The Roles And Molecular Mechanism Of Polarity Protein Par3 On Tumor Growth And Metastasis In Colorectal Cancer

Part1 The Expression and Clinical Pathological Significances of Par3 in Colorectal cancerObjective:To study the expression of Par3 in CRC tissues and analyze the correlation of Par3 expression and clinicopathologic features of CRC.Methods:A total of 45 pairs of CRC tissue samples including cancer tissues and corresponding adjacent tissues were collected from Gastrointestinal surgery of Union Hospital during Sep.2014 and Dec.2016.Real time PCR?western blot and IHC were used to detect the expression of Par3 in CRC tissues and their corresponding adjacent tissues.Moreover,the correlation of Par3 expression and clinicopathologic features including TNM stage,patients age,gender,tumor size and distant metastasis was analyzed.Finally,the data from TCGA database was used to analyze the prognostic value of Par3 expression in CRC by Kaplan-Meier.Results:The results of qRT-PCR and Western blot showed,the expression of Par3 in CRC tissues was lower than in the corresponding adjacent tissues(P<0.05).Furthermore,the results of IHC showed,compared with the CRC tissues,the expression of Par3 in normal tissues was strong(P<0.05).Loss of Par3 was positively related with T stage and N stage(P<0.05),but not with distant metastasis,tumor differentiation,patient age and gender(P<0.05).Compared to the patients with Par3 positive expression,the rate of 5-year OS and DFS was decreased in CRC patients with Par3 negative expression(P<0.05).Conclusions:Par3 is down-expressed in CRC tissues.Loss of Par3 correlates with the T stage,N stage and poor prognosis in CRC patients.Part2 The effects of par3 on the malignant behaviors of colorectal cancerObjective:To study the effects of polarity protein Par3 on cell growth,migration and invasion of CRC cell lines.Methods:Lentiviral vector with Par3 shRNA was transfected into SW480 and HCT116 cells to establish stable cell line with down regulation of Par3.Then,CCK8 assay,Colony-forming assay,Transwell assay,capillary tube-like formation assay and tumor formation in nude mice were used to study the effects of Par3 on malignant behaviors of CRC cells.Results:The results of western blot and qRT-PCR showed sh-Par3 obviously decreased the expression of Par3 in SW480 and HCT116 cells(P<0.05).The cell growth assays revealed that Par3 silencing increased CRC cell growth in vivo and in vitro(P<0.05).The results of Transwell assay and Pulmonary metastasis assay showed down-regulation of Par3 obviously increased the ability of cell migration and invasion(P<0.05),Moreover,loss of Par3 promoted tube formation of tumor in vivo and in vitro(P<0.05).Conclusions:Loss of Par3 obviously promotes cell growth,tube formation,cell migration and invasion in CRC cells.Part3 The mechanism of Par3 regulating malignant behaviors of colorectal cancer cells via MYH9Objective:To explore the molecular mechanism of Par3 regulating malignant behaviors of colorectal cancer cellsMethods:After lentiviral vector with Par3 shRNA was transfected into SW480 and HCT116 cells,western blot,qRT-PCR and Co-IP were used to detect the effects of Par3 on MYH9 expression.SiMYH9 was transfected into cells with Par3 down-expression.Then,CCK8 assay,Colony-forming assay,Transwell assay and immunofluorescence assay were used to study the effects and molecular mechanism of Par3/MYH9 complex on cell growth,migration and invasion in CRC cells.Results:In SW480 and HCT116 cells,loss of Par3 up-regulated the expression of MYH9(P<0.05).The result of Co-IP assay revealed Par3 could bind directly to MYH9.The CCK8 and colony formation assays showed down-regulation of MYH9 decreased the cell growth of cells lacking Par3(P<0.05).Meanwhile,MYH9 silencing inhibited cell migration and invasion by Transwell assay(P<0.05).The results of western blot and immunofluorescence assay showed loss of Par3 down-regulated the expression of tight junction protein Zo-1 via up-regulating MYH9 expression.Moreover,Par3 silencing promoted the expression and phosphorylation of Stat3.Conclusions:In CRC cells,loss of Par3 promotes cell growth,migration and invasion via up-regulation of MYH9.Meanwhile,Par3/MYH9 complex regulates the formation of tight junction and the activation of Stat3,which are involved in the development of malignant behaviors of colorectal cancer cells...