The Effect Of Smad7sirna On Generation And Invasion In Escc Cell Line Ec-1

Esophageal cancer is one of the most common cancers throughout the world and it is a highly aggressive neoplasm.Smad7 belongs inhibitory Smads,which counteract TGF-β1/Smad pathway through binding to transforming growth factor-βreceptor.TGF-β/Smad signaling pathway is initiated by their association with a type-Ⅱreceptor serine-threonine kinase,which then recruits and transphosphorylates a type-receptor,also a transmembrane serine-threonine kinase.Trans- phosphorylation of the typeⅠreceptor by the typeⅡreceptor takes place on several residues in a glycine-serine-rich juxtamembrane domain and leads to activation of the receptor.This activated typeⅠreceptor phosphorylates two C-terminal serine residues of Smad 2 or Smad3,inducing complex formation with Smad4 and then translocation into the nucleus.There the complexes interact with other transcription factors to induce or repress transcription of a number of target genes.Smad7 is located in chromatin 18q21.1,between MADR2 and DPC4.MADR2 and DPC4 code Smad2 protein and Smad4 protein.The abnormal expression of Smad7 effect the reaction of cell with TGF-βand promote the cell malignant change. Expressions of Smad7 were elevated in cancer of colon,pancreatic carcinoma, endometrial cancer,mammary adenocarcinoma etc.Now,We have seen little report about the expressions and function of Smad7 in ESCC.In this study,we will detect the expression of Smad7,TSRⅡand Smad3 in ESCC tissue and relationship of the expression of Smad7 with TβRⅡand Smad3.we will survey the levels of Smad7,TβRⅡand Smad3 with RT-PCR and Western-blot after the transfection of Smad7 siRNA in ESCC cell line EC-1.Inverted microscope, MTT and Boyden chamber methods will be used to detect cell morphous, proliferation ability and migration ability.Materials and methods1.Expressions of Smad7,TβRⅡand Smad3 proteins were detected in 100 cases of surgically resected by the method of immunohistochemistry,which including carcinoma tissues and corresponding normal tissues.2.Cell morphological changes were observed in EC-1 cells which was treated with Smad7siRNA.3.RT-PCR method and Western-blot analysis were adopted to examine the expression of Smad7,TβRⅡ,Smad3 mRNA and protein in EC-1 cells which was treated with Smad7 siRNA in 24h,48h,72h.4.MTT method was adopted to investigate the proliferation of the EC-1 cells in 24h,48h,72h after the transfection of Smad7 siRNA.5.Boyden chamber experiment in vitro was used to detect the invasion ability of the EC-1 cells in 24h,48h,72h after the transfection of Smad7 siRNA.6.Statistical analysis:All the dates were analyzed by SPSS 13.0 statistical package, the count information calculated the positive rate,enumeration date are expressed by standard deviation((?)±s).The comparison of positive rates uses the Chi-square,the mean of two groups uses the t-test.The mean of more groups use the ANVOA.The relation of two variable groups is analyzed by the Kendall correlation analysis.The level of significant difference is a=0.05.Results1.The positive rate of expressions of Smad7 protein were higher significantly in carcinoma tissues 80.0%than that in normal tissues 58.0%(P＜0.01).The positive rate of expressions of Smad7 in deeper invasion group 89.1%were significantly higher than that in superficial invasion group 63.9%(P＜0.01).The positive rate of expressions of Smad7 in group with lymph node 100.0%were significantly higher than that in group without lymph node 74.4%(P＜0.05).2.The positive rate of expressions of TβRⅡprotein were lower significantly in carcinoma tissues 42.0%than that in normal tissues 82.0%(P＜0.01).The positive rate of expressions of 1βRⅡin deeper invasion group 37.5%were significantly lower than that in superficial invasion group 50.0%(P＜0.05).The expression of TβRⅡis related to grade.The positive rate of expressions in gradeⅢ(18.2%) were significantly lower than that in gradeⅠ(60.0%)(P＜0.05).The positive rate of expressions of TβRⅡin group with lymph node 18.2%were significantly lower than that in group without lymph node 48.7%(P＜0.05).3.The positive rate of expressions of Smad3 protein were lower significantly in carcinoma tissues 68.0%than that in normal tissues 87.0%(P＜0.01).The positive rate of expressions of Smad3 in group with lymph node 27.3%were significantly lower than that in group without lymph node 79.5%(P＜0.05).4.There were negative correlations between the positive rate of Smad7 protein expression and that of TβRⅡ,Smad3 protein expression in cancer tissues(P＜0.05). positive correlations between the positive rate of TβRⅡprotein expression and that of Smad3 protein expression in cancer tissues(P＜0.01).5.After transfection of Smad7siRNA,transfected cells display distinct biological behaviour,such as shape becoming shrink and round,and more grains.But control group cells were fusiform shape,adherence growth,and had clear layout among cells.6.RT-PCR results showed that the expressions of Smad7 mRNA in Smad7siRNA group were obviously lower than that in control group in 24h,48h,72h(P＜0.05).The expression of Smad7 gene mRNA decreased with different time in Smad7siRNA group(P＜0.05).After transfection,the expression of TβRⅡ,Smad3 gene mRNA increased with different time(P＜0.05).7.Western-blot results showed that the level of Smad7 protein was declined by Smad7 siRNA with different time(P＜0.05).Meanwile,The results in Western-blot indicate that the expression of TβRⅡ,Smad3 protein was increased with different time(P＜0.05).8.MTT results:Compared with control groups,the ability of EC-1 cell growth was decreased obviously in Smad7siRNA group in 24h,48h,72h(P＜0.05).9.Boyden chamber results:Compared with control groups,the number of EC-1 cells traversed Matrigel was decreased obviously in Smad7siRNA group in 24h,48h,72h(P＜0.05).Conclusions1.The expressions of Smad7 were increased and expression of TβRⅡ,Smad3 was decreased in ESCC tissue.Abnormal expression of the Smad7,TβRⅡand Smad3 gene have the close relation to carcinogenesis of ESCC.2.Smad7siRNA has significant inhibition effects on the expression of Smad7 mRNA and protein.The inhibition effects were specific and time-depended.It offer a new strategy and technique for gene therapy of Smad7 in ESCC.3.Smad7siRNA could inhibited the proliferation and invasion of EC-1 cells.The result showed that Smad7 in ESCC could take active effect in preventing the malignant progress of ESCC,the Smad7 could be an adjunctive therapy target to the ESCC.4.There were negative correlations between the positive rate of Smad7 protein expressions and that TβRⅡ,Smad3 protein expression in ESCC tissue,positive correlations between the positive rate of TβRⅡprotein expression and that of Smad3 protein expression in cancer tissues.The expression of TβRⅡ,Smad3 protein was increased after transfection of Smad7siRNA.Smad7 may be induce the cell to appear the counteract of TGF-β.