The Function And Mechanism Of LncRNA-DANCR On Promoting Colorectal Tumor Growth

Introduction:Colorectal cancer(CRC)is the third most common type of cancer and the fourth leading cause of cancer-associated mortality worldwide.Multiple factors are involved in the occurrence and development of colon cancer,including uncontrolled cell proliferation,angiogenesis and suppression of apoptosis.Long non-coding RNAs(lncRNAs)have no protein-coding abilities but may act as key regulators to control biological and pathological processes.lncRNA differentiation antagonizing non-protein coding RNA(DANCR)functioned as a oncogene in diverse cancers.However,the biological functions and significance of DANCR in colon cancer have not yet been established.Methods:We collected 20 pairs of normal tissues and cancer tissues adjacent to colorectal cancer,and 84 colorectal cancer tissues containing 5-year survival of patients in our hospital.Then,RNA-FISH was used to determine DANCR in colorectal cancer(CRC)tissues and adjacent normal tissues.Furthermore,the correlation between DANCR and patient prognosis was analyzed based on the expression of DANCR.Quantitative PCR was used to detect the expression of normal human colorectal epithelial cells and multiple colorectal cancer tumor cells,while RNA-FISH was employed to detect the distribution of DANCR in CRC cells.To investigate the functional role of DANCR on the growth of colorectal cancer cells,we employed lentiviral-mediated shRNA targeting DANCR to infect SW480 and HCT15 cells.And CCK-8 experiments and clone formation experiments were performed in vitro.Apoptosis was determined by flow cytometry and Hoechst staining in SW480 and HCT15 cells that were stably infected with lenti-shDANCR.In in vivo study we established a subcutaneous xenograft model using SW480 and HCT15 cells that stably knocked out of DANCR,and then plotted tumor growth curves and calculated tumor weight.On the basis of clarifying the role of DANCR in promoting the growth of colorectal cancer,Western blotting was performed to detect the expression of Caspase 3,Caspase 8,Caspase 9,P53,Cyto C,Bcl-2 and Bcl-xL in SW480 and HCT15 cells.Furthermore,the expression of Caspase 3,p53 and cyto C in SW480 and HCT15 subcutaneous xenografts was also detected by immunohistochemical staining.Results:1)DANCR upregulated in CRC tissues and correlated with poor prognosis of patients:RNA-FISH results of 20 pairs of colorectal cancer tissues and adjacent normal tissues indicated that DANCR is highly expressed in colorectal cancer tissues;DANCR was highly expressed in colorectal cancer tumor cells and mainly located in cytoplasm;further survival analysis indicated that patients with colorectal cancer with high DANCR expression are often accompanied with poor prognosis(5-year survival).2)Knockout of DANCR inhibited CRC cell growth by promoting apoptosis:Stably infection of SW480 and HCT15 cells by lentiviral-mediated shDANCR significantly inhibited DANCR expression in SW480 and HCT15 cells;stable knockout of DANCR expression significantly inhibited the proliferation and clonal formation of CRC,and promoted the apoptosis of CRC cells.Knockout of DANCR expression impaired CRC tumor growth via promoting tumor cell apoptosis in the subcutaneous transplantation of colorectal cancer in vivo.3)Knockout of DANCR promoted the expression of mitochondria-associated apoptotic proteins and p53 protein:knockout of DANCR promoted the expression of Caspase 3,Caspase 8 and Caspase 9 proteins in SW480 and HCT15 cells,and more Caspase 3 positive cells were observed in SW480 and HCT15 subcutaneous xenograft tumor tissues that were stably infected lenti-shDANCR.Knockout of DANCR promoted the expression of p53 proteins in SW480 and HCT15 cells,and more p53 positive cells were observed in SW480 and HCT15 subcutaneous xenograft tumor tissues that were stably infected lenti-shDANCR.Knockout of DANCR promoted the expression of Cyto C protein and inhibited Bcl-2 and Bcl-xL expression in SW480 and HCT15 cells,and more Cyto C positive cells were observed in SW480 and HCT15 subcutaneous xenograft tumor tissues that were stably infected lenti-shDANCR.Conclusion:In the present study,it was demonstrated that lncRNA DANCR was overexpressed in malignant tissues of CRC patients.Furthermore,the expression of DANCR was inversely correlated with the prognosis of patients with CRC.RNA-FISH indicated that DANCR mostly located in cytoplasm of human CRC cancer cells.Silencing DANCR significantly inhibited cell proliferation,colony formation and tumor growth via inducing mitochondria-related apoptosis.The present study provides evidence of the functional role and mechanism of DANCR in colon cancer,which suggested that DANCR would be a potential therapy and prognosis predicting target for CRC.