The Role Of S100A4 In The Infiltration, Metastasis And Its Influence To The Growth Of Esophageal Squamous Cell Carcinoma

The incidence and fatality of Malignant tumors increase year by year,which have seriously threaten human health.The invasion and the metastasis are common bionomics of the malignant tumor which play an important role in the treatment and the prognosis of patients.They are the hot topics that scholars are studying nowadays and the nodus of tumor clinical treatment,esophageal carcinoma is one of the most common malignant tumors in our country especially in Henan Province.Therefore,it is significant to have a discussion about the mechanism of invasion and metastasis of esophageal carcinoma.The process of invasion and metastasis of tumor is a complex course which involves many complex mechanisms,including the adherency,movement,invasion of tumor cells and the angiogenesis.S100A4 belongs to the family of calcium ion bond protein which is an active tumor metastasis regulatory factor secreted by the tumor cell and the tumor interstitial cells.It has been confirmed that there are S100A4 high expression in many kinds of malignant tumors.S100A4 not only exists in the intracellular in the form of non-covalence same type dimer,but also is secreted to the extracellular and exists in the intercellular substance in the form of covalence dimer with extracellular function.The S100A4 existing in the intercellular substance is the foundation of the signal conduction between cells,the mutual relation and the interaction between the cell and the cell matrix.In addition,it is also the pathological foundation of the invasion and metastasis of tumor cells.Thus,S100A4 which has been classified as the metastasis related protein is an very important biomarker to predict the prognsis of the tumors.Nowadays,it is believed that S100A4 protein united with many kinds of target proteins can exert its intracellular and extracellular function to regulate the progress of cell cycle,change the cell adhesivity and the movement ability,increase the survival ability of tumor cells,and can adjust the ability of infiltration and metastasis of tumor cells.Suppressing the expression of the S100A4 protein with siRNA or the antisense oligonucleotides technology can drop the locomotor activity of tumor cells about 2 times.In addition,the S100A4 can influence the expression and activity of matrix metalloproteinase and the inhibitor of matrix metalloproteinase,destroys the integrity of basilar membrane and causes metastasis of tumor cells.E-cadherin is important in keep the adhesion between the normal cells and inhibition of the invasion.Its functional incapacitation is critial to the invasion of tumor.E-cadherin as anti-metastasis gene is well-known.It was reported that there was negative correlation between the expression of S100A4 and E-cadherin in non-minicelllung cancer and uterine cervix cancer.This provided a new way to prevent the metastasis of tumor through suppressing the expression of the S100A4.It has been confirmed by the foreign scholars that there are over expression of S100A4 in the esophageal squamous cell carcinoma(ESCC) detected by immunohistochemical method which concerned with the infiltration and metastasis of tumor,and these studies suggest that patients with the over expression of S100A4 protein have bad prognosis.But it is unclear the role of S100A4 in the infiltration and metastasis,whether which concerned with the expression of MMP-2 and E-cadherin? We well study this in the following reasearch.In our research,we have studied the molecular mechanism further and the role of S100A4 in the process of metastasis of esophageal scale cancer:1.We applied the immunohistochemistry technology, Western Blotting and the RT-PCR methods to examine the expression of S100A4, MMP-2 and the E-cadherin simultaneously and analyzed their relevance in ESCC organization and the ESCC cell lines EC-1,Eca109,EC9706,TE-1.The Boyden chamber was used to observe the invasiveness for 4 kinds of ESCC cell EC-1,Eca109, EC9706,TE-1 and their relation with S100A4 in vitro.2.Using RNAi technology to silence the S100A4 gene in the ESCC cell and observing the influence to invasiveness of the ESCC cell and its regulative function to MMP-2,and the expression of E-cadherin in vitro.MTT methods was used to observe the influence of S100A4siRNA to the multiplication of ESCC cells.3.By constructing of the athymic mouse model,we observed the growth of transplantation tumor with transfection cells..This experiment was divided into three parts as follows.Part one The expression of S100A4 in ESCC,its relationship with the expression of MMP-2 and E-cadherin and the relationship with the potential invassiveness of cell line of ESCCMethods1.Immunohistochemical method was used to detect the expression of S100A4 protein, MMP-2 protein and E-cadherin protein in the tissues of ESCC and the relatively normal tissue besides the tumor in 100 samples.Analyzing the relationship between the expression of S100A4 protein and MMP-2 as well as the E-cadherin protein.2.Western Blotting and immunocytochemical methods were applied to examine the expression of S100A4 protein in 4 kinds of ESCC cell(EC-1,EC9706,Eca109,TE-1). RT-PCR technology was used to examine the expression of S100A4 mRNA in 4 kinds of ESCC cell.3.the Boyden cab was used to determine the ability of invasion of 4 kind of ESCC cells(EC-1,EC9706,Eca109,the TE-1) in vitro,examining the relationship between the expression of S100A4 and them.4.statistical treatment:SPSS13.0 software was used,positive ratio of the enumeration data was statisticed with x~2 tests(chi-square);The measurement data was manifested with X±S;The comparison between two groups of mean values with T-test(t-test) and multi-group mean values with variance analysis(ANVOA);The relevant analysis used the Pearson's related analytic method.size of testα=0.05.Results1.The results of S100A4,MMP-2 and E-cadherin protein expression in relation to clinicopathological parameters in esophageal squamous cell carcinoma1.1.The expression of S100A4 protein in esophageal squamous cell carcinomaS100A4 is cytoblaslema coloration in the ESCC cells,but it is coloured in cytoblaslema and nucleus in epithelium mucosae of esophagus,almost all the positive cells scatter in the tissues of ESCC,only a few assemble in some positions.Vascular smooth muscle cells and endothelial cells are also found to be coloured in interstitial substance,and lymphocyte and fibrocellular was coloured more strongly in cytoblaslema.The positive rate is 52.00%in the ESCC which is obviously higher than the expression rate 26.00%(P=0.001) in the esophagus normal mucous membrane organization.There is significant difference between two groups.The positive rate of S100A4 in the high,middle and low differentiation ESCC is 36.00%,51.56%, 90.91%,respectively and there is diffference,too(P=0.04);In the ESCC infiltrates to in-depth(deep myo-level and external coat periplast),the positive rate is 59.38% which is obviously higher than 38.89%(P=0.049) in the shallow layer.(mucous membrane and shallow myo-level).The positive rate of the expression of S100A4 protein(69.50%) in the lymph node is higher than that in the non-lymph node (40.00%),and the difference has statistics significance(P=0.004).1.2.The expression of MMP-2 protein in esophageal squamous cell carcinoma.The positive expression of MMP-2 in cancer cell mainly locates in cytoplast and partially in mesenchymal.The positive rate in the ESCC organization is 67.00% which is obviously higher than the rate in normal mucous membrane organization of esophagus which is 31%(P=0.001).There is significant difference among groups different differentiated degree(P=0.027).The positive rate of the expression of MMP-2 protein in high,middle and poorly differentiation ESCC is 48.00%,70.31%, and 90.91%,respectively.The difference of the positive rate of the expression of MMP-2 protein in the in-depth ESCC 78.13%and 47.22%in the shallow layer ESCC is significant which has remarkable statistics significance(P=0.002).The positive rate of the expression of MMP-2 protein(86.96%) in the metabasis lymph node is higher than 47.00%in the non- metabasis lymph node group and there were the statistics significance(P=0.020).1.3.The expression of E-cadherin protein in esophageal squamous cell carcinoma.E-cadherin located in the cell membrane in the normal mucous membrane organization detected by immunocytochemical.However,it located in the cytoplast in cancer organization.The expression of E-cadherin in the cancer organization(44.00%) is obviously lower than its level(86.00%) in the normal tissue.The difference of the expression has statistics significance(P=0.001).The positive rate of the expression of E-cadherin protein in the high,middle and poorly differentiated ESCC was 64.00%, 40.63%and 18.18%(P=0.026),respectively.The E-cadherin protein expression rate reduces gradually with the increasing invasion depth of the cancer cell.The positive rate of its expression was 39.06%in the ESCC invaded deeply was lower than the rate for the invaded shallowly which is 52.78%,but there is no statistics significance (P=0.185).The positive rate of E-cadherin protein(26.87%) with the lymph node metabasis is lower than the rate(49.35%) withnot and the difference has statistics significance(P=0.049).1.4.The relevant analysis about the expression of S100A4 proteins,MMP-2 protein and E-cadherin proteinIn the ESCC organizations,the correlation coefficient between the expression of S100A4 protein and the MMP-2 protein is 0.262(P=0.009).The correlation coefficient between the expression of S100A4 protein and the E-cadherin protein is-0.237(P=0.018);The correlation coefficient between the expression of MMP-2 protein and the E-cadherin protein is -0.363(P=0.001).The S100A4 protein and the MMP-2 protein are connected positively obviously.The S100A4 protein and the MMP-2 protein assume the inverse correlation relations with the E-cadherin protein.2.The expression of S100A4 in 4 kind of esophageal carcinoma cell lines(EC-1, Eca109,EC9706,TE-1) 2.1.Expression of S100A4 proteins in four ESCC cell linesThe results of immunocytochemical method shows that there is expression of the S100A4 protein in 4 cell lines,the positive expression mainly locates in the cytoplasma with faint yellow and the yellowish brown color,and partially concerned with the nucleus coloration in the Eca109 cell line.The image analysis applied by the HPIAS-1000 high clear imagery processing system shows that the average light density(the OD value) is EC-1(0.358±0.016),Eca109(0.326±0.025), EC9706(0.310±0.019),TE-1(0.285±0.017).Comparion of four groups there were significant difference(F=17.589,P=0.001).The comparison between Eca109 and EC9706 show that there is no statistics significance(P=0.134),the other comparisons between two groups has significant difference(P＜0.05).The results of the Western Blotting indicated that,there were the expression of S100A4 protein in 4 kinds of esophageal carcinoma cell lines.The expression of S100A4 protein in EC-1 is(0.897±0.053) which is higher than(0.806±0.058) in Eca109,(0.720±0.068) in EC9706,and(0.645±0.089) in TE-1.there was significant difference(P=0.001).The comparison between EC9706 and TE-1 shows that there is no statistics significance(P=0.073),the other comparisons between two groups has significant difference(P＜0.05).2.2.The expression of S100A4 mRNA in four ESCC cell lines.RT-PCR results showed that there were the goal bandings of S100A4 mRNA in 4 kinds of cell lines.The semi-quantitative analysis showed that the relative expression quantity of S100A4 mRNA compared with EC-1(0.894±0.021),Eca109(0.890±0.022), EC9706(0.842±0.028),TE-1(0.812±0.040).There was significant difference (P=0.001),The comparison between EC1 and Eca109 show that there is non-statistics significance(P＞0.05),the other comparisons between two groups has significant difference(P＜0.05).3.The examination of the invasiveness for 4 kinds of ESCC cell in vitro.The number of cells passed through the filter membrane was used to indicate the invasiveness in the Boyden attack cab experiment.In 4 kind of ESCC cell,EC-1 cells had the greatest number(91.00±17.44),followed by Eca109(83.60±12.93),EC9706 (79.00±11.29),TE-1(61.80±11.10).The differences between four groups had significance(P=0.001).The comparison between EC1 and Eca109 showed that there is no statistics significance(P=0.575),the other comparisons between two groups has the significant difference(P＜0.05).The locomotory capacity of 4 cell lines were as the following:EC-1(274.28±33.24),Eca109(251.95±32.71),EC9706 (223.32±56.81),TE-1(200.12±18.15),and there were significant difference between each group(P=0.016).4.The relationship between the expression of S100A4 protein,S100A4 mRNA and the invasiveness of ESCC cell in vitroThe relevant analysis demonstrated that the correlation coefficient between the expression of S100A4 protein and the invasion of these cell in vitro was 0.573 (P＜0.05).And the correlation coefficient between S100A4 mRNA expression and the invasiveness of these cells in vitro was 0.832(P=0.001).There was positive correlation between them.Thus,it indicated that there was close correlation between the expression of S100A4 and the invasiveness of ESCC cell in vitro.Part two The infulence of S100A4siRNA on the Multiplication and Invasiveness of ESCC Cell In VitroMethods1.EC-1 cells transfected with 50nM chemical synthesis S100A4siRNA as Transfected group,EC-1 cells transfected with transfected reagent as control group.2.After transfection with S100A4siRNA,RT-PCR and Western the Blotting were used to detect the expression of S100A4 in mRNA and protein at 0,24,48 and 72h.3.The Boyden cab was used to detect the invasion of EC-1 cells after transfection at 0,24,48 and 72h in vitro.4.After transfected with S100A4siRNA,RT-PCR and Western Blotting were used to detect the expression of MMP-2 and E-cadherin mRNA and protein at 0,24,48 and 72h. 5.MTT experiment was used to detect the influence of S100A4siRNA on the cytostasis of EC-1 cells.6.statistical treatment:Using the SPSS11.0 software to process data,The measurement data was manifested with(?)±S;The comparison between two group of mean values with T-test(t-test) and multi-group mean values with variance analysis (ANVOA);The relevant analysis was used the Pearson's related analytic method,size of testα=0.05.Results1.The comparison of cell growth condition before and after transfectedAfter transfection with S100A4 siRNA into the EC-1 cells,the morphous of these cells were observed under inverted microscope.The degree of cell fusion goes down;in the butcher the pellet increases;But the control group's cell pastes the wall to grow,the cell outline is clear,the intercellular space structure is close and the growth is exuberant.2.S100A4siRNA effectively silenced the expression of S100A4 gene in EC-1 cells.Results of Western Blotting indicated that,afte transfection expression of S100A4 protein reduced.Results of S100A4/β-actin protein were:0 hours (0.658±0.017),24 hours(0.508±0.010),48 hours(0.436±0.008),72 hours (0.247±0.006).After statistics analysis,there were significant difference between 24h,48h,72h after tranfection with control group in each times and 0h after tranfection(P＞0.05).Transfected after 72h,the effectiveness of inhibition was optimization,the expression of S100A4 reduced 62.46%.Results of semi-quantitative mRNA of S100A4/β-actin after transfection is:0 hours(0.775±0.014),24 hours(0.634±0.006),48 hours(0.498±0.008),72 hours (0.233±0.012).After statistics analysis,there were significant difference between 24h,48h,72h after tranfection with control group in each times and 0h after tranfection (P＞0.05).Transfected after 72h,the effectiveness of inhibition was optimization,the expression of S100A4 reduced 69.94%.Which indicated that there were high degree of specificity and time dependence in the suppression of S100A4 siRNA to S100A4. 3.Influence of S100A4siRNA on the invasiveness and locomotory capacity of ESCC cell in vitro.After tranfection,the invasiveness and motoricity of EC-1 cells reduced gradually after 48h.After 72h,the invasiveness of EC-1 cells reduced 60.66%,the motoricity of EC-1 cells reduced 60.80%.The results are as the fllowings,invasiveness in vitro:0 hours(85.46±15.10),24 hours(76.86±18.64),48 hours(59.39±7.47),72 hours (33.62±7.82);locomotory capacity in vitro:0 hours(270.33±27.11),24 hours (264.55±34.24),48 hours(163.13±34.15),72 hours(105.96±6.64).4.S100A4siRNA suppressed the expression of MMP-2 in EC-1 cellsThe results of RT-PCR and Western Blotting showed that:after transfected with S100A4siRNA the expression of protein and mRNA of MMP-2 reduced gradually. The results of MMP-2/β-actin mRNA are as the followings:0h(1.004±0.058),24h (0.969±0.077),48h(0.622±0.035),72h(0.275±0.078),there was significant difference between 48h,72h and control group(P<0.05).The results of MMP-2/β-actin:0h(0.864±0.054),24h(0.710±0.041),48h(0.549±0.041),72h (0.288±0.050),there was significant difference between 24,48h,72h and control group(P<0.05).S100A4siRNA reduced the expression of MMP-2 proteion early than MMP-2 mRNA,which indicated that S100A4 not only influence the transcription of MMP-2 mRNA,but also influence the synthesis of protein after transcription.5.S100A4siRNA suppresses the expression of E-cadherin in EC-1 cellsAfter transfection with S100A4siRNA,the results of RT-PCR and Western Blotting showed that:the expression of protein and mRNA of E-cadherin increased gradually.there was no significant changes at 0h and 24h,however there was significant difference between 48h,72h and control group.The results of E-cadherin /β-actin mRNA:0h(0.318±0.013),24h(0.334±0.023),48h(0.476±0.022),72h (0.586±0.013);The results of E-cadherin /β-actin:0h(0.289±0.012),24h (0.294±0.020),48h(0.479±0.014),72h(0.625±0.022).6.Suppression of S100A4siRNA on the multiplication of ESCC cells in vitroThe MTT results showed that:after transfection reproductive activity of these cells were inhibited effectively,after transfected 48h,OD of transfection group,control group was(0.487±0.019),(0.571±0.019)(P＜0.05).after transfected 72h,OD of transfection group,control group was(0.488±0.008),(0.681±0.013) (P＜0.05).Ttransfected after 48h,72h,inhibited ratio to these cells is 14.71%,28.34% respectively.Which indicated that S100A4 siRNA can inhibit the reproductity of ESCC Cell.part three Research on the effect of S100A4siRNA to the growth of transplants tumor of esophageal carcinoma in the body of athymic mouse.Methods1.The establishment of the modle of heterogenic transplantation tumor of esophageal carcinoma in the body of athymic mouse:Take the logarithm growth period EC-1 cell, adjust cell density to(1×10~7/ml),hypodermic vaccinates 0.2ml in each bare mouse's foreleg right armpit back,constructs the model of bare mouse transplant tumor.2.The animals successfully constructed were divided into 3 groups at random: S100A4siRNA group(A),irrelevant sequence siRNA(B),control group(C).There are 5 athymic mice in each group.Drugs was injected with 50nM S100A4siRNA,50nM control siRNA and PBS nearby the tumors to treat 1 time every other day, altogether 14 times.3.Observe the change of the tumor volume of athymic mice everyday(Vernier caliper was used),and curve the growth of tumor.In the end all athymic mice were executed,and the weight of tumor were measured,the formula is:Tumor suppression rate=(quantity of control group tumor-quantity of experimental group tumor)/ quantity of control group tumor×100%.4.Tumor organization of each group of athymic mice were carried on the convention pathological slice and dyed with HE;Examine the expression of S100A4,MMP-2 in each group and the expression of E-cadherin protein.5.Examine the expression S100A4,MMP-2 and E-cadherin mRNA of each group with RT-PCR. 6.statistical treatment:Using the SPSS11.0 software to process data,The measurement data were manifested with(?)±S;The comparison between two groups of mean values with T-test(t-test) and multi-group mean values with variance analysis(ANVOA);The relevant analysis used the Pearson's related analytic method. size of testα=0.05.Results1.Tumors were succeeded in the hypodermic of 15 athymic mice,the EC-1 cell line heterotransplantation rate is completely 100%,formed the tumor after 1 week.2.The comparison of the weight and volume of tumors stripped out of the nude mice in three groups at the end of treamentBefore getting treatment,there were no significant difference in the tumor volume(P>0.05).After dealing with different methods,volume of tumors of A group were smaller than B and C groups(P<0.05),and there was no difference between B and C groups.We weighed those tumors after killing these mice,Weight of tumors of A group were smaller than B and C groups(P<0.05),there were siginifant difference between them(P<0.01),But the deffenence between B and C groups had no statistics significance(P>0.05).3.The influence on morphology of cells after transfected with S100A4siRNAObserving under the microscope,we found that the tumor cells of A group were smaller and the nuclear chromatins were sparse.In addition the tumor giant cells were rare,and the obvious plot and the laminated necrosis stove were found in the tumor organization.We also find that the size of tumor cells of B group and C group were irregular,and there were a lot of nuclear chromatin,the nucleus were large and irregular,the tumor giant cells were common,and the heterogeneous type was obvious.4.The expression of S100A4,MMP-2 and E-cadherin protein in three groupsThe results of immunocytochemsty were observed under 5 highpower field randomedly,in each field we obersved 200 positive cells.Quantity of the expression of S100A4 protein were as the flowings:A group(68±4),B group(123±14),C group (135±18);The expression of S100A4 in A group were lower than that in B group and C group(P＜0.05),Compared the expression of B group and C group,we found there was no obvious statistics difference(P＞0.05).Which indicated that S100A4siRNA was injected besides the tumor can effectively inhibit the expression of S100A protein;quantity of the expression of MMP-2 protein in A group were (76±8),which was lower than B group(134±9)and C group(142±7).The expression of the E-cadherin protein in the tumor of A group(68±6) was higher than B and C groups(P＜0.05).The results is uniformed with the results of vitro.5.The expression of S100A4,MMP-2 and E-cadherin mRNA in three groupsThe result of RT-PCR showed that quantity of the expression of S100A4 mRNA were as the flowings:A group(0.334±0.027),B group(0.689±0.035),C group (0.702±0.047);quantity of the expression of MMP-2 mRNA were as the flowings:A group(0.572±0.084),B group(0.804±0.173),C group(0.922±0.451).The expression of S100A4 and the MMP-2mRNA in A group was lower than that in B group and C group(P＜0.05).Compared the expression of B group and C group,we found there was no obvious statistics difference(P＞0.05).The expression of the E-cadherin mRNA in the tumor of A group(0.516±0.274) was higher than B group (0.327±0.033) and C group(0.284±0.057)(P＜0.05). Conclusion1.There is high expression of S100A4 in the tissue of ESCC and its cell lines,which was positive correlation with the invasiveness of tumor cells.2.The results of expremient in vitro suggests that EC-1 cell lines have the highist invasion in the 4 different cell lines.The expression of S100A4 concerned with malignant degree and invasiveness of tumor.3.S100A4siRNA can effectively inhibit the expression of S100A4,invasion and locomotory capacity of tumor cells.4.There is positive correlation between S100A4 and MMP-2,but there is negative correlation between S100A4 and E-cadherin,which indicated that there were synergistic effect to the influence on the infiltration,metastasis of ESCC between S100A4,MMP-2 and E-cadherin.S100A4siRNA can effectively silence the expression of S100A4 gene,suppresses the expression of MMP-2,but the expression of E-cadherin increased,which indicated that S100A4 may adjust the expression of MMP-2 and E-cadherin through some way.5.S100A4 has the ability of inhibition of the cell proliferation.6.The model of heterogenic transplantation tumor of esophageal carcinoma in athymic mice demonstrats that,S100A4siRNA can effectively reduce the expression of S100A4 protein,and suppress the growth transplantation tumor,which indicats S100A4 RNA may become new targat gene for the treatment of esophageal carcinoma.