miR-34a靶向调控NOTCH1基因对SW480细胞增殖的影响

Inhibitory effects of miR-34a on NOTCH1 gene expression and SW480 cell proliferation

目的探讨miR-34a靶向调控NOTCH1基因表达而对结肠癌SW480细胞增殖的影响。方法通过生物信息学预测,NOTCH1为miR-34a特异性靶基因。构建含miR-34a结合位点的NOTCH1基因3'-UTR域荧光素酶报告载体。通过荧光素酶报告载体系统检测miR-34a与NOTCH1的3'-UTR相互作用对荧光素酶活性的影响;免疫印迹技术检测miR-34a对NOTCH1蛋白表达的影响。采用MTT法及流式细胞检测转染miR-34a对SW480细胞增殖的影响。结果经过酶切及基因测序鉴定,NOTCH1基因3'-UTR序列的双荧光素酶报告重组质粒构建成功;荧光素酶结果显示在SW480细胞中加入miR-34a的类似物和重组载体,荧光素酶的活性是只加入空载体的SW480组53.4%(P=0.003 8);而在HEK293细胞中加入miR-34a的抑制物和重组载体,荧光素酶的活性是只加入空载体的HEK293组145%(P=0.002 1),说明miR-34a有与NOTCH1的3'-UTR位点相结合。免疫印迹结果显示在SW480细胞中加入miR-34a的类似物,NOTCH1蛋白的表达水平是未处理SW480组下降53.6%(P<0.05);而在HEK293细胞中加入miR-34a的抑制物,NOTCH1蛋白的表达水平较未处理HEK293组升高78.9%(P=0.03),说明miR-34a负性调控NOTCH1蛋白的表达。miR-34a过表达的SW480细胞较未处理的SW480的生长速度明显减慢(P<0.05),且阻滞在G0～G1期,说明miR-34a过表达后能抑制SW480细胞增殖。结论 miR-34a负性靶向调控NOTCH1基因的表达而抑制SW480细胞的增殖。

Objective To analyze the effect of miR-34a on NOTCH1 gene expression and SW480 cell proliferation.Methods NOTCH1 was predicted to be the specific target gene of miR-34a by bioinformatics.The dual luciferase vector containing 3′-UTR of NOTCH1 gene was constructed,and the 3′-UTR was regarded as the binding site of miR-34a.The effect of miR-34a interaction with the 3′-UTR of NOTCH1 on luciferase activity was detected with a dual luciferase assay system.The expression level of NOTCH1 protein affected by miR-34a was detected by Western blotting.The proliferation of SW480 cells transfected with miR-34a was measured by MTT assay and flow cytometry.Results The dual luciferase recombinant vector containing the 3′-UTR of NOTCH1 gene was successfully constructed and verified by enzyme digestion and gene sequencing.The luciferase activity significantly reduced to 53.4% in the SW480 cells co-transfected with the recombinant vectors and miR-34a mimics as compared with the SW480 cells transfected with empty vectors（P=0.003 8）,while the luciferase activity significantly was enhanced to 145% in the HEK293 cells co-transfected with the recombinant vectors and miR-34a inhibitors as compared with the HEK293 cells transfected with empty vectors（P=0.002 1）.The results of the luciferase assay revealed that miR-34a could negatively regulate the luciferase activity by interacting with the 3′-UTR of NOTCH1.Western blotting results demonstrated that the NOTCH1 protein level in the SW480 cells transfected with miR-34a mimics decreased by 53.6% as compared with the untreated SW480 cells（P0.05）,while the NOTCH1 protein level in the HEK 293 cells transfected with miR-34a inhibitors increased by 78.9% as compared with the untreated HEK293 cells（P=0.03）.NOTCH1 protein expression was negatively regulated by miR-34a.The growth of SW480 cells transfected with miR-34a was much slower than that of the untreated SW480 cells,and the cells were arrested at G0-G1 phase,suggesting miR-34a overexpression could inhibit the proliferation of SW480 cells.Conclusion miR-34a negatively targetedly regulates the expression of NOTCH1 and inhibits the proliferation of SW480 cells.