Effect Of Neuropilin-2 On Biological Behavior Of Colon Cancer Cell Line HT-29

Objectives To evaluate the effect of Neuropilin-2(NRP-2)silencing on HT-29 such as proliferation,apoptosis and other biological behavior changes.Methods The fluorescent siRNA was transfected to HT-29 cell line by liposome.After 48 hours of conventional culture,the transfection efficiency of HT-29 cell lines was observed by fluorescence microscope.The colon cancer cell was divided into three groups,which can be named as transfection group(transfer siRNA-NRP-2),meaningless sequence group(transfer nonsense sequence)and blank control group(transfer PBS).Using quantitative real-time PCR(qPCR)to detect the expression of NRP-2 mRNA in different groups of cells.Acridine orange/propranidine iodide(AO/PI)method was used to detect the apoptosis of HT-29 by dye staining.The OD value of living cells in each group was measured by MTT to evaluate cell proliferation.The expression of proliferation-associated protein Ki-67 in different groups of cells was detected by immunocytochemical staining.The scratch healing rate of different groups of cells was detected by the scratch repair experiment of monolayer cells to determine the migration ability of the cells.The Transwell chamber invasion assay determines the invasiveness of tumor cells by measuring the number of tumor cells which are traversing the membrane.Every experiment was repeated 3 times in each group.Results 1 Fluorescent siRNA was transfected into HT-29 cells with liposome Lipofectamine 2000 for 48 h.The transfection efficiency of the cells was over 80% under fluorescence microscope.2 The results of qPCR showed that the relative expression of NRP-2 mRNA in transfection group was significantly lower than which in meaningless sequence group and blank control group,and the difference was statistically significant(P < 0.05).3 The results of double staining of acridine orange/pyridine iodide showed that the apoptosis level of transfected cells was significantly higher than meaningless sequence group and blank control group,and the difference was statistically significant(P < 0.05).4 The results of MTT showed that compared with meaningless sequence group and blank control group,when the cells were cultured for 24 h,48h and 72 h,the proliferation ability of transfected group cells was time dependent,and the difference was statistically significant(P < 0.05).5 The results of immunocytochemical staining showed that compared with the meaningless sequence group and the blank control group,the expression of Ki-67 protein was significantly decreased in the transfected group,the difference was statistically significant(P < 0.05).6 The results of scratch repair of monolayer cells showed that after 24 h,compared with meaningless sequence group and blank control group,the rate of scratch healing of transfected cells decreased,and the difference was statistically significant(P < 0.05).7 The results of Transwell chamber invasion test showed that compared with the meaningless sequence group and the blank control group,the number of cells passing through the membrane in the transfected group was significantly reduced,and the difference was statistically significant(P < 0.05).Conclusions 1 Silencing NRP-2 can promote apoptosis,inhibit cell proliferation,migration and invasion of HT-29 cell.The decrease of Ki-67 protein expression may be one of the factors contributing to the decrease of cell proliferation.2 It provides a theoretical basis for NRP-2 to become a new research direction for colon cancer targeted therapy.