[1]王 婷a,白 彬b,许红月a,等.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[J].现代检验医学杂志,2025,40(01):24-31.[doi:10.3969/j.issn.1671-7414.2025.01.05]
　WANG Tinga,BAI Binb,XU Hongyuea,et al.Experimental Study on the Mechanism of LncRNA NEAT1 Promoting Proliferation and Metastasis of Myeloma Cells Through miR-195-5p/CEBPA Axis[J].Journal of Modern Laboratory Medicine,2025,40(01):24-31.[doi:10.3969/j.issn.1671-7414.2025.01.05]
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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()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第40卷
期数:
2025年01期
页码:
24-31
栏目:
论著
出版日期:
2025-01-15

文章信息/Info

Title:
Experimental Study on the Mechanism of LncRNA NEAT1 Promoting Proliferation and Metastasis of Myeloma Cells Through miR-195-5p/CEBPA Axis
文章编号:
1671-7414（2025）01-024-08
作者:
王 婷a; 白 彬b; 许红月a; 张学勇a
（山东省第一医科大学附属聊城市第二人民医院a. 血液内科；b. 病理科，山东聊城 252600）
Author(s):
WANG Tinga;  BAI Binb;  XU Hongyuea;  ZHANG Xueyonga
（a. Department of Hematology; b. Department of Pathology, the Second People’s Hospital of Liaocheng, Shandong First Medical University, Shandong Liaocheng 252600, China）
关键词:
多发性骨髓瘤; 核富集丰富的转录本1; 微小RNA-195-5p; CCAAT 增强子结合蛋白α; 增殖
分类号:
R733.3；R730.43
DOI:
10.3969/j.issn.1671-7414.2025.01.05
文献标志码:
A
摘要:
目的 探究长链非编码RNA（LncRNA）核富集丰富的转录本1（NEAT1）通过miR-195-5p/CCAAT 增强子结合蛋白α（CEBPA）轴对骨髓瘤细胞增殖和转移的影响及机制。方法 ①收集2021 年3 月～ 2023 年2 月在山东省第一医科大学附属聊城市第二人民医院血液内科住院的40 例多发性骨髓瘤（MM）患者和10 例健康骨髓供者的骨髓单个核细胞（BMNC）及MM 细胞系U266，RPMI 8226，NCI-H 929 和MM.1S，RT-qPCR 和Western blot 检测细胞中NEAT1，miR-195-5p，CEBPA mRNA 和蛋白水平。② U266 细胞分为过表达NEAT1 组（NEAT1 组）及其对照组（NC 组）、敲低NEAT1 表达组（sh-NEAT1 组）及其对照组（sh-NC 组）、过表达NEAT1 和miR-195-5p 组（NEAT1+miR-195-5p 组）及其对照组（NEAT1+miR-NC 组）、过表达miR-195-5p 和CEBPA组（miR-195-5p+CEBPA组）及其对照组（miR-195-5p+NC组）。CCK-8 和5- 乙炔基-2’- 脱氧尿苷（EdU）染色检测细胞增殖能力；Transwell 实验检测细胞迁移和侵袭能力；RT-qPCR 检测细胞中NEAT1，miR-195-5p，CEBPA mRNA 水平；Western blot 检测细胞中CEBPA，Ki67，增殖细胞核抗原（PCNA），基质金属蛋白酶（MMP）-2，MMP-9，磷脂酰肌醇3 激酶（PI3K），p-PI3K，蛋白激酶B（AKT），p-AKT，雷帕霉素靶蛋白（mTOR），p-mTOR 蛋白水平。③ 20 只裸鼠分为敲低NEAT1 表达组（sh-NEAT1 组）及其对照组（sh-NC组），每组各10 只。裸鼠皮下注射相应已转染的U266 细胞悬液，四周后测定移植瘤各项指标差异。结果 ①与正常BMNC 相比，MM 患者和MM 细胞系中NEAT1，CEBPA mRNA 和蛋白水平升高，miR-195-5p 水平降低，差异具有统计学意义（t=11.697，9.272，4.352，11.639，均P<0.05）。②过表达NEAT1 后，与NC 组比较，NEAT1 组细胞NEAT1，CEBPA mRNA 和蛋白水平升高，miR-195-5p 水平降低（t=12.825，5.874，13.893，4.797）；细胞培养72h A 值、EdU 阳性率升高，细胞迁移、侵袭数增多（t=9.425，5.632，8.841，5.364）；Ki67，PCNA，MMP-2，MMP-9，p-PI3K/PI3K，p-AKT/AKT，p-mTOR/mTOR 蛋白水平升高（t=14.227，7.743，7.348，7.803，8.714，8.629，7.359），差异具有统计学意义（均P<0.05）；敲低NEAT1 表达后，与sh-NC 组比较，sh- NEAT1 组细胞NEAT1，CEBPA mRNA 和蛋白水平降低（t=5.776，5.001，4.503），miR-195-5p 水平升高（t=4.456），细胞迁移、侵袭能力降低（t=6.204，8.792），差异具有统计学意义（均P<0.05）；过表达miR-195-5p 能够部分逆转过表达NEAT1 对细胞上述指标的影响，差异具有统计学意义（t=4.356 ～ 10.809，均P<0.05）；过表达CEBPA 能够部分逆转过表达miR-195-5p 对细胞上述指标的影响，差异具有统计学意义（t=4.329 ～ 14.452，均P<0.05）。③敲低NEAT1 表达能够抑制裸鼠移植瘤生长，差异具有统计学意义（t=5.175 ～ 18.190，均P<0.05）。结论 LncRNA NEAT1 通过靶向miR-195-5p/CEBPA 表达促进MM 细胞增殖和转移，其可能通过激活PI3K/AKT/mTOR 通路发挥作用。
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.

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备注/Memo

备注/Memo:
基金项目：山东省医药卫生科技发展计划项目（202203040878）。
作者简介：王婷（1983-），女，硕士，副主任医师，研究方向：浆细胞疾病的病生理及临床诊治，E-mail：wangting131@163cn.com.cn。
通讯作者：张学勇（1969-），男，本科，主任医师，研究方向：难治性贫血，E-mail：xueyong221@163cn.com.cn。
更新日期/Last Update: 2025-01-15