LncRNA PVT1对弥漫大B细胞淋巴瘤细胞活性的影响及其机制  

Effect of long non-coding RNA plasmacytoma variant translocation gene 1 on the viability of diffuse large B-cell lymphoma cells and its mechanism

作　　者：路晓辉[1] 李文永[1] 王孟林 陈香莉 LU Xiaohui;LI Wenyong;WANG Menglin;CHEN Xiangli(Lymphoma Hematopoietic Stem Cell Transplantation Center,Jiaozuo People’s Hospital,Jiaozuo 454150,China)

机构地区：[1]焦作市人民医院淋巴瘤造血干细胞移植中心,河南焦作454150 [2]河南省人民医院血液内科

出　　处：《青岛大学学报（医学版）》2024年第3期381-387,共7页Journal of Qingdao University(Medical Sciences)

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20191341)。

摘　　要：目的 探究长链非编码RNA(LncRNA)浆细胞瘤变体异位基因1(PVT1)对弥漫大B细胞淋巴瘤(DLBCL)细胞生物学行为的影响,并分析其潜在机制。方法 收集41例DLBCL病人和15例淋巴结反应性增生(RLH)病人的组织标本,体外培养人正常B淋巴细胞GM12878和人DLBCL细胞(OCI-Ly3、U2932、TMD8),对TMD8细胞进行转染,将其分为control组(只转染Lipofectamine-2000)、si-NC组(转染si-NC)、inhibitor-NC组(转染inhibitor-NC)、si-PVT1组(转染si-PVT1)、miR-145-5p inhibitor组(转染miR-145-5p inhibitor)、si-PVT1+miR-145-5p inhibitor组(转染si-PVT1和miR-145-5p inhibitor)。应用qRT-PCR方法检测各组细胞PVT1 mRNA和miR-145-5p表达,Western Blot方法检测CDK6蛋白表达,CCK-8法检测TMD8细胞增殖,流式细胞术检测TMD8细胞周期变化,Transwell实验检测TMD8细胞迁移和侵袭能力,RNA pull down和双荧光素酶报告基因法验证PVT1、miR-145-5p与细胞周期蛋白依赖性激酶6(CDK6)的靶向关系。结果 DLBCL组织PVT1 mRNA、CDK6蛋白的表达水平高于RLH组织,miR-145-5p表达低于RLH组织(t=14.264~24.445,P<0.05)。与GM12878细胞比较,OCI-Ly3、U2932、TMD8细胞中PVT1 mRNA、CDK6蛋白表达均增加,miR-145-5p表达均减少(F=69.557~234.718,P<0.05)。6组细胞PVT1 mRNA、miR-145-5p、CDK6蛋白表达及增殖率、G0/G1期细胞比例、S期细胞比例、迁移和侵袭细胞数差异有统计学意义(F=25.589~319.150,P<0.05);与control组比较,si-PVT1组细胞PVT1 mRNA、CDK6蛋白、增殖率、S期细胞比例、迁移和侵袭数量降低,miR-145-5p表达、G0/G1期细胞比例升高(P<0.05),miR-145-5p inhibitor组呈相反变化(P<0.05);下调miR-145-5p表达可减弱敲低PVT1对TMD8细胞恶性生物学行为的抑制作用(P<0.05)。过表达PVT1 mRNA增高CDK6蛋白表达、细胞增殖率、S期细胞比例、迁移和侵袭数量,降低miR-145-5p表达、G0/G1期的细胞比例(F=38.025~327.887,P<0.05)。miR-145-5p是PVT1的靶基因,且miR-145-5p可靶向下调CDK6表达。结论 敲低PVT1可Objective To investigate the effect of long non-coding RNA(LncRNA)plasmacytoma variant translocation gene 1(PVT1)on the biological behavior of diffuse large B-cell lymphoma(DLBCL)cells and its potential mechanism.Methods Tissue specimens were collected from 41 patients with DLBCL and 15 patients with lymph node reactive hyperplasia(RLH),and normal human B lymphocytes GM12878 and human DLBCL cells(OCI-Ly3,U2932,TMD8)were cultured in vitro.TMD8 cells were transfected and divided into control group(transfected with Lipofectamine-2000 alone),si-NC group(transfected with si-NC),inhibitor-NC group(transfected with inhibitor-NC),si-PVT1 group(transfected with si-PVT1),miR-145-5p inhibitor group(transfected with miR-145-5p inhibitor),and si-PVT1+miR-145-5p inhibitor group(transfected with si-PVT1 and miR-145-5p inhibitor).The qRT-PCR method was used to measure the mRNA expression levels of PVT1 and miR-145-5p in each group of cells;Western Blot was used to measure the protein expression level of cyclin-dependent kinase 6(CDK6);CCK-8 assay was used to measure the proliferation of TMD8 cells;flow cytometry was used to measure the change in cell cycle of TMD8 cells;Transwell assay was used to measure the migration and invasion abilities of TMD8 cells;RNA pull-down and dual-luciferase reporter assay were used to verify the targeting relationship between PVT1,miR-145-5p,and CDK6.Results The mRNA expression level of PVT1 and the protein expression level of CDK6 in DLBCL tissue were significantly higher than those in RLH tissue,and the expression level of miR-145-5p in DLBCL tissue was significantly lower than that in RLH tissue(t=14.264-24.445,P<0.05).Compared with GM12878 cells,OCI-Ly3,U2932,and TMD8 cells had significant increases in the mRNA expression level of PVT1 and the protein expression level of CDK6 and a significant reduction in the expression level of miR-145-5p(F=69.557-234.718,P<0.05).There were significant differences between the six groups of cells in PVT1,miR-145-5p,CDK6,proliferation rate,the proportion of ce

关 键 词：淋巴瘤 大B细胞 弥漫性 RNA 长链非编码 浆细胞瘤变体异位基因1 miR-145-5p 细胞周期蛋白依赖激酶6 

分 类 号：R733.4[医药卫生—肿瘤]