miR-126调控肺鳞癌细胞增殖的作用及机制研究

Effect and novel mechanism of miR-126 on proliferation of lung squamous carcinoma SK-MES-1 cells

目的探讨miR-126在调控肺鳞癌细胞SK-MES-1增殖中的作用及机制。方法取10对肺鳞癌组织和癌旁组织,利用q PCR检测各个组织中miR-126的表达水平;合成并将miR-126 mimic和NC转染进入SK-MES-1细胞后,q PCR检测miR-126在上述细胞中的表达,并用MTS检测12、24、48 h时细胞的活力。构建野生型和突变型(nuclear receptor coactivator,NCo A) 7 3’UTR插入p MIR-REPORTTM luciferase vector载体,并将其与pRL-TK质粒共转染进入SK-MES-1细胞,之后分别将等量的miR-126和NC再转染进入SK-MES-1细胞,利用双荧光素酶报告基因检测试剂盒检测萤火虫和海肾荧光素酶活性。应用q PCR和western blot检测转染miR-126 mimic后不同时间点NCo A7 mRNA和蛋白以及芳基烃受体核转运子(Aryl hydrocarbon receptor nuclear translocator,ARNT)蛋白的变化。应用co-IP检测NCo A7与ARNT蛋白的相互作用。转染ARNT siRNA以及NC 48 h后,应用q PCR和western blot检测ARNT mRNA和蛋白的表达,并用MTS评估其对SK-MES-1细胞增殖的抑制作用。结果肺鳞癌组织中miR-126的表达水平显著低于癌旁组织(P<0.05)。转染miR-126 mimic 12、24和48 h后,SK-MES-1细胞中miR-126的表达水平均显著高于0 h组(P<0.05),而NCo A7 mRNA和蛋白、ARNT蛋白的表达水平显著低于0 h组(P<0.05)。荧光素酶报告基因结果显示野生型NCo A7 3’UTR质粒和miR-126 mimic共转染组的相对荧光素酶活性较野生型NCo A7 3’UTR质粒和NC共转染组明显的降低(P<0.05)。Co-IP的结果显示NCo A7与ARNT蛋白之间有相互作用关系。应用ARNT siRNA转染后较NC组可显著下调ARNT mRNA和蛋白表达水平(P<0.05),其中S1和S2序列siRNA沉默效果较好。MTS结果显示转染S1和S2后12、24、48 h的细胞增殖水平显著低于NC组(P<0.05)。结论 miR-126在肺鳞癌中的表达水平较低。上调肺鳞癌细胞SK-MES-1中miR-126表达后,通过其下调靶点NCo A7的表达,进而可抑制ARNT介导的细胞增殖。miR-126可作为潜在的治疗肺鳞癌的靶点。

Objective To investigate the role and mechanism of miR-126 on proliferation of SK-MES-1.Method qPCR was used to detect the relative expression of miR-126 in lung squamous carcinoma tissue and adjacent tissue(n=10).The sequence of miR-126 mimic and negative control(NC)were synthesized.SK-MES-1 cells had been transfected respectively with miR-126 mimic and NC for 48 h,then qPCR and MTS had been used to evaluate the expression of miR-126 in these cells and the cell viability at 12,24 and 48 h timepoint,respectively.The wild-type and mutation of NCoA 3’UTR was inserted into the plasmid of pMIR-REPORTTM luciferase vector repectively,which was co-transfected into HPASMCs with pRL-TK plasmid.miR-126 mimic and NC was transfected into these cells,which had been transfected pMIR-REPORTTM luciferase vector and pRL-TK plasmid.Firefly and Renilla reniformis luciferase activities were measured 48 h later.SK-MES-1 cells were transfected with miR-126 mimic,and the expression of NCoA7 mRNA and protein,and ARNT protein were detected by qPCR and western blot.Co-IP was used to analyze the interactive ARNT with NCoA7.SK-MES-1 cells were treated with ARNT siRNA and NC for 48 h,then qPCR,western blot and MTS had been used to evaluated the expression of ARNT mRNA and protein,and these cells’viability.Result The level of miR-126 was lower in lung squamous carcinoma tissue than in adjacent tissue(P<0.05).The expression of miR-126 was higher after miR-126 mimic transfected into SK-MES-1 cells for 12,24,and 48 h than at 0h group(P<0.05),whereas,the expression of NCoA7 and ARNT protein were significantly decreased at 12,24,and 48 h group,compared to 0 h(P<0.05).The relative lucifierase activity in wild type NCoA7 3’UTR/miR-126 mimic group was lower than in wild type NCoA7 3’UTR/NC group(P<0.05).Co-IP showed that the interactive NCoA7 with ARNT protein.The expression of ARNT mRNA and protein was lower after three siRNAs oilgos transfected into SK-MES-1 cells for 48 h than NC group,and S1 and S2 siRNA was better to silence ARNT gene.Additionally,the proliferation rate was notably decreased in S1 and S2.Conclusion miR-126 was down-regulation in lung squamous carcinoma tissue.Overexpression miR-126 could suppress NCoA7,result in blockage ARNT mediated celluar proliferation.Inhibition miR-1322 might be as a novel strategy for hypoxia PAH and cor pulmonary treatment.Up-regulation miR-126 might be as a novel strategy for lung squamous carcinoma therapy.