Canopy2(CNPY2),A Novel Secreted Protein,Influence The Growth And Development Of Colorectal Carcinoma

Objective:The morbidity and mortality of tumor diseases have increased consistently in the world. Angiogenesis is a major cause to support the growth and development in tumor.Normally, there is a balance between pro-angiogenesis and anti-angiogenesis to maintain the demands of blood in normal organs and tissues. During the progression of tumor, the growth of tumor tissues is enhanced excessively and parted with abundant angiogenesis for supplying the demands of nutritive materials. Proangiogenic factors have a crucial role in the development of tumor. Recently there is a report that CNPY2 is a novel secreted pro-angiogenic factor that promotes the angiogenesis from the aorta ring. It suggests CNPY2 might involve the etiology of tumor by improving the angiogenesis to promote the growth and invasion of tumor. Colorectal carcinoma(CRC) is a main type of human tumor diseases. The morbidity and mortality of CRC are the 3rd and 4th in all tumor disease,respectively. Same to other tumors, the mechanism of CRC is still unclear. We have defined the expression of CNPY2 in CRC patients and it has shown the levels of CNPY2 in the cancer tissues and plasmas from patients are increased significantly than the para-normal tissues and healthy control. It suggests that CNPY2 involves the growth and development of CRC but it should to be explored in further.Hence, this study was focus on the role of the novel secreted CNPY2 in the growth and development of CRC.The effects of CNPY2 in the human colorectal tumor cell 116(HCT116) was evalued by determination of proliferation, migration/invasion and apoptosis in vitro and in vivo by injection cells into nude mice to make the xenografts.Methods:1.Determination the levels of CNPY2 in the CRC patientsThe samples of tissues were harvest from the CRC patients when they were treated by surgery randomly. The tumors and para normal tissues were harvested separated. Moreover,the plasmas of patients before the surgeries and healthy control were also collected. The RNAs and proteins in tissues were abstracted for RT-PCR and WB assays, respectively.The tissues also were fixed by formalin solution for IHC staining of CNPY2. ELISA was performed to measure the levels of CNPY2 in the plasmas from CRC patients and healthy controls.2.Establishment the stable transfected plasmid-sh RNA HCT116 cell linesThe plasmids of p RFP-C-RS-sh RNA(short hairpin RNA)-human CNPY2 and–control were transfected to HCT116, respectively. After 21 days with the selection of puromycin and limited dilution, the stable transfected cell lines with sh RNA-CNPY2 and sh RNA–control were established, which were named HCT116 sh RNA-CNPY2 and HCT116 sh RNA-control, respectively.3.Effects of CNPY2 in the bioactivities of HCT116 in vitroBy using the HCT116 sh RNA-CNPY2 and HCT116 sh RNA-control, the growth, migration and apoptosis were determined with CCK8, colony formation, scratch assay and flow cytometry of Annexin V and PI double staining, respectively.4.Effects of CNPY2 in the xenografts in vivoThe HCT116 sh RNA-CNPY2 or HCT116 sh RNA-control was injected subcutaneous into the underarm of nude mice. The growth of xenografts were observed and recorded. After 21 days, the xenografts were harvested. The weight and volume of xenografts were meansured.Then the immunohistochemical staining of proliferating cell nuclear antigen(PCNA),CD105 and TUNEL were performed to value the proliferation, angiogenesis and apoptosis of xenografts, respectively.Results:1.The levels of CNPY2 were significant increased in the tumor tissues than para-normal tissues of CRC patients(P<0.01). The levels of CNPY2 in the plasmas from patients were increased dramatically as folds as 1.72 than healthy controls.2.Knocking down the endogenouse CNPY2 gene expression in HCT116 resulted in the inhibition of cellular growth and migration whereas enhance the apoptosis induced by oxaliplatin.3.In the xenografts,knocking down of CNPY2 prevented the growth and angiogenesis of tumor but promote the apoptosis in vivo.Conclusion:CNPY2 might act a crucial role in the growth and development of CRC by enhancing the proliferation, migration, angiogenesis and preventing the apoptosis. Here we demonstrated the novel secreted CNPY2 involved the bioactivities of HCT116 and directed the studies for exploring the mechanisms of CNPY2 in CRC. The increasing levels of CNPY2 in the tumor tissues and plasmas presents a highlight of CNPY2 to be a novel target of therapy and biomarker of prognosis in CRC, especially in the CRC patients who are resistant the chemotherapy in terms of knockingdown CNPY2 improving the apoptosis induced by oxaliplatin.