摘要
目的:探究miR-30c调控骨肉瘤细胞增殖及转移的机制。方法:通过TargetScan、MiRGen等数据库分析预测miR-30c可能的靶基因,双荧光素酶报告基因实验验证候选靶基因与miR-30c的靶向相关性,构建U2OS细胞miR-30c mimic/inhibitor细胞系,定量实时聚合酶链反应(qRT-PCR)及蛋白质印迹法(WB)验证miR-30c对候选靶基因表达的影响。收集2016年5月至2018年5月于新疆医科大学第六附属医院接受骨肉瘤切除术的37例患者的癌组织与癌旁组织,qRT-PCR及WB检测miR-30c与候选靶基因在体内组织中表达相关性,免疫组化检测组织水平候选靶基因的表达分布。CCK8、Transwell小室实验分别用于检测miR-30c与候选靶基因对U2OS细胞增殖及转移能力的影响。结果:数据库筛选预测miR-30c与SIRT1存在靶向结合的位点,双荧光素酶报告实验显示野生型SIRT1报告基因质粒共转染miR-30c mimic后荧光素酶活性较对照组受到抑制(P<0.001),U2OS细胞内miR-30c高表达抑制SIRT1蛋白表达而对SIRT1 mRNA表达无影响。体内组织学水平检测显示miR-30c与SIRT1蛋白表达呈负相关(P=0.006),免疫组化结果表明SIRT1高表达于miR-30c低表达的肿瘤组织中。过表达miR-30c抑制U2OS细胞增殖及转移能力,miR-30c mimic共转染SIRT1真核表达载体后,细胞增殖及转移能力较对照组差异无统计学意义。结论:miR-30c可能通过负调控SIRT1的表达参与骨肉瘤细胞U2OS的增殖及转移。
Objective:To investigate the effects of miR-30 c on the proliferation and metastasis of Osteossarcoma(OS)cells.Methods:TargetScan and MiRGen database were used to predicate the target genes of miR-30 c.Dual luciferase reporter gene assays were used to detect the targeting of miR-30 c and the candidate target genes.The overexpression and inhibition of miR-30 c were induced with miR-30 c mimics and miR-30 c inhibitor.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blotting were used to detect the expression of candidate gene after transfection.Form May 2016 to May 2018,36 cases of tumor tissues and adjacent tissues were isolated from OS patients after surgery in the Sixth Affiliated Hospital of Xinjiang Medical University.qRT-PCR and Western blotting were used to detect the correlation of miR-30 c and the candidate gene in vivo.Immunohistochemistry was used to detect the expression distribution of candidate gene in the tumor tissues.CCK-8,transwell invasion and migration assays were conducted to assess the effects of miR-30 c and the target gene on cell proliferation and metastasis of U2 OS cells.Results:MiRGen predicted that SIRT1 contains the binding site with miR-30 c.The results of dual luciferase assay system showed that the luciferase activity was inhibited when co-transfection of miR-30 c mimic with SIRT1-Wild Type plasmid vector compared to control(P<0.001).Overexpression of miR-30 c in U2 OS cells inhibited SIRT1 protein translation but had no effect on SIRT1 mRNA expression.miR-30 c was negatively correlated with SIRT1 protein expression in the tumor tissues in vivo(P=0.006).Immunohistochemistry results indicated that SIRT1 was concentrated in tumor tissues with low expression of miR-30 c.Overexpression of miR-30 c suppress the ability of cell proliferation and metastasis.After co-transfection with miR-30 c mimic and SIRT1 eukaryotic expression vector,the difference of cell proliferation and metastasis ability have no statistical significance compared with the control group.Conclusion:miR-30 c may participate in the proliferation and metastasis of U2 OS cells by negatively regulating the expression of SIRT1.
作者
舒莉
巨啸晨
柴浩
杨德勇
孙荣鑫
SHU Li;JU Xiaochen;CHAI Hao;YANG Deyong;SUN Rongxin(Department of Joint Surgery,the Sixth Affiliated Hospital of Xinjiang Medical University,Urumqi 830002,China)
出处
《现代医学》
2020年第7期817-823,共7页
Modern Medical Journal
基金
新疆维吾尔自治区自然科学基金资助项目(2017D01C257)
