MiR-29c Influence The Expression Of Inflammatory Cytokines By Targeting Tristetraprolin Under High Glucose Conditions In Mice Podocyte

BackgroundDiabetic nephropathy(DN), as one of the serious complications of diabetes mellitus, is widely popular in the global world, and the most common cause of the end-stage renal disease(ESRD). Multiple factors contribute to the development of diabetic nephropathy, however, the pathogenesis is still not fully understood. There is growing evidence that activated inflammation engages to the pathogenesis of diabetic nephropathy.Tristetraprolin(TTP) is a RNA binding protein, which promotes mRNA decay by binding with adenosine/uridine-rich element(ARE) in 3'-untranslated region(3'-UTR). It has been confirmed that TTP could regulate a variety of inflammatory cytokines, such as IL-6 and TNF-?, mediating the degradation of the mRNA. Therefore, TTP is also called “anti-inflammatory protein”. With the increasing of urinary protein, serum and urinary levels of IL-6 and IL-18 were significantly elevated, but those of TTP were significantly decreased in patients with DN. Decreased TTP expression might occur prior to the increase in IL-6 and IL-18, which demonstrated that TTP may involve in the development of diabetic nephropathy.MicroRNAs, which are short noncoding RNAs that regulate gene expression by posttranscriptional mechanisms, consist of 20-22 nucleotides. MiRNAs regulate gene expression by perfect or imperfect base pairing to the 3'UTR of specific target mRNAs, which leads to mRNA degradation and/or translational repression. Evidence showed miR-29c promotes the development of diabetic nephropathy, and its knockdown prevents the progression of diabetic nephropathy. Our previous study using miRNA arrays found that miR-29c is differentially expressed in the blood, urine and kidney tissues of patients with DN. But whether mi R-29c is involved in the inflammatory response of DN is still unclear.We applied TargetScan software finding that TTP may be a target of miR-29c. Therefore, we hypothesised that miR-29c influence the expression of inflammatory cytokines by targeting TTP under high glucose conditions in mice podocyte. ObjectiveThe experiment is intended to study the expression of miR-29c, TTP, IL-6, TNF-? under high glucose conditions, and those of after alternative miR-29c expression. To observe whether miR-29c involves in the inflammation response in hyperglycemic conditions, and identify whether TTP is the target of miR-29c. All those may provide a new theoretical basis for the diagnostic and therapeutic procedure for DN. MethodsConditionally immortalized mice podocyte cell line in vitro were adopted.1. podocytes were cultured in vitro and divided into groups as follows:(1)normal group(5.6mmol/L D-glucose);(2)High glucose group(25 mmol/L D-glucose);(3)Hypertonic group(5.6mmol/L D-glucose+19.4 mmol/L D-mannitol). The corresponding indicators were measured at different time. Quantitive real time-PCR was used to detect the expression of mi R-29c and mRNA expression of TTP, IL-6 and TNF-?. Western blot and ELISA was used to detect the protein expression of TTP and IL-6, TNF-? respectively. To observe the expression of miR-29c, TTP and inflammatory cytokines IL-6, TNF-? under high glucose conditions.2. Wild and mutative TTP 3'UTR-pmirGLO plasmid was constructed. Then the plasmid and miR-29c mimics were respectively co-transfected in mice podocyte. Cells were lysed after 36 h. Dual luciferase reporter assay kit was used to detect the activity of luciferase to verify whether TTP is the target of miR-29c.3. miR-29c mimics and inhibitor were transfected into podocytes under normal glucose conditions, normal control group and transfection control group were set up.The corresponding indicators were measured at different time. Quantitive real time-PCR was used to detect the expression of miR-29c and mRNA expression of TTP, IL-6 and TNF-?. Western blot and ELISA was used to detect the protein expression of TTP and IL-6, TNF-? respectively. To observe the expression of TTP and inflammatory cytokines IL-6, TNF-? after alteration of miR-29c by using miR-29c mimics and inhibitor.4. Podocytes were cultured in vitro and divided into groups as follows:(1)Normal control group(5.6mmol/L D-glucose);(2)High glucose group(25 mmol/L D-glucose);(3)High glucose miR-29c inhibitor transfection group(25mmol/L D-glucose+miR-29c inhibitor);(4)High glucose transfection control group(25mmol/L D-glucose+miR-control). Quantitive real time-PCR was used to detect the expression of miR-29c and mRNA expression of TTP, IL-6 and TNF-?. Western blot and ELISA was used to detect the protein expression of TTP and IL-6, TNF-? respectively. To observe the expression of TTP and inflammatory cytokines IL-6, TNF-? after alteration of miR-29c under high glucose conditions. Results1. The expression of miR-29c, TTP and inflammatory cytokines IL-6, TNF-? in mice podocyte under high glucose conditions.Compared with the normal control group, the expression of miR-29c were significantly increased in high glucose group; The protein and mRNA expression of TTP were decreased, whereas those of IL-6, TNF-? were increased in high glucose group(P<0.05).2. TTP is the target of miR-29c.Wild and mutative TTP 3'UTR-pmirGLO plasmid was constructed successfully. Then the plasmid and miR-29c mimics were respectively co-transfected in mice podocyte. Dual luciferase reporter assay kit showed the luciferase activity of wild TTP 3'UTR and miR-29c mimics co-transfected group were decreased compared with that of mutative group(P<0.05).Compared with the normal control group, the protein and mRNA expression of TTP were increased in miR-29c inhibitor transfection group, whereas decreased in miR-29c mimics transfection group(P<0.05).3. The expression of inflammatory cytokines IL-6, TNF-? after alteration of miR-29c expression.Compared with the normal control group, the protein and mRNA expression of IL-6 and TNF-? were decreased in miR-29c inhibitor transfection group, whereas increased in miR-29c mimics transfection group(P<0.05).4. The expression of TTP and inflammatory cytokines IL-6, TNF-? after alteration of miR-29c expression under high glucose conditions.Compared with the normal control group, the protein and mRNA expression of TTP were decreased, whereas those of IL-6 and TNF-? were increased in high glucose group(P<0.05). Compared with high glucose group, the protein and mRNA expression of TTP were increased, whereas those of IL-6 and TNF-? were decreased in high glucose miR-29c inhibitor transfection group(P<0.05). Conclusion1. The expression of miR-29c and inflammatory cytokines are influenced under high glucose condition in mice podocyte.2. TTP is the target of miR-29c.3. miR-29c influence the expression of inflammatory cytokines by targeting TTP under high glucose conditions in mice podocyte.