| Objective: To investigate the effect of Bortezomib on renal tubular epithelial-mesenchymal transition(EMT),and to determine whether Smad ubiquitin regulatory factor 2(Smurf2)expression induced by tumor necrosis factor-α(TNF-α)can be reversed by Bortezomib in human renal tubular epithelial cells(HK-2).Methods: The HK-2 cells were divided into control group(cultured with DMEM/F12),TNF-α group(cultured with TNF-α),Bortezomib group(cultured with Bortezomib)and experimental group(treated with Bortezomib and TNF-α together).Each group of cells was treated with the corresponding drug and cells were collected.Real-time PCR(RT-PCR)and Western blot were used to detect the mRNA and protein expression of fibronectin(FN),Smurf2,E-cadherin,and α-smooth muscle actin(α-SMA)respectively;observing the cell morphology changes in each group under inverted microscope;using cell scratch test and invasion experiment to observe the changes of cell migration and invasive ability.In addition,we also stimulated HK-2 cells with TNF-α for 0-24 h,then detected the phosphorylation of key mediators in different intracellular signaling pathways by Western blot to confirm the signaling pathway that HK-2 cells can be activated by TNF-α.Then,HK-2 cells were pretreated with the inhibitors of above-mentioned pathways,and then stimulated with Bortezomib and/or TNF-α for 24 to 48 h to investigate the molecular mechanisms involved in the intervention of Bortezomib.Results: 1.Compared with the control group,the expression of FN in the TNF-α group was significantly higher in mRNA and protein level(P<0.05).Compared with the TNF-α group,bortezomib inhibited the expression of FN induced by TNF-α(P <0.05);bortezomib-treated group compared with the control group,the change of FN was not statistically significant(P> 0.05).2.Compared with the control group,E-cadherin was significantly decreased in HK-2 after treatment with TNF-α,and α-SMA was significantly increased(P<0.05);compared with TNF-α group,co-stimulation with bortezomib reversed the expression of E-cadherin and α-SMA induced by TNF-α(P<0.05),but the expression of E-cadherin and α-SMA did not change significantly in the bortezomib treated group compared with the control group(P>0.05).3.Compared with the TNF-α-treated group,the morphology of HK-2 was still cobblestone-like after intervention with bortezomib;however,the cell morphology did not change significantly in the bortezomib-treated group compared with the control group.4.Compared with the control group,TNF-α treatment could increase the migration and invasive ability of HK-2 cells(P< 0.05).Compared with TNF-a group,the changes could be reversed after adding Bortezomib(P<0.05).Compared with the control group,there was no significant change in cell migration and invasive ability in the Bortezomib treatment group(P>0.05).5.Compared with the control group,TNF-α can increase the expression of Smurf2 in mRNA and protein level(P<0.05);bortezomib can inhibit the increase of Smurf2 induced by TNF-α(P<0.05);Compared with the control group,the expression of Smurf2 did not change significantly in the bortezomib-treated group(P>0.05).6.Compared with the control group,TNF-α could activate the Akt/mTOR/p70S6 k pathway and increase the phosphorylation levels(P<0.05);co-stimulation with Bortezomib could inhibit the phosphorylation of pathway activated by TNF-α(P<0.05).However,there was no significant change in the pathway between Bortezomib group and control group.Compared with TNF-α group,the addition of Bortezomib or MK2206 both inhibited the expression of pathway phosphorylation,FN,E-cadherin,α-SMA and Smurf2 induced by TNF-α(P<0.05),and the difference between the two interventions was not statistically significant(P>0.05);however,there was no significant difference in the results between the MK2206 group and the control group.Conclusion: Bortezomib could partially reverse expression of Smurf2 induced by TNF-α by inhibiting the Akt/mTOR/p70S6 k pathway,thereby partially reversing renal tubule EMT in HK-2 cells induced by TNF-α. |
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