Association of miRNA-196b-5p and miRNA-99a-5p with autophagy and apoptosis in multiple myeloma cells
10.3760/cma.j.issn.0253-2727.2018.09.013
- VernacularTitle: miRNA-196b-5p和miRNA-99a-5p对多发性骨髓瘤细胞自噬及凋亡的影响
- Author:
Jin SHANG
1
;
Zhizhong CHEN
;
Zhihong WANG
;
Tiannan WEI
;
Wenbing WU
;
Weimin CHEN
Author Information
1. Fujian Medical University Shengli Clinical Medical College, Department of Hematology, Fujian Provincial Hospital, Fuzhou 350001, China
- Publication Type:Journal Article
- Keywords:
Multiple myeloma;
MicroRNAs;
Autophagy;
Apoptosis
- From:
Chinese Journal of Hematology
2018;39(9):766-772
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the relationship between miRNA-196b-5p and miRNA-99a-5p expression and autophagy and apoptosis in multiple myeloma cells.
Methods:Human myeloma cell line U266 and normal CD138+ plasma cells were selected as the research objects. The subjects were divided into 45 cases of multiple myeloma patients and 40 healthy controls. The expression of miRNA-196b-5p and miRNA-99a-5p was measured by real-time quantitative PCR, and Western blot was used to determine the expression of autophagy related protein LC3-Ⅱ, LC3-Ⅰ, P62, Beclin-1 expression, apoptosis related protein CL caspase3, CL caspase7, Bcl-2, Bax, and TGF-β/Smad pathway associated proteins TGF-β1, Smad2/3, p-Smad3 and Smad7. The cell apoptosis rate was determined by flow cytometry. The correlation between miRNA expression level and clinical characteristics of multiple myeloma patients was analyzed.
Results:Compared with normal plasma cells, the expression of miRNA-196b-5p in myeloma cells increased significantly (0.43±0.15 vs 2.44±0.63 or 2.02±0.85, all P<0.001), the expression of miRNA-99a-5p was significantly decreased (1.87±0.61 vs 0.62±0.15 or 0.80±0.33, P<0.001), LC3-Ⅱ/LC3-Ⅰ increased significantly (P<0.05), Beclin-1 expression increased significantly (P<0.05), P62 expression decreased significantly (P<0.05). The expression of Bax, CL caspase3 and CL caspase7 decreased significantly (P<0.05), and the expression of Bcl-2 increased significantly (P<0.05) and apoptosis rate significantly decreased (P<0.05). After transfected with miRNA-196b-5p mimic or miRNA-99a-5p inhibitor, the LC3-Ⅱ/LC3-Ⅰ of CD138+ plasma cells increased significantly (P<0.05), the expression of Beclin-1 increased significantly (P<0.05), P62 expression decreased significantly (P<0.05), and the apoptosis rate significantly decreased (P<0.05). However, after autophagy inhibitor of 3-MA was administered, the apoptotic rate of the above reaction system did not change significantly (P>0.05). The expression of miRNA-196b-5p and miRNA-99a-5p was significantly correlated with DS and ISS stage in multiple myeloma patients (P<0.05).
Conclusion:miRNA-196b-5p and miRNA-99a-5p are closely related to the clinical characteristics of patients with multiple myeloma. The overexpression of miRNA-196b-5p and down regulation of miRNA-99a-5p could inhibit the apoptosis of myeloma cells by up regulation of autophagy, and the mechanism is related to the activation of the TGF-β/Smad signaling pathway.
