Expression Of RhoC And Its Regulators RhoGDIβ RhoGDIγ In Lung Squamous Cell Cancer And Adenocarcinoma

INTRODUCTIONThe Rho family of small GTPase is a subgroup of the Ras superfamily of GTPases.The Ras homologous sub-family of low molecular mass(approx 21 kDa) GTP-binding proteins encompasses Rho(e.g.,RhoA,B,C),Rac and Cdc42 proteins. Among the Rho GTPases are molecular switches that use a simple biochemical strategy to control complex cellular processes.They cycle between two conformational states: one bound to GTP('active' state),the other bound to GDP('inactive' state).Further interest in Rho GTPases has been fuelled by the realization that they regulate many other signal transduction pathways in addition to those linked to the actin cytoskeleton. Recent studies suggest that overexpression of RhoC may promote tumour cells invasion and miragation.Guananine dissociation inhibitors(GDIs),which stabilize either the GDP or GTP-bound state,are considered as negative regulator.And in some tumours RhoGDI were found overexpressed or underexpressed.Whether the abnormal expression of RhoC is regulated by RhoGDI and what are they relationship between clinicopathological characteristics are not clear.Therefore,we design to examine the expression of RhoC,RhoGDIβand RhoGDIγin lung cancer samples and analyze the relationship between them and clinicopathological characteristics,in order to discuss the function and significance in lung cancer.METERAL and METHODS1.Material (1)Forty fresh lung cancer samples with corresponding noncancerous tissue were obtained from the First Affiliated Hospital of China Medical University between 2006 and 2007.(2)Human lung cancer cells BE1,LH7,A549 were obtained from Beijing Medical University.Human bronchial epithelial cell HBE was purchased from Zhongnan University Xiangya laboratory.2.ReagentsThe primary antibody anti-human RhoC goat polyclonal antibody and anti-human RhoGDIβ,RhoGDIγgoat polyclonal antibody were produced in Santa Cruz Co.S-P kit was purchased from Fuzhou Maixin Biocompany.DAB agent kit and anti-humanβ-actin rabbit polyclonal antibody were bought from Zhongshan Biotechnology and Beijing Zhongshan Golden Bridge Biotechnology Co,Ltd.3.Method(1)Western BlotFresh tissue(1-2g)or cells(1*10~7)in 5 volumes of lysis buffer was homogenized, treated with ultrasonic wave and centrifuged for 30 minutes.Then it followed by the whole procedure,including electrophoresis,transfer,blocking,incubation with primary antibody and enzyme conjugated compound,and dipping with DAB.It was determined as positive case that the special bands appeared in PVDF membrane,and then examine the gray value of the bands.(2)Reverse transcription-polymerase chain reactionThe expressions of RhoC,RhoGDIβand RhoGDIγmRNA were detected with reverse transcription-polymerase chain reaction.4.Statistical analysisThe statistical software SPSS 13.0 was applied for data analysis.Chi-square and t-test were used to study the relationship between RhoC/RhoGDIβ,RhoGDIγexpression and clinicopathological characteristics and prognosis of patients, respectively.P＜0.05 was significative in statistic.RESULTS1.Expression of RhoC,RhoGDIβand RhoGDIγprotein in various lung carcinoma cells and HBEThe expression level of RhoC,RhoGDIβand RhoGDIγprotein in various lung carcinoma cells were significantly higher than in HBE.The expression of RhoC and RhoGDIγprotein in BE1(the cell with high metastasis capability)was significantly higher than in LH7(the cell with low metastasis capability).But the expression of RhoGDIβprotein in LH7(the cell with low metastasis capability)was significantly higher than in BE1(the cell with high metastasis capability).2.Expression of RhoC,RhoGDIβand RhoGDIγmRNA in various lung carcinoma cells and HBEThe expression level of RhoC,RhoGDIβand RhoGDIγmRNA in various lung carcinoma cells were significantly higher than in HBE.The expression of RhoC and RhoGDIγmRNA in BE1(the cell with high metastasis capability)was significantly higher than in LH7(the cell with low metastasis capability).But the expression of RhoGDIβmRNA in LH7(the cell with low metastasis capability)was significantly higher than in BE1(the cell with high metastasis capability).3.Expression of RhoC,RhoGDIβand RhoGDIγprotein in Lung Squamous Cell Carcinoma and Adenocarcinoma and it's neighboring noncancerous tissueAs a result of the Western Blot analysis on 40 cases of fresh Lung Squamous Cell Carcinoma and Adennocarcinoma and their neighboring noncancerous tissue,both RhoC,RhoGDIβand RhoGDIγproteins were detected in cancer and corresponding paracarcinoma tissue.But the level of RhoC,RhoGDIβand RhoGDIγexpression were higher in cancer tissue than in paracarcinoma.The level of RhoC and RhoGDIγ expression were higher in cancer tissue with lymph node metastasis than with no lymph node metastasis.And the level of RhoGDIβexpression was opposite with them.4.Expression of RhoC,RhoGDIβ,RhoGDIγmRNA in lung squamous cell carcinoma and adennocarcinoma and it's neighboring noncancerous tissueAs a result of RT-PCR analysis on 30 cases of fresh Lung Squamous Cell Carcinoma and Adennocarcinoma and their neighboring noncancerous tissue,both RhoC,RhoGDIβ,RhoGDIγmRNA were detected in cancer and corresponding paracarcinoma tissue.But the level of RhoC,RhoGDIβ,RhoGDIγexpression were higher in cancer tissue than in paracarcinoma.The level of RhoC,RhoGDIγexpression were higher in cancer tissue with lymph node metastasis than with no lymph node metastasis.And the level of RhoGDIβexpression was opposite with them.5.The relationship between expression of RhoC / and RhoC /RhoGDIγExamine the gray value of the bands with Person correlation analysis found that RhoC/RhoGDIβand RhoC/RhoGDIγhad no obvious relationship.CONCLUSION1.The level of RhoC,RhoGDIβand RhoGDIγprotein expression were higher in cancer tissue than in paracarcinoma.The level of RhoC and RhoGDIγexpression positively correlate with lymph node metastasis,and the level of RhoGDIγnegatively correlates with lymph node metastasis.2.The level of RhoC,RhoGDIβand RhoGDIγmRNA expression were higher in cancer tissue than in paracarcinoma.The level of RhoC and RhoGDIγexpression positively correlate with lymph node metastasis,and the level of RhoGDIβnegatively correlates with lymph node metastasis.3.The expression of RhoC and RhoGDIγprotein in BE1 was significantly higher than in LH7.But the expression of RhoGDIβprotein in LH7 was significantly higher than in BE1.4.The expression of RhoC and RhoGDIγmRNA in BE1 was significantly higher than in LH7.But the expression of RhoGDIβprotein in LH7 was significantly higher than in BE1.5.Expression of RhoC /RhoGDIβand RhoC /RhoGDI had no obvious relationship.