LncRNA DANCR 调节 miR-656/BMPR1A 轴对脑胶质瘤细胞恶性行为的影响

Effect of LncRNA DANCR on the immune microenvironment of glioma cells by regu- lating the miR-656/BMPR1A axis

摘要

目的：探讨长链非编码 RNA（long non-coding RNA，LncRNA）分化拮抗非蛋白编码 RNA（differentiation an- tagonizing non-protein coding RNA，DANCR）调节微小 RNA-656（miR-656）/骨形态发生蛋白受体1A（bone morphoge-netic protein receptor type 1A，BMPR1A）轴对脑胶质瘤细胞免疫微环境的影响。方法：实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction，qRT-PCR）检测脑胶质瘤细胞中 DANCR、miR-656、BMPR1A 的表达水平。通过转染或共转染至 U87 细胞，分为沉默（short hairpin，sh）-DANCR 组、过表达（plasmid cytome galoirus， pc）DNA 3.1DANCR 组、阴性对照（negative control，NC）sh 组、pcDNA 3.1 组、sh-DANCR＋miR-656 抑制剂组、sh-DANCR＋NC 抑制剂组、sh-DANCR＋pcDNA 3.1 BMPR1A 组、sh-DANCR＋pcDNA 3.1 组，并以未处理的 U87 细胞为空白组。CCK8 检测 U87 细胞增殖；Transwell 实验检测侵袭与迁移；流式细胞仪检测细胞凋亡；qRT-PCR 检测细胞中 DANCR、miR-656、BMPR1A mRNA 表达；DANCR 与 miR-656 靶向关系通过双荧光素酶验证；BMPR1A 及免疫逃逸因子[程序性死亡受体1（programmed death-1，PD-1）和程序性死亡配体1（programmed death-ligand 1，PD-L1）]通过 Western blot 检测。结果：U87、A172、LN229和U251细胞较NHA 细胞中 DANCR、BMPR1A mRNA 表达显著增加，miR-656 表达显著降低（P<0.05）；与空白组、sh-DANCR 组相比，sh-DANCR 组 U87 细胞增殖率，侵袭、迁移数，DANCR、BMPR1A mRNA、BMPR1A、PD-1、PD-L1 表达显著降低，凋亡率、miR-656 表达显著增加（P<0.05）；与pcDNA 3.1 组相比，pcDNA 3.1 DANCR 组 U87 细胞增殖率，侵袭、迁移数，DANCR、BMPR1A mRNA、BMPR1A、PD-1、PD-L1 表达显著增加，凋亡率、miR-656 表达显著降低（P<0.05）；与 sh-DANCR＋NC 抑制剂组相比，sh-DANCR＋miR-656 抑制剂组 U87 细胞增殖率，侵袭、迁移数，BMPR1A mRNA、BMPR1A、PD-1、PD-L1 表达显著增加，凋亡率、miR-656 表达显著降低（P<0.05），DANCR 表达的差异无统计学意义（P>0.05）；与 sh-DANCR＋pcDNA 3.1 组相比，sh-DANCR＋pcDNA 3. 1 BMPR1A 组 U87 细胞增殖率，侵袭、迁移数，BMPR1A mRNA、BMPR1A、PD-1、PD-L1 表达显著增加，凋亡率显著降低（P<0.05），miR-656 表达、DANCR 表达差异无统计学意义（P>0.05）。DANCR、miR-656 具有靶向负调节关系。结论：LncRNA DANCR 调节 miR-656/BMPR1A 轴改善脑胶质瘤细胞免疫微环境，抑制癌细胞恶性行为发展。

Abstract

Objective：To investigate the effect of long non-coding RNA（LncRNA）differentiation an- tagonizing non-protein coding RNA（DANCR）on the immune microenvironment of glioma cells by regu- lating the miR-656/bone morphogenetic protein receptor type 1A（BMPR1A）axis. Methods：The ex- pression levels of DANCR，miR-656 and BMPR1A in glioma cells were detected by quantitative real-time polymerase chain reaction（qRT-PCR）.The U87 cells were transfected or co-transfected to form the fol- lowing groups：sh-DANCR（transfected with sh-DANCR），overexpression（pcDNA 3.1）DANCR （transfected with pcDNA 3. 1 DANCR），NC sh（transfected with negative control sh），pcDNA 3.1 （transfected with pcDNA 3.1 vector），sh-DANCR＋miR-656 inhibitor（co-transfected with sh-DANCR and miR-656 inhibitor），sh-DANCR＋NC inhibitor（co-transfected with sh-DANCR and NC inhibi- tor），sh-DANCR＋pcDNA 3.1 BMPR1A（co-transfected with sh-DANCR and pcDNA 3.1 BMPR1A）， and sh-DANCR＋pcDNA 3.1 （co-transfected with sh-DANCR and pcDNA 3.1 BMPR1A）.The un-treated U87 cells were used as the blank group.The proliferation of U87 cells was detected by CCK-8； invasion and migration were detected by Transwell assay；apoptosis was detected by flow cytometry；the expression of DANCR，miR-656，and BMPR1A mRNA in cells was detected by qRT-PCR；the targeting relationship between DANCR and miR-656 was verified by dual-luciferase；and BMPR1A and immune escape factors[programmed death receptor 1 （PD-1）and programmed death-ligand 1 （PD-L1）]were detected by Western blot. Results：The mRNA expressions of DANCR and BMPR1A in U87，A172， LN229 and U251 cells were significantly increased，while the expression of miR-656 was significantly de- creased compared with those in NHA cells (P<0.05).Compared with the blank group and sh-DANCR group，the proliferation rate，invasion，migration number，DANCR，BMPR1A mRNA，BMPR1A，PD-1， PD-L1 expression of U87 cells in the sh-DANCR group were obviously reduced，while the apoptosis rate and miR-656 expression were obviously increased (P<0.05).Compared with the pcDNA 3.1 group， the proliferation rate，invasion，migration number，DANCR，BMPR1A mRNA，BMPR1A，PD-1，and PD-L1 expression of U87 cells in the pcDNA 3. 1 DANCR group were obviously increased，while the apoptosis rate and miR-656 expression were obviously reduced (P<0.05).Compared with the sh-DANCR＋ NC inhibitor group，the proliferation rate，invasion，migration number，BMPR1A mRNA，BMPR1A， PD-1，and PD-L1 expression of U87 cells in the sh-DANCR＋miR-656 inhibitor group were obviously in- creased，and the apoptosis rate and miR-656 expression were obviously reduced (P<0.05) ，while the expression of DANCR was not obvious (P>0.05).Compared with the sh-DANCR＋pcDNA 3.1 group， the proliferation rate，invasion，migration number，BMPR1A mRNA，BMPR1A，PD-1，and PD-L1 ex- pression of U87 cells in the sh-DANCR＋pcDNA 3. 1 BMPR1A group obviously increased，and the apop- tosis rate obviously decreased (P<0.05) ，while here was no statistically obvious difference in miR-656 expression and DANCR expression (P>0.05).DANCR and miR-656 had a targeted negative regulatory relationship. Conclusion：LncRNA DANCR improves the immune microenvironment of glioma cells and inhibits the malignant behavior development of cancer cells by regulating the miR-656/BMPR1A axis.

关键词

LncRNA DANCR / miR-656/BMPR1A 轴 / 脑胶质瘤细胞 / 免疫微环境 / 恶性行为发展

Key words

LncRNA DANCR / MiR-656/BMPR1A axis / Glioma cells / Immune microenvironment / Malignant behavior development

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基金资助

温州市科技局课题(Y2023462)