miR-373靶向调控同源异型盒基因B3表达影响子宫内膜癌Ishikawa细胞生物学特性的实验研究

Experimental study of the effect of miR-373 on the biological characteristics of endometrial carcinoma Ishikawa cell by targeting the expression of homobox gene B3

目的探讨微小RNA(miR)-373靶向调控同源异型盒基因B3(HOXB3)表达对子宫内膜癌Ishikawa细胞增殖、侵袭和凋亡的影响。方法本研究起止时间为2018年2月至2019年6月,将体外培养的Ishikawa细胞分为抑制剂阴性对照(inhibitorNC)组(转染inhibitor-NC)、miR-373抑制剂(inhibitor)组(转染miR-373 inhibitor)、miR-373 inhibitor+siNC组(共转染miR-373 inhibitor和NC siRNA)和miR-373 inhibitor+siHOXB3组(共转染miR-373 inhibitor和HOXB3 siRNA),采用实时荧光定量PCR检测Ishikawa细胞中miR-373的表达,双荧光素酶报告基因实验检测miR-373和HOXB3的靶向关系,采用免疫印迹法检测Ishikawa细胞中HOXB3蛋白的表达,细胞计数(CCK-8)法、Transwell实验和膜联蛋白V-FITC(Annexin V-FITC)/碘化丙啶(PI)双染法检测各组细胞增殖、侵袭和凋亡能力。结果HOXB3是miR-373的靶基因。与inhibitor-NC组相比,miR-373 inhibitor组、miR-373 inhibitor+siNC组和miR-373 inhibitor+siHOXB3组细胞中miR-373表达水平[(1.00±0.11)比(0.32±0.02)/(0.36±0.03)/(0.34±0.03)]和细胞增殖活力[24 h(0.63±0.04)比(0.41±0.03)/(0.43±0.03)/(0.52±0.03);48 h(0.97±0.06)比(0.68±0.05)/(0.72±0.06)/(0.85±0.05);72 h(1.35±0.11)比(0.88±0.08)/(0.92±0.07)/(1.12±0.09)]、穿膜细胞数[(85.26±8.72)个比(37.64±2.85)个/(40.55±3.23)个/(61.38±3.64)个]均明显降低,而细胞凋亡率[(8.75±1.23)%比(25.64±3.38)%/(28.48±3.26)%/(14.25±2.02)%]和细胞中HOXB3蛋白的表达水平[(0.27±0.03)比(0.77±0.06)/(0.73±0.06)/(0.51±0.03)]均明显升高(P<0.05);且miR-373 inhibitor+siHOXB3组中各指标的变化强度明显低于miR-373 inhibitor组(P<0.05);而miR-373 inhibitor+siNC组和miR-373 inhibitor组之间无明显差异(P>0.05)。结论下调miR-373表达可通过靶向HOXB3抑制子宫内膜癌Ishikawa细胞增殖、侵袭并促进细胞凋亡。

Objective To investigate the effects of micro(miR)-373 targetinghomologous box gene B3(HOXB3)expression on the proliferation,invasion and apoptosis of Ishikawa cells in endometrial cancer.Methods from February 2018 to June 2019.Ishikawa cells cultured in vitro were divided into inhibitor-negative control group(transfection inhibitor-NC),inhibitor group(transfection of miR-373 inhibitor),miR-373 inhibitor+siNC group(co-transfection of miR-373 inhibitor and NC siRNA)and miR-373 inhibitor+siHOXB3 group(co-transfection of miR-373 inhibit+siHOXB3).The expression of miR-373 in Ishikawa cells was detected by real-time fluorescent quantitative PCR,the targeting relationship between miR-373 and HOXB3 was tested by double luciferase reporter gene assay,the expression of HOXB3 protein in Ishikawa cells was measured by western blotting.Cell proliferation,invasion and apoptosis were detected by cell counting(CCK-8),Transwell assay and Annexin V-FITC/propidium iodide(PI)double staining method separately.Result HOXB3 was the target gene of miR-373.Compared with the inhibitor-NC group,the expression level of miR-373[(1.00±0.11)vs.(0.32±0.02)/(0.36±0.03)/(0.34±0.03)],the cell proliferation activity[24 h(0.63±0.04)vs.(0.41±0.03)/(0.43±0.03)/(0.52±0.03);48 h(0.97±0.06)vs.(0.68±0.05)/(0.72±0.06)/(0.85±0.05);72 h(1.35±0.11)vs.(0.88±0.08)/(0.92±0.07)/(1.12±0.09)]and the number of penetrating cells[(85.26±8.72)vs.(37.64±2.85)/(40.55±3.23)/(61.38±3.64)]decreased significantly in miR-373 inhibitor group,miR-373 inhibitor+siNC group and miR-373 inhibitor+siHOXB3 group,while the apoptosis rate[(8.75±1.23)%vs.(25.64±3.38)%/(28.48±3.26)%/(14.25±2.02)%]and the expression level of HOXB3 protein[(0.27±0.03)vs.(0.77±0.06)/(0.73±0.06)/(0.51±0.03)]increased significantly(P<0.05).The change intensity of each index in miR-373 inhibitor+siHOXB3 group was significantly lower than that in miR-373 inhibitor group(P<0.05),but there was no significant difference between miR-373 inhibitor+siNC group and miR-373 inhibitor group(P>0.05).Conclusion Downregulation of the expression of miR-373 can inhibit the proliferation,invasion and apoptosis of endometrial cancer Ishikawa cells by targeting HOXB3.