| Objective:To study the sorting nexin(SNX)17 may effect pancreatic cancer cells through activating the epidermal growth factor receptor(EGFR)pathway,and effect the invasion、migration and moreover,the proliferation ability in these cells.Methods:We collected three cases of human pancreatic cancer tissues and para-carcinoma normal tissues,all came from the Department of Hepatic-Biliary-Pancreatic Surgery,The Affiliated Hospital of Guizhou Medical University since Jan.2021 to Mar.2021.The relative expression diversity of SNX17 in pancreatic cancerous tissues and adjacent normal tissues were examined by immunohistochemistry(IHC).The relative expression level of SNX17 in pancreatic cancer cell lines was detected by RT-q PCR.The pancreatic cells were transfected with si RNA in order to knockdown SNX17(the cells were divided into group NC,group SNX17-si1,group SNX17-si2),and the efficiency was verified by RT-q PCR and Western blot.CCK-8 and cell cloning assay were used to examine cell proliferation efficiency.Cell invasion ability was examined by scratch assay,meanwhile migration and invasion capacity was examined by Transwell assay.The correlation expression of EGFR、ERK(extracellular regulated protein kinases)and p-ERK(phosphorylated extracellular signal-regulated kinase)in SNX17-knockdown cancer cells was detected by Western blot.Finally,co-transfected SNX17-knockdown si RNA and EGFR-overexpression plasma in pancreatic cancer cells were used for revert experiments(the cells were grouped into group NC,group SNX17-si1,group EGFR-overexpression + SNX17-si1).T-test was used for analysis the variance of inter-group,and the LSD-t test was applied for analysis the variance of pair-to-group.The mean ± standard deviation was used to represent the measurement data,and the difference was statistically significant when p < 0.05.Results:Immunohistochemically analysis showed the relative expression of SNX17 in pancreatic cancerous tissues was higher than the normal tissues(5.750 ± 1.323,t =4.345,p < 0.05),the differences between these numbers were statistically significant.RT-q PCR showed us that the relative expression of SNX17 in MIA Pa Ca-2(Human Pancreatic Carcinoma Cell Line-2)(19.240 ± 2.048,t = 8.844,p < 0.05)and PANC-1(Human Pancreatic Carcinoma-1)(5.796 ± 1.256,t = 3.712,p < 0.05)cells was increased,the differences were statistically significant.CCK-8 technology verified that SNX17-knockdown reduced the proliferation efficiency of cells,the relative OD level at 450 nm of group SNX17-si1 and SNX17-si2 was decreased(0.707 ± 0.059、t = 12.060,0.346 ± 0.075、t = 4.642;1.109 ± 0.052、t = 21.440,0.602 ± 0.047、t =12.750;p < 0.01),the differences between these data were reach statistical significance.The plate clone assay and the scratch experiment showed SNX17-knockdown reduced the number of cell colonies and invasion ability.Transwell assay results claimed that SNX17-knockdown reduced the ability of migration and invasion in pancreatic cells.The migration data showed that the number of cells passed through the membrane in the SNX17-si1 and SNX17-si2 group decreased(666.300 ± 14.400、t = 46.260,574.300 ± 10.790、t = 53.220;129.300 ± 4.333、t =29.850,93.670 ± 3.064、t = 15.450,per visual field),the differences between these data were statistically significant.Meanwhile,the invasion assay showed the number of cells decreased(137.500 ± 20.120、t = 6.836,100.800 ± 17.350、t = 5.807;324.500 ± 7.911、t = 41.020,289.800 ± 10.190、t = 28.430;p < 0.01,per visual field),the differences were statistically significant in these data.Western blot confirmed the protein expression of EGFR was decreased in SNX17-knockdown cells,the relative expression level of EGFR protein in SNX17-si1 and SNX17-si2 group was lower than NC group(0.477 ± 0.032、t = 14.920,p < 0.01,0.278 ± 0.042、t = 6.640,p < 0.05;0.853 ± 0.053、t = 16.130,p < 0.01,0.826 ± 0.050、t = 16.430,p < 0.01),the differences were reach statistical significance.The relative expression level of ERK protein in SNX17-si1 and SNX17-si2 group has no statistic difference compared to the NC group,the differences weren’t reach statistical significance.The relative expression level of p-ERK protein in SNX17-si1 and SNX17-si2 group was lower than NC group(0.840 ± 0.098、t = 8.584,0.468 ± 0.110、t = 4.247,p <0.05;0.458 ± 0.034、t = 13.420,0.201 ± 0.014、t = 14.22,p < 0.01),the differences were reach statistical significance.In the functional reply experiment,CCK-8 、transwell and scratch assay proved co-transfection of EGFR-overexpression and SNX17-si1 reversed the reduction of proliferation,invasion and migration capacity due to SNX17-knockdown in pancreatic cancer cells.Western Blot confirmed the protein expression of EGFR was highly increased in pancreatic cancer cells who were co-transfected with EGFR-overexpression and SNX17-si1,the EGFR protein expression in co-transfected group increased by(0.712 ± 0.010、t = 70.220,p <0.01;1.002 ± 0.027、t = 37.580,p < 0.01)compared to group SNX17-si1,the differences were statistically significant.The relative expression level of ERK protein in co-transfection group has no statistic difference compared to the group SNX17-si1,the differences weren’t reach statistical significance.The p-ERK protein expression in co-transfected group increased by(0.175 ± 0.012、t = 14.56,p < 0.01;0.084 ± 0.015、t = 5.561,p < 0.01)compared to group SNX17-si1,the differences were statistically significant.Conclusions:In pancreatic cancer cells MIA Pa Ca-2 and PANC-1,SNX17 promotes the proliferation,invasion and migration ability,and realizes its function by affecting EGFR pathway. |
PDF Full Text Request