The Effect Of CDC42 Overexpression In The Mechanism Of Invasion And Metastasis In Cholangiocarcinoma

BackgroundCholangiocarcinoma is a common type of cancer,and with the development of medical science,in recent years of China,more cases are found continuously,espesily in extrapotar Cholangiocarcinoma.Cholangiocarcinoma is a type of digestive cancer,it charactered by growing at its primary position and invasive development,invasion and metastasis are the major reason to death.The major treatment for Cholangiocarcinoma is still surgery,but there are many small cancer metastasis in lymph nodes and other orgens,so it is evitable.But can't guarantee the cholangiocarcinoma in the distance,lymph nodes and small metastases eradication,postoperative recurrence and metastasis is difficult to avoid,The situation of bile duct carcinoma in the surgical removal of the effect and prognosis evaluation,so further study of cholangiocarcinoma invasion and metastasis of the clinical features and molecular mechanism,for the diversification of clinical patients with cholangiocarcinoma treatment and prognosis to provide technical and theoretical support is necessary.Previous study found that 67 LR cholangiocarcinoma cells express(QBC939,RBE)the synapse formation obviously than normal bile duct epithelial cells,and mRNA level increased significantly,too.67 LR is a kind of non-integrin receptor,to express more in gastrointestinal epithelial cell surface,involved in the cell and basement membrane and extracellular matrix adhesion function.Study shows that multiple tissues increased 67 LR expression in tumor,through a variety of mechanisms to promote cancer cell invasion and metastasis.The lab early experiments have found 67 LR expression in cholangiocarcinoma,significantly promote cancer cell invasion.We guess: in 67 increase in the number of synapse formation LR high expression in cell lines,whether for 67 LR express promoted the synapse formation or start,promoted the influence the formation of synaptic signaling molecule.In tumor cells,synapse formation and Rho is GTP enzyme made closely related.RHO GTP enzyme is a kind of signal molecule,Ras related made in tumor can appear abnormal activation or inhibition.Rho GDP coupling GTP enzymes can be made to the activation of GTP form,made active forms of molecules can be combined with a certain number of effect,signaling cascade amplifier,thus regulate various cellular processes.Cdc42 is one of the members of Rho family protein(RhoGTPase).Rho family members as long as there is Rho A,RhoB,including RhoC,Rac1,Rac2,Rac4,Cdc42,and many other molecules,the group members molecules for important signal in middle molecule in cells,such as Rho A induced the formation of the tension of fiber,Rac1 induced cell plate pseudopodia and the formation of the cell membrane wrinkle wall,Cdc42 and involved in the formation of the cells invade the foot,and participate in the restructuring of the actin cytoskeleton,evolution,gene transcription,cell cycle and cell adhesion and cell movement,etc.In recent years,the study found that Rho family molecules associated with the occurrence of tumor,appear in breast cancer,head and neck tumor molecular Rho family high expression,promote the tumor invasion and metastasis.We will focus on 67 LR express the emergence of the increase in the number of cell synaptic(invapodia),investigate Cdc42 expression patterns in the synaptic Whether 67 LR expressed by influencing the Cdc42,thereby promoting cholangiocarcinoma cell invasion and metastasis.Objectives:Using IHC,real-time pcr,Western Blot method from tissue protein and m RNA level testing Cdc42 protein expression level,Cdc42 in cholangiocarcinoma is discussed and the expression level of clinical pathological parameters correlation and Cdc42 role and significance in the prognosis of patients with cholangiocarcinoma assessment.And study 67 LR regulation Cdc42 affect bile duct cancer cell invasion and metastasis.Methods:1.According to the immunohistochemical staining positive cells number and staining intensity of half quantitative analysis on the expression of Cdc42,adopt the method of immunohistochemical staining for 87 cases of cholangiocarcinoma specimens,23 cases of metastatic lymph nodes,and specimens of 18 cases of metastases(2005-2010 treatment in patients with cholangiocarcinoma resection specimens),contrast metastases and the primary carcinoma in Cdc42 expression differences.2.Cdc42 protein expression and clinicopathological parameters of cholangiocarcinoma correlation analysis,Cdc42 expression quantity and survival of patients with cholangiocarcinoma correlation analysis by Kaplan Meier-method and the log-rank test;Cox multivariate linear regression analysis to all involved in the clinical pathological indicators,assess its overall survival and disease-free survival in patients with cholangiocarcinoma.3.Built Cdc42 interference and expression vector,transfection(QBC939 and RBE),cholangiocarcinoma cells intervention Cdc42 expression;Cdc42 by wound healing test,Transwell invasion experiment,researches on the cholangiocarcinoma cell migration,the influence of the ability of invasion and metastasis.4.Using immunohistochemical observation of 87 cases of cholangiocarcinoma tissue expression of LR,67 LR analysis and correlation of Cdc42 expression.Build 67 LR interference and expression vector,transfection cholangiocarcinoma cells(QBC939 and RBE);By real-time PCR,western blot experiments,research on cholangiocarcinoma cells 67 LR express the influence of the expression of CDC42 intervention;Coprecipitation immunized with chromatin(Ch IP)experiment,research CDC42 in the cholangiocarcinoma cells can be combined with 67 LR gene promoter.cholangiocarcinoma cells by western blot experiments,research intervention 67 LR expression and the expression of CDC42 activity.Results:1.Definite diagnosis of cholangiocarcinoma resection in tissue samples(87 cases),34 cases of carcinoma tissue(39%)presented CDC42 cytoplasm staining positive;4 cases of paracarcinoma were positive,the others were negative;Prompt cholangiocarcinoma CDC42 abnormal expression in statistically significant(p < 0.01).Analysis of CDC42 express tip: correlation with clinical pathological features people CDC42 positive expression were significantly associated with lymph node metastasis,primary focal TNM classification and distant metastasis.and with the patient gender,age,degree of differentiation,has nothing relation.32 cases of lymph node metastases,25 cases(78%)appeared CDC42 staining positive,positive rate is significantly higher than primary tumors(39%).18 cases of distant metastases,13 cases(75%)showed CDC42 staining positive,the positive rate is higher than the original site;These results indicate that: the cholangiocarcinoma in CDC42 abnormal expression is associated with tumor invasion and metastasis.2.Kaplan-Meieranalysis showed that CDC42 postoperatively in patients with positive expression of overall survival time was significantly lower than CDC42 negative patients(log-rank p = 0.002);And the tumor recurrence significantly earlier than CDC42 patients with negative expression(log-rank p = 0.002).Cox model analysis hint: CDC42 abnormal expression is judge patients postoperative survival and recurrence time after the relatively independent risk factor(HR=2.047,95%CI=1.414-3.581,p=0.002;HR=1.892,95%CI=1.315-3.203,p=0.003).Results indicate: this section CDC42 abnormal expression may be a potential molecular marker that estimate the prognosis of patients with cholangiocarcinoma.3.Cholangiocarcinoma cells(QBC939,RBE)wound healing experiment,Transwell invasion,interference Cdc42 express significant inhibition of cancer cell invasion and migration ability.Overexpression of Cdc42 significantly enhance the capacity of cancer cell invasion and migration.This part experiment hints: Cdc42 significantly promote the invasion and metastasis of cholangiocarcinoma.4.Of all 87 cancerous tissue samples,67 LR showed positive staining of 35 cases.67 LR protein expression was closely associated with Cdc42(c2 test).Continues slices showed 67 LR expressed accordance with Cdc42 at the same slice in cholangiocarcinoma.5.In vivo,cell groups of QBC939 and RBE,the level of m RNA and protein did not change after knockdown 67 LR in QBC939,the level of mRNA and protein did not change after upregulated 67 R in RBE.Chromatin Immunoprecipitation showed 67 LR had no connection with Cdc42 at gene promoter region,but using western blot,we found that when knockdown 67 LR in QBC939,the activation of Cdc42 decreased,when upregulated 67 R in RBE,the activation of Cdc42 increased.So,we could conclude that 67 LR upregulated the activation of Cdc42 to promote the invasion and metastasis of cholangiocarcinoma.Conclusions:Cdc42 was highly expressed in cholangiocarcinoma using immunohistochemistry,real time PCR,westernblot and so on.The Cdc42 protein expression was closely associated with Lymph nodes and distant metastasis,recurrence,and invasion and prognosis.67LR maybe regulate the active Cdc42 to promote invasion and metastasis of cholanginocarcinoma.