KIF18B通过激活Wnt/βcatenin信号通路促进子宫内膜癌细胞增殖和转移的实验研究

Experimental Study of KIF18B Promoting Endometrial Cancer Cell Proliferation and Metastasis by Activating Wnt/β-catenin Signaling Pathway

目的 探讨驱动蛋白家族成员18B(kinesin family member18b,KIF18B)在子宫内膜癌组织中的表达,及促进子宫内膜癌细胞增殖和转移的作用机制。方法 收集子宫内膜癌组织50例和正常子宫内膜组织30例,采用免疫组织化学染色检测KIF18B表达水平,分析KIF18B表达与子宫内膜癌患者临床病理特征的关系及对患者预后的影响。KIF18B小干扰RNA(siRNA)转染子宫内膜癌细胞Ishikawa,分为si-NC组和si-KIF18B组,蛋白免疫印迹法检测KIF18B siRNA的转染效果;噻唑蓝比色法(MTT)检测细胞的增殖能力;Transwell实验检测细胞的转移能力;双荧光素酶报告基因实验检测各组细胞中的Wnt/β-连环蛋白(β-catenin)信号通路活性;蛋白免疫印迹法检测Wnt/β-catenin信号通路相关蛋白Wnt3a和β-catenin,及其下游增殖相关蛋白cMYC,细胞周期蛋白D1(cyclinD1)和转移相关蛋白基质金属蛋白酶2(matrix metalloproteinase,MMP-2)、基质金属蛋白酶7 (matrix metalloproteinase-7,MMP-7)的表达。结果KIF18B在子宫内膜癌组织中的阳性表达率高于正常子宫内膜组织(70.00%vs 46.67%),差异有统计学意义(χ2=4.301,P=0.038)。FIGO分期Ⅲ~Ⅳ期、有淋巴结转移患者KIF18B阳性表达率高于FIGO分期Ⅰ~Ⅱ期(88.89%vs47.82%)、无淋巴结转移患者(86.36%vs 57.14%),差异有统计学意义(χ2=9.972, 5.009;P=0.002,0.025)。KIF18B高表达组五年总生存率低于KIF18B低表达组(34.29%vs 66.67%),差异有统计学意义(χ2=4.305,P=0.038)。siKIF18B组KIF18B表达低于si-NC组(0.15±0.04 vs 1.01±0.03),差异有统计学意义(t=29.790,P<0.001)。第2~5天si-KIF18B组细胞增殖率低于si-NC组,差异均有统计学意义(t=3.267~11.080,均P<0.05)。si-KIF18B组细胞穿膜数目低于si-NC组(39.67±4.52个vs 74.33±4.51个),差异有统计学意义(t=9.402,P<0.001)。si-KIF18B组细胞中荧光素酶相对活性低于si-NC组(0.41±0.04 vs 1.00±0.03),差异有统计学意义(t=20.438,P<0.001)。si-KIF18B组Wnt3a,β-catenin,cMYC,cyclinD1,MMP-2和MMP-7表达低于si-NC组,差异均有统计学意义(t=4.695~36.509,均P <0.05)。结论 KIF18B在子宫内膜癌组织中表达增加,下调KIF18B可以抑制子宫内膜癌细胞的增殖和转移,KIF18B可能通过激活Wnt/β-catenin信号通路参与子宫内膜癌的发生、发展。

Objective To investigate the expression of kinesin family member18b(KIF18B)in endometrial carcinoma and its mechanism of promoting proliferation and metastasis of endometrial carcinoma cells.Methods 50 cases of endometrial cancer tissue and 30 cases of normal endometrial tissue were collected.Immunohistochemical staining was used to detect the expression level of KIF18B,and the relationship between KIF18B expression and clinical pathological parameters of endometrial cancer patients was analyzed,as well as its impact on patient prognosis.KIF18B small interfering RNA(siRNA)was transfected into endometrial cancer cell line Ishikawa and divided into si-NC group and si-KIF18B group.The transfection effect of KIF18B siRNA was detected by Western blotting.The proliferation ability of the cells was detected by thiazole blue colorimetry.Transwell assay was used to detect the ability of cell metastasis.The Wnt/β-catenin signaling pathway activity in each group was detected by double luciferase reporter gene experiment.Wnt/β-catenin signaling pathway related proteins Wnt3a andβ-catenin,as well as downstream proliferation-related proteins cMYC,cyclinD1 and transfer related proteins matrix metalloproteinase(MMP-2)and MMP-7 were detected by Western blotting.Results The positive expression rate of KIF18B in endometrial cancer tissue was higher than that in normal endometrial tissue(70.00%vs 46.67%),and the difference was statistically significant(χ^(2)=4.301,P=0.038).The positive expression rate of KIF18B in patients with FIGO stage III~IV and lymph node metastasis was higher than that in patients with FIGO stage I~II(88.89%vs 47.82%)and no lymph node metastasis(86.36%vs 57.14%),and the difference was statistically significant(χ^(2)=9.972,5.009,P=0.002,0.025).The overall 5-year survival rate of the KIF18B high expression group was lower than that of the KIF18B low expression group(34.29%vs 66.67%),the difference was statistically significant(χ^(2)=4.305,P=0.038).The expression of KIF18B in the si-KIF18B group was lower than that in the si-NC group(0.15±0.04 vs 1.01±0.03),and the difference was statistically significant(t=29.790,P<0.001).On the 2nd to 5th day,the cell proliferation rate of the si-KIF18B group was lower than that of the si-NC group,and the differences were statistically significant(t=3.267~11.080,all P<0.05).The number of cells penetrating the membrane in the si-KIF18B group was lower than that in the si-NC group(39.67±4.52 vs 74.33±4.51),the difference was statistically significant(t=9.402,P<0.001).The relative activity of luciferase in the si-KIF18B group cells was lower than that in the si-NC group(0.41±0.04 vs 1.00±0.03),and the difference was statistically significant(t=20.438,P<0.001).The expressions of Wnt3a,β-catenin,cMYC,cyclinD1,MMP2 and MMP7 in si-KIF18B group were lower than those in si-NC group,and the differences were statistically significant(t=4.695~36.509,all P<0.05).Conclusion The expression of KIF18B was increased in endometrial cancer tissues,and down-regulation of KIF18B could inhibit the proliferation and metastasis of endometrial cancer cells.KIF18B may participate in the occurrence and development of endometrial cancer by activating Wnt/β-catenin signaling pathway.