抑制MDC1基因蛋白表达对裸鼠食管癌细胞移植瘤大小及放疗敏感程度的影响
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摘要
目的分析抑制MDC1基因蛋白表达对裸鼠食管癌细胞移植瘤大小及放疗敏感程度的影响。方法取54只SPF级BALB/C雄性裸鼠,构建食管癌动物模型。通过MDC1 mRNA序列,对有效的干扰序列与阴性对照序列进行设计并合成,且与载体p SIH1-H1-cop GFP生成重组质粒。裸鼠随机分为6组,分别为空白对照组(Eca109细胞未进行任何处理)、阴性转染组(转染阴性Eca109细胞)、单一照射组(Eca109细胞仅进行放射线照射)、阳性转染组(转染阳性ECA09细胞)、阴性转染+照射组(转染阴性Eca109细胞联合放射线照射)、阳性转染+照射组(转染阳性ECA09细胞联合放射线照射),每组各9只。采用蛋白印迹法与实时荧光定量PCR法对MDC1蛋白及mRNA表达量进行检测,取MDC1阳性转染Eca109细胞、阴性转染的Eca109细胞,分别将其接种于裸鼠。记录各组裸鼠移植瘤照射后1周、2周、3周、4周的体积变化情况,并用流式细胞仪对裸鼠移植瘤组织中细胞周期分布及细胞凋亡情况进行检测,且根据蛋白印迹法对各组裸鼠移植瘤组织中CHK2、CHK2-T68、CHK1表达情况进行检测,并将所得数据纳入统计学分析。结果p MDC1-shRNA质粒成功构建,且Eca109细胞得以转染,取得MDC1稳定转染的Eca109细胞。接种的裸鼠均成活,且于接种后7 d左右裸鼠爪下移植瘤形成。经15 Gy照射后,单一照射组、阴性转染+照射组裸鼠移植瘤生长速度较空白对照组明显减缓(P <0. 05),阳性转染+照射组裸鼠移植瘤生长速度较其它各组均明显减缓(P <0. 05),生长抑制率明显增高(P <0. 05)。照射前,各组裸鼠移植瘤体积的比较,并无显著差异(P> 0. 05);照射后1周、2周、3周、4周,空白对照组裸鼠移植瘤增长较明显,阳性转染+照射组裸鼠移植瘤增长较缓慢,与单一照射组、阴性转染+照射组裸鼠移植瘤体积明显缩小(P <0. 05)。各组裸鼠移植瘤组织中细胞周期分布及细胞凋亡率的比较,并无显著差异(P> 0. 05)。与对照组、单一照射组比较,阳性转染+照射组裸鼠移植瘤组织中CHK2-T68磷酸化水平显著下降(P <0. 05),而CHK2与CHK1蛋白表达水平较接近(P> 0. 05)。结论 RNA干扰下调MDC1基因蛋白表达具有提高裸鼠食管癌细胞放疗敏感程度的作用,主要体现在阻滞放射线照射后裸鼠移植瘤的肿瘤体积增长方面。
Objective To analyze the effect of inhibiting the expression of MDC1 gene on the size and radiosensitivity of transplanted esophageal cancer cells in nude mice. Methods The effective interference sequence and negative control sequence were designed and synthesized by MDC1 gene sequence,and the recombinant plasmid was generated with vector p SIH1-H1-copGFP. The expression of MDC1 protein and RNA was detected by Western blotting and real-time fluorescence quantitative PCR. MDC1 was transfected into positive and negative Eca109 cells,respectively,and inoculated into nude mice. The volume changes of transplanted tumors in nude mice after irradiation were recorded. The cell cycle distribution and apoptosis in transplanted tumors were detected by flow cytometry. The expression of CHK2,CHK2-T68 and CHK1 in transplanted tumors of nude mice was detected by Western blotting,and the data were included in the statistical analysis. Results The pMDC1-shRNA plasmid was successfully constructed and transfected into Eca109 cells. Eca109 cells stably transfected with MDC1 were obtained. The nude mice inoculated survived,and the transplanted tumors formed under the claws of nude mice about 7 days after inoculation. After 15 Gy irradiation,the growth rate of transplanted tumors in single irradiation group and negative transfection + irradiation group was significantly slower than that in blank control group( P < 0. 05). The growth rate of transplanted tumors in positive transfection + irradiation group was significantly slower than that in other groups( P < 0. 05),and the growth inhibition rate was significantly increased( P < 0. 05). Before irradiation,there was no significant difference in the volume of transplanted tumors between groups( P > 0. 05). At 1,2,3 and 4 weeks after irradiation,the growth of transplanted tumors in nude mice in the blank control group was more obvious,while that in the positive transfection + irradiation group was slower,and the volume of transplanted tumors in nude mice in the negative transfection + irradiation group was significantly smaller than that in the single irradiation group( P < 0. 05). There was no significant difference in cell cycle distribution and apoptotic rate between groups( P > 0. 05).The phosphorylation level of CHK2-T68 in transplanted tumor tissue of nude mice in positive transfection + irradiation group decreased significantly( P < 0. 05),while the expression level of CHK2 and CHK1 protein was similar( P > 0. 05). Conclusion The down-regulation of MDC1 gene expression by RNA interference can improve the radiosensitivity of nude mice esophageal cancer cells,mainly in blocking the growth of tumor volume in nude mice after irradiation.
Objective To analyze the effect of inhibiting the expression of MDC1 gene on the size and radiosensitivity of transplanted esophageal cancer cells in nude mice. Methods The effective interference sequence and negative control sequence were designed and synthesized by MDC1 gene sequence,and the recombinant plasmid was generated with vector p SIH1-H1-copGFP. The expression of MDC1 protein and RNA was detected by Western blotting and real-time fluorescence quantitative PCR. MDC1 was transfected into positive and negative Eca109 cells,respectively,and inoculated into nude mice. The volume changes of transplanted tumors in nude mice after irradiation were recorded. The cell cycle distribution and apoptosis in transplanted tumors were detected by flow cytometry. The expression of CHK2,CHK2-T68 and CHK1 in transplanted tumors of nude mice was detected by Western blotting,and the data were included in the statistical analysis. Results The pMDC1-shRNA plasmid was successfully constructed and transfected into Eca109 cells. Eca109 cells stably transfected with MDC1 were obtained. The nude mice inoculated survived,and the transplanted tumors formed under the claws of nude mice about 7 days after inoculation. After 15 Gy irradiation,the growth rate of transplanted tumors in single irradiation group and negative transfection + irradiation group was significantly slower than that in blank control group( P < 0. 05). The growth rate of transplanted tumors in positive transfection + irradiation group was significantly slower than that in other groups( P < 0. 05),and the growth inhibition rate was significantly increased( P < 0. 05). Before irradiation,there was no significant difference in the volume of transplanted tumors between groups( P > 0. 05). At 1,2,3 and 4 weeks after irradiation,the growth of transplanted tumors in nude mice in the blank control group was more obvious,while that in the positive transfection + irradiation group was slower,and the volume of transplanted tumors in nude mice in the negative transfection + irradiation group was significantly smaller than that in the single irradiation group( P < 0. 05). There was no significant difference in cell cycle distribution and apoptotic rate between groups( P > 0. 05).The phosphorylation level of CHK2-T68 in transplanted tumor tissue of nude mice in positive transfection + irradiation group decreased significantly( P < 0. 05),while the expression level of CHK2 and CHK1 protein was similar( P > 0. 05). Conclusion The down-regulation of MDC1 gene expression by RNA interference can improve the radiosensitivity of nude mice esophageal cancer cells,mainly in blocking the growth of tumor volume in nude mice after irradiation.
引文
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[13] Lee JH,Park SJ,Hariharasudhan G,et al. ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability[J]. Nat Commun,2017,8(1):903.
[14] Bi JP,Huang A,Liu T,et al. Expression of DNA damage checkpoint53BP1 is correlated with prognosis,cell proliferation and apoptosis in colorectal cancer[J]. Int J Clin Exp Pathol,2015,8(6):6070-6082.
[15]梁宗英,侯继申,张召,等.大蒜素干预下SD大鼠血清对食管癌EC109细胞增殖活性的影响[J].临床和实验医学杂志,2015,14(19):1577-1579.
[2] Liang N,Song X,Xie J,et al. Effect of galectin-3 on the behavior of Eca-109 human esophageal cancer cells[J]. Mol Med Rep,2015,11(2):896-902.
[3] Wang ZM,Kang YH,Yang X,et al. Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro[J]. Dis Esophagus,2016,29(1):54-61.
[4] Li YJ,Zhou JH,Du XX,et al. Dihydroartemisinin accentuates the anti-tumor effects of photodynamic therapy via inactivation of NF-κB in Eca109 and Ec9706 esophageal cancer cells[J]. Cell Physiol Biochem,2014,33(5):1527-1536.
[5] Gill MR,Menon JU,Jarman PJ,et al. 111In-labelled polymeric nanoparticles incorporating a ruthenium-based radiosensitizer for EGFRtargeted combination therapy in oesophageal cancer cells[J].Nanoscale,2018,10(22):10596-10608.
[6] Oronsky B,Scicinski J,Reid T,et al. RRx-001,a novel clinicalstage chemosensitizer,radiosensitizer,and immunosensitizer,inhibits glucose 6-phosphate dehydrogenase in human tumor cells[J]. Discov Med,2016,21(116):251-265.
[7] Oronsky B,Scicinski J,Ning S,et al. RRx-001,A novel dinitroazetidine radiosensitizer[J]. Invest New Drugs,2016,34(3):371-377.
[8] Ochs F,Somyajit K,Altmeyer M,et al. 53BP1 fosters fidelity of homology-directed DNA repair[J]. Nat Struct Mol Biol,2016,23(8):714-721.
[9] Yun H,Shi R,Yang Q,et al. Over expression of hRad9 protein correlates with reduced chemosensitivity in breast cancer with administration of neoadjuvant chemotherapy[J]. Sci Rep,2014,4(1):7548.
[10]李一鸣,李恒,孙菡,等.迷迭香酸衍生物RAD-9调控人食管癌EC9706细胞增殖、凋亡的作用观察及机制探讨[J].临床和实验医学杂志,2018,17(21):2275-2280.
[11] Pfeiffer A,Luijsterburg MS,Acs K,et al. Ataxin-3 consolidates the MDC1-dependent DNA double-strand break response by counteracting the SUMO-targeted ubiquitin ligase RNF4[J]. EMBO J,2017,36(8):1066-1083.
[12] Lee JH,Park SJ,Kim SW,et al. c-Fos-dependent miR-22 targets MDC1 and regulates DNA repair in terminally differentiated cells[J].Oncotarget,2017,8(29):48204-48221.
[13] Lee JH,Park SJ,Hariharasudhan G,et al. ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability[J]. Nat Commun,2017,8(1):903.
[14] Bi JP,Huang A,Liu T,et al. Expression of DNA damage checkpoint53BP1 is correlated with prognosis,cell proliferation and apoptosis in colorectal cancer[J]. Int J Clin Exp Pathol,2015,8(6):6070-6082.
[15]梁宗英,侯继申,张召,等.大蒜素干预下SD大鼠血清对食管癌EC109细胞增殖活性的影响[J].临床和实验医学杂志,2015,14(19):1577-1579.