miR-222通过靶向RB1促进视网膜母细胞瘤细胞生长与侵袭

miR-222 promotes retinoblastoma cell proliferation and invasion by targeting RB1

背景与目的:视网膜母细胞瘤基因1(retinoblastoma 1,RB1)能够抑制多种肿瘤的发生、发展,且与细胞周期、分化、衰老、凋亡及生长抑制等调控密切相关。该研究旨在明确miR-222是否通过靶向RB1表达而促进视网膜母细胞瘤细胞的生长与侵袭,进一步揭示miR-222促瘤作用的分子机制。方法:将miR-222(miR-222模拟物)+RB1-wt(野生型RB1的3’-非翻译区的荧光素酶报告载体)、miR-NC(无关序列对照)+RB1-wt、miR-222+RB1-mut(突变型RB1的3’-非翻译区的荧光素酶报告载体)及miR-NC+RB1-mut共转染人视网膜母细胞瘤细胞株Y79,并采用单光子检测荧光素酶活性。采用蛋白[质]印迹法(Western blot)检测RB1表达水平的改变。将miR-222与miR-NC、RB1(pc DNA3.1-RB1)与vector(pc DNA3.1)、miR-222+RB1及miR-NC+vector转染Y79细胞,MTS检测细胞生长增殖活性,Transwell侵袭实验检测Y79细胞生长与侵袭能力的影响。结果:与miR-NC+RB1-wt组比较,共转染miR-222+RB1-wt组的荧光素酶活性强度降低了约56.67%(P<0.05)。与miR-NC比较,miR-222组RB1蛋白水平显著下调(P<0.05)。转染miR-222组细胞生长速度显著高于miR-NC组(P<0.05)。与pc DNA3.1组比,pc DNA3.1-RB1组可显著抑制Y79细胞的生长(P<0.05),而miR-222+pc DNA3.1-RB1组和miR-NC+pc DNA3.1组比较,细胞生长速度差异无统计学意义(P>0.05)。转染miR-222组穿过基底膜的细胞数分别为(193±10),与对照组(144±11)比较能明显加快Y79细胞的穿膜能力,差异有统计学意义(P<0.05)。而miR-NC+pc DNA3.1组和miR-222+pc DNA3.1-RB1组比较,穿过基底膜的细胞数差异无统计学意义(P>0.05)。结论:miR-222通过靶向调控RB1表达而促进视网膜母细胞瘤细胞的生长与侵袭。

Background and purpose： A large number of studies have showed that retinoblastoma gene 1 （RB1） can inhibit the occurrence and development of many tumors, including neuroblastoma, small cell lung cancer, osteosarcoma, pancreatic cancer, breast cancer and so on. RB1 is also closely related to the regulation of cell cycle, differentiation, senescence, apoptosis, growth inhibition, etc. The goal of this article is to elucidate whether miR-222 promotes cell proliferation and invasion by targeting RB1, further to explore the molecular mechanism that miR-222 functions as an oncogene in retinoblastoma cells. Methods： miR-222 （miR-222 mimics） and RB1-wt, miR-NC and RB1-wt, miR-222 and RB1-mut, miR-NC （a controlled miR-222 mimics） and RB1-mut were co-transfected into Y79 cells, and luciferase activity was detected by single photon. Retinoblastoma cells were transfected with miR-222 mimics and miR-NC, and the expressions of RB1 protein were detected by Western blot. Retinoblastoma cell proliferation assays were performed by MTS assay when miR-222, miR-NC, RB1 （pcDNA3.1-RB1）, vector （pcDNA3.1）, miR-222＋RB1 and miR-NC＋vec- tor were transfected into Y79 cells. The growth and invasion ability of Y79 cells with ectopic expression of miR-222 were evaluated by MTS and Transwell invasion assays. Results： This study demonstrated that miR-222 could promote the luciferase activity of RB1-wt. The expression levels of luciferase reporter gene activity in Y79 cells after transfection with miR-222＋RB1-wt were higher than those in the negative control cells （miR-NC＋RB1-wt） （P〈0.05）. The protein expression levels of RB1 in Y79 cells after transfection with miR-222 were lower than those in miR-NC （P〈0.05）. Overexpression of RB1 inhibited the proliferation of retinoblastoma cells. miR-222 promoted the proliferation of retinoblastoma cells through targeting RB1 （P〈0.05）. Moreover, there was no significant difference between the cell survival rates of Y79 which were transfected with miR-222＋pcDNA3.1-RB1 and miR-NC＋pcDNA3.1 （P〉0.05）. After transfection with miR-222 mimics for 48 h, Transwell invasion assay showed that the number of cells through the basement membrane was （193±10）. Compared with the control group （144±11）, it could significantly accelerate the invasion of Y79 cells （P〈0.01）. There was no significant difference between the number of cells through the basement membrane which were transfected with miR-222＋pcDNA3.1-RB1 and miR-NC＋pcDNA3.1 （P〉0.05）. Conclusion： miR-222 promotes cell proliferation and invasion by targeting RB1 expression in retinoblastoma cells.