The Role Of Mir10 In The Pathogenesis Of DKD Through TGF-β1/Smad3 Pathway

Objective:Diabetes mellitus（DM）is a complex chronic metabolic disease caused by long-term high blood sugar.The main reason is the disorder and loss of function of B cell of islet of Langerhans.Diabetic kidney,heart,retina and peripheral nerve are in hyperglycemia environment for a long time,which will affect the physiological function of organs and even lead to organ failure.Diabetic kidney disease（DKD）is one of the important complications of diabetic microangiopathy.Hyperglycemia can cause glomerular,tubule and interstitial lesions by causing glomerular basement membrane thickening and glomerular basement membrane dilation,leading to renal damage^[1].In recent years,studies have shown that chronic low-grade inflammatory response,fibrosis,oxidative stress,neovascularization,endothelial dysfunction and so on play an important role in the occurrence and development of DKD,until the final cause of glomerulosclerosis and interstitial fibrosis^[2]MicroRNA（MIR）is a kind of small non coding RNA.It is highly conserved in different species and highly specific in different stages of development.Mir10 has been shown to play a regulatory role in inflammation,fibrosis and tumor migration^[3].Mir10 is widely expressed in adult mice,with the highest expression in kidney,muscle,lung and liver.Many studies have confirmed that mir10 expression in fibrotic tissue increased.In the mouse model of pulmonary fibrosis,mir10 may play a regulatory role in the activation of fibroblasts and collagen deposition through TGF-β1 signaling pathway^[4].In addition,mir10 can also promote tumor migration through EMT.The role of TGF-βin fibrosis has been confirmed^[5].TGF-β1 promotes fibrosis in the following ways:1)TGF-β1 directly induces ECM synthesis through Smad3 dependent way.2)TGF-β1 inhibits ECM degradation by inhibiting MMP and inducing MMP inhibitors such as metalloproteinase inhibitors.3)TGF-β1 plays a key role in the process of transforming into myofibroblasts through the endothelial cells of EMT,endmt and the peripheral cells of MMT.4)TGF-β1 acts directly on different types of renal cells,induces glomerular proliferation and eliminates the damage of Tec,podocyte and endothelial cells,which may lead to more serious renal damage and fibrosis.TGF-β1 is secreted initially in an inactive form and binds to receptors（TGF-βRIor TGF-βRII）to form dimers that activate Smad2 and Smad3.Smad2/3/4 forms a complex,translocates into the nucleus and initiates transcription that promotes fibrosis.TGF-β1 can directly induce the synthesis of extracellular matrix（ECM）by Smad3 dependent way.Smad family is a downstream factor of TGF-β1,and Smad3is activated in DKD patients and animal models of renal fibrosis.Smad3 is an important transmitter of TGF-β/Smads signal,regulating cell proliferation,differentiation and death^[6].In diabetic mice,Smad3 and collagen deposition were significantly reduced after TGF-β1 gene knockout compared with those of wild-type mice with renal damage.Further study of Smad3 Gene in knockout mice showed that collagen deposition was significantly lower than that in wild-type mice with kidney damage.It can be seen that Smad3 plays an important role in DKD renal fibrosis as a downstream factor of TGF-β1^[7].As a downstream factor of TGF-β,connective tissue growth factor（CTGF）can further mediate fibroblast activation and ECM formation,and become a target for the treatment of fibrosis.It has been proved that NF-κB and TGF-β1/Smad3signaling pathway work together to induce CTGF protein expression,promote the secretion of fibrogenic factors including type I collagen and FN,and play an important role in the process of liver fibrosis^[8-9].The mechanism of mir10 in the pathogenesis of DKD has not been reported in the literature.Therefore,the expression of mir10,TGF-β1,Smad3 and CTGF in rat mesangial cells（RMCS）cultured with high glucose was observed.Using cell transfection technology to regulate the expression of mir10,we observed whether mir10 can regulate TGF-β1/Smad3 pathway and participate in the development of RMCS fibrosis induced by high glucose,in order to find new molecular mechanism of DKD,and provide new ideas for the early diagnosis and treatment of DKD.Methods:RMCS were divided into normal glucose control group（NG,containing 5.5mmol/L glucose）,mannitol osmotic pressure control group（OC,containing 5.5mmol/L glucose and 24.5 mmol/L mannitol）and high glucose group（Hg,containing 30 mmol/L glucose）.After 24 hours of culture,the level of mir10 was detected by QRT PCR and Western blot The protein expression of TGF-β1,Smad3and CTGF was detected by blotting.Then RMCS were divided into normal glucose control group（NG）,normal glucose inhibition group（5.5mmol/l glucose+mir10 inhibitor in）,high glucose group（Hg containing 30mmol/L glucose）,high glucose inhibition group（30mmol/L glucose+mir10 inhibitor Hg+in）.Western blot was used to detect the protein expression of TGF-β1,Smad3 and CTGF.Results:1.After 24 hours of high glucose culture of RMCS,the content of mir10increased,and the level of fibrosis factors（TGF-β1,Smad3,CTGF）increased(P>The expression of TGF-β1,Smad3 and CTGF decreased when mir10 was inhibited in RMCS under high glucose condition.（all P<0.05）.Conclusions:1.High glucose could induce the increase of mir10 content and the synthesis of TGF-β1,Smad3 and CTGF in RMCS in vitro;2.There was no significant difference in the inhibition of mir10 on TGF-β1,Smad3 and CTGF in RMCS under normal glucose conditions;3.The inhibition of mir10 in RMCS under high glucose conditions could decrease the expression of TGF-β1,Smad3 and CTGF.It is speculated that mir10 can regulate TGF-β1/Smad3 signaling pathway and participate in DKD fibrosis in high glucose environment.