miR-222通过靶向RB1促进视网膜母细胞瘤细胞生长与侵袭

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(pcDNA3.1-RB1)与vector(pcDNA3.1)、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pcDNA3.1组比，pcDNA3.1-RB1组可显著抑制Y79细胞的生长(P<0.05)，而miR-222+pcDNA3.1-RB1组和miR-NC+pcDNA3.1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+pcDNA3.1组和miR-222+pcDNA3.1-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。

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

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, osteosar-coma, pancreatic cancer, breast cancer and so on. RB1 is also closely related to the regulation of cell cycle, differentia-tion, 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 prolifera-tion of retinoblastoma cells through targeting RB1 (P<0.05). Moreover, there was no signiifcant 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 signiifcantly accelerate the invasion of Y79 cells (P<0.01). There was no signiifcant 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.