<italic style="font-style: italic">SPAG</italic>6 promotes proliferation and drug resistance in B-ALL cells through the NF-κB/ TNF-α pathway

Shirui PAN; Jie LUO; Jing LUO; Jiaxiu YIN; Haiqiu ZHAO; Rong SU; Lin LIU

doi:10.16016/j.2097-0927.202407086

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SPAG6 promotes proliferation and drug resistance in B-ALL cells through the NF-κB/ TNF-α pathway

Basic Medicine | 更新时间：2026-01-09

- SPAG6 promotes proliferation and drug resistance in B-ALL cells through the NF-κB/ TNF-α pathway
- Journal of Army Medical University Vol. 46, Issue 24, Pages: 2723-2735(2024)
- 作者机构：
  
  1.400016 重庆，重庆医科大学附属第一医院血液内科
  2.400016 重庆，重庆医科大学实验研究中心
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(82070130)
- DOI：10.16016/j.2097-0927.202407086
  CLC： R341;R730.23;R733.7
- Received：22 July 2024，
  
  Revised：2024-11-15，
  
  Published：30 December 2024
- 稿件说明：
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Shirui PAN, Jie LUO, Jing LUO, et al. SPAG6 promotes proliferation and drug resistance in B-ALL cells through the NF-κB/ TNF-α pathway[J]. Journal of Army Medical University, 2024, 46(24): 2723-2735.
DOI：

Shirui PAN, Jie LUO, Jing LUO, et al. SPAG6 promotes proliferation and drug resistance in B-ALL cells through the NF-κB/ TNF-α pathway[J]. Journal of Army Medical University, 2024, 46(24): 2723-2735. DOI： 10.16016/j.2097-0927.202407086.

摘要

目的

探究精子相关抗原6(sperm-associated antigen 6

SPAG

6)对急性B淋巴细胞白血病(B-cell acute lymphoblastic leukemia

B-ALL)细胞增殖及耐药的相关作用机制。

方法

纳入2019年1月至2023年12月重庆医科大学附属第一医院血液内科收治的56例B-ALL患者和15例缺铁性贫血(iron-deficiency anemia

IDA)患者

将B-ALL患者作为实验组

IDA患者作为对照组。收集B-ALL患者骨髓组织并提取其骨髓单个核细胞

RT-qPCR检测

SPAG

6的mRNA表达情况并对B-ALL患者进行分层分析

分为新诊断病例组(New-diag)、完全缓解组(complete remission

CR)、微小残留病(minimal residual disease

MRD)阳性组(MRD+)和复发组(Relapse); 使用慢病毒感染构建

SPAG

6基因过表达(

SPAG

6-OE)和敲低(

SPAG

6-KD)的B-ALL细胞系

CCK-8和基于甲基纤维素的集落形成实验检测各组细胞的存活、增殖和成集落潜能; CCK-8检测化疗药物柔红霉素(daunorubicin

DNR)和甲氨蝶呤(methotrexate

MTX)处理后细胞存活率(可反映其药物敏感性和IC50);从TARGET数据库获取B-ALL患者的mRNA-seq图谱

通过基因集富集分析(gene set enrichment analysis

GSEA)探索与

SPAG

6相关的信号通路并进行实验验证; NF-κB激活剂PMA和抑制剂BAY-11-7082作用后

通过CCK-8检测细胞存活率以及对化疗药物的敏感性

Western blot检测通路相关蛋白NF-κB P65、p-NF-κB P65、TNF-α等的表达情况; 构建小鼠异种移植瘤模型

-/+敲低

SPAG

6组腹腔注射生理盐水作为对照组

-/+敲低

SPAG

6组腹腔注射DNR作为实验组

免疫组化检测NF-κB信号通路在组织中的表达情况。

结果

B-ALL患者

SPAG

6 mRNA表达明显高于IDA组(

0.05)

同时与CR组比较

SPAG

6高表达于MRD+组(

=0.001)和复发组(

=0.003)。对比不同

SPAG

6表达水平细胞对化疗药物的反应

CCK-8实验证实

SPAG

6促进B-ALL细胞增殖(

0.05)

并降低其对DNR和MTX的敏感性(

均

0.05);机制上

GSEA结果提示NF-κB通路在B-ALL富集

实验证实

SPAG

6通过激活NF-κB/TNF-α通路增强B-ALL细胞对化疗药物的耐药。在体内

敲低

SPAG

6明显减小小鼠移植瘤体积(

0.05)

并在联合DNR治疗组观察到肿瘤体积额外减小(

0.05)。此外

免疫组化结果提示联合DNR治疗组NF-κB P65和p-IKBα水平降低。

结论

SPAG

6通过NF-κB/TNF-α通路促进B-ALL细胞增殖

降低其化疗敏感性

提示

SPAG

6与B-ALL患者的治疗效果和预后密切相关。

Abstract

Objective

To elucidate the underlying mechanisms of sperm-associated antigen 6(

SPAG

6) in the proliferation and drug resistance of B-cell acute lymphoblastic leukemia (B-ALL).

Methods

A total of 56 B-ALL patients and 15 iron-deficiency anemia (IDA) patients admitted in the First Affiliated Hospital of Chongqing Medical University from January 2019 to December 2023 were recruited and served as the experimental and control groups

respectively.Bone marrow mononuclear cells (BMMNCs) were derived from the bone marrow tissues of the experimental group.According to the results of qRT-PCR for the expression of

SPAG

6 in the obtained BMMNCs

the B-ALL patients were stratified into newly-diagnosed (New-diag) group

complete remission (CR) group

minimal residual disease Possitive (MRD+) group

and relapse group.Lentiviral vectors were used to construct B-ALL cells with

SPAG

6 overexpression and knockdown.CCK-8 assay and methylcellulose-based colony formation experiment were employed to assess the survival

proliferation

and clonogenic potential of the cells with varying

SPAG

6 expression levels.After f B-ALL cells were treated with the chemotherapeutic drugs

daunorubicin (DNR) and methotrexate (MTX)

CCK-8 assay

flow cytometry and Western blot analysis were applied to detect cell viability (drug sensitivity and IC50)

cell apoptosis

and protein levels of apoptosis-related molecules Bax

Caspase-3 and Bcl-2

respectively.The mRNA-seq profiles of B-ALL patients were obtained from the TARGET database

and gene set enrichment analysis (GSEA) was conducted to explore

SPAG

6-associated signaling pathways

which were subsequently validated experimentally.After treatment with the NF-κB agonist Phorbol 12-myristate 13-acetate (PMA) and the antagonist BAY-11-7082

cell viabi

lity and sensitivity to chemotherapeutic drugs were assessed with CCK-8 assay

and the expression of pathway-related proteins NF-κB P65

p-NF-κB p65

IKBα

p-IKBα

TNF-α

and EPO was detected by Western blot analysis.A mouse xenograft tumor model was constructed in

SPAG

-/+

knockdown mice to observe the effect of DNR on tumor growth and the expression of the NF-κB signaling pathway in the tumor tissues with immunohistochemical assay.

Results

The mRNA level of

SPAG

6 was significantly higher in the B-ALL patients (

0.05)

and in the MRD group (

=0.001) and R group (

=0.003) than the CR group.

SPAG

6 overexpression and knockdown in B-ALL cells confirmed that

SPAG

6 promoted the proliferation (

0.05) and decreased the sensitivity to DNR and MTX (

0.05).

SPAG

6 resisted chemotherapy-induced apoptosis in B-ALL cells by down-regulating pro-apoptotic proteins Bax and Caspase-3(

0.05)

and up-regulating the expression of anti-apoptotic protein Bcl-2(

0.05).GSEA analysis suggested that the NF-κB pathway was enriched in B-ALL cells

and our experiments confirmed that

SPAG

6 enhanced the chemoresistance of B-ALL cells to chemotherapy by activating the NF-κB/TNF-α pathway.In

in vivo

experiments

knocking

SPAG

6 down significantly reduced the volume of transplanted tumors (

0.05)

and an additional decrease in tumor growth was observed in the DNR-combined treatment group (

0.05)

with reduced levels of NF-κB P65 and p-IKBα by immunohistochemical assay.

Conclusion

SPAG

6 promotes the proliferation of B-ALL cells and reduces their chemosensitivity

via

the NF-κB/TNF-α pathway

suggesting a close association

SPAG

6 with the therapeutic outcomes and prognosis in B-ALL patients.

关键词

Keywords

references

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⁰

SPAG6 promotes proliferation and drug resistance in B-ALL cells through the NF-κB/ TNF-α pathway

DOI：10.16016/j.2097-0927.202407086

摘要

Abstract

关键词

Keywords

references