serum and high glucose?25 mmol/L?.L)After the medium was cultured for 48 hours,logarithmic growth phase cells were used for detection of lnc RNAs.Using the Arraystar human Lnc RNA chip V4.0 chip,including 40,173 Lnc RNAs and 20,730 protein-encoding transcripts,detect normal sugar group?5.6 mmol/L,n=3?and high glucose group?25 mmol/L,n=3?HK-2 differentially expressed lnc RNAs and m RNAs in cells to construct differential expression profiles.By performing GO analysis and KEGG Pathway analysis on differential m RNAs,real-time quantitative PCR verification of partially differentially expressed lnc RNAs,m RNAs,and related pathway markers was performed at the level of clinical specimens and cells,and the lnc RNA?NR₀22011?and its related gene TXNIP were found in high glucose.The underlying mechanisms of fibrosis in HK-2 cells were preliminary investigated.Results 1.The number of expressed lnc RNAs in HK-2 cells of high glucose group was higher than that in normal glucose group HK-2 cells.There were 1356 lnc RNAs with up-regulated expression and 978 lnc RNAs with down-regulated expression.Greater than 2 times?P<0.05?.Compared with the normal group,there were 2056 differentially expressed m RNAs,of which 1201 were up-regulated and 855 were down-regulated,and the fold change was greater than 2 fold?P<0.05?.2.GO analysis of differentially expressed m RNAs revealed that the biological processes were mainly concentrated in response processes,cell stress,cell surface receptor regulation,cell metabolism,cycle,chromatin assembly,DNA replication and other processes;molecular functions were mainly concentrated in DNA binding,calcium,iron ion regulation,protein binding,transcription factor binding and so on.KEGG Pathway analysis showed that gene regulation mainly focused on metabolic pathways such as type 1 diabetes metabolic pathway,MAPK pathway,ECM receptor pathway,FOXO pathway,inflammatory pathway,redox regulatory pathway,cell cycle,and RNA transport.3.Real-time fluorescence quantitative PCR validated some of the differentially expressed lnc RNAs.The cell-level validation results showed that the expression of TCONS₀0014969,NR₀22011,T379873,T183728 in high glucose group HK-2 cells increased,and the expression of ENST00000602697,NR₀26949,T256378 decreased;clinical specimen kidney The tissue results showed that the expression of TCONS₀0014969 and NR₀22011 increased in the kidney tissue of diabetic patients,and the expression of T379873,ENST00000602697,and NR₀26949 decreased in diabetic kidney tissue and the differences were statistically significant.4.Lnc RNA?NR₀22011?was up-regulated in HK-2 cells with different sugar concentrations and time,and its mechanism of action was initially explored.The results showed that Lnc RNA?NR₀22011?has no protein coding ability,which is consistent with the basic conditions for long-chain non-coding RNA research.Fluorescence in situ hybridization further confirmed that lnc RNA?NR₀22011?was mainly located in the cytoplasm.Western blot and real-time fluorescence quantitative PCR results showed that lnc RNA?NR₀22011?and TXNIP expression changes in each group of cells: high glucose treatment of HK-2 cells lnc RNA?NR₀22011?and TXNIP expression increased,TXNIP increased inhibition of TRX protein expression,increased FN,Col IV protein expression,further overexpression or interference with lnc RNA?NR₀22011?expression,found that TXNIP,FN,Col IV m RNA and protein expression were increased/decreased,TRX m RNA and protein expression decreased/increased.Conclusion 1 High-glucose cultured HK-2 cells showed significant changes in lnc RNA expression profiles.2 lnc RNA?NR₀22011?may be involved in hyperglycemia-induced renal tubular epithelial cell fibrosis injury via TXNIP.