| Part I Study on the role of mi R-155 in regulating TGF-β1-induced podocyte injuryObjective:To investigate the changes of expression and stability of mi R-155 after mi R-155 mimics and mi R-155 inihibitor transfected into podocytes as well as the effects of mi R-155 expression changes on podocyte marker protein in the process of TGF-β1-induced podocyte injuryMethods:（1）The conditionally immortalized mouse podocytes line MPC5 was cultured in vitro,and the podocyte differentiation and maturation was identified by cell immunofluorescence staining;（2）Podocytes were treated with 12ng/m L TGF-β1 for 72h to construct a podocyte injury model,and RT-q PCR and Western blot were used to detect the expression of mi R-155,CD2AP and synaptopodin,respectively.（3）Immunofluorescence microscopy was used to observe the fluorescence effect after transfection of different concentrations of Cy3 mi R-155 NC at different doses of Lipofectamine TM 2000 for 24 hours,and then RT-q PCR was used to detect the mi R-155 expression after transfection of mi R-155mimics and inhibitor at different concentrations and different times.（4）The cells were randomly divided into tnormal control group,TGF-β1 group,mimics+TGF-β1 group,mimics NC+TGF-β1 group,inhibitor+TGF-β1 group and inhibitor NC+TGF-β1 group.The expression of mi R-155 was detected by RT-q PCR,podocin,CD2AP and synaptopodin were detected by RT-q PCR and Western blot,and the fluorescence expression and distribution of CD2AP were observed by immunofluorescence staining.Results:The immunofluorescence of CD2AP and synaptopodin showed a characteristic distribution of podocyte differentiation and maturation.After TGF-β1 intervention,the expression of mi R-155 increased and the expression of CD2AP,synaptopodin protein decreased（P<0.01）.A certain dose of Lipofectamine TM2000 was sufficient to saturate mi RNA at different concentrations,which could successfully transferred different concentrations of Cy3 mi R-155 NC into podocytes,and the transfection efficiency was more than 80%.The results of RT-q PCR showed that after transfection of mi R-155 mimics and mi R-155 inhibitor for 24 hours,the difference was statistically significant compared with the negative control group（NC）.The expression of mi R-155 in podocytes increased significantly（P<0.01）when the concentration of mi R-155 was 80 nmol/m L,and decreased significantly（P<0.01）when the concentration of mi R-155 inhibitor was 150 nmol/m L（P<0.01）.After mi R-155 mimics or mi R-155 inihibitor transfecting podocytes interfered by TGF-β1 for 48 and 72 hours,mi R-155expression significantly increased or decreased（P<0.01）.The expression of mi R-155increased in the mi R-155 mimics+TGF-β1 group while comparing with those in the TGF-β1group and NC+TGF-β1 group（P<0.01）,but the m RNA and protein expressions of podocin,CD2AP,and synaptopodin decreased（P<0.01）.However,the effect of mi R-155 inihibitor transfected on podocytes was opposite to mi R-155 mimics.The immunofluorescence results showed that the expression of CD2AP protein was consistent with the results of Western blot and RT-PCR,and its distribution also changed with the expression of mi R-155.Conclusion:mi R-155 can participate in the regulation of podocyte injury induced by TGF-β1,and its specific mechanism needs to be further exploredPart II Mi R-155 regulates TGF-β1-induced podocyte injury throughα3β1/FAK/Rho A/ROCK pathwayObjective: To study the role of integrin α3β1/ FAK/ Rho A/ ROCK cytoskeletal protein signaling pathway in TGF-β1 injured podocytes;to investigate whether Mi R-155 regulates TGF-β1-induced podocyte cytoskeletal protein,slit diaphragm protein injury,and the effects on TGF-β1 induced podocyte migration and F-actin cytoskeletal disorder through the integrinα3β1/ FAK/ Rho A/ ROCK cytoskeletal protein pathway.Methods:（1）Western blot was used to detect the time effect of activation of FAK /Rho A / ROCK pathway at different time points after podocyte stimulation by 12 ng/m L of TGF-β1;（2）Cells were randomly divided into normal control group,TGF-β1 treatment group,FAK inhibitor+TGF-β1 group and Rho A inhibitor C3 + TGF-β1 group,and Western blot was used to detect the effects of TGF-β1,FAK and Rho A inhibitors on the α3β1/ FAK/ Rho A/ROCK cytoskeletal protein pathway.（3）The interaction between β1 and FAK、β1 and Rho A was detected by immunoprecipitation assay.（4）The effect of Rho A inhibitor on mi R-155 expression induced by TGF-β1 was detected by RT-q PCR;（5）The cells were randomly divided into the normal control group,TGF-β1 group,mimics+TGF-β1 group,mimics NC+TGF-β1 group,inhibitor+TGF-β1 group,inhibitor NC+TGF-β1 group and C3 + TGF-β1group.RT-q PCR,Western blot,scratch test,Transwell test and cytoskeleton immunofluorescence staining were used to detect the expression of mi R-155 and the activation of α 3 β 1 / FAK / Rho A / ROCK pathway induced by TGF-β 1 by Rho A inhibitor C3,and proteinα-actinin-4,nephrin damage and effects of podocyte migration and F-actin cytoskeletal disorders were marked.Results:（1）Compared with 0h,the phosphorylation levels of FAK and ROCK1activity-related proteins MYPT began to increase after 30 minutes of TGF-β1 stimulation,and reached a higher level at 12 hours,and Rho A activity also increased significantly after 30 min of TGF-β1 stimulation,and it increased the most significantly after 1 hour of TGF-β1stimulation,and the difference was statistically significant（P<0.01）.（2）After inhibiting FAK y397 phosphate site or Rho A GTP,TGF-β1 activation of α3β1/ FAK/Rho A/ROCK cytoskeletal protein pathway was blocked,so the expression levels of p-FAK,active Rho A and ROCK significantly decreased,the expression levels ofβ1 increased,and the difference was statistically significant（P<0.01）.（3）Immunoprecipitation showed that TGF-β1 enhanced the interaction between β1 and p-fak,β1 and active Rho A;（4）The expression level of mi R-155 in the C3TGF-β1 group decreased significantly compared with that in the C3+TGF-1 group,and the difference was statistically significant（P<0.01）;（5）The over expression of mi R-155 could further enhance the activity of α3β1/ FAK/Rho A/ROCK induced by TGF-β1,and then triggered the decrease ofα-actinin-4 and nephrin expression,the increase of podocyte mobility and the obvious disorder of F-actin cytoskeleton;Rho A inhibitors,similar to mi R-155 inhibitor,not only inhibited Rho A signal transduction,but also down-regulated the expression of mi R-155.At the same time,they prevented TGF-β1 from activating α3β1/ FAK/Rho A/ROCK pathway,reversed TGF-β1-inducedα-actin-4 and nephrin expressions,podocyte migration and F-actin cytoskeleton disorder;all differences were statistically significant（P<0.01）.Conclusion:（1）TGF-β1 induces the activation of integrin α3β1/ FAK/ RhoA/ ROCK cytoskeleton pathway,which leads to the damage of podocyte marker protein,cell migration and cytoskeleton disorder,and there is a synergistic effect between signal pathway molecules;（2）Rho A signal pathway transduction is closely related to mi R-155 expression,mi R-155 can regulate TGF-β1 induced podocyte injury through the α3β1/ FAK/Rho A/ROCK pathway. |
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