LncRNA FGD5-AS1靶向miR-195-5p调控弥漫型大B细胞淋巴瘤细胞活性

LncRNA FGD5-AS1 regulates cell activity of diffuse large B-cell lymphoma by targeting miR-195-5p

目的:探讨FGD5-AS1靶向miR-195-5p调控弥漫型大B细胞淋巴瘤细胞增殖及凋亡的分子机制。方法:qRTPCR检测B淋巴母细胞IM-9与弥漫型大B细胞淋巴瘤细胞系OCI-Ly1、OCI-Ly4、OCI-Ly10中FGD5-AS1、miR-195-5p表达;si-NC、si-FGD5-AS1、miR-NC、miR-195-5p mimics、anti-miR-NC、anti-miR-195-5p、si-FGD5-AS1+anti-miR-NC、si-FGD5-AS1+antimiR-195-5p分别转染OCI-Ly1细胞,MTT检测细胞活力,流式细胞术检测细胞凋亡率,双荧光素酶报告基因实验检测FGD5-AS1与miR-195-5p的靶向关系,Western blot检测CyclinD1、Cleaved-caspase-3、β-catenin蛋白表达。结果:与IM-9细胞比较,弥漫型大B细胞淋巴瘤细胞系OCI-Ly1、OCI-Ly4、OCI-Ly10中FGD5-AS1表达升高(P<0.05),miR-195-5p表达降低(P<0.05);转染si-FGD5-AS1或miR-195-5p mimics,与阴性对照相比,细胞活力降低(P<0.05),凋亡率升高(P<0.05),CyclinD1、β-catenin蛋白水平降低(P<0.05),Cleaved-caspase-3蛋白水平升高(P<0.05);双荧光素酶报告基因实验证实FGD5-AS1可靶向结合miR-195-5p;OCI-Ly1细胞转染anti-miR-195-5p,与转染anti-miR-NC相比,细胞活力升高(P<0.05),凋亡率降低(P<0.05),CyclinD1、β-catenin蛋白水平升高(P<0.05),Cleaved-caspase-3蛋白水平降低(P<0.05);OCI-Ly1细胞转染si-FGD5-AS1与antimiR-195-5p,与转染si-FGD5-AS1、anti-miR-NC比较,细胞活力升高(P<0.05),凋亡率降低(P<0.05),CyclinD1、β-catenin蛋白水平升高(P<0.05),Cleaved-caspase-3蛋白水平降低(P<0.05)。结论:干扰FGD5-AS1表达可通过靶向miR-195-5p减弱弥漫型大B细胞淋巴瘤细胞增殖,并诱导其凋亡,其作用机制可能与抑制Wnt/β-catenin信号通路活化有关。

Objective:To explore molecular mechanism of FGD5-AS1 targeting miR-195-5p to regulate proliferation and apoptosis of diffuse large B cell lymphoma cells.Methods:qRT-PCR method was used to detect expressions of FGD5-AS1 and miR-195-5p in B lymphoblasts IM-9 and diffuse large B cell lymphoma cell lines OCI-Ly1,OCI-Ly4 and OCI-Ly10. si-NC,si-FGD5-AS1,miR-NC,miR-195-5p mimics,anti-miR-NC,anti-miR-195-5p,si-FGD5-AS1+anti-miR-NC,si-FGD5-AS1+anti-miR-195-5p were transfected into OCI-Ly1 cells respectively. MTT was used to detect cell viability,flow cytometry was used to detect apoptosis rate,dual luciferase reporter gene experiment was used to detect targeting relationship between FGD5-AS1 and miR-195-5p. Western blot was used to detect expressions of CyclinD1,Cleaved-caspase-3 and β-catenin proteins.Results:Compared with IM-9 cells,expression of FGD5-AS1 in diffuse large B cell lymphoma cell lines OCI-Ly1,OCI-Ly4,OCI-Ly10 was increased(P<0.05),and expression of miR-195-5p was decreased(P<0.05). When transfected with si-FGD5-AS1 or miR-195-5p mimics,compared with its negative control,cell viability was decreased(P<0.05),apoptosis rate was increased(P<0.05),protein levels of CyclinD1,β-catenin were decreased(P<0.05),and protein level of Cleaved-caspase-3 was increased(P<0.05). Dual luciferase reporter gene experiment confirmed that FGD5-AS1could target miR-195-5p. OCI-Ly1 cells transfected with anti-miR-195-5p,compared with transfection with anti-miR-NC,cell viability was increased(P<0.05),apoptosis rate was decreased(P<0.05),protein levels of CyclinD1,β-catenin were increased(P<0.05),and protein level of Cleaved-caspase-3 was decreased(P<0.05). Compared with transfection of si-FGD5-AS1 and anti-miR-NC in OCI-Ly1 cells,cell viability was increased(P<0.05),apoptosis rate was decreased(P<0.05),protein levels of CyclinD1 andβ-catenin were increased(P<0.05),and protein level of Cleaved-caspase-3 was decreased(P<0.05).Conclusion:Interfering with expression of FGD5-AS1 can reduce proliferation ability of diffuse large B cell lymphoma cells and induce apoptosis by targeting miR-195-5p,whose mechanism may be related to inhibition of Wnt/β-catenin signaling pathway.