The Role And Molecular Mechanism Of Cortactin In Proliferation And Metastasis Of Human Colorectal Cancer

OBJECTIVE(1)To assay the expression level of Cortactin(CTTN)in colorectal cancer tissues,and investigate the tumor biological function and molecular involved when the expression of CTTN up-regulate or down-regulate in colorectal cancer cells.(2)To explore the molecular mechanism of how the modulation and activation of CTTN was regulated in colorectal cancer cell.METHODS(1)We used the real-time qualified RT-PCR to detect the expression level of CTTN in 61 pairs colorectal cancer tissue samples.(2)The proliferation and motion ability of colorectal tumor cells were evaluated by CCK-8 assay and transwell assay respectively with the aid of siRNA targeting CTTN.(3)The CTTN stable expression or suppression cell line were established by lentiviral transfection.The effect of CTTN overexpression on colorectal cancer cell proliferation,migration,invasion and clonogenic ability were assayed by CCK-8 assay,transwell assay,and clone formation experiments.In vivo tumour xenotransplantation and nude mouse lung or live metastasis model were also performed to examine the effect of CTTN inhibition on colorectal cancer cell proliferation and metastasis.(4)The addition of EGF into medium to detected the proliferative effect of the upregulation of CTTN.And we used the CST-PathScan array to explore the downstream of EGFR signaling pathway.(5)The enhanced phosphorylation of ERK by the overexpression of CTTN was verified by WB.(6)By biological information analysis we presumed that PZR and CTTN may interact with each other,and the co-immunoprecipitation(CoIP)experiment further indicated these two proteins can associate with each other.(7)The tumor biological function of PZR in colorectal cancer was assayed by CCK-8?Transwell and clone formation experiments with the aid of RNAi or lentivirus plasmid.(7)The PZR expression in colorectal tumor tissue of 61 patients was detected by qRT-PCR.And the relationship between PZR expression and pathological parameters was analyzed with?~2 test.(8)The identification of PZR targeted molecules was found by WB.RESULTS(1)In our study CTTN positive associated with pathologic stage and lymph node metastasis in CRC.(2)CTTN promoted colorectal cancer cell migration,invasion and proliferation in vitro and in vivo.(3)CTTN increased the expression of EGFR,the phosphorylation level of ERK and potentized the proliferative effect of EGF.(4)CTTN may be involved in the EGF-induced EGFR downregulation.(5)PZR can interact with CTTN,and promoted the proliferation and motion of tumor cell.(5)The suppression of CTTN inhibited the biological fucnction of PZR(5)The expression of PZR increased the phosphorylation level of CTTN and ERK(6)The expression of PZR are increased in colorectal tumor tissue samples,and positive associated with TNM stage and lymph node metastasis.CONCLUSIONSCTTN and PZR were overexpressed in colorectal tumor and promoted colorectal cancer cell motion and proliferation by upregulate the expression of EGFR and phosphorylation level of ERK.PZR enhanced the biological function and phosphorylation level of CTTN.