摘要
目的探讨miR-31通过靶向SATB2对结肠癌上皮细胞HCT116焦亡的促进作用,以期为结肠癌发病机制的研究及治疗提供新的靶标。方法常规培养HCT116细胞,采用脂质体瞬时转染方法转染miR-31 mimic(miR-31模拟物)和miR-31 inhibitor(miR-31抑制剂)分别过表达和敲低miR-31,转染si-SATB2敲低SATB2的表达;另转染pEXPRB-Mam-SATB2质粒(过表达SATB2)和pEXP-RB-Mam质粒(空载体)。试验均设加入无血清、无青链霉素的DMEM的细胞作为对照,均重复3次。72 h后收集各组细胞,提取蛋白,Western blot方法检测SATB2及焦亡信号通路相关蛋白NLRP3、胱天蛋白酶(Caspase)-1、胃泌素蛋白D(gasdermin D,GSDMD)-N、IL-18和IL-1β的表达。结果与对照组相比,miR-31过表达组和敲低SATB2组细胞SATB2蛋白的表达水平均显著下降(t分别为4.677和4.463,P分别<0.01和<0.05),NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表达水平均显著升高(t=2.859~6.843,P<0.05或<0.01);敲低miR-31组SATB2蛋白表达水平显著升高(t=3.313,P<0.05),NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表达水平均显著下降(t=2.747~3.545,P<0.05或<0.01)。与对照组相比,空载体组SATB2、NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表达水平差异均无统计学意义(t=0.03310~0.8111,P均>0.05);SATB2过表达组SATB2蛋白表达水平显著升高(t=6.186,P<0.01),NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表达水平均显著下降(t=2.963~7.894,P<0.05或<0.01),与敲低miR-31组趋势一致。结论miR-31通过靶向SATB2上调HCT116细胞中经典的细胞焦亡信号通路蛋白的表达,促进细胞焦亡。
Objective To investigate the promoting effect of miR-31 on pyroptosis of colon cancer epithelial HCT116 cells by targeting SATB2 so as to provide a new target for study on the pathogenesis and therapy of colon cancer.Methods HCT116 cells were cultured routinely and transfected with miR-31 mimic and miR-31 inhibitor through liposome transient transfection method to overexpress and knockdown miR-31 respectively,and with si-SATB2 to knockdown the expression of SATB2.Meanwhile,the cells were transfected with pEXP-RB-Mam-SATB2(to overexpress SATB2)and pEXP-RB-Mam plasmid(empty vector)respectively.The cells cultured with serum-,penicillin-and streptomycin-free DMEM were used as control.All the tests were repeated for 3 times.The cells in various groups were harvested 72 h after culture,from which proteins were extracted.The expressions of SATB2 and pyroptosis signaling pathway related proteins NLRP3,Caspase-1,gasdermin D(GSDMD)-N,IL-18 and IL-1βwere determined by Western blot.Results Compared with those in control group,the protein expression level of SATB2 decreased significantly(t=4.677 and 4.463,P<0.01 and<0.05),while those of NLRP3,Caspase-1,GSDMD-N,IL-18 and IL-1βincreased significantly(t=2.859~6.843,P<0.05 or<0.01)in the cells with miR-31 overexpression and with SATB2 knockdown.However,the expression level of SATB2 protein increased significantly(t=3.313,P<0.05),while those of NLRP3,Caspase-1,GSDMD-N,IL-18 and IL-1βdecreased significantly(t=2.747~3.545,P<0.05 or<0.01)in the cells with miR-31 knockdown.Compared with those in control group,the expression levels of SATB2,NLRP3,Caspase-1,GSDMD-N,IL-18 and IL-1βshowed no significant differences in the cells transfected with empty vector(t=0.03310~0.8111,each P>0.05).However,the expression level of SATB2 in the cells with SATB2 overexpression increased significantly(t=6.186,P<0.01),while those of NLRP3,Caspase-1,GSDMD-N,IL-18 and IL-1βdecreased significantly(t=2.963~7.894,P<0.05 or<0.01),which was consistent with the trend of cells with miR-31 knockdown.Conclusion
作者
张文琴
都新新
张一楠
任彩佩
崔香丽
ZHANG Wen-qin;DU Xin-xin;ZHANG Yi-nan;REN Cai-pei;CUI Xiang-li(Department of Physiology,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第11期1310-1316,共7页
Chinese Journal of Biologicals
基金
山西省自然科学基金(201801D121311)
山西省“1331工程”重点学科建设计划经费(1331KSC).
