摘要
目的:探讨miRNA异常表达对小儿急性淋巴细胞白血病(ALL)细胞增殖能力的影响及其相关机制。方法:收集2018年7月至2021年3月于海南医学院第二附属医院就诊的ALL患儿和健康受试者各15例,对其骨髓细胞进行miRNA测序,并使用qRT-PCR进行验证。将miR-1294和miR-1294抑制分子(miR-1294-inhibitor)转染至Nalm-6细胞,通过CCK-8和集落形成实验检测Nalm-6细胞增殖情况,Western blot和ELISA检测Nalm-6细胞凋亡情况。对miR-1294进行生物学预测,寻找靶基因,使用荧光素酶报告实验进行验证。将si-SOX15转染至Nalm-6细胞,Western blot检测Wnt信号通路相关蛋白表达情况,并验证si-SOX15对Nalm-6细胞增殖和凋亡的影响。结果:与健康受试者相比,ALL患者骨髓细胞中有22个miRNA显著上调,其中miR-1294上调最为显著。此外,在ALL患者骨髓细胞中SOX15基因表达水平显著降低。与NC组相比,miR-1294组Wnt3a和β-catenin蛋白表达水平升高、细胞增殖速度增加、集落形成数量增多,而caspase-3蛋白表达水平和细胞凋亡率降低。与NC组相比,miR-1294-inhibitor组Wnt3a和β-catenin蛋白表达水平降低、细胞增殖速度减慢、集落形成数量减少,而caspase-3蛋白表达水平增加,细胞凋亡率升高。miR-1294与SOX15的3'UTR区存在碱基互补对,miR-1294直接靶向SOX15。在ALL细胞中,miR-1294和SOX15的表达呈负相关。与si-NC组相比,si-SOX15组Wnt3a和β-catenin蛋白表达水平升高、细胞增殖速度增加,而caspase-3蛋白表达水平和细胞凋亡率降低。结论:miR-1294能够靶向抑制SOX15表达,从而激活Wnt/β-catenin信号通路,促进ALL细胞增殖、抑制细胞凋亡,最终影响疾病进程。
Objective:To explore the effect of abnormal miRNA expression on the proliferation of pediatric acute lymphoblastic leukemia(ALL)cells and its related mechanism.Methods:15 children with ALL and 15 healthy subjects were collected from the Second Affiliated Hospital of Hainan Medical University from July 2018 to March 2021.MiRNA sequencing was performed on their bone marrow cells,and validated using qRT-PCR.MiR-1294 and miR-1294-inhibitory molecule(miR-1294-inhibitor)were transfected into Nalm-6 cells,and the proliferation of Nalm-6 cells was detected by CCK-8 and colony formation assays.Western blot and ELISA were used to detect apoptosis of Nalm-6 cells.Biological prediction of miR-1294 was performed to find the target gene,which was verified by luciferase reporter assay.Si-SOX15 was transfected into Nalm-6 cells,Western blot was used to detect the expression of Wnt signaling pathway-related proteins and to verify the effect of si-SOX15 on the proliferation and apoptosis of Nalm-6 cells.Results:Compared with healthy subjects,22 miRNAs were significantly upregulated in bone marrow cells of ALL patients,of which miR-1294 was the most significantly upregulated.In addition,the expression level of SOX15 gene was significantly reduced in bone marrow cells of ALL patients.Compared with the NC group,the miR-1294 group showed increased protein expression levels of Wnt3a andβ-catenin,faster cell proliferation,and more colony-forming units,while caspase-3 protein expression level and cell apoptosis were reduced.Compared with the NC group,the miR-1294-inhibitor group showed reduced protein expression levels of Wnt3a andβ-catenin,slower cell proliferation,and fewer colony-forming units,while caspase-3 protein expression level was increased and apoptosis rate was elevated.miR-1294 had a complementary base-pair with the 3'UTR region of SOX15,and miR-1294 directly targeted SOX15.The expression of miR-1294 was negatively correlated with SOX15 in ALL cells.Compared with the si-NC group,the si-SOX15 group showed increased protein expression levels of Wnt3a andβ-catenin,accelerated cell proliferation,and decreased caspase-3 protein expression level and cell apoptosis rate.Conclusion:MiR-1294 can target and inhibit SOX15 expression,thus activating the Wnt/β-Catenin signaling pathway to promote the proliferation of ALL cells,inhibit cell apoptosis,and ultimately affect the disease progression.
作者
岑红霞
蔡思铭
姜虹羽
廖赵妹
韩栋光
CEN Hong-Xia;CAI Si-Ming;JIANG Hong-Yu;LIAO Zhao-Mei;HAN Dong-Guang(Department of Pediatrics,The Second Affiliated Hospital of Hainan Medical University,Haikou 570311,Hainan Province,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2023年第2期344-351,共8页
Journal of Experimental Hematology
基金
海南省卫生计生行业科研项目(编号:18A200022)。
