分选蛋白17对胰腺癌细胞生物学性状的影响

Sorting nexin 17 affects biological behaviors of pancreatic cancer cells

目的探讨分选蛋白(SNX)17通过激活表皮生长因子受体(EGFR)影响胰腺癌细胞侵袭、增殖和迁移能力。方法采集3例人胰腺癌组织及癌旁组织样本,自2021年1月至2021年3月收集于贵州医科大学附属医院肝胆外科,用免疫组织化学(IHC)染色检测癌组织与癌旁组织中SNX17的表达量。用实时定量反转录聚合酶链反应(RT-qPCR)技术检测胰腺癌细胞系中SNX17的相对表达量;用空siRNA以及两种敲低SNX17的小干扰RNA(siRNA)分别转染上述细胞,分组为NC组、SNX17-si1组和SNX17-si2组。通过细胞计数试剂盒(CCK-8)、平板克隆实验检测胰腺癌细胞的增殖能力,Transwell及划痕实验检测胰腺癌细胞迁移能力。蛋白质印迹法(Western blot)检测在SNX17敲低的癌细胞中EGFR以及细胞外调节激酶(ERK)的相对表达量差异。最后在胰腺癌细胞中共转染siRNA以及EGFR的过表达质粒进行功能回复实验(分组为NC组、SNX17-si1组、EFGR-overexpression+SNX17-si1组),组间比较采用t检验,组内两两比较采用LSD-t检验。结果免疫组织化学检测结果显示癌组织中SNX17相对表达量高于癌旁组织(5.750±1.323,t=4.345,P<0.05),差异有统计学意义。RT-qPCR发现在胰腺癌细胞系中,人胰腺癌细胞-2(MIA PaCa-2,19.240±2.048、t=8.844,P<0.05)和人胰腺癌细胞-1(PANC-1,5.796±1.256,t=3.712,P<0.05)细胞中SNX17相对表达量显著高于其他胰腺癌细胞系,差异有统计学意义。CCK-8实验技术结果显示,SNX17-si1组、SNX17-si2组低于NC组吸光度值(0.707±0.059比0.346±0.075比1.109±0.052比0.602±0.047,t=12.060、4.642、21.440、12.750,P<0.01),差异有统计学意义。平板克隆结果显示转染组细胞集落数减少。划痕实验结果显示敲低SNX17降低细胞的侵袭能力。Transwell迁移实验结果显示,SNX17-si1组、SNX17-si2组单位视野穿过的细胞数低于NC组[(666.300±14.400)个比(574.300±10.800)个比(129.300±4.300)个比(93.700±3.100)个,t=46.260、53.220、29.850、15.450,P<0.01],差异有统计学意义。而侵袭实验结果表示,SNX17-si1组、SNX17-si2组单位视野穿过的细胞数低于NC组[(137.5±20.1)个比(100.8±17.4)个比(324.5±7.9)个比(289.8±10.2)个,t=6.836、5.807、41.020、28.430,P<0.01],差异有统计学意义。Western blot检测结果显示,SNX17-si1组与SNX17-si2组EGFR的相对表达量低于NC组(0.477±0.032比0.278±0.042比0.853±0.053比0.826±0.050,t=14.920、6.640、16.130、16.430,P<0.01),差异有统计学意义。在回复实验中,CCK-8、Transwell实验及划痕实验证明共转染EGFR-overexpression+SNX17-si1逆转敲低SNX17所导致的胰腺癌细胞增殖、侵袭和迁移能力的降低。最后用Western blot结果表明,共转染组比较单独转染SNX17-si1的组别,EGFR的相对表达量分别增高(0.712±0.010、t=70.220,P<0.05;1.002±0.027、t=37.580,P<0.05),差异均有统计学意义。结论SNX17通过影响EGFR促进胰腺癌细胞MIA PaCa-2和PANC-1的增殖、侵袭、迁移能力。

Objective To study the effects of knockdown of sorting nexin(SNX)17 on the invasion,migration and proliferation of pancreatic cancer cells by activating the epidermal growth factor receptor(EGFR)pathway.Methods We collected three cases of human pancreatic cancer tissues and para-carcinoma tissues from Department of Hepatic-Biliary-Pancreatic Surgery,the Affiliated Hospital of Guizhou Medical University during Jan.2021 to Mar.2021.The relative expression of SNX17 in cancer tissues and adjacent tissues was detected by immunohistochemistry(IHC).The relative expression level of SNX17 in pancreatic cancer cell lines was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The cells were transfected with siRNA to knock down SNX17(the cells were grouped into normal control group,SNX17-si1 group and SNX17-si2 group).Cell proliferation efficiency was detected by cell counting kit-8(CCK-8)and cell cloning assays.The cell invasion ability was detected by scratch assay,and cell migration and invasion ability was detected by Transwell assay.The relative expression of EGFR and extracellular regulated protein kinases(ERK)in SNX17-knockdown cancer cells was detected by Western blotting.Finally,pancreatic cancer cells co-transfected with SNX17-knockdown siRNA and EGFR-overexpression vector were used for revert experiments(the cells were grouped into normal control group,SNX17-si1 group,and EGFR-overexpression+SNX17-si1 group).T-test was used for inter-group comparison,and the LSD-t test was used for pair-to-group comparison.Results IHC showed the relative expression of SNX17 in cancer tissues was higher than adjacent tissues(5.750±1.323,t=4.345,P<0.05),the differences were statistically significant.RT-qPCR showed the relative expression of SNX17 in MIA PaCa-2(19.240±2.048,t=8.844,P<0.05)and PANC-1(5.796±1.256,t=3.712,P<0.05)cells was increased.CCK-8 assay verified that SNX17-knockdown reduced the proliferation efficiency of cells,and the absorbance of SNX17-si1 group and SNX17-si2 group 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 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 assay showed the number of cells penetrating the membrane in the SNX17-si1 group and SNX17-si2 group decreased[(666.300±14.400)cells/field,t=46.260,(574.300±10.790)cells/field,t=53.220;(129.300±4.333)cells/field,t=29.850,(93.670±3.064)cells/field,t=15.450].The invasion assay showed the number of cells decreased[(137.500±20.120)cells/field,t=6.836,(100.800±17.350)cells,t=5.807;(324.500±7.911)cells/field,t=41.020,(289.800±10.190)cells/field,t=28.430,P<0.01].Western blotting that the relative expression level of EGFR protein in SNX17-si1 group and SNX17-si2 group was lower than in normal control group(0.477±0.032,t=14.920,0.278±0.042,t=6.640;0.853±0.053,t=16.130,0.826±0.050,t=16.430;P<0.01).In the functional reply experiment,CCK-8,Transwell and scratch assays proved co-transfection of EGFR-overexpression and SNX17-si1 reversed the reduction of proliferation,invasion and migration of pancreatic cancer cells due to SNX17-knockdown.Western blotting confirmed that the EGFR protein expression in co-transfected group increased by(0.712±0.010,t=70.220,1.002±0.027,t=37.580,P<0.05)compared to SNX17-si1 group.Conclusion In pancreatic cancer cells MIA PaCa-2 and PANC-1,SNX17 promotes the proliferation,invasion and migration ability by affecting EGFR.