沉默HK2基因表达对乳腺癌MDA-MB231细胞放疗敏感性影响的体外研究

发布时间：2018-07-28 10:00

【摘要】：目的本实验利用乳腺癌体外细胞模型,探讨短发夹RNA(sh RNA)介导的己糖激酶2(hexokinase 2,HK2)基因沉默是否对乳腺癌细胞的放疗敏感性和放疗效果有影响。通过慢病毒介导的RNA干扰技术构建己糖激酶-2(HK2)低表达的稳定的乳腺癌MDA-MB231细胞株,然后利用构建的稳定细胞株观察利用sh RNA干扰技术使HK2基因表达水平降低与乳腺癌细胞MDA-MB231放射治疗敏感性之间的关系。方法利用慢病毒介导的短发夹RNA(sh RNA)干扰技术设计3个沉默HK2的sh RNA基因序列(h-HK2 sh RNA1、h-HK2 sh RNA2、h-HK2 sh RNA3)以及一个阴性对照序列(Negative sh RNA),将3沉默序列及一个阴性对照序列分别与质粒连接,然后转染人293T细胞,将靶基因植入慢病毒载体,随后转染乳腺癌MDA-MB231细胞系,通过抗生素嘌呤霉素筛选获得HK2低表达的乳腺癌MDA-MB231细胞系。采用RT-PCR及western blotting方法分别从m RNA和蛋白水平检测转染后的沉默效果。筛选出沉默效果最佳的稳定细胞株用于后续实验,后续实验分为3组:空白组(Control组,即不做任何处理),阴性对照组(Negative组,即转染阴性质粒组)以及沉默的实验组(HK2 sh RNA组,即转染稳定沉默HK2基因质粒的实验组)。1、将上述三组不同的细胞分别置于0、2、4、6、8Gy剂量的X线下照射,通过CCK-8实验观察三组细胞在上述不同剂量下的细胞增值情况。2、将上述三组细胞在6Gy剂量的X线下照射,然后利用流式细胞术(FCM)检测三组细胞在6 Gy X线照射下细胞的凋亡情况。以此来判断沉默HK2与乳腺癌细胞MDA-MB231放射治疗敏感性之间的关系。结果1、成功构建带有沉默HK2基因序列p HBLV-U6-Scramble-Zs Green-Puro的质粒。2、RT-PCR及western blot结果显示:三个沉默HK2的序列中,h-HK2-sh RNA1基因序列沉默的效果最好,沉默后HK2的m RNA相对表达量下降了89%。3、三组细胞分别在0、2、4、6、8Gy的X线照射后,三组细胞的存活率在同一个剂量下h-HK2-sh RNA1组要明显低于Control组和Negative组(P0.05);并且,随着照射剂量的逐渐增高,三组细胞的存活率也逐渐下降,且h-HK2-sh RNA1组下降的幅度更大。4、流式细胞术结果显示:三组细胞在6Gy的X线照射下,凋亡率分别为Control组(29.1%),Negative组(29.4%)和h-HK2-sh RNA1组(59.9%),可以看出h-HK2-sh RNA1组的细胞凋亡率明显高于其余两组,且P0.05。结论利用慢病毒介导的RNA干扰技术有效的降低的乳腺癌细胞中HK2的表达水平,并成功的构建了带有沉默HK2基因序列的质粒,进而感染乳腺癌MDA-MB231细胞株,得到稳定转染HK2的乳腺癌细胞株。通过放射治疗后,沉默细胞组增值率明显下降,凋亡率明显升高,说明HK2与乳腺癌的放疗敏感性有关,沉默HK2可增加乳腺癌对放射治疗的敏感性。
[Abstract]:Objective to investigate whether short hairpin RNA (sh RNA) mediated hexokinase 2 (hexokinase 2 HK2) gene silencing has an effect on the radiosensitivity and the effect of radiotherapy on breast cancer cells. A stable breast cancer MDA-MB231 cell line with low expression of hexokinase 2 (HK2) was constructed by lentivirus-mediated RNA interference technique. Then the relationship between the expression of HK2 gene and the sensitivity of breast cancer cell line to MDA-MB231 radiotherapy was observed by using sh RNA interference technique. Methods using lentivirus mediated short hairpin RNA (sh RNA) interference technique, three shRNA sequences (h-HK2 sh RNA1h-HK2 sh RNA2h-HK2sh RNA3) of silencing HK2 and one negative control sequence (Negative sh RNA), were designed to connect with the plasmids, respectively. Then human 293T cells were transfected and the target gene was implanted into the lentivirus vector. Then the breast cancer MDA-MB231 cell line was transfected with the target gene. The low expression HK2 MDA-MB231 cell line was obtained by antibiotic purine mycin screening. The silencing effect of transfection was detected by RT-PCR and western blotting from m RNA and protein levels, respectively. The stable cell lines with the best silencing effect were selected for further experiment. The following experiments were divided into three groups: blank group (Control group, that is, without any treatment), negative control group (Negative group, that is, transfection negative plasmid group) and silent experimental group (HK2 sh RNA group). The experimental group transfected with stable silencing HK2 gene plasmids). The proliferation of three groups of cells at different doses was observed by CCK-8 assay. The above three groups of cells were irradiated under the X-ray of 6Gy dose, and then the apoptosis of the three groups was detected by flow cytometry (FCM) under 6 Gy X-ray irradiation. In order to determine the relationship between silent HK2 and breast cancer cell MDA-MB231 radiotherapy sensitivity. Results 1. The results of RT-PCR and western blot showed that among the three sequences of silencing HK2, the silencing of h-HK2-sh RNA1 gene sequence was the best. The relative expression of m RNA in HK2 decreased 89% after silencing. The survival rate of h-HK2-sh RNA1 group was significantly lower than that of Control group and Negative group (P0.05), and the survival rate of the three groups was significantly lower than that of Control group and Negative group at the same dose (P0.05), and the cell survival rate of the three groups was significantly lower than that of Control group and Negative group (P0.05), and the survival rate of the three groups was significantly lower than that of Control group and Negative group (P0.05). The survival rate of the three groups also decreased gradually, and that of the h-HK2-sh RNA1 group was much larger. The results of flow cytometry showed that the three groups of cells were exposed to 6Gy X ray irradiation. The apoptotic rates of Control group (29.1%) and h-HK2-sh RNA1 group (29.4%) were significantly higher than those of the other two groups (P 0.05). Conclusion Lentivirus mediated RNA interference technique can effectively reduce the expression of HK2 in breast cancer cells, and successfully construct a plasmid with silencing HK2 gene sequence, and then infect breast cancer MDA-MB231 cell line. Breast cancer cell lines stably transfected with HK2 were obtained. After radiotherapy, the proliferation rate and apoptosis rate in silencing group decreased significantly, indicating that HK2 was related to the radiosensitivity of breast cancer, and silencing HK2 could increase the sensitivity of breast cancer to radiotherapy.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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