SPAG6通过NF-κB/TNF-α途径促进B-ALL细胞增殖和耐药

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

目的探究精子相关抗原6(sperm-associated antigen 6,SPAG6)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SPAG6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SPAG6基因过表达(SPAG6-OE)和敲低(SPAG6-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SPAG6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SPAG6组腹腔注射生理盐水作为对照组,-/+敲低SPAG6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SPAG6 mRNA表达明显高于IDA组(P<0.05),同时与CR组比较,SPAG6高表达于MRD+组(P=0.001)和复发组(P=0.003)。对比不同SPAG6表达水平细胞对化疗药物的反应,CCK-8实验证实SPAG6促进B-ALL细胞增殖(P<0.05),并降低其对DNR和MTX的敏感性(P均<0.05);机制上,GSEA结果提示NF-κB通路在B-ALL富集,实验证实SPAG6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SPAG6明显减小小鼠移植瘤体积(P<0.05),并在联合DNR治疗组观察到肿瘤体积额外减小(P<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SPAG6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SPAG6与B-ALL患者的治疗效果和预后密切相关。

Objective To elucidate the underlying mechanisms of sperm-associated antigen 6(SPAG6)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 SPAG6 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 SPAG6 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 SPAG6 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 SPAG6-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 viability 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 SPAG6-/+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 SPAG6 was significantly higher in the B-ALL patients(P<0.05),and in the MRD group(P=0.001)and R group(P=0.003)than the CR group.SPAG6 overexpression and knockdown in B-ALL cells confirmed that SPAG6 promoted the proliferation(P<0.05)and decreased the sensitivity to DNR and MTX(P<0.05).SPAG6 resisted chemotherapy-induced apoptosis in B-ALL cells by down-regulating pro-apoptotic proteins Bax and Caspase-3(P<0.05),and up-regulating the expression of anti-apoptotic protein Bcl-2(P<0.05).GSEA analysis suggested that the NF-κB pathway was enriched in B-ALL cells,and our experiments confirmed that SPAG6 enhanced the chemoresistance of B-ALL cells to chemotherapy by activating the NF-κB/TNF-αpathway.In in vivo experiments,knocking SPAG6 down significantly reduced the volume of transplanted tumors(P<0.05),and an additional decrease in tumor growth was observed in the DNR-combined treatment group(P<0.05),with reduced levels of NF-κB P65 and p-IKBαby immunohistochemical assay.Conclusion SPAG6 promotes the proliferation of B-ALL cells and reduces their chemosensitivity via the NF-κB/TNF-αpathway,suggesting a close association of SPAG6 with the therapeutic outcomes and prognosis in B-ALL patients.