[1]朱春霞,王利,罗小华,等.精子相关抗原6基因以非P53依赖方式促进TRAIL诱导的骨髓增生异常综合征细胞凋亡[J].陆军军医大学学报(原第三军医大学学报),2018,40(03):198-204.
 ZHU Chunxia,WANG Li,LUO Xiaohua,et al.Sperm-associated antigen 6, independent on P53, regulates cell apoptosis of myelodysplastic syndromes induced by TRAIL[J].J Amry Med Univ (J Third Mil Med Univ),2018,40(03):198-204.
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精子相关抗原6基因以非P53依赖方式促进TRAIL诱导的骨髓增生异常综合征细胞凋亡(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第03期
页码:
198-204
栏目:
基础医学
出版日期:
2018-02-15

文章信息/Info

Title:
Sperm-associated antigen 6, independent on P53, regulates cell apoptosis of myelodysplastic syndromes induced by TRAIL
作者:
朱春霞王利罗小华刘林
重庆医科大学附属第一医院血液内科
Author(s):
ZHU Chunxia WANG Li LUO XiaohuaLIU Lin

Department of Hematology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China

关键词:
精子相关抗原6基因P53基因TRAILRNA干扰骨髓增殖异常综合征
Keywords:
sperm-associated antigen 6 P53 gene TRAIL RNA interference myelodysplastic syndrome
分类号:
R394-33; R394.3; R551.3
文献标志码:
A
摘要:

目的     研究精子相关抗原6(sperm-associated antigen 6,SPAG6)基因是否以P53依赖方式促进TRAIL诱导的骨髓增生异常综合征(myelodysplastic syndrome,MDS)细胞凋亡。方法     通过P53kd RNA慢病毒转染人结肠癌细胞株HCT116筛选P53基因沉默的最佳靶点,利用筛选出来的最佳靶点序列转染SKM-1细胞;加入嘌呤霉素提高转染效率,RT-PCR及Western blot 检验P53基因的干扰效果,构建SKM-1  P53kd细胞模型。用SPAG6kd RNA慢病毒载体转染SKM-1和SKM-1  P53kd细胞,RT-PCR及Western blot验证。用CCK-8法检测重组人可溶性TRAIL蛋白(rTRAIL)对SKM-1细胞的适宜作用浓度。Western blot检测rTRAIL处理后SKM-1和SKM-1  P53kd细胞株中Caspase-8、cleaved-PARP、PARP的表达水平。结果     构建了SKM-1  P53kd细胞模型,转染率在90%以上,P53基因在mRNA和蛋白表达水平较对照组均显著降低(P<0.01)。SPAG6kd RNA慢病毒载体成功感染SKM-1细胞和SKM-1  P53kd细胞,转染率均在80%以上,SPAG6基因表达较对照组明显降低(P<0.01)。随着rTRAIL浓度的增加,细胞生长逐渐被抑制,30 ng/mL TRAIL诱导的细胞凋亡与对照组差异没有统计学意义,100 ng/mL和300 ng/mL TRAIL显著诱导SPAG6kd细胞凋亡(P<0.05),而P53基因沉默与否并没有影响该结果,rTRAIL处理后细胞凋亡标志分子明显激活或水平升高,细胞中Caspase-8、cleavedPARP、PARP的表达水平在SKM-1和SKM-1 P53kd细胞中差异无统计学意义(P>0.05)。结论     SPAG6基因以非P53依赖方式促进TRAIL诱导的MDS细胞凋亡。

Abstract:

Objective     To investigate the sperm-associated antigen 6 (SPAG6) regulating cell apoptosis of myelodysplastic syndromes (MDS) induced by the TRAIL whether is dependent on P53. Methods     The P53-knock down RNA (P53kd RNA) lentiviral vector was transfected into HCT116 cells to obtain the most effective shRNA-containing plasmid. To construct SKM-1 P53kd cells model, the most effective interference sequence was transferred into SKM-1 cells, with the transfection efficiency being increased by purinomycin. Then the interference efficiency was determined by quantitative reverse transcription PCR (RT-PCR) and Western blotting. Next, the SPAG6kd RNA-expressing lentiviral vector was transferred into SKM-1 cells and SKM-1 P53kd cells, and the interference efficiency was verified by RT-PCR and Western blotting. Cell apoptosis was measured after they were treated with recombinant human sTRAIL (rTRAIL) of different concentrations by CCK-8 assay. The expression levels of PARP, cleaved PARP and Caspase-8 were measured by Western blotting after the cells were treated with rTRAIL. ResultsThe SKM-1 P53kd cell model was successfully constructed, with the transfection rate over 90%, and both RT-PCR and Western blotting indicated the significant decrease of P53 expression (P<0.01). The SPAG6kd RNA lentiviral vector was also successfully infected the SKM-1 cells and SKM-1 P53kd cells, and the transfection rate was more than 80%. The expression of the SPAG6 was decreased significantly after the transfection (P<0.01). With the concentration of TRAIL was increased, cell growth was inhibited gradually. When the concentration was 30 ng/mL, there was no difference in cell apoptosis between the treated cells and the control cells, but 100 and 300 ng/mL obviously induced cell apoptosis (P<0.05), and P53 knockdown showed no effect on the results. rTRAIL treatment increased the expression of apoptotic molecules and enhanced cell apoptosis, but there were no significant differences in the expression levels of PARP, cleaved PARP and Caspase-8 between the SKM1 cells and SKM-1 P53kd cells  (P>0.05). Conclusion     SPAG6 is not depend on P53 in regulating cell apoptosis of MDS induced by TRAIL.

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更新日期/Last Update: 2018-02-08