微小RNA-339-5p通过靶向鼠双微体基因调节乳腺癌MCF-7细胞p53肿瘤抑制通路的研究

MicroRNA-339-5p regulates the p53 tumor-suppressor pathway by targeting murine double minute 2

目的 观察微小RNA（miRNA,miR）-339-5p通过鼠双微体基因（MDM2）对乳腺癌MCF-7细胞p53肿瘤抑制通路调节的效果.方法 建立高通量筛选方法,将hsa-miR-339-5p前体miRNA、hsa-miR-192-5p前体miRNA、hsa-miR-605前体miRNA、小干扰RNA（siRNA） p53和siRNA MDM2转染至MCF-7、HEK293、ACNH和BJ-人端粒酶反转录酶（hTERT）细胞中,5-氟尿嘧啶（5-Fu）水溶液或雷公藤甲素处理转染细胞,Western blot和实时荧光定量聚合酶链反应（FQ-PCR）检测MDM2表达.结果 转染miR-339-5p后,MCF-7和HCT116细胞系中p53蛋白水平分别升高2.60倍及2.48倍;在无遗传毒性应激情况下,MDM2蛋白水平显著降低.5-Fu处理后,p53蛋白及目标MDM2和p21水平均上调.miR-339-5p与siRNA MDM2可降低MDM2 mRNA水平.MCF-7细胞株中,与对照组比较,MDM2mRNA水平均由于转染siRNA MDM2或miR-339-5p而分别降低至0.48±0.15和0.47±0.08.雷公藤甲素处理后,MDM2 mRNA水平下降更加明显,分别为0.19±0.05和0.23 ±0.10.p53敲除试验则表明miR-339前体水平升高不依赖于p53.MDM2基因3＆#39;端非编码区域（3＆#39;-UTR）包含有能与miR-339-5p结合的功能位点,正是这些位点的存在介导了转录本抑制.结论 miR-339-5p通过靶向MDM2调节乳腺癌MCF-7细胞p53肿瘤抑制通路.

Objective To study the effect of microRNA （miRNA,miR）-339-5p on the regulation of p53 tumor suppressor pathway in breast cancer MCF-7 cells by murine double minute 2 （MDM2）.Methods A high throughput screening method was created.miRNA mimics or small interfering RNAs （siRNAs）,including has-miR-339-5p pre-miR,has-miR-192-5p pre-miR,has-miR-145-5p pre-miR,has-miR-605 pre-miR,siRNA p53,siRNA MDM2 and control siRNA were transfected into MCF-7,HEK293,ACNH and BJ-human telomerase reverse transcriptase （hTERT） cell lines.The transfected cells were treated with 5-fluorouracil （5-Fu） or Triptolide.Western blotting and real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） were used to detect the expression of MDM2.Results miR-339-5p can enhance the expression of p53 protein.After transfection,the levels of p53 protein were increased by 2.60 times in MCF-7 cell lines and 2.48 times in HCT116 cell lines.And the level of MDM2 protein was significantly decreased in the absence of genetic toxicity,and the effect of MDM2 siRNA was similar to that of HCT116 cells.After 5-Fu treatment,the levels of p53 protein were increased in MCF-7 and which was similar in HCT116 cell lines.So did MDM2 and p21.miR-339-5p expression can strongly induce the transcription of BBC3,and the level of mRNA in CDKN1A is also significantly increased.miR-339-5p and MDM2 MDM2 decreased the level of siRNA gene mRNA.In MCF-7 cell line,gene mRNA level was significantly decreased to 0.48 ± 0.15 by siRNA MDM2,and reduce to 0.47 ± 0.08 by miR-339-5p,compared with control group,siRNA gene was significantly decreased in 5-Fu cells.After treatment with triptolide,the level of mRNA gene decreased significantly to 0.19 ± 0.05 and 0.23 ± 0.10.p53 knock test showed that the miR-339 precursor levels were not dependent on p53.MDM2 gene 3＆#39;-UTR contained functional sites that could be combined with miR-339-5p,and it was the presence of these sites that mediated transcription of this inhibition.Conclusion miR-339-5p regulates the p53 tumor-suppressor pathway by targeting MDM2.miR-339-5p would play an active role in tumor formation and development,and may be used as a prognostic marker and therapeutic effect evaluation.