Study On The Regulatory Mechanism Of Smad7 On TGF-? Mediated EMT In Keloid Formation

Objective:Normal skin keratinocytes and keloid keratinocytes were cultured in vitro,and lentivirus was used to interfere with Smad7 gene and overexpression of Smad7 gene to regulate the epithelia-mesenchymal transformation,so as to elucidate the role and mechanism of keloid keratinocytes in obtaining anti-apoptotic characteristics,excessive proliferation and migrationMethods:1.Normal skin keratinocytes(NK cells),ear keloids and abdominal keloid keratinocytes(KK cells)were cultured from upper eyelid skin excision of healthy persons in the early stage of the department as experimental cells2.According to the purpose of Smad7 gene mRNA sequence,the gene synthesis Smad7 purpose,build Smad7 overexpressed lentivirus vector and Smad7 interference expression vector,build four possible interference through screening selection sequence Smad7shRNA slow virus vector and empty Smad7shRNA slow virus carrier,into e.coli DH5 alpha cells,transfection lentivirus packaging 293T cells.QPCR was used to detect the protein expression of Smad7 overexpressed lentivirus vector,to determine the effectiveness of Smad7 overexpression,and to screen the best interfering lentivirus3.The optimal interference and overexpression of lentivirus and Control vector were screened to infect NK and KK cells respectively.and the stable expression cell lines were screened with 1.5g/ml puromycin concentration.After the intervention,a total of 8 groups of cells were divided into the following groups:NK-control(NK cells in normal culture).NK-NC:(NK cells screened against lentivirus);NK-shSmad7(NK cells interfering with lentivirus screening);NK-mSmad7(NK cells expressing lentivirus screening);KK-control(KK cells in normal culture);KK-NC(KK cells screened against lentivirus);KK-shSmad7(KK cells that interfere with lentivirus screening);KK-mSmad7:(KK cells expressing lentivirus screening);The correlation between Smad7 and Smad3 was detected by Western-blot and QPCR.4.CCK-8 method was used to observe the difference of cell proliferation in 8 groups,the difference of cell apoptosis in 8 groups was detected by flow cytometry,and the difference of cell migration ability in 8 groups was detected by Transwell chamber.5.Western blot was used to detect the expression of key proteins(n-cadherin and Occludin)in epithelial-mesenchymal transition of NK cells and KK cells with stable expression and interference Samd7.Results:1.Primary KK and NK cells were successfully resuscitated,the Smad7 lentivirus-interfering vector was successfully transfected,and the Smad7 lenti virus-expressing vector was successfully transfected.Successfully established cut and increase expression of NK cells and KK Smad7,using QPCR detection Samd7 gene screening the best interference in KK cells and validate expression vector,slow virus expression vector transfection KK cell Samd7 genes increase(2694.706+900.541)times,slow virus carrier interference Samd7 gene transfection KK cells later(5.993+0.023)times,(P<0.01).2.QPCR and Western Blot were used to detect Samd7 and Samd3 expression in NK cells with stable expression and interference with Samd7 and in KK cells:it was suggested that Samd7 and Samd3 expression were negatively correlated.3.CCK-8 was used to detect cell proliferation.It was found that the stable expression of Smad7 could inhibit its proliferation in both NK cells and KK cells,while the stable interference with Smad7 could promote the proliferation of cells,and the trend of inhibition and promotion became more and more obvious with the extension of culture time.4.Transwell chamber was used to detect the differences in NK cells with stable expression and interference with Samd7 and the migration ability of KK cells:the number of migrating cells in each group of KK cells was higher than that in the NK group,which was consistent with the characteristic that keloid growth exceeded the original trauma range.The stable expression of Smad7 can inhibit the migration ability of NK cells and KK cells.Stable interference with Smad7 can enhance the migration ability of NK cells and KK cells.The inhibition and promotion trend was more obvious in KK cells.5.Flow cytometry was used to detect the differences in NK cells and KK cell apoptosis with stable expression and interference with Samd7.Overexpression of Smad7 in normal skin keratinocytes can promote cell apoptosis,while interference with Smad7 can slow cell apoptosis.In keloid keratinocytes,Smad7 can also be overexpressed to promote cell apoptosis,and its promoting efficiency is greater than that of normal skin.6.Western blot was used to detect the expression of NK cells with stable expression and interference with Samd7,as well as the expression of key proteins in the intermediate transformation of KK cells:Expression of the epithelial cell signature protein Occludin was significantly reduced in NK cells and KK cells after infection by shSmad7 lentivirus(P<0.01),The expression of N-cadherin,a marker protein in the stromal cells,was opposite to that in Occludin.Conclusion:By down-regulating Smad7 gene expression in keloid keratinocytes,TGF-?-mediated epithelial-mesenchymal transition was promoted and the cells exhibited more mesenchymal characteristics.The TGF-?-mediated epithelial-mesenchymal transition in keloid keratinocytes could be inhibited by Up-regulating the expression of Smad7 gene,thereby inhibiting its proliferative and migratory capacity and promoting apoptosis.