Influence Of Rac1 Gene On The Biological Behavior Of Colorectal Cancer

Colorectal cancer is a common malignant tumor and the incidence of colorectal cancer ranks fourth in China.In recent years,remarkable progress has been made in the treatment of colorectal cancer,however the prombles associated with high metastasis and recurrence limit the outcome of colorectal cancer pantients.Hence,it is urgent need to find a new treatment strategy.The method of gene therapy is one of hot spots in cancer research.Tumor invasion and metastasis are very complicated multi-step process,in which a number of structural barriers composed with extracellular matrix and basement membrane must be broken through,and then a new cell colony with exuberant proliferation in distant parts is established.The whole process is precisely controlled by a number of signal transduction pathways and mutual cross-linking of signal transduction network in which each of signal transduction pathways is quite complex.Invasion and metastasis of tumor cells require the capacity of migration of tumor cell itself apart from extracellular protease degradation of surrounding tumor. The occurrence and progress of tumor are the results of out-of-control process of apoptosis and cell cycle regulation.Generally speaking,the cell cycle is divided into pre-DNA synthesis（G1 phase）,DNA synthesis phase（S phase）,DNA synthesis of late（G2 phase） and cell mitosis phase（M phase）.It is interesting to find the process of cell cycle regulation,activation of extracellular protease and movement of cellular have shared many of the same signal transduction pathways.The process of cell movement require complete cytoskeleton which include the plate formation of pseudopodia,the evection and the contraction of cell body.The process of cell cycle also require a complete cytoskeleton apart from require tubulin and cell actin microfilaments to past cross the G1-S phase checkpoint to complete a full cell cycle.Rho protein with GTP activity belongs to Ras superfamily of small molecular weight monomer-binding protein（including Rho,Rac1,and Cdc42）,and plays an important role in the occurrence and development of tumor.In particular,Rho protein and their downstream effect molecules has become a very promising new target for tumor therapy through a multifaceted process involved in a variety of signal transduction pathway such as the malignant tumor transformation,invasion, metastasis and angiogenesis.Rac1（Ras-related C3 botulinum substrate protein） is an important member of Rho family and the role of Rac1 is reported to controll the cell growth,cytoskeletal reorganization and activation of protein kinase.It is reported that Rac1 also plays an important role in cell migration,cell-cell,cell-extracellular matrix adhesion,cell division and cell anoikis.Rac1 protein exists in two forms: inactivation status（Rac1-GDP） and activation status（Rac1-GTP）,and play biological effects based on the cytoskeleton or target proteins as a signal converter or the molecular switch in the cell signal transduction pathway.Tiam1 is an upstream regulatory molecule of Rac1,that is,T lymphoma invasion and metastasis-inducing factor and PAK1 is the downstream effector molecules of Rac1,which is called as P21-activated kinase 1.The different roles of Rac1 are regulated by different signal transduction pathway.It is reported there is closely relationship between Rac1 and certain tumors, which posses the invasion and metastasis.The relationship is showed that:firstly,the relationship is Rac1 and cytoskeleton,Main function of Rac1 is to regulate the reconstruction of actin cytoskeleton and the shaping the muscle actin,myosin contraction and the adhesion of focal adhesion.Some growth factors,such as platelet-derived growth factor（PDGF）,epidermal growth factor（EGF） and insulin can activate the activation of Rac1 to induce the formation actin layer-shaped footplate and the formation of membrane folds.Rac1 protein has played an important role in the process of the collection and reconciliation of actin cytoskeleton,such as the division of cytoplasm,phagocytosis and cell migration.In addition,the function of Rac1 protein is related to calcium-cadherin and E-selectin-mediated cell-cell contacts and integrin-extracellular matrix contacts,cell transformation,reduced coenzymeⅡ（NADPH） oxidase activity,NO generation,phospholipase activity and even cell apoptosis and survival.Secondly,the relationship is the Rac1 and cell apoptosis and cell cycle.Rho family small G proteins regulate the progress of cell cycle and cell movement through direct impacted on gene transcription and the reorganization of cell actin and tubulin.Rac1 is one of the members of Rho family.On the one hand, Rac1 can regulate cyclin D1 in the G1 phase of cell cycle;on the other hand,Rac1 can also regulate the contraction and formation of actin-myosin through Rac1-mediated the myosin light chain phosphorylation modification which result in affecting the formation of actin cytoskeleton.Because of the cross-regulation in the cell cycle and cell movement,the movement of cells can be slowed down by blocking cell cycle progression,which further to illustrates the cell cycle related to the cell movement.Taking into account the important role of the cytoskeleton and cell dynamics in the cell movement and cell cycle progression,it is conceivable that the regulation of cytoskeleton are caused as a result of changes of cell cycle and cell movement,finally,Rac1 take part in the malignant transformation and progress of tumor through regulation signal transducer and activator of transcription-3 and signal transducer and activator of transcription-5 resulting in the regulation of cell migration, proliferation and epithelial to mesenchymal cell transition.We are how to control the activity of Rac1 in order to achieve the purpose of the treatment of tumors.Firstly,Rac1 is inhibited by C-terminal of Rac1 protein isoprenylated.C-terminal of Rac1 protein has a structure of CAAX box,which determines what kind of isoprene residues in it.Protein isoprenylation need the right positioning in cell and GTPase function.Rac1 protein can be modified by farnesyltransferase and geranyl geranyl modification,but the Rho and Cdc42 GTPases can only modified by esterification geranyl geraniol.Secondly,the activity of GEF is inhibited.The best GEF inhibitor is only to inhibit certain Rac1 protein without affecting the activity of other subtypes.Methods for this purpose are the adoption of a selective inhibitor of protein compounds to inhibit the activity of activator protein,as well as by chemical synthesis of drugs with a direct role of Rac1 protein to block the exchange of GDP to GTP,thereby blocking the downstream effector response.Thirdly,the activity of Rac1 effects of protein is inhibited.Rac1 protein regulate different downstream effector produce the different reaction, therefore,the most speciality is the combination of Rac1 and its effects blocked or the inhibition activity of Rac1 protein.RNA interference is to import a section of double-stranded RNA（dsRNA） into cell through experimental means and the host cell respond quickly to the dsRNA so that the dsRNA will be cutted by their endonuclease Dicer in the cytoplasm into much small fragment of RNA with specific cut length and structure which is called siRNA. The siRNA can prevent efficiently particular gene from expression in vivo through promote mRNA degradation of specific genes so that cell-specific gene phenotype is deleted,which meet certain experimental or therapeutic purposes.Therefore,Rac1 mRNA and Rac1 protein were detected by RT-PCR,immunohistochemistry and Western blot methods respectively in tissues of wax block and fresh of colorectal cancer.The preliminary relationship between the Rac1 and colorectal cancer was investigated firstly.Then colorectal cancer cell line were used to be a study object and Rac1 gene was silenced by RNAi,and the changes of cytoskeleton,invasion, movement,and cell apoptosis and cell cycle changes were investigated in colorectal cancer cell after silencing Rac1 gene.It is our objective of this study to provide new ideas for gene therapy of colorectal cancer.PartⅠRac1 expressed in colorectal Tumors and its clinical significanceObjective:To detect the expression of Rac1 mRNA and Rac1 protein in human colorectal cancer tissues,and then to evaluate the relationship between the Rac1 gene and colorectal cancer and the relationship between Rac1 gene and colorectal cancer clinical pathological signidicance.Methods:Diagnosed wax block of colorectal carcinoma,colorectal adenoma and normal colorectal tissues collected of human,Rac1 protein was detected by immunohistochemical in these tissues.Fresh human colorectal carcinoma,colorectal adenoma tissues and normal colorectal tissues collected brfore the expression of mRNA and Rac1 protein detected using RT-PCR and Western blot in above-mentioned tissues.Results:Overexpression of Rac1 protein was found in colorectal cancer tissues, weakly expression of Rac1 did in colorectal adenoma tissues less of that in normal colorectal tissues in wax block tissues.There was significantly difference（x²= 126.69,P=0.000） in Rac1 expression among three colorectal tissues by Kruskal-Wallis Test.Further analysis showed that Rac1 protein is closely correlated with colorectal cancer differentiation,Dukes’ stage and lymph node metastasis（r=0.281, P=0.015;r=0.350,P=0.002;r=0.314,P=0.006）.There is statistically significant （F=1065.84,P=0.000） in the expression of Rac1mRNA among three fresh tissues by One Way ANOVA test.At the same time there is also statistically significant （F=540.88,P=0.000） in the expression of Rac1 protein among three fresh tissues by One Way ANOVA test.Conclusion:Rac1 protein can be used as valuable indicators to evaluate the degree of malignancy in colorectal cancer;Rac1 protein closely correlated with colorectal cancer differentiation,invasion depth,Dukes’ stage and lymph node metastasis and had no correlation with the patient’s age,sex,tumor location and tumor size.PartⅡExpression of Rac1 in colorectal cancer cell and the const-ruction of stable LoVo cell line with silenced Rae1Objective:To detect the expression of Rac1 mRNA and protein in human colorectal cancer cell lines,to build a stable LoVo cell line transfected with Rac1-shRNA plasmid expression vector,and to explore the relationship between Rac1 gene and human colorectal cancer cell proliferation in vitro.Methods:The expression of Rac1 mRNA and Rac1 protein was detected by RT-PCR and Western blot in five kinds of cell lines of colorectal cancer（LoVo,SW480, SW620,SW1116 and HT29）.A stable LoVo cell line transfected with Rac1-shRNA plasmid expression vector was constructed.The proliferation of LoVo^Rac1-shRNA cells were evaluated by MTT test and cell plate cloning experiment. Results:Both Rac1mRNA and Rac1 protein had high expression in five kinds of colorectal cancer cells.A stable LoVo cell line transfected with Rac1-shRNA plasmid expression vector was constructed successfully.The expression of Rac1 mRNA and Rac1 protein were decreased in transfected LoVo cells detected by RT-PCR and Western blot respectively.MTT test and cell plate cloning experiments showed that the proliferation of LoVo cells transfected with Rac1-shRNA plasmid expression vector became slow.Conclusion:Rac1 protein was over expressed in five colorectal cancer cell lines; Rac1 gene can promote the proliferation of LoVo cells in vitro.The proliferation of LoVo cells was slowed down after silencing Rac1.PartⅢThe relationship of Rac1 and invasion and metastasis in LoVo cellsObjective:To detected the expression of Rac1 mRNA and Rac1 protein in LoVo cell lines transfected with Rac1-shRNA plasmid.To observed LoVo cell morphology and cytoskeleton changes in colorectal cancer cell lines after Rac1 gene silenced.To evaluated the invasion and migration of changes of LoVo cell on condition of Rac1 gene silenced and enhanced respectively.Methods:The expression of Rac1 mRNA and Rac1 protein were detected by RT-PCR and Western blot in LoVo cells transfected with Rac1-shRNA plasmid.The Factin and cell changes in pseudopods of transfected LoVo cells with Rac1-shRNA vector plasmid were observed by confocal microscopy.The invasion and migration of changes in LoVo cells were observed by Transwell and Wound healing test in LoVo cells were transfected with Rac1-shRNA,Rac1 dominant negative（Rac1-N17） and Rac1 activation plasmid（Rac1-L61） respectively.Results:The expression of Rac1 mRNA and Rac1 protein was decreased in LoVo cells transfected with Rac1-shRNA.Compared with the control group,LoVo cells transfected with Rac1-shRNA showed that cross-linked F-actin network significantly were reduced,disordered,and the cell membrane pseudopod did or disappered. Monolayer scratch after 24 h,compared with the control group,the number of cell migration was obviously diminished in Rac1-shRNA group and Rac1-N17 group while increased cell migration in Rac1-L61 group.The numbers of cell migration in each group separately are 106±8 in Rac1-shRNA group,117±11 in Rac1-N17 group,369±15 in Rac1-L61 group and 312±12 in control group.There is statistically significant（F=146.4,P=0.000） in the number of cell migration by One Way ANOVA test among three groups.The numbers of cell invasion experiments in each group were counted as follow:34±4 in Rac1-shRNA group,42±5 in Rac1-N17 group,86±7 in Rac1-L61 group and 73±6 in control group.There is also statistically significant（F=48.55,P=0.000） in the number of cell invasion by One Way ANOVA test among three groups.Conclusion:Rac1 gene silenced in LoVo cells via RNAi resulted in follow:It inhibited the formation of cross-linked network of F-actin and pseudopod of cell membrane in LoVo cells.It inhibited the invasion and migration of of LoVo cells in vitro.Rac1 gene up-regulated via L61 plasmid enhanced the invasion and migration in LoVo cells.PartⅣThe relationship of Rac1 and cell cycle and apoptosis in LoVo cellsObjective:To detected the expression of Rac1 mRNA and Rac1 protein in LoVo cell lines transfected with Rac1-shRNA plasmid.The relationships between Rac1 gene and cell cycle and between Rac1 gene and apoptosis after deleting Rac1 were exploredMethods:The expression of Rac1 mRNA and Rac1 protein were detected by RT-PCR and Western blot in LoVo cells transfected with Rac1-shRNA plasmid.The change of LoVo cell cycle was observed by flow cytometry with single-PI staining method.LoVo cell apoptosis was detected by flow cytometry using Annexin V-FITC & PI double dying methods.Results:The expression of Rac1 mRNA and Rac1 protein was decreased in LoVo cells transfected with Rac1-shRNA.LoVo cells transfected with Rac1-shRNA plasmid showed that cell cycle delay in the G₀-G₁ phase,The numbers of cells in G₀-G₁ phase were significantly increased（74.63±4.40） while reduced in S period （13.20±1.77） detected by flow cytometry.By One Way ANOVA analysis,the difference was significant（F=26.256,P=0.001） among the three groups in the G1 phase;There is also statistically significant differences（F=78.893,P=0.000） at S phase.Apoptosis rate of LoVo cells was 0.251±0.026 in group transfected with Rac1-shRNA plasmid detected by flow cytometry while was 0.094+0.011 and 0.098+0.013 in control and mock group.By One Way ANOVA analysis there was statistically significant difference（F=62.786,P=0.000） in apoptosis rate of LoVo cells among the three groups.Conclusion:Rac1 protein was not expressed by RNAi deleted Rac1 gene in LoVo cells.The progression of cell cycle was inhibited and LoVo cells were blocked in the G0-G1 phase after silencing Rac1 gene.Apoptosis of LoVo cells were increased with deleted Rac1 via RNAi.