脯氨酸一谷氨酸一亮氨酸富集蛋白1对甲状腺癌细胞增殖和周期的影响及其机制

Effects of proline arginine rich end leucine rich repeat protein 1 on proliferation and cycle of thyroid cancer cells and its molecular mechanisms

目的探讨脯氨酸-谷氨酸-亮氨酸富集蛋白1(PRELP1)对甲状腺癌细胞增殖和周期的影响及其机制。方法采用蛋白质印迹法(Western blot)分析人甲状腺正常细胞系NTHY-ORI 3-1、甲状腺癌细胞CAL-62细胞和SW579细胞PRELP蛋白表达水平;采用对照组和PRELP1过表达慢病毒感染甲状腺癌细胞CAL-62细胞,建立对照组和PRELP1组细胞。采用细胞计数试剂盒(CCK-8)、克隆形成实验和体外移植瘤实验分析两组细胞的增殖情况;采用流式细胞数分析两组细胞周期和凋亡变化;采用Western blot分析增殖、周期和凋亡相关蛋白表达水平。组间比较采用t检验。结果甲状腺癌细胞CAL-62细胞和SW579细胞PELP1蛋白表达水平(1.72±0.12、2.24±0.22)明显高于人甲状腺正常细胞系NTHY-ORI 3-1(1.10±0.16),差异有统计学意义(t=7.628、10.390,P<0.05)。对照组细胞48 h吸光度值(1.91±0.05)明显高于PRELP1 KD组(1.22±0.06),差异有统计学意义(t=20.660,P<0.05)。对照组细胞克隆形成数量[(122.17±17.32)个]明显高于PRELP1 KD组[(91.17±10.14)个],差异有统计学意义(t=13.124,P<0.05)。对照组细胞肿瘤体积[(769.33±69.48)mm^(3)]明显高于PRELP1 KD组[(511.17±35.97)mm^(3)],差异有统计学意义(t=8.082,P<0.05)。对照组细胞G_(0)/G_(1)期比例[(35.36±2.02)%]明显低于PRELP1 KD组[(41.14±1.96)%],差异有统计学意义(t=5.019,P<0.05)。对照组细胞S期比例[(31.53±1.59)%]明显低于PRELP1 KD组[(25.81±1.53)%],差异有统计学意义(t=6.328,P<0.05)。对照组细胞G_(2)期比例[(27.91±1.66)%]低于PRELP1 KD组[(23.09±1.72)%],差异有统计学意义(t=4.939,P<0.05)。对照组细胞凋亡比例[(5.75±1.25)%]明显低于PRELP1 KD[(28.67±3.23)%],差异有统计学意义(t=16.180,P<0.05)。对照组细胞细胞核增殖抗原(Ki-67)蛋白和Cyclin D1蛋白表达水平(1.20±0.10、1.24±0.07)明显高于PRELP1 KD组(0.71±0.04、0.75±0.06),差异有统计学意义(t=10.700、12.420,P<0.05)。对照组细胞Caspase-3蛋白(0.90±0.03)明显低于PRELP1 KD组(1.31±0.07),差异有统计学意义(t=13.070,P<0.05)。结论PRELP1在甲状腺癌细胞中呈低表达,并参与了甲状腺癌细胞的增殖、周期和凋亡的发生过程。

Objective To investigate the effect of proline arginine rich end leucine rich repeat protein 1(PRELP1)on the proliferation and cell cycle of thyroid cancer cells and its molecular mechanism.Methods The expression of PRELP protein in NTHY-ORI 3-1,CAL-62 and SW579 cells were analyzed by Western blotting.The control group and PRELP1 overexpression lentivirus were used to infect thyroid cancer cell CAL-62 cells,and the control group and PRELP1 group cells were established.Cell counting kit-8(CCK-8),clone formation test and in vitro tumor transplantation test were used to analyze the proliferation of the two groups of cells.The changes of cell cycle and apoptosis were analyzed by flow cytometry.The expression level of proliferation,cycle and apoptosis related proteins were analyzed by Western blotting.Results The expression level of PELP1 protein in CAL-62 cells and SW579 cells of thyroid cancer(1.72±0.12,2.24±0.22)was significantly higher than that in NTHY-ORI 3-1 cells(1.10±0.16)of normal human thyroid cells(t=7.628,10.390,P<0.05).The 48h absorbance value A(1.91±0.05)of cells in the control group was significantly higher than that of cells in the PRELP1 KD group(1.22±0.06,t=20.660,P<0.05).The number of cell clones in the control group[(122.17±17.32)cells]was significantly higher than that in the PRELP1 KD group[(91.17±10.14)cells,t=13.124,P<0.05].The tumor volume[(769.33±69.48)mm^(3)]in the control group was significantly higher than that in the PRELP1 KD group[(511.17±35.97)mm^(3),t=8.082,P<0.05].The proportion of G_(0)/G_(1) phase cells in the control group[(35.36±2.02)%]was significantly lower than that in the PRELP1 KD group[(41.14±1.96)%,t=5.019,P<0.05].The proportion of S phase cells in the control group[(31.53±1.59)%]was significantly lower than that in the PRELP1 KD group[(25.81±1.53)%,t=6.328,P<0.05].The proportion of G_(2) phase cells in the control group[(27.91±1.66)%]was lower than that in the PRELP1 KD group[(23.09±1.72)%,t=4.939,P<0.05].The percentage of apoptosis in the control group[(5.75±1.25)%]was significantly lower than that in the PRELP1 KD group[(28.67±3.23)%,t=16.180,P<0.05].The expression levels of proliferation cell nuclear antigen(Ki-67)protein and Cyclin D1 protein in control group cells(1.20±0.10,1.24±0.07)were significantly higher than those in PRELP1 KD group cells(0.71±0.04,0.75±0.06,t=10.700,12.420,P<0.05).Caspase-3 protein in control group(0.90±0.03)was significantly lower than that in PRELP1 KD group(1.31±0.07,t=13.070,P<0.05).Conclusion PRELP1 is low expressed in thyroid cancer cells and participates in the process of proliferation,cycle and apoptosis of thyroid cancer cells.