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放射治疗(放疗)是乳腺癌治疗的重要手段之一,而肿瘤细胞对放射的不敏感性是影响放射治疗的重要因素。随着研究的不断深入,越来越多的证据表明, 乳腺癌细胞中特异或差异性表达的一些微小RNA(microRNAs,miRNAs)在放疗抵抗过程中发挥着重要作用,而干扰或过表达这些miRNAs可逆转肿瘤细胞对放疗的抵抗,增加放疗的敏感性[1-3]。miRNA(miR)-125b与肿瘤进展密切相关miRNA,在细胞增殖、转移、凋亡和耐药等方面发挥着重要作用[4]。miR-125b在乳腺癌等中异常低表达,被证实在乳腺癌的发生发展和放疗过程过程中发挥着积极作用[5-6],但其在肿瘤细胞放射增敏过程中的机制却知之甚少。己糖激酶-2(HK2)是糖酵解过程中的一种关键限速酶,被证实沉默其表达可增强乳腺癌细胞的放疗敏感性[7]。本研究通过生物信息学软件预测发现,HK2可能是miR-125b的潜在靶基因。因此,猜测miR-125b可能通过靶向抑制HK2表达在乳腺癌细胞中发挥放射增敏作用,故设计实验加以验证。本研究以乳腺癌MCF-7细胞为研究对象,通过观察miR-125b和HK2的靶向关系,探讨miR-125b增强乳腺癌MCF-7细胞放射敏感性的分子机制。
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RT-PCR检测结果显示,转染miR-125b模拟物后MCF-7细胞中miR-125b的表达水平(4.25±0.16)较miR-NC组(0.96±0.07)显著升高(t=32.63,P<0.01)。
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RT-PCR和Western blotting检测结果显示,转染miR-125b模拟物后MCF-7细胞中HK2 mRNA和蛋白的表达水平分别为0.35±0.03和0.27±0.03,较miR-NC组中HK2 mRNA和蛋白的表达水平(1.02±0.09和0.88±0.07)均显著降低(tmRNA=12.23,P<0.01;t蛋白=13.87,P<0.01)(见图 1)。
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TargetScan生物信息学软件预测结果显示,HK2的3′UTR中存在与miR-125b互补的结合位点,详见表 1。同时,双荧光素酶报告基因实验结果显示,共转染miR-125b模拟物和HK2-WT质粒的MCF-7细胞的荧光素酶活性较共转染miR-NC和HK2-WT质粒的荧光素酶活性显著降低(P<0.01);但与共转染miR-NC和HK2-MUT质粒相比,共转染miR-125b模拟物和HK2-MUT质粒的MCF-7细胞的荧光素酶活性无显著改变(P>0.05)(见表 2)。
基因 互补的核苷酸序列 结合位点 HK2 5′....ACA GCC AAA UAA AAC CUC AGG GA...3′ 1370-1377 of HK2 miR-125b 3′......AGU GUU CAA UCC CAG AGU CCC U......5′ 3′ UTR 表 1 miR-125b与HK2 3′ UTR的结合位点
分组 荧光素酶活性 miR-NC+HK2-WT组 1.12±0.09 miR-125b+HK2-WT组 0.53±0.04** miR-NC+HK2-MUT组 1.08±0.07 miR-125b+HK2-MUT组 0.99±0.06 F 48.84 P <0.01 MS组内 0.005 q检验:与miR-NC + HK2-WT组比较* *P<0.01 表 2 各MCF-7细胞的荧光素酶活性的比较(x±s)
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RT-PCR检测得到miR-NC组、miR-125b组和miR-125b+HK2组细胞中HK2 mRNA的相对表达量分别为1.00±0.06、0.34±0.02和0.65±0.03,Western blotting检测得出HK2蛋白的相对表达量分别为1.02±0.08、0.27±0.02和0.45±0.03(见图 2);各组细胞间HK2 mRNA和蛋白的表达差异有统计学意义(FmRNA=200.27,P<0.01,MS组内=0.002;F蛋白=179.18,P<0.01,MS组内=0.003);与miR-NC组相比,HK2 mRNA和蛋白的表达水平在miR-125b组细胞中显著降低(P<0.01),而miR-125b+HK2组与miR-125b组相比显著升高(P<0.01)。根据打击多靶模型拟合MCF-7细胞的生存曲线见图 3,各组细胞的放射相关参数见表 2。与miR-NC组相比,miR-125b组细胞的存活分数明显降低(P<0.01),放射增敏比SER为1.44;与miR-125b组相比,miR-125b+HK2组细胞的存活分数明显升高(P<0.01),细胞增敏比为0.69。给予4 Gy剂量X射线照射后,流式细胞仪检测miR-NC组、miR-125b组和miR-125b+HK2组的细胞凋亡率分别为(7.58±0.12)%、(20.64±0.23)%和(13.26±0.13)%;与miR-NC组相比,miR-125b组细胞凋亡率明显升高(t=86.31,P<0.01);而miR-125b+HK2组细胞的凋亡率较miR-125b组显著降低(t=47.91,P<0.01)(见图 4)。
分组 D0/Gy Dq/Gy SF2 SER miR-NC 2.47 3.48 0.91 miR-125b 1.26 1.35 0.49 1.44 miR-125b+HK2 1.82 1.58 0.62 0.69 表 3 各组细胞的放射生物学参数比较
miR-125b靶向调控HK2表达增强乳腺癌MCF-7细胞放射敏感性的研究
Study on the mechanism of miR-125b targeting HK2 expression to enhance radiosensitivity of breast cancer MCF-7 cells
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摘要:
目的探讨miR-125b靶向调控HK2对乳腺癌MCF-7细胞放射敏感性的影响。 方法将miR-125b模拟物及其阴性对照转染至乳腺癌MCF-7细胞,分别记为miR-125b组和miR-NC组,采用RT-PCR检测MCF-7细胞中miR-125b和HK2 mRNA的表达,Western blotting检测HK2蛋白的表达;双荧光素酶报告基因实验检测miR-125b和HK2的靶向关系。另外将miR-125b模拟物和pcDNA3.1-HK2质粒共转染至MCF-7细胞中,记为miR-125b+HK2组,通过克隆形成实验和流式细胞仪检测miR-NC组、miR-125b组和miR-125b+HK2组细胞的存活分数和凋亡率。 结果与miR-NC组相比,转染miR-125b模拟物后MCF-7细胞中miR-125b表达升高(P < 0.01),而HK2 mRNA和蛋白的表达降低(P < 0.01)。双荧光素酶报告基因实验证实HK2是miR-125b的潜在靶基因。与miR-NC组相比,miR-125b组细胞的存活分数降低、凋亡率升高(P < 0.01);与miR-125b组相比,miR-125b+HK2组存活分数降低明显升高而凋亡率降低(P < 0.01)。 结论miR-125b可通过靶向调控HK2表达增强乳腺癌MCF-7细胞的放射敏感性。 Abstract:ObjectiveTo investigate the effects of miR-125b targeting HK2 expression on the radiosensitivity of breast cancer MCF-7 cells. MethodsThe miR-125b mimics and its negative control were transfected into breast cancer MCF-7 cells, and divided into the miR-125b group and miR-NC group, respectively.The expression levels of miR-125b and HK2 mRNA in MCF-7 cells were detected using RT-PCR, and the expression of HK2 protein was detected by Western blotting.The targeting relationship between miR-125b and HK2 was detected using double luciferase reporter gene assay.The mimic of miR-125b and pcDNA3.1-HK2 plasmid were co-transfected into MCF-7 cells(miR-125b+HK2 group), and the survival fraction and apoptotic rate of cells in three groups were detected by clone formation assay and flow cytometry. ResultsAfter transfecting miR-125b mimics, compared with the miR-NC group, the expression level of miR-125b in MCF-7 cells increased(P < 0.01), and the expression levels of HK2 mRNA and protein decreased(P < 0.01).The results of double luciferase reporter gene assay confirmed that the HK2 was a potential target gene of miR-125b.Compared with the miR-NC group, the survival fraction and apoptotic rate of cells in miR-125b group decreased and increased, respectively(P < 0.01).Compared with the miR-125b group, the survival fraction and apoptotic rate in miR-125b + HK2 group significantly increased and decreased, respectively(P < 0.01). ConclusionsMiR-125b can enhance the radiosensitivity of breast cancer MCF-7 cells by targeting HK2 expression. -
Key words:
- breast neoplasms /
- miR-125b /
- HK2 /
- radiosensitivity
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表 1 miR-125b与HK2 3′ UTR的结合位点
基因 互补的核苷酸序列 结合位点 HK2 5′....ACA GCC AAA UAA AAC CUC AGG GA...3′ 1370-1377 of HK2 miR-125b 3′......AGU GUU CAA UCC CAG AGU CCC U......5′ 3′ UTR 表 2 各MCF-7细胞的荧光素酶活性的比较(x±s)
分组 荧光素酶活性 miR-NC+HK2-WT组 1.12±0.09 miR-125b+HK2-WT组 0.53±0.04** miR-NC+HK2-MUT组 1.08±0.07 miR-125b+HK2-MUT组 0.99±0.06 F 48.84 P <0.01 MS组内 0.005 q检验:与miR-NC + HK2-WT组比较* *P<0.01 表 3 各组细胞的放射生物学参数比较
分组 D0/Gy Dq/Gy SF2 SER miR-NC 2.47 3.48 0.91 miR-125b 1.26 1.35 0.49 1.44 miR-125b+HK2 1.82 1.58 0.62 0.69 -
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