| BackgroundEsophageal cancer(EC)is one of the top ten malignant tumors in humans.China is the country that a highest incidence and mortality of esophageal cancer.Esophageal squamous cell carcinoma(ESCC)is predominant in China.With the improvement of socioeconomic status,lifestyle and medical level,the morbidity and mortality rate of esophageal cancer in China decreased year by year,but the 5-year survival rate of patients was still very low,and tumor invasion and metastasis is the major cause of recurrence and mortality in cancer patients.In recent years,the studies have found that epithelial-mesenchymal transition(EMT)not only plays an important role in the physiological processes such as embryonic development,wound healing,but also the biological processes in invasion and metastasis of epithelial cell-derived malignant tumor cells.Matrix metalloproteinase(MMPs),a class of endopeptidase family that is found to depend on metal calcium(Ca2 +)and zinc ion(Zn2 +).Its main function is to almost degrade the extracellular matrix of various protein components.Among them,MMP-9,also known as gelatinase B,as a key enzyme for degrading extracellular matrix and basement membrane,plays an important role in the tumor invasion and metastasis and interstitial angiogenesis.In addition to the ability of MMPs to remodel ECM,some of these molecules,such as MMP-3,MMP-9,MMP-14,and MMP-28 are well known to directly induce EMT or EMT-related processes.This has been confirmed in a variety of epithelial cells.Transforming growth factor(TGF)-β1 is one of the key factors to regulate EMT,which is widely involved in the development of human malignant tumor EMT.More importantly,studies have reported that MMP-9 is involved in TGF-β-induced EMT in glomerular endothelial cell.This suggests that MMPs play an important role in TGF-β-mediated EMT.Up to now,whether MMP-9 is involved in the EMT process of esophageal squamous cell carcinoma and whether MMP-9 is involved in TGF-β1-induced EMT progression of esophageal squamous cell carcinoma has not been reported.In order to solve these problems,this study will be studied from the following four parts.Part I Expression of MMP-9,Snail,TGF-β1 and EMT in esophageal squamous cell carcinoma tissues: the correlation and climical significance ObjectiveTo study the relationship of MMP-9,Snail,TGF-β1 and E-cadherin in esophageal squamous cell carcinoma and adjacent normal tissues and their relationship with clinicopathological parameters.Methods1.The expression of MMP-9,Snail,TGF-β1 and E-cadherin proteins was detected by immunohistochemistry in 77 cases of human ESCC tissues and corresponding normal esophageal mucosa tissues.2.To investigate the correlation of MMP-9,Snail,TGF-β1 and E-cadherin with clinicopathological features including gender,age,histological grade,invasion depth,lymph node metastasis and TNM staging were analyzed in ESCC tissues.Furthermore,the correlation of MMP-9 protein expression with Snail,TGF-β1 and E-cadherin were analyzed in ESCC tissues.Results1.The expression of MMP-9(74.03% vs 31.17%),Snail(68.83% vs 25.97%)and TGF-β1(71.43% vs36.36%)of ESCC obviously increased(P<0.01),and E-cadherin(44.16% vs 100.0%)level significantly decreased than normal esophageal epithelial(P<0.01).2.The expression of MMP-9 protein was positively associated with histological grade(I vs II vs III: 58.33% vs 77.27% vs 100.0%,P<0.05),tumor invasion depth(T1 vs T2 vs T3: 42.86% vs 65.0% vs 88.37%,P<0.01),lymph node metastasis(no vs yes: 67.21% vs 100.0%,P<0.01)and TNM staging(I vsII vsIII: 66.67% vs 67.39% vs 100.0%,P<0.05),but not correlated with gender,age and tumor histological grade(P>0.05).The expression of Snail protein was positively associated with tumor invasion depth(T1 vs T2 vs T3: 35.71% vs 75.00% vs 76.74%,P<0.01),lymph node metastasis(no vs yes: 60.66% vs 100.0%,P<0.01),but not correlated with gender,age,tumor histological grade and TNM staging(P>0.05).The expression of TGF-β1 protein was positively associated with lymph node metastasis(no vs yes: 65.57% vs 93.75%)and TNM staging(I vs II vs III: 46.7% vs 74.0% vs 87.5%)(P<0.05),but not correlated with gender,age,tumor histological grade,tumor invasion depth and tumor invasion depth(P>0.05).The expression of E-cadherin protein was negatively correlated with tumor invasion depth(T1 vs T2 vs T3:71.43% vs 55.0% vs 30.23%),lymph node metastasis(no vs yes: 50.82% vs 18.75%)(P<0.05),but not correlated with gender,age and tumor histological grade(P>0.05).3.In ESCC tissues,MMP-9 protein expression was positively correlated with Snail(γs=0.292,P<0.01);MMP-9 protein expression was negatively correlated with E-cadherin(γs=-0.295,P<0.01);Snail protein expression was negatively correlated with E-cadherin(γs=-0.241,P<0.01).MMP-9 protein expression was positively correlated with TGF-β1(γs=0.327,P<0.01);TGF-β1 protein expression was negatively correlated with E-cadherin(γs=-0.432,P<0.01).Part II The role of MMP-9 in esophageal squamous cell carcinoma EMT and its effect on invasion and migration ObjectiveTo investigate the effect of MMP-9 on epithelial-mesenchymal transition and invasion and migration of esophageal squamous cell carcinoma and its possible mechanism.Methods1.Effects of MMP-9 up-regulation on EMT and invasion and migration of esophageal squamous cell carcinoma: exogenous recombinant human MMP-9(rMMP-9)protein stimulated esophageal squamous cell carcinoma cell line EC-1 at different time(0 h,24 h,48h),the mRNA and protein levels of E-cadherin and Vimentin were detected by Real-time PCR,Western blot and Immunofluorescence,the morphological alterations of cells were observed under an inverted phase contrast microscope,and cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.2.The effect of MMP-9 down-regulation on EMT and invasion and migration of esophageal squamous cell carcinoma: MMP-9 shRNA was transfected into EC-1 cell line for 48 hours,the mRNA and protein levels of E-cadherin and Vimentin were detected by Real-time PCR,Western blot and Immunofluorescence,the morphological alterations of cells were observed under an inverted phase contrast microscope,and cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.3.After the expression of MMP-9 was up-regulated / down-regulated,the mRNA and protein levels of Snail was detected by Real-time PCR and Western blot.4.After EC-1 cells with Snail siRNA transfection were treated with rMMP-9,the mRNA and protein levels of E-cadherin and Vimentin were detected by Real-time PCR and Western blot,cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.Results1.Effects of up-regulation of MMP-9 on EMT and invasion and migration of esophageal squamous cell carcinoma: After EC-1 cells were treated with rMMP-9,the morphological changes of the cells were changed,from epithelial phenotype to mesenchymal cell phenotype;compared with blank control cells(0h),E-cadherin mRNA[after treatment 24 h :(0.62±0.07 vs 1.00±0.00)、after treatment 48h(0.48±0.09 vs 1.00±0.00),all P<0.01] and protein [after treatment 24 h :(0.51±0.0.07 vs 0.67±0.05),P<0.05 、 after treatment 48h(0.43±0.09 vs 0.67±0.05),P<0.01]were significantly downregulated,Vimentin mRNA[after treatment 24 h :(1.30±0.08 vs 1.00±0.00)、after treatment 48h:(1.58±0.09 vs 1.00±0.00),all P<0.01] and protein [after treatment 24 h :(0.66±0.07 vs 0.50±0.07),P<0.05 、 after treatment 48h:(0.77±0.06 vs 0.50±0.07),P<0.01] were significantly upregulated.Cell immunofluorescence results were consistent with the results of Western blot.cell invasiveness(113±12 vs 75±9)and motility(0.66±0.05 vs 0.43±0.07)were significantly enhanced(P<0.05).2.Effects of down-regulation of MMP-9 on EMT and invasion and migration of esophageal squamous cell carcinoma: after EC-1 cells were stably transfected with MMP-9 shRNA,compared with negative control cells and blank control cells,the morphological changes of the cells transfected with MMP-9 shRNA group were changed from long spindle or interstitial phenotype to epithelial cell phenotype.The cell adhesion was enhanced and the connection between cells and cells became close.MMP-9 mRNA(0.36±0.05 vs 0.92±0.07 vs 1.00±0.00)and protein(0.48±0.05 vs 0.64±0.06 vs 0.72±0.08)were significantly inhibited(P<0.01),E-cadherin mRNA(1.75±0.09 vs 0.90±0.07 vs 1.00±0.00)and protein(0.80±0.06 vs 0.57±0.05 vs 0.62±0.08)were significantly upregulated(P<0.01),Vimentin mRNA(0.70±0.08 vs 0.91±0.05 vs 1.00±0.00)and protein(0.48±0.03 vs 0.63±0.04 vs 0.68±0.07)were significantly downregulated(P<0.05).Cell immunofluorescence results were consistent with the results of Western blot.cell invasiveness(49±5 vs 78±4 vs 82±8)and motility(0.25±0.07 vs 0.43±0.08 vs 0.44±0.05)were significantly reduced(P<0.05).3.Effects of MMP-9 on the expression of Snail:After EC-1 cells were treated with rMMP-9,compared with blank control cells,Snail mRNA[after treatment 24 h :(1.55±0.08 vs 1.00±0.00)、after treatment 48h:(2.29±0.11 vs 1.00±0.00),all P<0.01] and protein [after treatment 24 h :(0.69±0.04 vs 0.56±0.08),P<0.05、after treatment 48h:(0.80±0.07 vs 0.56±0.08),P<0.01]were significantly upregulated.After EC-1 cells were stably transfected with MMP-9 shRNA,compared with negative control cells and blank control cells,Snail mRNA(0.65±0.08 vs 0.90±0.08 vs 1.00±0.00)and protein(0.54±0.08 vs 0.69±0.04 vs 0.72±0.06)were significantly downregulated(P<0.01).4.Effects of down-regulation of Snail on EMT and invasion and migration of esophageal squamous cell carcinoma: Compared with blank control cells,Snail mRNA(0.43±0.04 vs 1.00±0.00)and protein(0.31±0.05 vs 0.51±0.07)were significantly downregulated(P<0.01),E-cadherin mRNA(1.49±0.07 vs 1.00±0.00)and protein(0.82±0.06 vs 0.66±0.03)were significantly upregulated(P<0.01),Vimentin mRNA(0.56±0.08 vs1.00±0.00)and protein(0.37±0.05 vs 0.52±0.06)were significantly downregulated(P<0.01).cell invasiveness(42±8 vs 82±4)and motility(0.21±0.09 vs 0.41±0.05)were significantly reduced(P<0.05).5.Effects of down-regulation of Snail on MMP-9-mediated EMT and invasion and migration of esophageal squamous cell carcinoma: after EC-1 cells with Snail siRNA transfection were treated with rMMP-9,compared with rMMP-9 alone group cells,E-cadherin mRNA(0.72±0.06 vs 0.44±0.09)and protein(0.57±0.04 vs 0.45±0.05)were increased(P<0.01);Vimentin mRNA(0.73±0.09 vs 1.36±0.08)and protein(0.44±0.05 vs 0.80±0.04)were decreased(P<0.01).cell invasiveness(61±10 vs 121±9)and motility(0.34±0.06 vs 0.59±0.04)were reduced(P<0.01).Part III The effect of MMP-9 on TGF-β1-induced EMT and invasion and metastasis in human esophageal squamous cell carcinoma cells ObjectiveTo investigate the effect of MMP-9 on TGF-β1-induced EMT and invasion and migration in human esophageal squamous cell carcinoma cells.Methods1.After EC-1cells were treated with TGF-β1(10ng/ml)at different times(0 h,24 h,48 h),the morphological alterations of cells were observed under an inverted phase contrast microscope,the mRNA and protein levels of E-cadherin and Vimentin were detected by Real-time PCR,Western blot and Immunofluorescence,and cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.2.After EC-1cells were treated with GM6001,the morphological alterations of cells were observed under an inverted phase contrast microscope,the mRNA and protein levels of E-cadherin and Vimentin were detected by Real-time PCR,Western blot and Immunofluorescence.3.After the EC-1 cells were treated with TGF-β1,the mRNA and protein levels of MMP-9 was detected by Real-time PCR,Luciferase reporter assay,gelatin zymography and Western blot.4.After EC-1 cells with MMP-9 shRNA transfection were treated with TGF-β1,the mRNA and protein levels of E-cadherin and Vimentin were detected by Real-time PCR and Western blot,cell motility and invasiveness were detected by Matrigel invasion assay and scratch wound healing assay.Results1.TGF-β1 induced EMT in esophageal squamous cell carcinoma: After EC-1 cells were treated with rMMP-9,the morphological changes of the cells were changed,from epithelial phenotype to mesenchymal cell phenotype;compared with blank control cells,E-cadherin mRNA[after treatment 24 h :(0.65±0.04 vs 1.00±0.00)、after treatment 48h:(0.46±0.08vs1.00±0.00)] and protein [after treatment 24 h :(0.51±0.03 vs 0.67±0.06)、after treatment 48h(0.39±0.06 vs 0.67±0.06)were significantly downregulated(P<0.01),Vimentin mRNA[after treatment 24 h :(1.27±0.12 vs 1.00±0.00)、after treatment 48h(1.40±0.07 vs 1.00±0.00),all P<0.01] and protein [after treatment 24 h :(0.65±0.07 vs 0.53±0.06),P<0.05 、 after treatment 48h(0.73±0.06 vs 0.53±0.05),P<0.01] were significantly upregulated.Cell immunofluorescence results were consistent with the results of Western blot.2.TGF-β1-induced EMT is abrogated by MMP inhibitor GM6001:Following the treatment of cells with this inhibitor for 48 h,we observed that TGF-β1-induced EMT was abrogated.EC-1 cells generally maintained endothelial cobblestone shape,but some cells showed fibroblastic spindle-shaped morphology.Real-time PCR and western blot analysis showed that GM6001 inhibited the TGF-β1-induced decrease in E-cadherin expression [mRNA:(0.62±0.04 vs 0.44±0.07),P<0.05;protein:(0.55±0.03 vs 0.44±0.06),P<0.05]and increase in vimentin expression [mRNA: 1.22±0.03 vs 1.42±0.07,P<0.05;protein:(0.71±0.03 vs 0.82±0.05),P<0.01] compared with those in the TGF-β1-treated cells.Cell immunofluorescence results were consistent with the results of Western blot.3.TGF-β1 increase the expression and activity of MMP-9 in ESCC: Gelatin zymography showed that MMP-9 activity is significantly increased following the treatment with TGF-β1.However,after the cells were treated with GM6001,MMP-9 activity was shown to be reduced.After the EC-1 cells were treated with TGF-β1,compared with the control group(0h),the expression of MMP-9 mRNA [24h group(1.25 ± 0.10 vs 1.00 ± 0.00),48 h group(1.42 ± 0.07 vs 1.00 ± 0.00)] was significantly increased in a time-dependent manner(P<0.01).The results of luciferase activity showed that there was no significant difference in the expression of MMP-9 between the two groups in the experiment of transfection of internal plasmid pGL3-Basic(P> 0.05).The activity of MMP-9(17.26 ± 3.69 vs 36.06 ± 5.27)in the TGF-β1 stimulated group was significantly higher than that in the blank group(P<0.05).Compared with the control group(0h),the expression of MMP-9 protein [24h group(0.64±0.08 vs 0.52±0.04),P<0.05 48 h group(0.77±0.08 vs 0.52±0.04),P<0.01] was significantly increased in a time-dependent manner.4.MMP-9 expression knockdown attenuates TGF-β1-induced EMT and inhibits cell invasiveness and migration: After EC-1 cells with MMP-9 shRNA transfection were treated with TGF-β1,compared with blank control cells,MMP-9 mRNA(0.42±0.05 vs 1.00±0.00)and protein(0.31±0.04 vs 0.57±0.06)were significantly downregulated(P<0.01).E-cadherin mRNA(1.53±0.08 vs 1.00±0.00)and protein(0.80±0.05 vs 0.67±0.06)were significantly upregulated(P<0.01),Vimentin mRNA(0.57±0.08 vs 1.00±0.00)and protein(0.37±0.05 vs 0.53±0.08)were significantly downregulated(P<0.01).compared with TGF-β1 group cells,E-cadherin mRNA(0.71±0.06 vs 0.46±0.08)and protein(0.58±0.05 vs 0.45±0.06)were increased(P<0.05);Vimentin mRNA(0.70±0.04 vs 1.40±0.07)and protein(0.42±0.04 vs 0.82±0.03)were decreased(P<0.01).cell invasiveness(58±6 vs 119±7)and motility(0.36±0.04 vs 0.64±0.06)were reduced(P<0.01).Part IV The effect of MMP-9 blockade on cell proliferation and EMT of xenograft tumors with esophageal squamous cell carcinoma in nude mice ObjectiveTo study the effect of MMP-9 shRNA on EMT of esophageal squamous cell carcinoma in vivo.Methods1.ESCC xenograft tumor model was established,EC-1 cells were stably transfected with MMP-9 shRNA,EC-1 cells were stably transfected with antisense shRNA,and blank control EC-1 cells were injected right fore axillary subcutaneous into nude mice.The tumor volume was measured and the growth curve was generated.2.After the experiment,the nude mice were removed,the tumor was removed,and the volume and weight were measured.3.HE staining was used to observe the histological morphology of xenograft tumors.Real-time,PCR and immunohistochemistry were used to detect the expression of MMP-9,Snail,E-cadherin,Vimentin,mRNA and protein in different groups of transplanted tumors.Results1.The xenograft tumors model of ESCC in the nude mouse was established successfully.2.At the end of experiment,compared to negative control group and blank control group,the average volume(624.69±136.85 vs 1118.19±254.85 vs 1280.74±371.78)and weight(g)(0.58±0.13 vs 0.94±0.17 vs 1.04±0.20)of MMP-9 shRNA group xenograft tumors were significantly reduced(P<0.05).3.Compared to negative control group and blank control group,the expression of MMP-9 mRNA(0.48±0.06 vs 0.92±0.06 vs 1.00±0.00)and protein(positive cell numbers/200 cells)(82±7 vs 114±6 vs 125±9)were significantly inhibited(P<0.01);the expression of Snail mRNA(0.60±0.10 vs 0.96±0.05 vs 1.00±0.00,P<0.05)and protein(positive cell numbers/200 cells)(87±8 vs 124±4 vs 132±10,P<0.01)were significantly downregulated in EC-1 cells stably transfected with MMP-9 shRNA,and moreover,E-cadherin mRNA(1.48±0.08 vs 0.94±0.08 vs 1.00±0.00,P<0.01)and protein(134±12 vs 91±32 vs 87±12,P<0.05)were significantly upregulated,Vimentin mRNA(0.70±0.06 vs 0.95±0.03 vs 1.00±0.00,P<0.01)and protein(68±6 vs 102±44 vs 122±8,P<0.05)were significantly downregulated.Conclusions1.The high expression of MMP-9 in ESCC tissues is positively correlated with Snail and TGF-β1,and negatively correlated with E-cadherin,suggesting that three of them have synergistic effects in the EMT process of esophageal squamous cell carcinoma.2.MMP-9 is involved in EMT of esophageal squamous cell carcinoma.MMP-9 can promote the invasion and migration of esophageal squamous cell carcinoma through EMT pathway.3.MMP-9 affects the EMT process and invasion and metastasis of esophageal squamous cell carcinoma by regulating Snail.4.MMP-9-mediated EMT of esophageal squamous cell carcinoma was regulated by TGF-β1.5.MMP-9 shRNA can effectively inhibit the activity of MMP-9,can down regulate the expression of Snail and inhibit the occurrence of EMT in nude mice with squamous cell carcinoma of esophagus. |
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