SPAG6基因沉默和地西他滨对SKM-1细胞凋亡和PTEN甲基化的影响  

Effects of SPAG6 silencing and decitabine treatment on apoptosis and phosphatase and tensin homolog methylation in SKM-1 cells

作　　者：罗洁[1,2] 牟佼[1,2] 刘林 Luo Jie;Mu Jiao;Liu Lin(Department of Hematology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Experimental Research Center,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院血液科,400016 [2]重庆医科大学实验研究中心,400016

出　　处：《中华血液学杂志》2021年第12期1005-1010,共6页Chinese Journal of Hematology

基　　金：国家自然科学基金(82070130、81570109)。

摘　　要：目的探讨SPAG6基因沉默和地西他滨在体内外对SKM-1细胞凋亡和PTEN启动子甲基化的作用。方法慢病毒载体转染SKM-1细胞沉默SPAG6基因的表达,地西他滨处理细胞后CCK-8检测细胞存活率,流式细胞术检测细胞凋亡,Western blot和甲基化特异性PCR检测PTEN的蛋白表达和甲基化情况,并构建非肥胖糖尿病/重度联合免疫缺陷(NOD/SCID)小鼠异体移植瘤模型,TUNEL法观察肿瘤组织凋亡,免疫组化检测PTEN在组织中表达情况。结果慢病毒转染后SPAG6干扰组SPAG6基因被成功沉默;CCK-8检测结果提示地西他滨处理能够降低SKM-1细胞的存活率,同时流式细胞术检测显示地西他滨处理组的细胞凋亡率[(17.35±3.37)%]高于未处理组[(5.09±2.06)%],且SPAG6基因沉默联合地西他滨处理组的凋亡率最高[(36.34±4.00)%];地西他滨处理后DNMT1的表达降低而PTEN表达升高,同时PTEN启动子甲基化程度降低,而经地西他滨处理后的SPAG6干扰组的PTEN去甲基化引起的蛋白表达升高效果最明显;NOD/SCID小鼠异体瘤移植模型中地西他滨组的肿瘤体积明显小于生理盐水组,而地西他滨处理SPAG6干扰组的小鼠肿瘤体积最小,且经TUNEL检测发现其凋亡程度最高(阳性比值为3.57±0.48)。结论SPAG6基因沉默在SKM-1细胞中能增强地西他滨诱导的凋亡和PTEN去甲基化作用,并且在NOD/SCID异体瘤移植模型小鼠中能够增强地西他滨的抗肿瘤特性。Objective To investigate the effects of SPAG6 silencing and decitabine on apoptosis and phosphatase and tensin homolog(PTEN)methylation in SKM-1 cells in vitro and in vivo.Methods SKM-1 cells were transfected with a lentiviral vector to silence the expression of SPAG6.Cell survival rate was detected by CCK8 after treatment with decitabine,and cell apoptosis was detected by flow cytometry.Protein expression and methylation of PTEN were detected using Western blot and merozoite surface protein(MSP).An non-obese diabetic/severe combined inmunodeficiency disease(NOD/SCID)mice xenograft tumor model was established,and the apoptosis and PTEN expression of tumor tissue were observed through terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)and immunohistochemistry(IHC),respectively.Results After lentivirus transfection,SPAG6 in the interference group was silenced successfully.CCK8 results indicated that the cell survival rate of SKM-1 cells treated with decitabine decreased.Flow cytometry showed that the apoptosis rate of cells treated with decitabine[(17.35±3.37)%]was higher than that of the untreated group(5.09%±2.06%)and the apoptosis rate of the SPAG6 silencing combined with the decitabine treatment group was the highest[(36.34±4.00)%].After treatment with decitabine,the expression of DNMT1 decreased,while the expression of PTEN increased,and the promoter methylation degree of PTEN also decreased.Moreover,the increased protein level caused by PTEN demethylation was the most obvious in the SPAG6 in the interference shRNA group treated with decitabine.In NOD/SCID mice,the tumor volume of the decitabine group was significantly smaller than that of the placebo group,and the tumor volume of the SPAG6 silencing combined with the decitabine treatment group was the smallest.Additionally,the apoptosis rate was the highest(the positive ratio was 3.57±0.48).Conclusion SPAG6 silencing may enhance the apoptosis level and the effect of PTEN demethylation in SKM-1 cells and enhance the antitumor effect of deci

关 键 词：骨髓增生异常综合征 精子相关抗原6 PTEN基因 地西他滨 

分 类 号：R551.3[医药卫生—血液循环系统疾病]