Expression Of RECK And Its Relationship With The Invasion And Metastasis Of Esophageal Squamous Cell Carcinoma

Esophagus cacinoma is one of the most common malignancy in our country,and Henan province also has higher morbility.It's mortality reside forth position in our country,whereas it's invasion and metastasis is the first cause of invoke esophagus cancer sufferer's obituary,so discussing the esophague cacinoma invasion and metastasis mechanism became one of the investgative hotspot.Tumor's occurrence and development are the course of multifactor,multistep, multistage and polygene alterative.Oncogene activation and tumor suppressor gene inactivation are the material basis of human tumor formation and development.Previous research found that the mutations and deletions of some tumor suppressor genes,such as p53,Rb,p16 and so on,are relate to malignancy occurrence.A new anti-oncogene named RECK(reversion inducing cysteine rich protein with Kazal motifs,RECK)was found in recent years.The expression of RECK closely relate to tumorous invasion,metastasis and angiogenesis.In recent years,there are several relevant studies about the relation between RECK and tumor invasion and metastasis in the fields of liver cancer,pancreatic cancer,breast cancer and lung cancer.Researches show that RECK gene's expression were negative correlate to tumorous invasiveness and the sufferers whose RECK gene higher expression have better prognoses than those with lower expression quantity.This gene's deletions or(and) mutation and depressing expression of mRNA or protein relate to tumorous occur,invasion,metastasis and histology grade.The mechanism may be that RECK regulated negatively tumour growth,invasion,metastasis through restraining the secretion and activity of MMP-2,MMP-9 and MT1-MMP.In addition, RECK can inhibit tumor growth,invasion and metastasis by inhibition of tumor angiogenesis.There have no other report both in China and abroad so far except the relevant articles pulished by our team before.In order to discuss RECK gene's relation to esophagus squama carcinogenesis and invasion,metastasis for more,sought early detection esophagues squama carcinogenesis and soakage transitionary indices and found available approach to restrain esophagus squama carcinogenesis and soakage transitionary,this topic firstly used RT-PCR, hybridization in situ and immunohistochemistry SP combine detected the expression of esophagus cancer verdure excision specimen's RECK,MMP-9mRNA and protein.We use immunohistochemistry SP detected blood vssel endotheliocyte growth factor in esophague squama,adjacentatypical hyperplasia tissue and normal esophagus mucous membrane tissue.At the same time,use resist CD105 antibody detected microvascular density(MVD) in 62 cases' esophagus squama carcinoma,and discussed the relationship among RECK, VEGF and MVD,revealed the relation of RECK and esophagus cancer invasion and metastasis.On this foundation,we constructed RECK's eukaryon expression vector successfully,then transfected it into esophagus cancer cell EC9706,use RT-PCR, hybridization in situ combine detected esophagus cancer cell EC9706's RECK, MMP-9mRNA expression level,respectively;Immunohistochemistry SP combined Western blot detected esophagus cancer verdure excision specimen's RECK,MMP-9 protein's expression level;The transfected cell's invasion ability was determined by Boyden-chamber model in vitro.In the final,apply nude mice lotus tunour experiment, combined use hybridization in situ,RT-PCR,immunohistochemistry detected the expression of RECK,MMP-9 at nude mice transplantation tumor,we in depth explorate upper regulate RECK's expression and activity restrain esophagus cancer invade in multi-aspect,such as gene,protein,cellular localization and cellular biology of tumor behavior,vitro invade cell experiment,in vivo nude mice achieve tumour experiment and so on.We try to illuminate the meaning in RECK's expression at esophagus squama carcinogenesis and development,invasion and metastasis.For more illuminate RECK gene repress esophagus squama carcinogenesis and development,invasion and metastasis's mechanism whether related to down regulate the expression of VEGF,depress MVD in tumour.Providing fundamental basis for deeply discuss esophagus carcinogenesis,invasion and metastasis mechanism and soght restrain esophagus squama carcinogenesis, developmental way.This study includes the following four parts.The first part:The clinicopathological significance of the expression of RECK in esophageal squamaous cell carcinomaMethods:1.Detected RECK gene protein expression in 62 cases esophageal squamous cell carcinoma tissue(ESCC),31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by immunohistochemical, then observed morphology.2.Detected RECK gene's mRNA expression in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by RT-PCR,then observed morphology.3.Detected RECK gene's mRNA expression in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by hybridization in situ,then observed morphology.4.Statistical treatment:Statistics analysis was performed by SPSS 11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.Results:1.RECK mRNA and protein masculine expression in esophagus cancer tissue mostly locate in normal esophagus mucous membrane squamous cells' cytoplast,no or lower expression in tumor cell.2.Combine RT-PCR and hybridization in situ detect resulting:RECK mRNA expression level depress by turns in normal esophagus mucous membrane tissue,adjacent atypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).The result of immunohistochemical display:RECK mRNA expression level depress by turns in normal esophagus mucous membrane tissue, adjacentatypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).3.RECK mRNA masculine expression rate in infiltrate to deep depth and lymphatic metastasis' esophagus cancer tissue signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).RECK protein masculine expression rate in infiltrate to deep depth and lymph node metastasis' esophagus cancer tissue signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).4.RECK mRNA masculine expression rate in poorly differentiated esophagus cancer signifcant depress compare to well,moderately differentiated esophagus cancer,the difference make statistics sense(P＜0.05).RECK protein masculine expression rate in poorly differentiated esophagus cancer signifcant depress compare to well,moderately differentiated esophagus cancer,the difference make statistics sense(P＜0.05).5.RECK mRNA's expression was not related esophagus cancer sufferer's sex,age, general style and tumorigenes is position.The second part:The study on MMP-9,VEGF,CD105's expression in ESCC tissue and the relation to RECK geneMethods:1.Detected MMP-9 mRNA expression and relation in 62 eases ESCC tissue(ESCC), 31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by RT-PCR.2.Detected MMP-9 mRNA expression and relation in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by hybridization in situ. 3.Detected MMP-9,VEGF's protein expression and relation 62 cases ESCC tissue(ESCC),31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by immunohistochemical,then observed morphology.4.Detected CD105 protein expression and relation in 62 cases ESCC tissue by immunohistochemical,then observed morphology.5.Statistical treatment:Statistics analysis was performed by SPSS11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.Results:1.The expression of MMP-9 mRNA and protein mainly located in the cytoplasm of tumor cells and no or lower expression in the normal esophageal squamous mucosal epithelial cells.The expression of MMP-9 mRNA and protein in three groups was negatively correlated with the expression of RECK(P＜0.05).2.MMP-9 mRNA masculine expression rate in infiltrate to deep depth and lymphatic metastasis' esophagus cancer tissue siguifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).3.The expression MMP-9 protein and mRNA was significantly decreased in poorly differentiated esophageal cancer compare to well,moderately differentiated ones.The difference among groups make statistics sense(P＜0.05).4.The expression of VEGF protein mainly located in the cytoplasm of tumor cells. The expression level step up by turns in normal esophagus mucous membrane tissue, adjacent atypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).Esophagus squama carcinoma tissue VEGF protein expression was related to esophagus squama carcinoma tissue soakage depth and lymphatic metastasis,VEGF protein masculine expression mostly locate in esophagus cancer cells' cytoplast.5.CD105 protein masculine expression mostly locate in tumour interstitial blood vessel endotheliocyte's kytoplasm.The masculine expression rate in esophagus cancer tissue infiltrated to deep depth and with lymphatic metastasis signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis.The difference among groups make statistics sense(P<0.05).The protein expression is negatively correlated with the expression of RECK and positively correlated with the expression of MMP-9(P<0.05).6.RECK mRNA's expression was not related esophagus cancer sufferer's sex,age, general style and tumorigenesis position(P>0.05).The third part:The influence of biology behaviour on Human RECK gene molecular cloning and construction of eukaryotic expression bearer to human esophagus cancer strain EC9706Methods:1.Total RNA was extracted from from esophagus tissue,RECK cDNA was isolated by using reverse transcription-polymerase chain reaction(RT-PCR);use restrict enzyme Kpn I and Not I two enzyme cut PCR offspring and pcDNA3 plasmid,then use T4 DNA Ligase joint,translate colibacilus JM109 competent call,PCR demonstrat converter,pick masculine clone intermediate culture,abstract there plasmid,proceed Kpn I and Not I two enzyme cut demonstrat construct RECK genic eukaryon expression bearer pcDNA3-RECK and judged by enzyme cut.2.Adopt liposome Lipofectamine2000 mediate eukaryon expression bearer pcDNA3 -RECK import esophagus cancer EC9706 cell in vitro culture.3.Detect pro and post transfection RECK and MMP-9 in human esophagus cancer cell strain EC9706 mRNA's expression by RT-PCR,hybridization in situ.4.Detect pro and post transfection RECK and MMP-9 in human esophagus cancer cell strain EC9706 protein's expression by immunocytochemistry,Western blot.5.Cell count protract each group cellular growth curve and observe RECK gene's impact on cell multiplication capable.6.Boyden chamber experiment count each group worn membranous cell count and compare transfect RECK gene varied to cell invade capable.7.Statistical treatment:Statistics analysis was performed by SPSS 11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.Results:1.Amplified idiosyncratic 4.4Kb gene fragment through PCR.Gene piecewise size accord with anticipate.The RECK gene sequencing and BLAST analysis proved the exactness of clone human RECK gene order,adopt enzyme cut pcDNA3-RECK got 5.4 kb and 4.4kb two fragment.2.PCR judge,enzyme cut judge pcDNA3-RECK reform bearer,resulting display: PCR amplificate fragment and Kpn I and Not I two enzyme slab segment size match with design.3.DNA sequencing result display:The construct eukaryon expression bearer's purpose gene nucleotidesequence perfect agreement with GeneBank's predict part of gene, and in the right direction.4.After transfected pcDNA3-RECK eukarya expression bearer,EC9706 cells all can stable express RECK mRNA and protein.5.Boyden chamber extraneous invade experimental result display:Compare with blank control group,EC9706pcDNA3-RECK group notable decrease in through Matrigel gluey's cell(P＜0.05),but pcDNA3 was no distinct difference in vacancy plasmid group and blank control group(P＞0.05)The fourth part:The research of inhibitory action and mechanism on RECK gene to nude mice transplantation tumor's vegetalMethods:1.Separately inject pro,post transfection and vancancy plasmid group's esophagus cancer cell strain EC9706 into nude mice subsurface,compared each groups nude mice's tumor size and metastasis scale.2.Detect each group's nude mice tissue's RECK,MMP-9 mRNA expression by RT-PCR,hybridization in situ.3.Detect each group's nude mice turnout tissue's RECK,MMP-9,VEGF genie protein expression by immunohistochemical method.4.Statistical treatment:Statistics analysis was performed by SPSS11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.Results:1.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor volume significant larger than EC9706/pcDNA3-RECK group's,compare among groups had difference in statistic sence(P＜0.05).2.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor RECK gene protein and mRNA expression significant lower than EC9706/pcDNA3 -RECK's expression,compare among groups had difference in statistic sence(P＜0.05).3.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor MMP-9 gene protein and mRNA expression significant higher than EC9706/pcDNA3 -RECK's expression,compare among groups had difference in statistic sence(P＜0.05).4.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor VEGF gene protein expression significant higher than EC9706/pcDNA3-RECK's expression,compare among groups had difference in statistic sence(P＜0.05).Conclusion1.The expression of RECK protein and mRNA is lower in esophagus squama carcinoma tissue.2.RECK may be a important biological markers of invasion and metastasis of ESCC.3.RECK play an important role in the tumor evolution.4.Successfully constructing RECK genie eukaryon expression bearer peDNA3-RECK, and importing human esophageal cancer EC9706 cell lines.Selecting a stable cell line laid foundation for further research about the biological functions of RECK and target therapy on RECK.5.pcDNA3-RECK can be increased RECK protein and mRNA expression of EC9706 cell.6.RECK can influence tumor invasion and metastasis by inhibiting MMP-9.7.Using Boyden chamber technology to detect the change of invasion in pro and post transfected plasmid groups and blank control group.8.RECK can anti tumor invasion and metastasis by inhibiting MMP-9.