The Effect Of Neuropilin-1 On Biological Behavior Of Colon Cancer Cell Line HT-29

Objectives After the si RNA silencing the expression of NRP-1,investigate the effects of NRP-1 on proliferation, apoptosis, migration and invasion of human colon cancer cell line HT-29.Methods Specification of cultured cell line HT-29, the fluorescence si RNA of NRP-1 was transfected into HT-29 cell line by Lip-2000. After 48 h, the efficiency of transfection of HT-29 cell lines was observed by fluorescence microscopy. On the basis of successful transfection, transfect the different substances into colon cancer cell line HT-29 by Lip-2000.Then the colon cancer cell lines were divided into three distinct groups: transfection group, meaningless sequence group and blank control group. The relative expression of m RNA in three groups of cells was detected by real-time fluorescence quantitative PCR. The expression of Ki67 protein in three groups of cells was detected by immunocytochemistry. MTT assay was used to detect the proliferation of the cells in the optical density of the three groups Flow cytometry was used to detect the distribution of cell cycle in the three groups. To detect the apoptosis of acridine orange / propidium iodide method of cells in three groups. Cell migration and invasion assay were detected in three groups of cells migration and invasion. Repeat the experiment 3 times.Results 1 After Lip-2000 transfected with fluorescent si RNA 48 h, The ratio of the number of cells and the total number of cells transfected into si RNA was observed under fluorescence microscope. Three times tests show that the transfection efficiency can reach more than 80%. 2 Real time fluorescent quantitative PCR results display : compared with the control group, the relative expression of NRP-1 m RNA in transfected cells was significantly decreased,the difference was statistically significant(P < 0.05). 3 According to the results of immunocytochemistry staining: compared with the control group, the expression of Ki-67 protein in transfected group was significantly lower than that in control group, the difference was statistically significant(P < 0.01). 4 MTT experimental results show that: at 24 h, 48 h, 72 h three time points, compared with the control group, the proliferation ability of transfected cells was significantly lower than that of the control group, the difference was statistically significant(P < 0.01). 5 Cell cycle test results showed by flow cytometry: Compared with the blank control group, the cell cycle arrest in the G1 phase of the cell cycle was significantly blocked in the transfected group, the proportion of S phase was significantly decreased, the difference was statistically significant(P < 0.05). 6 Acridine orange / propidium iodide experiments showed that: compared with the blank control group, the apoptosis of transfected cells was significantly increased, the difference was statistically significant(P < 0.01). 7 Cell migration experiments showed that: in transfected group than the control group on the cell membrane matrix through the number on the back of the chamber to reduce cells, the difference was statistically significant(P < 0.01). 8 Cell migration experiments showed that: in transfected group than the control group on the cell membrane matrix through the number on the back of the chamber to reduce cells, the difference was statistically significant(P < 0.01).Conclusions 1 The proliferation, migration, invasion of colon cancer cells were significantly decreased and the level of apoptosis was significantly increased after transfection of si RNA NRP-1. 2 NRP-1 may be a new target of colon cancer therapy. 3 si RNA of NRP-1 may be a new therapeutic means for the treatment of colon cancer.