miR-370-3p和FOXM1 mRNA对人成骨肉瘤细胞增殖、迁移、侵袭的影响观察及其靶向关系验证

Effects of miR-370-3p and FOXM1 mRNA on proliferation,migration,and invasion of human osteosarcoma cells and verification of their targeting relationship

目的观察骨肉瘤组织中miR-370-3p、FOXM1 mRNA的表达,观察miR-370-3p和FOXM1 mRNA对人成骨肉瘤细胞系MG63增殖、迁移及侵袭的影响,并验证其靶向关系机制。方法①观察骨肉瘤及癌旁组织miR-370-3p及及其下游靶基因叉头框蛋白M1(FOXM1)mRNA表达:37例骨肉瘤患者,均行肿瘤切除术,术中保存骨肉瘤及癌旁组织,采用实时定量PCR法检测骨肉瘤及癌旁组织miR-370-3p、FOXM1 mRNA,分析骨肉瘤组织FOXM1 mRNA表达与miR-370-3p表达的相关性。采用Kaplan-Meier曲线、Log-Rank检验分析miR-370-3p表达与骨肉瘤患者临床病理参数及预后关系。②观察miR-370-3p过表达及FOXM1抑制表达的人成骨肉瘤细胞系MG63增殖、迁移及侵袭的影响:取MG63细胞分为1~4组,分别转染miR-370-3p mimic(促进miR-370-3p表达)、mimic NC(空载体质粒)、空白对照组及FOXM1 siRNA干扰质粒(FOXM1基因敲除质粒)。分别于转染0、24、48、72、96 h采用CCK-8法观察各组细胞增殖情况,采用划痕实验观察各组细胞迁移情况,采用Transwell小室观察各组细胞侵袭能力。③预测MG63细miR-370-3p与FOXM1靶向关系构建pmirGLO-FOXM1-野生型/突变型质粒,将MG63细胞分为A、B、C、D组,分别用1μL pmirGLO-FOXM1-WT+200 nmol miR-370-3p mimic、1μL pmirGLO-FOXM1-WT+mimic NC、1μL pmirGLO-FOXM1-MUT+200 nmol miR-370-3p mimic、1μL pmirGLO-FOXM1-MUT+200 nmol mimic NC转染。转染24 h时采用双荧光素酶报告检测系统观察各组细胞荧光活性。结果①与癌旁组织比较,骨肉瘤组织miR-370-3p相对表达量低、FOXM1 mRNA相对表达量高(t=4.66,3.41;P均<0.05)。随miR-370-3p表达量升高,骨肉瘤组织FOXM1 mRNA表达量呈下降趋势(R2=0.6481,P=0.1284)。miR-370-3p表达与骨肉瘤患者临床分期、远端转移情况有关(χ2=4.430、6.027;P均<0.05)。miR-370-3p低表达的骨肉瘤患者预后比miR-370-3p高表达患者差(P<0.05)。②2、3组相比,转染48、72、96 h时1、4组细胞OD450值均降低(P均<0.05)。与2、3组相比,转染24 h时1、4组划痕愈合率低、穿膜细胞数少(P均<0.05)。③与B组比较,A组细胞荧光活性降低(P<0.05);与D组比较,C组细胞中荧光活性变化不明显(P>0.05)。结论骨肉瘤组织中miR-370-3p表达降低、FOXM1 mRNA表达升高。促进miR-370-3p表达及FOXM1抑制表达的MG63细胞增殖、侵袭及迁移能力均降低。骨肉瘤细胞中miR-370-3p可能通过靶向调控FOXM1表达来抑制MG63的增殖、迁移及侵袭。

Objective To observe the expression of miR-370-3p and FOXM1 mRNA in the osteosarcoma tissues,to observe the effects of miR-370-3p and FOXM1 mRNA on the proliferation,migration,and invasion of human osteosarcoma cell line MG63,and to verify their targeting relationship.Methods①The mRNA expression of miR-370-3p and its downstream target gene FOXM1 were detected in the osteosarcoma and adjacent tissues.All 37 patients with osteosarcoma underwent tumor resection,and osteosarcoma and adjacent tissues were preserved during surgery.Real-time quantitative PCR was used to detect miR-370-3p and FOXM1 mRNA expression in osteosarcoma and adjacent tissues,and the correlation between FOXM1 mRNA expression and miR-370-3p expression in osteosarcoma tissues was analyzed.Kaplan-Meier curve and Log-Rank test were used to analyze the relationship between miR-370-3p expression and clinicopathological parameters and prognosis of patients with osteosarcoma.②Observing the effects of miR-370-3p overexpression and FOXM1 inhibition on the proliferation,migration and invasion of human osteosarcoma cell line MG63:MG63 cells were divided into groups 1 to 4:miR-370-3p mimic(promoting miR-370-3p expression),MIMIC NC(no-loaded plasmid),blank control group and FOXM1 siRNA interference plasmid(FOXM1 gene knockout plasmid)were transfected into MG63 cells,respectively.At 0,24,48,72 and 96 h after transfection,cell proliferation was observed by CCK-8 method,cell migration was observed by Scratch test,and cell invasion ability was observed by Transwell chamber.③Predicting the targeted relationship between miR-370-3p and FOXM1:PmirGLO-FOXM1-wild-type/mutant plasmids were constructed by predicting the targeted relationship between MG63 miR-370-3p and FOXM1,and MG63 cells were divided into groups A,B,C and D,which were transfected with 1μL pmirglo-FOXM1-WT+200 nmol miR-370-3p mimic,1μL pmirglo-FOXM1-WT+mimic NC,1μL pmirglo-FOXM1-MUT+200 nmol miR-370-3p mimic,and 1μL pmirGLO-FOXM1-MUT+200 nmol mimic NC,respectively.At 24 h after transfection,the fluorescence activity of each group was observed by dual luciferase reporting assay.Results①Compared with the adjacent tissues,the relative expression levels of miR-370-3p was down-regulated,while FOXM1 mRNA in the osteosarcoma tissues was up-regulated(t=4.66,3.41;both P<0.05).With the increase of miR-370-3p expression,FOXM1 mRNA expression in the osteosarcoma tissues showed a downward trend(R2=0.6481,P=0.1284).The miR-370-3p expression was correlated with clinical stage and distal metastasis in patients with osteosarcoma(X2=4.430,6.027;both P<0.05).The prognosis of osteosarcoma patients with lower miR-370-3p expression was worse than that of patients with higher ones(P=0.0395).②Compared with groups 2 and 3,OD450 values of cells in the groups 1 and 4 decreased at 48,72 and 96 h after transfection(all P<0.05).Compared with groups 2 and 3,the scratch healing rate and the number of transmembrane cells in the groups 1 and 4 were lower at 24 h after transfection(all P<0.05).③Compared with group B,the fluorescence activity of cells in group A decreased(P<0.05).Compared with group D,there was no significant change in fluorescence activity in the group C(P>0.05).Conclusions The expression of miR-370-3p is down-regulated,while FOXM1 is up-regulated in osteosarcoma tissues.The proliferation,invasion and migration of MG63 cells with promotion of miR-370-3p and inhibition of FOXM1 expression decrease.MiR-370-3p in osteosarcoma cells may inhibit the proliferation,migration and invasion of MG63 cells by targeting FOXM1 expression.