摘要
目的探讨miR-433抑制Notch1和PAX6表达对视网膜母细胞瘤(RB)恶变的影响。方法选取2015年1月至2020年1月间西安市第四医院收治的行手术切除并经病理检查确诊的RB组织50例(未接受放疗前),同时取正常视网膜组织50例。采用qRT-PCR方式分析RB细胞与正常视网膜组织中miR-433表达水平,采用MTT法分析miR-433、miR-433表达抑制(miR-433 Ctrl)和miR-433过表达(anti-miR-433)对RB细胞系增殖与凋亡的影响,采用Western blotting法检测miR-433、miR-433 Ctrl和anti-miR-433对蛋白Notch1和PAX6表达的影响。结果Notch1和PAX6在正常视网膜组织中表达阳性率为20.0%和24.0%,在RB组织中表达阳性率为80.0%和76.0%,组间比较,差异均有统计学意义(均P<0.05)。同正常视网膜组织相比,RB细胞中miR-433的表达水平更低,提示miR-433可能在RB的发育中发挥了重要作用。将RB细胞系分别使用miR-433、miR-433 Ctrl和anti-miR-433进行转染,并于转染24h、48h、72h及96h时使用MTT检测法,分析RB细胞系增殖凋亡情况。细胞增殖方面,anti-miR-433抑制RB细胞增殖,miR-433 Ctrl促进RB细胞增殖,24h、48h、72h和96h时,miR-433 Ctrl组RB细胞增殖>miR-433组>anti-miR-433组(P<0.05)。细胞凋亡方面,anti-miR-433提高RB细胞凋亡水平,miR-433 Ctrl会提高RB细胞抗凋亡能力,组间比较,24h、48h、72h和96h时,miR-433 Ctrl组RB细胞活性>miR-433组>antimiR-433组(P<0.05)。开展蛋白质印迹分析发现,anti-miR-433组RB细胞Notch1和PAX6蛋白表达水平最低,其次为miR-433组,miR-433 Ctrol组最高。结论miR-433可特异性地作用于Notch1和PAX6蛋白靶基因,以影响RB细胞的增殖、凋亡、迁移和侵袭。
Objective To explore the effect of inhibition of Notch1 and Pax6 expression with Mir-433 on malignant transformation of retinoblastoma(RB).Methods Fifty RB tissues confirmed by pathologic findings were collected from patients undergoing surgery for RB before radiotherapy at Xi'an Fourth Hospital from January 2015 to January 2020.Meanwhile,50 healthy retinal tissues were collected.The difference in miR-433 expression level was analyzed between RB tissues and normal retinal tissues by qRTPCR,and the effects of miR-433,miR-433 Ctrl and miR-433 overexpression on the proliferation and apoptosis of RB cell lines were analyzed by MTT assay.The effects of miR-433,miR-433 Ctrl and anti-miR-433 on the expression of Notch1 and PAX6 were detected by Western blotting.Results The positive expression rate of Notch1 and PAX6 was 20.0%and 24.0%,respectively in normal retina and 80.0%and 76.0%,respectively in RB(all P<0.05).Compared with normal retina,the expression of miR-433 was low in RB indicating miR-433 plays an important role in the development of RB.RB cell lines were transfected using miR-433,miR-433 Ctrl and anti-miR-433,respectively and the proliferation and apoptosis of RB cell lines were detected at 24,48,72 and 96 h using MTT assay.In terms of proliferation,anti-miR-433 may inhibit proliferation of RB cells,miR-433 Ctrl may increase the proliferation ability of RB cells.At 24,48,72 and 96 h,proliferation of RB cells was higher in the miR-433 Ctrl group than in the miR-433 group and higher in the miR-433 group than in the anti-miR-433 group.With regard to apoptosis,anti-miR-433 may promote the apoptosis of RB cells and miR-433 Ctrl may improve anti-apoptotic ability of RB cells.In compassion,viability of RB cells was higher in miR-433 Ctrl group than in miR-433 group and higher in miR-433 group than in anti-miR-433 group.Western blot analysis found that the expression of Notch1 and PAX6 was the lowest in the anti-miR-433 group followed by miR-433 group and the expression of Notch1 and PAX6 was the highest in miR-433 Ctrl.Conclusion miR-433 can specifically act on the target genes of Notch1 and PAX6 proteins to influence the proliferation,apoptosis,migration and invasion of RB cells by regulating the expression of these proteins.
作者
田蕴霖
白淑玮
邵娟
TIAN Yun-lin;BAI Shu-wei;SHAO Juan(Department of Ophthalmology,Xi'an People's Hospital/Xi'an Fourth Hospital,Xi'an 710000,China)
出处
《中国肿瘤临床与康复》
2021年第4期454-457,共4页
Chinese Journal of Clinical Oncology and Rehabilitation
