The Effect Of ANXA2 On The Development Of Colorectal Cancer And The Preliminary Study Of Signal Pathway

Background and ObjectiveColorectal cancer is one of the most common gastrointestinal cancer. It is a complicated pathological process in which multi gene and multi risk factors were involved and the incidence is increasing year by year. According to the summation of prior research, current clinical trials of colorectal cancer include surgery, chemotherapy, radiotherapy, thermotherapy, immunotherapy and traditional Chinese medicinal therapy, in which surgery is prior to others. While colorectal cancer is a malignancy with a 5-year survival of 50% due to poor diagnosis, metastasis, resistence to chemotherapy and toxicity. Thus the need for early diagnosis and advanced therapy is crucial. It is well known that Annexin A2 (ANXA2) is a tumor associated gene correlates with the oncogenesis and development of malignancies. Annexin A2 (ANXA2) is ubiquitously expressed in various cancers and might be involved in the process of cell proliferation, migration and invasion of tumors. Gene deletion and gene inhibition are most commonly utilized approaches in the study of gene function, eapecially the former is one of the most useful and frequently used genome editing technologies with high efficiency and specificity in the elimination of target gene expression.Therefore, ANXA2 deleted caco2 cell line were obtained by employing traditional gene knockout technology in this study. And the expression of ANXA2 deleted caco2 cell line was examined before we determine the effects of ANXA2 on colorectal cancer. To further investigate the role of ANXA2 played in caco2 cell line, the cell proliferation and migration were detected in cells without ANXA2 expression. In addition, two-demensional electrophoresis and Mass spectrometry analysis were utilized so as to further explore the changes of proteome between ANXA2 deleted caco2 cell line and wild type of caco2 cell line, which could contribute to the further investigation of ANXA2 associated genes and signal pathways involved in the development and pathology of colorectal cancer as well as the role of ANXA2 in the process of colorectal cancer development.Furthermore, tPA is a gene whose fuction is reported to be relevant to the ANXA2, to explore the roles tPA played during the development of colorectal cancer, we trying to construct a tissue plasminogen activator (tPA or PLAT) deleted cell line by employing transcriptional activator-like effector nucleases (TALEN), and expect to explore the interanction and potential regulatory mechanism of both gene in colorectal cancer development by comparing the ANXA2 deleted caco2 cell line and tPA deleted caco2 cell line, which might bring new theoretical and experimental prospectives and ideas for diagnosis and therapy of colorectal cancer.Methods1. Identification of ANXA2 deletion in caco2 cell line (ANXA2-/- caco2) (Western blot assay).2. Determination of cell viability and cell proliferation in ANXA2 deleted caco2 cell line at different time points (MTT assay, Inverted fluorescence microscope imaging system).3. Detection of the cell motility and migratory ability in ANXA2 deleted caco2 cell line at different time points (Wound healing assay, Transwell chambers assay).4. Utilization of proteomics approaches to study protein expression changes in caco2 cell line without ANXA2 expression (Two-Demensional electrophoresis, Mass spectrometry analysis).5. Construction of tPA deleted caco2 cell line (TALEN).Results1. The deletion of ANXA2 was further confirmed by the Western blot and no ANXA2 expression was detected in ANXA2 deleted caco2 cell line.2. MTT assay revealed that the ability of cell proliferation was significantly decreased in the ANXA2 deleted caco2 cell line (**P< 0.01).3. The results of Wound healing assay indicated that the migratory ability of the cells was decreased markedly in ANXA2 deleted caco2 cell line, especially when it was 48h after the wound were made (*P< 0.05). The transwell chanbers assay consistently showed that ANXA2 significantly enhanced the migration ability of caco2 cell line illustrate that 11 protem were down regulated and 4 protein were up regulated in ANXA2 deleted cell group compared with wild type cell group. The mass spectrometiy analysis indicated that the 15 proteins were actin cytoplasmic 1 (ACTB)、actinin alpha 4 (ACTN4)、keratin type I cytoskeletal 10(KRT10)、BiP protein (HSPA5)、alpha-tubulin (a-tubulin)、mitochondrial heat shock 60kD protein 1 variant 1 (HSPD1)、thyroid hormone binding protein precursor (p55)、mitochondrial ATP synthase H+transporting F1 complex beta subunit (ATP5B)、Human Enolase 1 (Henol)、Keratin 18 (KRT18)、 Keratin 19 (KRT19)、cyclin-dependent kinase 7 (CDK7)、alpha-tubulin (a-tubulin)、 protein disulfide isomerase-related protein 5 (PDIA 5、keratin 1 (KRT1), among which 11 proteins were down regulated and 4 proteins were up regulated after ANXA2 deletion.5. During the construction of tPA deleted caco2 cell line by utilizing TALEN,10 monoclones were obtained from tPA TALEN trasfected caco2 cerls. While the sequencing result showed that there were no mutations occurred in the genome sequence of all monoclones, that is to say we failed to construct a tPA deleted caco2 cell line by utilizing this TALEN kit, and the kit was supposed to be further optimized.ConclusionANXA2 is closely relavent to the growth, proliferation and migration of colorectal cancer caco2 cell line. The results showed that the cell proliferation and migratory ability of ANXA2 deleted caco2 cell line were significantly decreased compared with the wild type of caco2 cell line, suggested that ANXA2 maybe a novel target for gene therapy or mediacal therapy in colorectal cancer. Besides, the result of two-demensional electrophoresis revealed the role ANXA2 played in the proteomics of colorectal cancer. The results also provide a novel theoretical and experimental prospective for a further study on mechanism of protein interactions and gene function during the oncogenesis and development of colorectal cancer. In addition, the deletion of tPA by utilizing TALEN were failed to construct a tPA deleted colorectal cancer caco2 cell line, which indicated that the kit and the process of TALEN still needed optimization for the construction of a tPA deleted cell line.