Expression Of RhoC Gene And Its Correlation With Invasion, Metastasis In Esophageal Squamous Cell Carcinoma

Esophageal cancer, one of the most aggressive carcinomas of the gastrointestinal tract, is the sixth most frequent cause of cancer deaths worldwide, while the cause of death of the patients with esophageal carcinoma was evoked by invasion and metastasis of cancer. Therefore, to explore the mechanism of the esophageal carcinoma invasion and metastasis is of great importance in the Esophagus carcinoma therapy.RhoA, RhoB and RhoC are a kind of small GTP-binding proteins, and participate in tumor's invasion, metastasis and angiogenesis. Recently, it is well documented that high expression of the RhoC gene were correlated with invasion and metastasis of several different tumors, including breast cancer, melanoma, pancreatic cancer, colon carcinoma, carcinoma of urinary bladder, and non-small cell lung cancer, etc. Until now domestic and foreign still did not have the systematic research report.s related the relations RhoC and invasion,metastasis of esophageal squamous cell carcinoma (ESCC).In order to delve into the relationship between the expression of RhoC in esophageal squamous carcinoma and invasion, metastasis, and seek for available approach to inhibit the occurrence, invasion and metastasis of esophagus squamous carcinogenesis, in the present study, expressions of RhoC mRNA and protein were detected in ESCC, adjacent atypical hyperplasia and normal esophagus mucous membrane tissues by RT-PCR, hybridization in situ and immunohistochemistry SP methods, subsequently, expression of vessel endodermis growth factor(VEGF) was detected using immunohistochemistry SP in these tissues. Meanwhile, micro vascular density (MVD) was detected using CD 105 antibody in 62 cases of ESCC,and the correlation between the expression of RhoC and VEGF and MVD was analyzed. Based on the results above-mentioned, interfering expression vector pRNAT-U6.1-siRhoC was successfully constructed and stably transfected into esophagus cancer cell EC9706 in order to establish RhoC gene "knock weak" (knockdown) cell model.Further the expression of RhoC was detected using RT-PCR, hybridization in situ,Western blot and immunohistochemistry methods in EC9706 cells, respectively. Expression of VEGF was detected by immunohistochemistry method in EC9706 cells, and then invasion ability of EC9706 cells was determined by Boyden-chamber model in vitro. Finally, the expression of RhoC was detected in xenografted human esophageal squamous cell carcinoma by in situ hybridization, RT-PCR and immunohistochemistry, respectively, and then expression of VEGF in nude mice transplantation tumor was detected by immunohistochemistry method. In vivo, we observed the role of RhoC gene in nude mice transplantation tumor and effect of RhoC gene expression on the expression of VEGF, which will elucidate the significance of RhoC expression in the occurrence and development of ESCC. Further, the relationship between RhoC gene and angiogenesis was illuminated, which will provide the theoretical basis for molecular target therapy of ESCC. This study includes the following three parts. Part I:Expressions of RhoC, VEGF and CD105 in esophageal squamous cell carcinoma tissues and their significanceMethods:1. Using RT-PCR,in situ hybridization, immunohistochemistry to detect the expression of RhoC gene in 62 cases of esophageal squamous cell carcinoma,31 cases of adjacent dysplasia and 62 cases of normal esophageal mucosa.2. VEGF protein expression was detected in 62 cases of esophageal squamous cell carcinoma,31 cases of adjacent dysplasia and 62 cases of normal esophageal mucosatissues by immunohistochemistry.Simultaneously,micro vascular density (MVD) was determined using CD 105 antibody.3.Statistical analysis:Statistics analysis was used by SPSS13.0 software,using chi-square test,T test and variance analysis and Spearmanm,test standardα=0.05.Results:1. Expression of the RhoC gene was no or lower in normal esophagus mucous membrane tissue compared to ESCC. RhoC mRNA and protein expression were mainly localized in in cytoplast of ESCC.2. RhoC mRNA and protein expression level gradually increased from normal esophagus mucous membrane tissue, adjacent atypical hyperplasia tissue to ESCC tissue, and there was significan difference among three groups (P<0.05).3. Expressions of RhoC mRNA and protein in deep layer invaded and lymphatic metastasis groups were significantly higher than those in infiltrate shallow layer group and without lymphatic metastasis group,and there was significan difference among three groups (P<0.05).4. The expressions of RhoC mRNA and protein in poor differentiated ESCC tissues were markedly higher than that in well differentiated tissues, and the differences were statistically significant (P<0.05)5. Expressions of RhoC mRNA and protein were not related to sex and age of the patients with esophagus cancer (P>0.05). PartⅡ:Construction of interference expression vector pRNAT-U6.1-siRhoC and its influence of the down-regulation of RhoC gene expression on cell biology behavior of esophageal squamous cell carcinoma cell line EC9706 cellsMethods:1. Three pairs of anti-sense oligonucleotide fragments and a pair of non-sense sequence was designed and annealed, and then was introduced into RNA interfering expression vector pRNAT-U6.1, and the recombinant expression vector pRNAT-U6.1-siRhoC was successfully constructed.2. EC9706 cells stably expressing pRNAT-U6.1-siRhoC1 were screened using G418.3. The mRNA and protein expressions of the RhoC gene were investigated after transfection with pRNAT-U6.1-siRhoCl by RT-PCR, Western blotting, in situ hybridization and immunocytochemistry, respectively.4. VEGF level was investigated by immunocytochemistry in the EC9706 cells transfected with pRNAT-U6.1-siRhoCl.5. Characteristics of cell invasion of the EC9706 cells transfected with pRNAT-U6.1-siRhoCl were determined using Boyden Chamber extraneous invade experiment.6. Statistics analysis was performed by SPSS13.0 software,including chi-square test and one-way analysis of variance, test standardα=0.05.Results:1.Three siRNA expression vector pRNAT-U6.1-siRhoCl, pRNAT-U6.1-siRhoC2 and pRNAT-U6.1-siRhoC3 were successfully constructed using gene recombination technology,in which pRNAT-U6.1-siRhoC1 was screened as most inhibitory effect.2. Expression levels of the RhoC gene in EC9706 cells transfected with pRNAT-U6.1-siRhoClwas significantly lower than that in cells transfected with pRNAT-U6.1-siC and control cells (P<0.05). 3. Expression levels of VEGF in EC9706 cells transfected with pRNAT-U6.1-siRhoCl was significantly lower than that in cells transfected with pRNAT-U6.1-siCl and control cells (P<0.05).4. The results of Boyden Chamber extraneous invade experiment showed that the number of EC9706 cells transfected with pRNAT-U6.1-siRhoCl notably decreased in through Matrigel gluey's cell compared to blank control group (P< 0.05),There was no distinct difference in vacancy plasmid group and blank control group (P>0.05). Part III:Influence of pRNAT-U6.1-siRhoCl on the growth of xenografted human esophageal squamous cell carcinomaMethods:1. Transplantable tumors were produced using EC9706 cells and the transfected EC9706 cells with pRNAT-U6.1-siRhoCl and vacancy plasmids, respectively, in nude mice.2. Expressions of the RhoC gene in each group nude mice tissue were detected by RT-PCR, hybridization in situ and immunohistochemistry, respectively.3. Expressions of the VEGF gene in each group nude mice tissue were detected by immunohistochemistry.4. Statistical analysis:The data was performed by chi-square test, t test and one-way analysis of variance using SPSS version 13.0. In all statistical analyses, test standardα=0.05.Results:1. As indicated in the growth curve of tumors, RhoC-siRNA obviously inhibited the growth of transplantable tumors compared to control and untranfected group (P <0.05).2. Expression levels of mRNA and protein of the RhoC gene were markedly lower in EC9706 cells transfected with pRNAT-U6.1-siRhoCl compared to control and untransfected groups (P<0.05).3. Expression levels of mRNA and protein of the VEGF gene in EC9706 cells transfected with pRNAT-U6.1-siRhoCl were significantly lower than those in control cells and cells transfected with vacancy plasmid (P<0.05).Conclusion1. High levels of RhoC protein and mRNA were observed in esophageal squamous cell carcinoma,were closely associated with histological grade,invasion and metastasis of ESCC,suggesting that RhoC maybe become an important biological marker of invasion and metastasis of ESCC. 2. High expression of RhoC gene can up-regulate the expression of VEGF,further promote the angiogenesis in ESCC tissues.3. Interfering expression vector pRNAT-U6.1-siRhoCl was successfully constructed, and introduced into EC9706 cells.EC9706 cells stably expressing pRNAT-U6.1-siRhoCl were successfully established,which will lay a foundation for further studing the biological function of RhoC and RhoC target therapy.4. Down-regulation of RhoC gene can decrease the invasive ability of EC9706 cells in vitro, and down-regulate the expression of VEGF protein.5. RhoC-siRNA obviously inhibites the growth of transplantable tumors and down-regulate the expression of VEGF protein in xenografted human esophageal squamous cell carcinoma.6. Down-regulation of RhoC gene can effectively inhibit the invasive and metastasis ability of EC9706 cells in vitro and in vivo, which will provide the new idea and theoretical basis for target gene therapy of ESCC.