The Mechanism Research Of LncRNA CASC2 Mediating Diabetic Nephropathy Inflammation And Fibrosis

Diabetes is a metabolic disease characterized by high blood sugar,usually caused by insufficient insulin production or an ineffective response to insulin,and is a major health problem worldwide.Diabetic nephropathy(DN)is one of the most common chronic complications of diabetes and the main cause of end-stage renal disease.About 40%of diabetic patients are affected by DN.Diabetes often leads to microvascular damage,especially in the kidney,but also can cause diabetic nephropathy.With the improvement of Chinese people’s living standard,the incidence of diabetes is also on the rise.Therefore,the study of new ideas for the treatment of diabetic nephropathy is becoming an increasingly important topic in the current field of diabetes.Long non-coding RNA(lncRNA)is a class of non-coding RNA molecules with a length of more than 200nt.Although lncRNA has no protein-coding function,with the deepening of research,it has been found that lncRNA plays a key role in physiological and pathological regulation of organisms,especially in tumors and metabolic diseases.The important role of lncRNA in the development of DN has attracted more and more attention.Studies have shown that lncRNA can participate in the pathophysiological process of multiple renal cell injuries in DN through various mechanisms such as DNA methylation,histone modification,miRNA competition inhibition,and activation signaling pathway,and play a regulatory role in epigenetic,transcriptional,and post-transcriptional gene expression.Therefore,to explore the differential expression of lncRNA in the pathological process of DN and its related regulatory molecular mechanism is of potential application value for the early diagnosis,targeted therapy and prognosis assessment of DN.More and more studies have shown that lncRNA CASC2,as a tumor suppressive long chain non-coding RNA,plays a key role in different types of tumors.In recent years,with the deepening of research,it has been found that CASC2 not only plays a regulatory role in tumors,but also plays a key role in diabetic nephropathy,a metabolic disease,and participates in the regulation of chronic renal failure in diabetic patients.Importantly,CASC2 is abnormally expressed in diabetic nephropathy cell models and affects hyperglycose-induced cell proliferation,extracellular matrix accumulation,and oxidative stress by regulating the miR-133b/FOXP1 signaling axis.The bioinformatics analysis revealed that CASC2 and Suppressors of cytokine signalling(SOCS2)and miR-144 had latent binding sites.It has been shown that SOCS2 was play a key regulatory role in inflammation and cancer.It has also been reported that the overexpression of SOCS2 in chronic kidney disease is related to the incidence of diabetic nephropathy.The overexpression of SOCS2 in the kidney can alleviate the phenomena of glomerular ultrafiltration,glomerular hypertrophy,abnormal inflammatory reaction and renal fibrosis caused by streptozotocin,and significantly reduce the expression levels of nephritic proteins and fibrogenic proteins.This study revealed that CASC2 may regulate the progression of diabetic nephropathy by regulating the miR-144/SOCS2 signaling axis.In this study,we constructed a mouse model and a cell model of diabetic nephropathy to analyze the differential expression of COSC2.At the same time,the regulatory mechanism of CASC2/miR-144/SOCS2 signal axis and its regulatory effect on renal mesangial cell apoptosis,inflammation and fibrosis induced by high glucose were detected by combining biological information and various molecular cell experimental techniques.Part Ⅰ The expression of lncRNA CASC2 in diabetic nephropathyObjective:To preliminarily determine the function of CASC2 in diabetic nephropathy by detecting the differential expression of lncRNA CASC2 in the DN mice models and the high glucose(HG)induced human renal mesangial cells(HRMC).Methods:Diabetic mice models was established to measure the body weight,blood glucose and urinary microprotein levels.PAS staining was used to observe the deposition of extracellular matrix in the kidney tissue of the diabetic mice.The expression level of CASC2 in the kidney tissues of diabetic mice was detected by Q-PCR.HRMCs were treated with high glucose to construct an in vitro model of diabetic nephropathy cells,and the differential expression of CASC2 in was detected by Q-PCR.Results:The body weight of diabetic mice was significantly higher than that of normal controls.In contrast,the urinary microprotein and blood glucose levels were significantly higher,and the deposition of extracellular matrix in kidney tissues was significantly increased.It is showed that DN mice model was successfully cnstructed.CASC2 expression was significantly down-regulated in kidney tissue of diabetic mice and HG-induced HRMCs.Conclusion:The diabetic mice models in vivo and HG-induced HRMCs in vitro were successfully constructed.The expression level of CASC2 was significantly down-regulated in kidney tissues of diabetic mice and HG-induced HRMCs.Part Ⅱ The function of lncRNA CASC2 in diabetic nephropathyObjective:HRMCs were stimulated by high glucose and the effects of overexpression of CASC2 on apoptosis,inflammation and fibrosis of HG-induced HRMCs were investigated.Methods:HRMCs were transfected with CASC2-overexpressed plasmids,then the transfection efficiency were measured by Q-PCR.The effect of CASC2 overexpression on cell apoptosis of HG-induced HRMCs was detected by flow cytometry assay.The effects of CASC2 overexpression on cell apoptotic-related proteins such as B-cell lymphoma-2(Bcl-2),cleaved caspase 3 and fibrosis-related proteins such as Fibronectin(FN),collagenⅣ(COI-Ⅳ),Transforming growth factor-β(TGF-β)were quantified by Western blot assay.The expressive effects of CASC2 overexpression on inflammatory cytokines such as Tumor necrosis factor-α(TNF-α),Interleukin-6(IL-6)and Interleukin-1β(IL-1β)were tested by Q-PCR assay.Results:CASC2 overexpression significantly reduced HG-induced apoptotic rate,cleaved caspase-3 protein level,while elevated Bcl-2 protein level.In addition,overexpression of CASC2 significantly decreased the expressions of inflammatory cytokines(TNF-α,IL-6,IL-1β)induced by high glucose.Simimlar trend was observed that CASC2 overexpression markedly inhibited the expression of fibrosis related proteins(FN,COL-Ⅳ and TGF-β1)caused by HG stiulation.Conclusion:Overexpression of CASC2 significantly alleviated apoptosis,inflammation and fibrosis progression of HG-induced HRMCs in vitro.Part Ⅲ LncRNA CASC2 negatively regulated miR-144 expressionObjective:To explore whether lncRNA CASC2 has a targeted regulation effect on miR-144.Methods:Bioinformatics software was used to analyze the potential targets sequence between CASC2 and miR-144.A double luciferase reporter vector wild-type(WT)-CASC2 sequences and mutat-type(MUT)-CASC2 sequences were constructed and tranfected into HRMCs cells,respectively.The binding relationship was detected by double luciferase reporter gene system.CASC2-overexpressed plasmids and CASC2-silenced siRNA were transfected into HRMCs,and the expression levels of miR-144 and CASC2 were detected by Q-PCR.Results:Overexpression of miR-144 decreased luciferase activity in cells containing WT-CASC2 plasmids,but had no significant effect on luciferase activity in cells containing MUT-CASC2 plasmids,suggesting that miR-144 was bound to the 3’-UTR of CASC2.Q-PCR results showed that CASC2 were successfully overexpressed and silenced in HRMCs,and significantly downregulated and upregulated the expression of miR-144,respectively.Conclusion:LncRNA CASC2 negatively regulated the expression of miR-144.Part Ⅳ miR-144 is involved in the progression of lncRNA CASC2-mediated diabetic nephropathyObjective:To investigate the effects of miR-144 on lncRNA CASC2-mediated apoptosis,inflammation and fibrosis in HG-induced HRMCs in vitro.Methods:CASC2 was overexpressed or co-overexpressed with miR-144 in HG-induced HRMCs,then the cell apoptosis was detected by flow cytometry.Western Blot was used to detect the expression levels of apoptosis-related proteins(Bcl-2,cleaved caspase 3)and Fibrosis associated proteins(FN,COI-Ⅳ,TGF-β1).The expression levels of inflammatory cytokines(TNF-α,IL-6 and IL-1β)were detected by Q-PCR.Results:Compared with HG group,overexpression of miR-144 significantly reversed the inhibition effects on apoptotic rate,cleaved-caspase 3 level,and the promotion effect on Bcl2 level mediated by CASC2 overexpression.Similarly,overexpression of miR-144 also eliminated the inhibitory effects of overexpression of CASC2 on the expressions of inflammatory factors(TNF-α,IL-6,IL-1β),and fibrosis related proteins(FN,Col-Ⅳ,and TGF-β1).Conclusion:miR-144 negatively affected the inhibitory effects of CASC2 on apoptosis,inflammation and fibrosis of HG-induced HRMCs.Part Ⅴ miR-144 negatively regulates the expression of SOCS2Objective:To investigate whether miR-144 and SOCS2 have targeted regulatory effects in HRMCs.Methods:Bioinformatics software was used to analyze the potential targeted sequences between of miR-144 and SOCS2 3’-UTR.Dual-luciferase reporter gene vectors containing SOCS2 wild-type(WT-SOCS2)and mutated type(MUT-SOCS2)3 ’-UTR were constructed,and the binding relationship was verified by the dual luciferase reporter gene system.miR-144 mimics or inhibitor were transfected into HRMCs,and the expression levels of miR-144 and SOCS2 were detected by Q-PCR.Results:Overexpression of miR-144 significantly decreased luciferase activity in WT-SOSC2 plasmids cells,but had no significant effect on luciferase activity in MUTSOSC2 plasmids cells.Moreover,Q-PCR results showed that miR-144 mimics and miR-144 inhibitor were successfully transferred into HRMCs,and the mRNA and protein levels of SOCS2 were significantly inhibited and increased,respectively.Conclusion:miR-144 negative regulated the expression of SOCS2 in HRMCs.Part Ⅵ SOCS2 is involved in lncRNA CASC2/miR-144 Axis mediated progression of diabetic nephropathyObjective:To investigate the effects of SOCS2 on apoptosis,inflammation and fibrosis of HRMCs mediated by CASC2/miR-144 axis in vitro.Methods:The gene co-expression technique was used to co-transfect CASC2,SOCS2 overexpressed vectors and miR-144 mimics in HG-induced HRMCs.Flow cytometry was employed to detect cell apoptotic rate.The expression levels of apoptosis-related proteins(Bcl-2,cleaved caspase-3),fibrosis-related proteins(FN,COI-Ⅳ,TGF-β1)were detected by Western Blot,The expression levels of inflammatory factors(TNF-α,IL-6,IL-1β)were detected by Q-PCR.Results:In CASC2 overexpressed HG-induced HRMCs,the promotive effects of miR-144 overexpression on apoptosis rate and cleaved-caspase 3 expression and the inhibitory effect on Bcl-2 expression were significantly alleviated by co-expression of SOCS2.Similarly,the induction effects of miR-144 overexpression on inflammatory factors(TNF-α,IL-6,IL-1β)and fibrosis related proteins(FN,Col-Ⅳ,TGF-β1)were also reversed by co-expression of SOCS2.Conclusion:Overexpression of SOSC2 significantly reversed the expression of pro-inflammatory cytokines,pro-apoptosis and pro-fibrosis responses mediated by miR-144 overexpression,and partially restored the protective effects of CASC2 upregulation on HG-induced HRMCs.Conclusion:The expression of CASC2 was significantly lower in diabetic mice model and HG-induced HRMCs.Overexpression of CASC2 obviously improved the fibrosis response by inhibiting apoptosis and inflammatory factor release of HG-induced HRMCs.Further investigation into its molecular mechanism revealed that CASC2 competitively induced the upregulation of SOCS2 as a molecule sponge of miR-144,and thus exert its anti-inflammatory,anti-fibrosis and anti-apoptotic functions.The results of our study might provide some theoretical reference for elucidating the pathogenesis of CASC2 in DN and developing new drug targets for inflammation-fibrosis therapy.