Study On The Mechanism Of PNN In The Invasion And Metastasis Of Colorectal Cancer

Background:Colorectal cancer occurs secondly in female and thirdly in male according to the globally epidemiological survey.unfortunately,the incidence of colorectal cancer,increased in our country,gradually increased towards the incidence of Europe and the united states.It is known to all that the metastasis of colorectal canceris closely related to the prognosis of patients.It is a pity that about 20%?40%cases are diagnosed with metastasis when they come to hospital.What is more,the metastasis is detected in liver even when patients receive radical operations and the incidence is about50%.The mortality of patients who undergo colorectal cancer with liver metastasis reaches as high as 45%?71%.Consequently,the 5-year survival rate of colorectal cancer has been hovering around 50%even when patients get radical surgical operation treatment.Therefore,it is extremely urgent to strengthen the basic research to understand the development mechanism of colorectal cancer and to find a reliable prediction index and effective therapeutic targets and means.And it is important to improve the cure rate of colorectal cancer as well as reduce the mortality rate.Tumor metastasis is a complex process which contains variation with multi-step and multi-stage.Each stage is influenced by many kinds of genes and proteins.Current studies suggest that metastatic tumor cells experienced a series of changes such as loss of adhesion characteristics,enhancement of athletic ability,expression of proteolytic enzymes and the formation of new blood vessels.when the metastasis occur,the enhancement of kinetism of tumor cell is essential for its invasion and metastasis.It is revealed that accurately changes of the shape and motion behavior of tumor cells happen when the cell get stimulationat any time.The tumor cell becomes flat or rounded,changesits polarity,becomes elongation and contraction or loses the connection with the surrounding cells.All those dynamic activities are required for the recombination of actin and tubulin.Pinin(PNN)also known as DRS,DRSP,SDK3,memA,located in 14q21.1,contains 9 exons sequences,only one transcript,and the CDS(coding sequence)length of 2154 base pairs(base pair bp).PNN is the earliest discovered as a desmosome related molecules(desmosome-associated molecule).It does not participate in the composition of desmosomal proteins,but has a close relation with mature desmosome.It is revealed that the protein PNN was vertical arrangement with filamentous keratin.Although it does not participate in binding of the intermediate filament and desmosomes,it can strengthen the connection of desmosomal proteins and intermediate filament and strengthen the adhesion between cell and cell.The protein PNN is not only detetable in the combination foci of desmosomes but also in the nucleus.Recenly,study assumed that PNN was tumor suppressor based on the analysis of structure of PNN.Moreover,it is suggested that PNN increased the adhesion of cell by interacting withthe E-box domain of E-cadherin and reverse CtBP-1 induced repression of E-cadherin expression.In addition,PNN is also involved in the process of shear precursor mRNA.As a member of the SR family,PNN can play different shear function through interaction with members of the other SR family.Here we put great emphasis on the studies which focus the the prediction of the possible role of PNN as a tumor suppressor through analysis its location and\structure.Another study reveals that PNN may play an important role in cell adhesion.It is these two points that indicate PNN may make great influenceson tumor occurrence and development.However,the exact influence of PNN on colorectal cancer is not detected,and the function of PNN in proliferation,invasion and metastasis was not reported previously.So the impact of PNN on colorectal cancer need to be further revealed.In this study,firstly,we will detect the mRNA level of PNN in normal and paired tumor tissue and analyze the expression of PNN in various tumor based on the GEO database.Secondly,we will detect the influence of PNN on colorectal cancer cell proliferation,invasion and metastasis by knocking down and overexpressing PNN in colorectal cancer cell in vitro and in vivo.Thirdly,we analyze the mechanism of PNN which is involved in the invasion and metastasis of colorectal cancer.Lastly,we compare PNN expression in normal and paired tumor paraffin embedded tissues and analyze the relationships between PNN level and clinical pathology tumor.All above,we may reveal the function and mechanism of PNN in colorectal cancer and provide a new target for the prognosis judgment and treatment of colorectal cancer.Objective:1.To reveal the function and mechanism f PNN in colorectal cancer both in vivo and in vitro.2.To detect PNN expression in normal tissue and paired tumor tissues of colorectal cancer and evaluate the value to predict the prognosis of colorectal cancer.3.To further clarify the mechanism and characteristics of colorectal cancer metastasis and provide new indicators and targets for the diagnosis and treatment of colorectal cancer.Methods:1.We detected the expression of mRNA expression of PNN in in normal tissues and the paired tumor tissues by real-time quantitative PCR technology and analyzed the relationship of PNN and tumor stage,differentiation and metastasis.2.We compared the expression of PNN in various malignant tumors based on the public microarray database(GEO database).3.In vitro experimental study was carried out to examine the impact of PNN on cell proliferation,clone formation,invasion and migration ability.4.Subcutaneous tumor formation and the tail vein injection experiments were carried out to detect the influence of PNN on tumor growth and metastasis that based on establishment of stable PNN interference and overexpression cell line.5.We then tried to evaluate the E-ca,DSG2,EGFR,p-EGFR,ERK variation in cell transfected with PNN-SiRNA and PNN overexpression plasma.6.Based on cell transfection technique combined with ERK pathway inhibitor treatment,cell with high expression of PNN were stimulated with ERK pathway inhibitor.Then,cell proliferation,clone formation,invasion and migration were detected.7.Expression of PNN in normal tissue and paired tumor tissue was detected bywestern blot expression.8 PNN expressions in normal tissue and paired tumor tissue in normal paraffin embedded tissue and paired tumor tissue by immunohistochemical and the relationship between PNN expression and T classification,differentiation,metastatic status and prognosis was analyzed.Results:1.mRNA expression level of PNN in colorectal cancer tissues(1)mRNA expression level of PNN in normal tissue and paired tumor tissues was evaluated by real-time quantitative PCR,the expression of PNN in tumor tissue is higher than that in normal tissue.(2)mRNA expression level of PNN is significantly correlated with T classification and metastatic status of tumor patients.2.Analysis of the expression of PNN in colorectal cancer and other tumor tissues based on public microarray database.(1)PNN showed a higher expression in colorectal cancer compared to the paired normal tissue based on the GEO database analysis.What is more,the expression of PNN is close to the metastatic status of patients with colorectal cancer.High expression of PNN in colorectal cancer showed a poor prognosis in 177 cases of colorectal cancer patients.(2)GEO database analysis results showed that the expression of PNN in tumor tissues are higher than that in normal tissue in gastric cancer,pancreatic cancer,prostate cancer and gastric cancer and is close to metastasis.3.Effect of interference and overexpression of PNN on biological behaviors of colorectal cancer cells.(1)The proliferation,clone formation,migration and invasion ability was suppressed in colorectal cancer cell line SW620 transfected with PNN interference RNA.(2)The proliferation,clone formation,migration and invasion ability was promoted in colorectal cancer cell line SW480 transfected with PNN overexpression plasma.4.Effect of interference and overexpression of PNN on tumor growth and metastasis in nude mice.(1)The tumor growth and metastasis ability were suppressed in colorectal cancer cell line SW620 transfected lentivirus with interference in nude mice.(2)Overexpression of PNN promotes subcutaneous tumor growth and metastasis in nude mice.5.Downstream regulatory mechanism of PNN.(1)Expression of DSG2 is suppressed in colorectal cancer cell line SW620 transfected with PNN SiRNA and in whichEGFR/ERK signal pathway is inhibited as well.(2)Expression of DSG2 is elevated in colorectal cancer cell line SW480 transfected with PNN overexpression plasma and in which EGFR/ERK signal pathway is activated as well.(3)ERK signaling pathway in the cells was significantly inhibited in PNN overexpression cell stimulated with ERK signaling pathway inhibitor compared with the control group which was stimulated with DMSO.(4)ERK signal pathway inhibitorinhibits the cellular biological behavior changes induced by PNN.6.The relationship between the expression of PNN and clinical pathology.(1)PNN shows a higher expression in fresh tumor tissues compared to that in matched normal fresh tissue.(2)Expression of PNN is higher in paraffin embedded colorectal cancer tissues than that in normal tissuesand levels of PNN is correlated to depth of tumor invasion,lymph node metastasis and distant metastasis.(3)Patients with high expression of PNN in colorectal cancer tissues show a poor prognosis.Conclusion:1.PNN expression is significantly increased in gastric cancer,pancreatic cancer,prostate cancer,ovarian cancer and colorectal cancer tissues.There is a close relationship between PNN expression levels and the prognosis of patients with colorectal cancer.2.The expression of PNN is associated with cell proliferation,clonal formation,migration and invasion abilities in vitro.In vivo PNN expression is associated with tumor growth and metastasis in nude mice.3.PNN promotes the expression of DSG2 so as to activate EGFR/ERK signaling pathway.4.The expression level ofPNN in tumor tissue of colorectal cancer patients is close to T stage,lymph node metastasis and distant metastasis andcan be used as a molecular markers for prognosis.