Experimental Study on the Mechanism of LncRNA NEAT1 Promoting Proliferation and Metastasis of Myeloma Cells Through miR-195-5p/CEBPA Axis
10.3969/j.issn.1671-7414.2025.01.05
- VernacularTitle:LncRNA NEAT1通过miR-195-5p/CEBPA轴促进骨髓瘤细胞增殖和转移机制的实验研究
- Author:
Ting WANG
1
;
Bin BAI
;
Hongyue XU
;
Xueyong ZHANG
Author Information
1. 山东省第一医科大学附属聊城市第二人民医院血液内科,山东聊城 252600
- Publication Type:Journal Article
- Keywords:
mulitple myeloma;
nuclear enriched abundant transcript 1;
miR-195-5p;
CCAAT enhancer binding protein α;
proliferation
- From:
Journal of Modern Laboratory Medicine
2025;40(1):24-31
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the effect and mechanism of long non-coding RNA (LncRNA) nuclear enriched abundant transcript 1 (NEAT1) on proliferation and metastasis of myeloma through miR-195-5p/CCAAT enhancer binding protein α (CEBPA) axis. Methods ① From March 2021 to February 2023,bone marrow mononuclear cells (BMNC) from 40 patients with multiple myeloma (MM) and 10 healthy bone marrow donors who were hospitalized in the Department of Hematology the Second People's Hospital of Liaocheng City,Shandong First Medical University and MM cell lines U266,RPMI 8226,NCI-H929 and MM.1S were collected. RT-qPCR and Western blot were used to detect NEAT1,miR-195-5p,CEBPA mRNA and protein levels in cells. ② U266 cells were divided into overexpressing NEAT1 group (NEAT1 group) and its control group (NC group),knockdown NEAT1 expression group (sh-NEAT1 group) and its control group (sh-NC group),overexpressing NEAT1 and miR-195-5p group (NEAT1+miR-195-5p group) and its control group (NEAT1+miR-NC group),and overexpressing miR-195-5p and CEBPA group (miR-195-5p+CEBPA group) and its control group (miR-195-5p+NC group). CCK-8 and EdU staining were used to detect cell proliferation ability. Transwell assay was used to detect cell migration and invasion ability. RT-qPCR was used to detect NEAT1,miR-195-5p,and CEBPA mRNA levels in cells. Western blot was used to detect CEBPA,Ki67,proliferating cell nuclear antigen (PCNA),matrix metalloproteinase (MMP)-2,MMP-9,phosphoinositide 3 kinase (PI3K),p-PI3K,protein kinase B (AKT),p-AKT,mechanistic target of rapamycin (mTOR),and p-mTOR protein levels. ③ Twenty nude mice were divided into knockdown NEAT1 expression group (sh-NEAT1 group) and its control group (sh-NC group),with ten mice in each group. Nude mice were subcutaneously injected with corresponding transfected U266 cell suspension,and the differences in various indicators of transplanted tumor were measured after 4 weeks. Results ① Compared with normal BMNC,NEAT1,CEBPA mRNA and protein levels in MM patients and MM cell lines were increased,while miR-195-5p level was decreased,and the differences were significant (t=11.697,9.272,4.352,11.639,all P<0.05). ② After overexpression of NEAT1,NEAT1,CEBPA mRNA and protein levels in cells were increased (t=12.825,5.874,13.893),while miR-195-5p level was decreased (t=4.797),the A value and EdU positive rate after 72 hours of cell culture were increased (t=9.425,5.632),the number of migration/invasion cells were increased (t=8.841,5.364),and Ki67,PCNA,MMP-2,MMP-9,p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR protein levels were increased (t=14.227,7.743,7.348,7.803,8.714,8.629,7.359),with significant differences (all P<0.05). After knockdown the expression of NEAT1,NEAT1,CEBPA mRNA and protein levels in cells were decreased (t=5.776,5.001,4.503),while miR-195-5p level was increased (t=4.456),the ability of cell proliferation,invasion and metastasis was decreased (t=6.204,8.792),with significant differences (all P<0.05). Overexpression of miR-195-5p could partially reverse the effects of overexpression of NEAT1 on the aforementioned cellular indicators,and the differences were significant (t=4.356～10.809,all P<0.05). Meanwhile,overexpression of CEBPA could partially reverse the effects of overexpression of miR-195-5p on the aforementioned cellular indicators,and the differences were significant (t=4.329～14.452,all P<0.05). ③ Knockdown NEAT1 expression could inhibit the growth of transplanted tumors in nude mice,and the differences were significant (t=5.175～18.190,all P<0.05). Conclusion LncRNA NEAT1 promotes proliferation,invasion and metastasis of MM cells by targeting miR-195-5p/CEBPA expression,which may act by activating the PI3K/AKT/mTOR pathway.
