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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 investigate the effect of miR-34a targeting the expression of NOTCH1 gene on the proliferation of colon cancer SW480 cells. Methods By bioinformatics prediction, NOTCH1 is a miR-34a specific target gene. Construction of NOTCH1 gene 3’-UTR luciferase reporter vector with miR-34a binding site. Luciferase reporter system was used to detect the effect of 3’-UTR interaction between miR-34a and NOTCH1 on luciferase activity. Western blotting was used to detect the effect of miR-34a on NOTCH1 protein expression. The effects of miR-34a on the proliferation of SW480 cells were detected by MTT assay and flow cytometry. Results After digestion and sequencing, the dual luciferase reporter gene of 3’-UTR sequence of NOTCH1 gene was successfully constructed. The luciferase results showed that miR-34a analogue and recombinant vector were added into SW480 cells, luciferase Of the HEK293 cells were 53.4% (P = 0.003 8) of the SW480 cells loaded with the empty vector. Inhibitors of miR-34a and recombinant vectors were added into the HEK293 cells. The luciferase activity was only 145% (P = 0.002 1), indicating that miR-34a binds to the 3’-UTR site of NOTCH1. Immunoblotting showed that the expression level of NOTCH1 protein in SW480 cells was 53.6% (P <0.05) lower than that in untreated SW480 cells when miR-34a was added to SW480 cells. In HEK293 cells, miR-34a inhibitor, NOTCH1 protein (P = 0.03), indicating that miR-34a negatively regulates the expression of NOTCH1 protein in HEK293 cells. The growth of SW480 cells overexpressing miR-34a was significantly slower than that of untreated SW480 cells (P <0.05), and the arrest was in G0-G1 phase, indicating that miR-34a could inhibit the proliferation of SW480 cells after overexpression of SW480 cells. Conclusion Negative targeting of miR-34a regulates the expression of NOTCH1 gene and inhibits the proliferation of SW480 cells.