SMAD7在多发性骨髓瘤中的表达及对细胞增殖和耐药的影响

doi:10.3969/j.issn.1674-8115.2022.07.003

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (7): 858-865.doi: 10.3969/j.issn.1674-8115.2022.07.003

• 论著 · 基础研究 • 上一篇

SMAD7在多发性骨髓瘤中的表达及对细胞增殖和耐药的影响

丁贺¹(), 曹杨琳², 何杨², 魏星³, 杨剑峰¹()

^1.苏州大学苏州医学院唐仲英医学研究院血液学研究中心，苏州 215123
^2.苏州大学附属第一医院江苏省血液研究所，苏州 215006
^3.苏州大学附属第一医院消化内科，苏州 215006

收稿日期:2022-03-07 接受日期:2022-06-24 出版日期:2022-07-28 发布日期:2022-09-04
通讯作者: 杨剑峰 E-mail:1838872960@qq.com;yangjianfeng@suda.edu.cn
作者简介:丁　贺（1997—），女，硕士生；电子信箱：1838872960@qq.com。
基金资助:
国家自然科学基金(81673096)

SMAD7 expression in multiple myeloma and its effect on cell proliferation and drug resistance

DING He¹(), CAO Yanglin², HE Yang², WEI Xing³, YANG Jianfeng¹()

^1.Hematology Center, Cyrus Tang Medical Institute, Suzhou Medical College of Soochow University, Suzhou 215123, China
^2.The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou 215006, China
^3.Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China

Received:2022-03-07 Accepted:2022-06-24 Online:2022-07-28 Published:2022-09-04
Contact: YANG Jianfeng E-mail:1838872960@qq.com;yangjianfeng@suda.edu.cn
Supported by:
National Natural Science Foundation of China(81673096)

摘要/Abstract

摘要：

目的·分析Sma和Mad相关蛋白7（Sma- and Mad-related protein 7，SMAD7）在多发性骨髓瘤（multiple myeloma，MM）中的表达，以及其对MM细胞的增殖和耐药的影响。方法·获取基因表达综合（gene expression omnibus，GEO）数据库中的3个数据集，分析SMAD7在健康捐献者和MM患者中的差异表达。通过收集8例健康捐献者和20例MM患者的骨髓标本，采用实时荧光定量PCR（quantitative real-time PCR，qPCR）检测骨髓CD138⁺细胞中SMAD7 mRNA的相对表达，并分析其与患者临床资料的关系。在MM细胞KMS11中过表达SMAD7后，通过CCK8实验、细胞周期实验观察SMAD7对MM细胞增殖的影响。采用不同浓度的硼替佐米处理过表达SMAD7的KMS11细胞后，观察SMAD7对MM细胞的耐药影响及细胞的凋亡情况。结果·数据集中SMAD7表达数据的分析显示，与健康捐献者相比，SMAD7 在MM患者中表达较高（均P<0.05）。qPCR检测结果显示，SMAD7 mRNA相对表达水平在MM患者中有上调（P=0.002），但未显示出与临床资料间存在关联。与对照细胞相比，过表达SMAD7后，KMS11的细胞活力均较高（均P<0.05）；且细胞周期分布亦发生了变化，S期细胞的比例有所下降（P=0.016）、G₂/M期细胞的比例有所上升（P=0.005）；而经硼替佐米处理后，过表达SMAD7细胞的药物敏感性及凋亡水平均较低。结论·SMAD7在MM中表达上调，过表达SMAD7可促进MM细胞的增殖和耐药能力。

Abstract:

Objective·To analyze the expression of Sma- and Mad-related protein 7 (SMAD7) in multiple myeloma (MM), and its effect on the proliferation and drug resistance of MM cells.

Methods·Three datasets from the Gene Expression Omnibus (GEO) database were obtained to analyze the differential expression of SMAD7 in healthy donors and MM. Bone marrow samples from 8 healthy donors and 20 MM patients were collected. The relative expression of SMAD7 mRNA in bone marrow CD138⁺ cells was detected by quantitative real-time PCR (qPCR), and the relationship between SMAD7 mRNA expression and clinical information of patients was analyzed. After overexpression of SMAD7 in MM cells KMS11, the effects of SMAD7 on the proliferation was observed by CCK8 assay and cell cycle assay. KMS11 cells overexpressing SMAD7 were treated with different concentrations of bortezomib, and the effect of SMAD7 on drug resistance and apoptosis of MM cells were observed.

Results·The analysis of SMAD7 expression data in the datasets showed that compared with healthy donors, SMAD7 expression in MM patients was higher (all P<0.05). The results of qPCR showed that the relative expression of SMAD7 mRNAwas up-regulated in MM patients (P=0.002), although no correlation with clinical information of patients was shown. Compared with the control cells, the cell viability of KMS11 was higher after overexpression of SMAD7 (all P<0.05); the change of cell cycle distribution showed that the proportion of cells in S phase decreased (P=0.016), and the proportion of cells in G₂/M phase increased (P=0.005); after bortezomib treatment, the drug sensitivity and apoptosis level of overexpression SMAD7 cells was lower.

Conclusion·The expression of SMAD7 is up-regulated in MM. Overexpression of SMAD7 can promote the proliferation and drug resistance of MM cells.

Key words: Sma- and Mad-related protein 7 (SMAD7), multiple myeloma (MM), cell proliferation, bortezomib

中图分类号:

R733.3

丁贺, 曹杨琳, 何杨, 魏星, 杨剑峰. SMAD7在多发性骨髓瘤中的表达及对细胞增殖和耐药的影响[J]. 上海交通大学学报（医学版）, 2022, 42(7): 858-865.

DING He, CAO Yanglin, HE Yang, WEI Xing, YANG Jianfeng. SMAD7 expression in multiple myeloma and its effect on cell proliferation and drug resistance[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 858-865.

图/表 6

表1 qPCR引物序列

Tab 1 Primer sequences for qPCR

Primer	Forward （5'→3'）	Reverse （5'→3'）
SMAD7	CCAACTGCAGACTGTCCAGA	TTCTCCTCCCAGTATGCCAC
GAPDH	GTGAAGGTCGGAGTCAACGG	GTTCTCAGCCTTGACGGTGC

图1 GSE5900（A）、GSE6477（B）、GSE13591（C）数据集中与MM相关的 SMAD7 的表达分析Note： HD—healthy donors; NdMM—newly diagnosed multiple myeloma.

Fig 1 Expression analysis of SMAD7 associated with MM in the GSE5900 (A), GSE6477 (B) and GSE13591 (C) datasets

图2 SMAD7 mRNA在健康捐献者及MM患者的骨髓CD138+ 细胞中的相对表达Note： HD—healthy donors.

Fig 2 Relative expression of SMAD7 mRNA in bone marrow CD138+ cells from healthy donors and MM patients

表 1 SMAD7 mRNA相对表达水平与MM患者临床资料的关系

Tab 1 Relationship between the relative expression of SMAD7 mRNA and clinical information of patients with MM

Clinical information	Relative expression of SMAD7 mRNA	P value
Gender		0.877
Male （n=13）	3.87 （0.73， 5.64）
Female （n=7）	3.35 （2.20， 4.86）
Age		0.247
<60 years （n=10）	2.52 （0.28， 4.77）
≥60 years （n=10）	3.96 （2.14， 5.56）
Plasma cell in BM^①		0.958
<10% （n=3）	3.92 （0.95， 4.01）
≥10% （n=16）	2.97 （0.81， 5.72）
Hb level^①		0.408
≥110 g·L^-1（n=10）	3.91 （0.84， 6.16）
<110 g·L^-1（n=8）	2.97 （0.53， 3.99）
Cr level^①		0.421
≥177 μmol·L^-1（n=17）	2.01 （0.10， 3.92）
<177 μmol·L^-1（n=2）	3.35 （1.33， 5.64）
LDH level		0.305
≥245 U·L^-1（n=8）	2.52 （0.35， 4.63）
<245 U·L^-1（n=12）	3.89 （2.02， 5.72）
Destruction of bone		0.699
Yes （n=7）	3.35 （1.33， 5.64）
No （n=13）	3.87 （0.33， 4.86）

图3 SMAD7对KMS11细胞增殖的影响Note： A. Detection of SMAD7 expression in KMS11 cells transfected with Vector or Lv-SMAD7 by Western blotting. B. Detection of the proliferation of KMS11 cells transfected with Vector or Lv-SMAD7 by CCK8. C. Detection of the cell cycle distribution of KMS11 cells transfected with Vector or Lv-SMAD7 by flow cytometry.

Fig 3 Effects of SMAD7 on proliferation of KMS11 cells

图4 SMAD7对经硼替佐米处理的KMS11细胞耐药的影响Note： A. After treatment with bortezomib at different concentrations, the survival rate of KMS11 cells transfected with Vector or Lv-SMAD7 was detected by CCK8 method. B. After treatment with 40 nmol/L bortezomib, the apoptosis of KMS11 cells transfected with Vector or Lv-SMAD7 was detected by flow cytometry.

Fig 4 Effects of SMAD7 on resistance of KMS11 cells treated with bortezomib

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SMAD7在多发性骨髓瘤中的表达及对细胞增殖和耐药的影响

SMAD7 expression in multiple myeloma and its effect on cell proliferation and drug resistance

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