锰型过氧化物酶与鼻咽癌放射敏感关系研究
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摘要
背景鼻咽癌流行于中国南方。低分化型是鼻咽癌最常见的病理组织型,对放射治疗敏感。常规放射治疗是鼻咽癌最常用和最有效的治疗。但是鼻咽癌五年生存率仅为50%左右,而失败主要原因是鼻咽癌组织对放射线的抵抗。增加照射剂量,因会引起周围组织的损害而受到限制。因此,提高疗效只能从增加肿瘤而不增加周围组织的辐射量,或者提高肿瘤组织相对于周围组织的放射敏感性入手。前者涉及改善放疗物理因素。后者涉及探索影响肿瘤与正常组织不同放疗差别的生物因素。步入分子医学时代,诸如免疫疗法和RNA干扰等靶向目标分子治疗等走向应用。对于鼻咽癌治疗,发现和应用有潜力的放射敏感相关生物分子更有意义。受离子辐射的细胞会产生自由基,起辐射诱导细胞毒作用。而锰型过氧化物酶(SOD2)则能歧化超氧阴离子自由基,降低哺乳动物细胞对放射的敏感性。
研究目的实验探索锰型过氧化物酶对鼻咽癌放射抵抗性的影响及其作用机制。
研究方法①应用集落形成实验检测鼻咽癌细胞株CNE1和CNE2放射前和不同剂量放射后的存活分数,应用多靶单击模型和线性二次模型拟合放射存活曲线,测算放射生物学参数D0,Dq,α,β和SF2,比较两细胞株的放射敏感性。
②应用水溶四氮唑盐微孔板实验测定细胞株CNE1与CNE2的总SOD和SOD2活性,测评SOD活性和SOD2活性与鼻咽癌细胞株放疗敏感或抵抗的关系。
③应用Western blot检测照射前和不同时间及不同剂量照射后细胞株CNE1和CNE2的SOD2蛋白表达,测评SOD2蛋白表达与鼻咽癌细胞株放疗敏感或抵抗的关系和随辐射剂量时间的变化。
⑤使用Gateway(?)-adapted expression vector构建表达miRNA对SOD2基因RNA干扰的质粒。
⑥采用脂质体转染法将构建的针对SOD2基因的miRNA干扰的质粒转染CNE1细胞,cck-8实验和FCM检测转染质粒对细胞增殖和细胞周期的影响。Western blot和RT-PCR检测瞬转细胞的SOD2的蛋白和mRNA表达变化。
⑦应用杀稻瘟素(Blasticidin)筛选转染细胞,建立针对SOD2基因表达miRNA干扰的CNE1细胞株,检测稳转细胞的SOD2的蛋白和mRNA表达情况。
⑧集落形成实验等测算和比较稳转SOD2基因沉默细胞的放射生物学参数,直接测评SOD2对鼻咽癌细胞放射抵抗的影响。
⑨x-线2Gy和4Gy辐射接受miRNA沉默SOD2基因治疗的CNE1细胞实验,测算细胞生存率,测评基因治疗对鼻咽癌细胞放射增敏作用。
结果①鼻咽癌细胞株放射生物学参数在CNE1和CNE2两细胞株放射生存曲线上,每一放射剂量点CNE1的细胞存活分数都高于CNE2。在放射敏感参数D0、Dq、SF2、α和MID CNE1与CNE2相比的分别为：高出50.97%、高出2.31倍、高出1.67倍、少3.35倍,P<0.05。
②鼻咽癌细胞CNE1与CNE2的SOD活性和SOD2活性在总SOD活性和SOD2活性水平细胞株CNE1比CNE2分别高出：63.69%和49.36%,P<0.05。
③鼻咽癌细胞CNE1与CNE2的SOD2蛋白表达细胞株CNE1的SOD2蛋白表达量是CNE2的量的1.94倍,P<0.05。受辐射,CNE1细胞的SOD2蛋白量增加(P<0.05),但是不随时间和剂量变化(P>0.05); CNE2细胞的SOD2蛋白量变化不明显(P>0.05)。
④构建靶向SOD2的miRNA载体质粒与转染CNE1细胞经过质粒DNA测序,质粒pcDNATM6.2-GW/EmGFP-imR-SOD2(411、424和700)构建成功。质粒转染鼻咽癌细胞CNE1,经检测EmGFP编码绿色荧光蛋白,转染成功。转染的细胞imR-SOD2411、imR-SOD2424、imR-SOD2700与未转染的细胞相比,SOD2的mRNA分别下调37%、52.19%、45.12%,P<0.05; SOD2蛋白分别下调13.43%、51.76%、41.36%,P<0.05。转染质粒pcDNATM6.2-GW/EmGFP-imR-SOD2424抑制细胞增殖生长25.87%,质粒pcDNATM6.2-GW/EmGFP-imR-SOD2700和阴性质粒抑制16.88%和17.15%。转染质粒对细胞周期影响不明显(P>0.05)。
⑤建立沉默SOD2的稳定转染细胞株经抗生素筛选获得稳定转染质粒pcDNATM6.2-GW/EmGFP-imR-SOD2700的CNE1细胞克隆。稳转imR-SOD2700的CNE1细胞与未转染的CNE1细胞相比,SOD2的mRNA分别下调72.18%,P<0.01; SOD2蛋白分别下调69.95%,P<0.05。
⑥沉默SOD2基因表达细胞放射生物学参数与基因治疗实验在放射生存曲线上,稳转质粒pcDNATM6.2-GW/EmGFP-imR-SOD2700的CNE1细胞与无转染的CNE1细胞相比每一放射剂量的生存分数减小,在放射生物参数SF2、D0、α前者比后者分别降低12.81倍、降低2.43、升高2.68倍,P<0.05。
⑦通过沉默SOD2基因-放射增敏治疗实验接受基因治疗敲除SOD2基因的CNE1细胞与未受干扰的CNE1细胞照射2Gy和4Gy的x-线细胞的生存率,基因治疗的CNE1细胞比未受治疗的CNE1细胞分别降低2.53倍和3.72倍。
结论鼻咽癌细胞株CNE1相比CNE2的放射抵抗性高。SOD和SOD2活性与鼻咽癌细胞株的放射抵抗有关,活性高的细胞的放射抵抗性大。SOD2蛋白表达与鼻咽癌细胞株的放射抵抗有关,蛋白表达高的细胞的放射抵抗性大。构建靶向SOD2的质粒,能够表达miRNA,下调SOD2基因表达,其中针对SOD2基因位点700至721的niRNA最有效。miRNA沉默SOD2的稳转细胞,SOD2基因mRNA和蛋白显著下调,放射抵抗变弱。总之,miRNA干扰沉默SOD2基因疗法可以应用于放疗抵抗的鼻咽癌,能使癌症放疗的放射敏感性增高。
Background Carcinoma of the nasopharynx is prevalent in the South China region. The poorly-differentiated subtype is most common histologic type of na-sopharyngeal carcinoma, which is radiosensitive. The most effective means of treatment is generally radiation therapy. The five-year survival rate of nasopha-ryngeal carcinomas is about 50% overall and failure to control the cancer is mainly due to a portion of radioresistant phenotype of nasopharyngeal carcinoma. To in-crease the dose of radiation that can be delivered to a tumor is limited by the damage caused to surrounding normal tissues and the consequent risk of com-plications. Hence, therapeutic efficacy can be improved either by increasing the effective radiation dose delivered to the tumor relative to that given to surround-ing normal tissues or by devising an approach that will increase the response of the tumor relative to that of the surrounding normal tissues. The former ap-proach implies improvement in the physical aspects of radiation therapy. The second approach involves exploiting biological factors that result in differences in the response of tumors and normal tissues to radiation therapy. Thanks to the advancements in genomics, proteomics and bioinformatics in recent decades, more understanding of the disease carcinogenesis and progression has been gained. In the era of molecular medicine, specific treatment to the potential target using tech-nologies such as immunotherapy and RNAi becomes formulating from bench to bedside application and thus makes a potential genetic marker of radiosensitivity discovery more meaningful for NPC management. Exposure of cells to ionizing radiation leads to the formation of reactive oxygen species that are associated with radiation-induced cytotoxicity. The antioxidant enzyme manganese super-oxide dismutase (SOD2) catalyzes the dismutation of the superoxide anions into hydrogen peroxide, to enhance the radioresistance of a mammalian cell.
Methods Two human nasopharyngeal carcinoma cell lines CNE1 and CNE2 were characterized using the clonogenic survival assay after irradiation. The multitar-get, single-hit cell survival equation and the linear-quadratic cell survival equation were fit to the radiation dose response data using the programme SigmaPlot9. Ra-diosensitivity parameters of the nasopharyngeal carcinoma cell lines (CNE1 and CNE2) obtained from the fitted data were D0(the slope of in the straight-line region), Dq(a measure of the size of the shoulder), a(a measure of the initial slope),β(a measure of the final slope), and SF2(surviving fraction at 2 Gy).
Activities of SOD2 gene on nasopharyngeal carcinoma cells were investigated in comparison with the relative radiosensitivity of NPC cell line and the relative radioresistance of NPC cell line. SOD enzymatic activity was assessed by the reduction of WST-1 using water-soluble tetrazolium salt microplate assay.
Expression of SOD2 gene on nasopharyngeal carcinoma cells was investigated in comparison with the relative radiosensitivity of NPC cell line and'the relative radioresistance of NPC cell line. Western blots were carried out essentially to assess SOD2 protein expression and the optic density (OD) value of each band was estimated by Quantity One(?) 1-D Analysis Software.
We have investigated the potential low expression of SOD2, through expres-sion of miRNA for RNAi using Gateway(?)-adapted expression vector in mam-malian cells, to attenuate the radioresistance of a nasopharyngeal carcinoma cell line. Using these as in vitro models, we have investigated whether SOD2 gene therapy may be suitable for the improvement of the effects of radiotherapy on nasopharyngeal carcinoma.
·Designed and synthesized three pairs of complementary DNA oligos for SOD2 messenger RNA, with each containing 4 nucleotide overhangs nec-essary for directional cloning.
·Annealed DNA oligos to generate three double-strand oligos.
·Clone the ds oligos into pcDNATM6.2-GW/EmGFP-imR expression vectors using T4 DNA Ligase.
·Transformed E.coli and analyzed colonies for the desired expression clone.
·Transfected the expression clone for transient and stable RNAi analysis.
·assessed protein expression and mRNA expression of SOD2 in the transfected cells using Western blot analysis and RT-PCR.
·assessed the effect of clonogenic cell kill of established stable cell line ex-pressing miRNA of SOD2 following exposure to ionizing radiation.
·determined if radiation efficiency increased with reduced expression SOD2 through miRNA expression construct transfer.
Results The radiobiological parameter of nasopharyngeal carcinoma cell lines Surviving fraction of CNE1 was higher than those of CNE2 at each dose point on radiation cell survival curves. The value of Do, Dq, a,β, SF2 of cell line CNE1 and CNE2 were 1.398 Gy,0.881 Gy,0.228 Gy-1,0.104 Gy-2,0.403 and 0.926 Gy, 0.266 Gy,0.763 Gy-1,0.066 Gy-2,0.151, respectively.
SOD enzyme activity and SOD2 expression of nasopharyngeal carcinoma cell lines The levels of total SOD activity and SOD2 activity in cell lines CNEl and CNE2 were 234.04 and 146.10 U/108cell,143.20 and 97.82U/108cell, respectively. Compared to CNE2 cells, levels of SOD2 protein expression were increased 1.94-fold in CNE1 cells.
Construction of the plasmid vectors and stable cell lines The plasmid vectors, pcDNATM6.2-GW/EmGFP-imR-SOD2(411,424 and 700), express miRNA for use in RNAi analysis of SOD2 gene in nasopharyngeal carcinoma cells. EmGFP en-codes Green Fluorescent Protein. Following plasmid/LipofectamineTM2000 trans-duction, transduced CNE1, uninduced cell and cell transfected with pcDNATM6.2-GW/EmGFP-imR-neg down-regulated the expression of SOD2 protein to 86.57%, 48.24%,58.64%,100% and 90% respectively; down-regulated the level of SOD2 mRNA to 63%,47.81%,54.88%,100% and 92% respectively. Transfected pcDNATM6.2-GW/Em GFP-imR-SOD2424 inhibited tumor cell proliferation. All transduced plasmids did not influence cell cycles.
Selected for stable cell line transfected with pcDNATM6.2-GW/EmGFP-imR-SOD2700 using Blasticidin. Assay for SOD2 gene knockdown, compare to unin-duced CNE1 cell and cell stably transfected with pcDNATM6.2-GW/EmGFP-imR-neg showed the level of SOD2 mRNA were down-regulated to 27.82%,100% and 98.28%; the protein expression of SOD2 gene were down-regulated to 30.05%, 100% and 94.88%.
The radiobiological parameter of SOD2 gene knockdown NPC cell lines Sur-viving fraction of SOD2 gene knockdown CNE1 was lower than those of uninduced CNE1 at each dose point on radiation cell survival curves. The value of Do, Dq, a, 0, SF2 of both were 1.446 Gy,0.856 Gy,0.256 Gy-1,0.085 Gy-2,0.407 and 0.927 Gy,0.276 Gy,0.790 Gy-1,0.043 Gy-2,0.153, respectively. The radiation cell sur-vival curves of cell stably transfected with pcDNATM6.2-GW/EmGFP-imR-neg was similar to that of uninduced CNE1 cell.
The effect of miRNA interference silencing SOD2 in CNEl cells on efficiency of radiation CNE1 cells transduced with miR-SOD2 or miR-neg control or parental CNE1 cells were irradiated at either 2 or 4 Gy. Cells transduced with miR-SOD2 demonstrated an increased efficiency of treatment of carcinoma cell with ionizing radiation as seen by a significant increase in colony number at both doses
Conclusions CNE1 is more radioresistance than CNE2. The levels of total SOD activity and SOD2 activity in CNEl cell were significantly(1.5-fold) higher than CNE2 cell. There is marked correlation observed between the activity of SOD, SOD2 and radiosensitivity of the nasopharyngeal carcinoma cell lines.
The protein expression of SOD2 in CNE1 cell is significantly (2-fold) higher than that of CNE2 cell. There is marked correlation observed between the quantity of SOD2 protein and radiosensitivity of the nasopharyngeal carcinoma cell lines. x-irradiation can result in increasing in total quantity of SOD2 protein.
We construct successfully the recombination plasmid vectors which target SOD2 gene, the plasmid vectors which express miRNA can down-regulate the expression of SOD2 gene efficiently, the miRNA of SOD2 gene loci from 700 to 721 is effective to down-regulate SOD2 gene.
In vitro, miRNA expression of SOD2 led to a decrease in mRNA and protein of SOD2 in transduced cell in comparison to contols, the Corresponding cell is very lower radioresistance than uninduced CNE1. The results presented suggest that miRNA interference silencing SOD2 gene therapy may be applicable to the radioresistant nasopharyngeal carcinoma, enabling radiosensitivity escalation in cancer radiotherapy.
研究目的实验探索锰型过氧化物酶对鼻咽癌放射抵抗性的影响及其作用机制。
研究方法①应用集落形成实验检测鼻咽癌细胞株CNE1和CNE2放射前和不同剂量放射后的存活分数,应用多靶单击模型和线性二次模型拟合放射存活曲线,测算放射生物学参数D0,Dq,α,β和SF2,比较两细胞株的放射敏感性。
②应用水溶四氮唑盐微孔板实验测定细胞株CNE1与CNE2的总SOD和SOD2活性,测评SOD活性和SOD2活性与鼻咽癌细胞株放疗敏感或抵抗的关系。
③应用Western blot检测照射前和不同时间及不同剂量照射后细胞株CNE1和CNE2的SOD2蛋白表达,测评SOD2蛋白表达与鼻咽癌细胞株放疗敏感或抵抗的关系和随辐射剂量时间的变化。
⑤使用Gateway(?)-adapted expression vector构建表达miRNA对SOD2基因RNA干扰的质粒。
⑥采用脂质体转染法将构建的针对SOD2基因的miRNA干扰的质粒转染CNE1细胞,cck-8实验和FCM检测转染质粒对细胞增殖和细胞周期的影响。Western blot和RT-PCR检测瞬转细胞的SOD2的蛋白和mRNA表达变化。
⑦应用杀稻瘟素(Blasticidin)筛选转染细胞,建立针对SOD2基因表达miRNA干扰的CNE1细胞株,检测稳转细胞的SOD2的蛋白和mRNA表达情况。
⑧集落形成实验等测算和比较稳转SOD2基因沉默细胞的放射生物学参数,直接测评SOD2对鼻咽癌细胞放射抵抗的影响。
⑨x-线2Gy和4Gy辐射接受miRNA沉默SOD2基因治疗的CNE1细胞实验,测算细胞生存率,测评基因治疗对鼻咽癌细胞放射增敏作用。
结果①鼻咽癌细胞株放射生物学参数在CNE1和CNE2两细胞株放射生存曲线上,每一放射剂量点CNE1的细胞存活分数都高于CNE2。在放射敏感参数D0、Dq、SF2、α和MID CNE1与CNE2相比的分别为：高出50.97%、高出2.31倍、高出1.67倍、少3.35倍,P<0.05。
②鼻咽癌细胞CNE1与CNE2的SOD活性和SOD2活性在总SOD活性和SOD2活性水平细胞株CNE1比CNE2分别高出：63.69%和49.36%,P<0.05。
③鼻咽癌细胞CNE1与CNE2的SOD2蛋白表达细胞株CNE1的SOD2蛋白表达量是CNE2的量的1.94倍,P<0.05。受辐射,CNE1细胞的SOD2蛋白量增加(P<0.05),但是不随时间和剂量变化(P>0.05); CNE2细胞的SOD2蛋白量变化不明显(P>0.05)。
④构建靶向SOD2的miRNA载体质粒与转染CNE1细胞经过质粒DNA测序,质粒pcDNATM6.2-GW/EmGFP-imR-SOD2(411、424和700)构建成功。质粒转染鼻咽癌细胞CNE1,经检测EmGFP编码绿色荧光蛋白,转染成功。转染的细胞imR-SOD2411、imR-SOD2424、imR-SOD2700与未转染的细胞相比,SOD2的mRNA分别下调37%、52.19%、45.12%,P<0.05; SOD2蛋白分别下调13.43%、51.76%、41.36%,P<0.05。转染质粒pcDNATM6.2-GW/EmGFP-imR-SOD2424抑制细胞增殖生长25.87%,质粒pcDNATM6.2-GW/EmGFP-imR-SOD2700和阴性质粒抑制16.88%和17.15%。转染质粒对细胞周期影响不明显(P>0.05)。
⑤建立沉默SOD2的稳定转染细胞株经抗生素筛选获得稳定转染质粒pcDNATM6.2-GW/EmGFP-imR-SOD2700的CNE1细胞克隆。稳转imR-SOD2700的CNE1细胞与未转染的CNE1细胞相比,SOD2的mRNA分别下调72.18%,P<0.01; SOD2蛋白分别下调69.95%,P<0.05。
⑥沉默SOD2基因表达细胞放射生物学参数与基因治疗实验在放射生存曲线上,稳转质粒pcDNATM6.2-GW/EmGFP-imR-SOD2700的CNE1细胞与无转染的CNE1细胞相比每一放射剂量的生存分数减小,在放射生物参数SF2、D0、α前者比后者分别降低12.81倍、降低2.43、升高2.68倍,P<0.05。
⑦通过沉默SOD2基因-放射增敏治疗实验接受基因治疗敲除SOD2基因的CNE1细胞与未受干扰的CNE1细胞照射2Gy和4Gy的x-线细胞的生存率,基因治疗的CNE1细胞比未受治疗的CNE1细胞分别降低2.53倍和3.72倍。
结论鼻咽癌细胞株CNE1相比CNE2的放射抵抗性高。SOD和SOD2活性与鼻咽癌细胞株的放射抵抗有关,活性高的细胞的放射抵抗性大。SOD2蛋白表达与鼻咽癌细胞株的放射抵抗有关,蛋白表达高的细胞的放射抵抗性大。构建靶向SOD2的质粒,能够表达miRNA,下调SOD2基因表达,其中针对SOD2基因位点700至721的niRNA最有效。miRNA沉默SOD2的稳转细胞,SOD2基因mRNA和蛋白显著下调,放射抵抗变弱。总之,miRNA干扰沉默SOD2基因疗法可以应用于放疗抵抗的鼻咽癌,能使癌症放疗的放射敏感性增高。
Background Carcinoma of the nasopharynx is prevalent in the South China region. The poorly-differentiated subtype is most common histologic type of na-sopharyngeal carcinoma, which is radiosensitive. The most effective means of treatment is generally radiation therapy. The five-year survival rate of nasopha-ryngeal carcinomas is about 50% overall and failure to control the cancer is mainly due to a portion of radioresistant phenotype of nasopharyngeal carcinoma. To in-crease the dose of radiation that can be delivered to a tumor is limited by the damage caused to surrounding normal tissues and the consequent risk of com-plications. Hence, therapeutic efficacy can be improved either by increasing the effective radiation dose delivered to the tumor relative to that given to surround-ing normal tissues or by devising an approach that will increase the response of the tumor relative to that of the surrounding normal tissues. The former ap-proach implies improvement in the physical aspects of radiation therapy. The second approach involves exploiting biological factors that result in differences in the response of tumors and normal tissues to radiation therapy. Thanks to the advancements in genomics, proteomics and bioinformatics in recent decades, more understanding of the disease carcinogenesis and progression has been gained. In the era of molecular medicine, specific treatment to the potential target using tech-nologies such as immunotherapy and RNAi becomes formulating from bench to bedside application and thus makes a potential genetic marker of radiosensitivity discovery more meaningful for NPC management. Exposure of cells to ionizing radiation leads to the formation of reactive oxygen species that are associated with radiation-induced cytotoxicity. The antioxidant enzyme manganese super-oxide dismutase (SOD2) catalyzes the dismutation of the superoxide anions into hydrogen peroxide, to enhance the radioresistance of a mammalian cell.
Methods Two human nasopharyngeal carcinoma cell lines CNE1 and CNE2 were characterized using the clonogenic survival assay after irradiation. The multitar-get, single-hit cell survival equation and the linear-quadratic cell survival equation were fit to the radiation dose response data using the programme SigmaPlot9. Ra-diosensitivity parameters of the nasopharyngeal carcinoma cell lines (CNE1 and CNE2) obtained from the fitted data were D0(the slope of in the straight-line region), Dq(a measure of the size of the shoulder), a(a measure of the initial slope),β(a measure of the final slope), and SF2(surviving fraction at 2 Gy).
Activities of SOD2 gene on nasopharyngeal carcinoma cells were investigated in comparison with the relative radiosensitivity of NPC cell line and the relative radioresistance of NPC cell line. SOD enzymatic activity was assessed by the reduction of WST-1 using water-soluble tetrazolium salt microplate assay.
Expression of SOD2 gene on nasopharyngeal carcinoma cells was investigated in comparison with the relative radiosensitivity of NPC cell line and'the relative radioresistance of NPC cell line. Western blots were carried out essentially to assess SOD2 protein expression and the optic density (OD) value of each band was estimated by Quantity One(?) 1-D Analysis Software.
We have investigated the potential low expression of SOD2, through expres-sion of miRNA for RNAi using Gateway(?)-adapted expression vector in mam-malian cells, to attenuate the radioresistance of a nasopharyngeal carcinoma cell line. Using these as in vitro models, we have investigated whether SOD2 gene therapy may be suitable for the improvement of the effects of radiotherapy on nasopharyngeal carcinoma.
·Designed and synthesized three pairs of complementary DNA oligos for SOD2 messenger RNA, with each containing 4 nucleotide overhangs nec-essary for directional cloning.
·Annealed DNA oligos to generate three double-strand oligos.
·Clone the ds oligos into pcDNATM6.2-GW/EmGFP-imR expression vectors using T4 DNA Ligase.
·Transformed E.coli and analyzed colonies for the desired expression clone.
·Transfected the expression clone for transient and stable RNAi analysis.
·assessed protein expression and mRNA expression of SOD2 in the transfected cells using Western blot analysis and RT-PCR.
·assessed the effect of clonogenic cell kill of established stable cell line ex-pressing miRNA of SOD2 following exposure to ionizing radiation.
·determined if radiation efficiency increased with reduced expression SOD2 through miRNA expression construct transfer.
Results The radiobiological parameter of nasopharyngeal carcinoma cell lines Surviving fraction of CNE1 was higher than those of CNE2 at each dose point on radiation cell survival curves. The value of Do, Dq, a,β, SF2 of cell line CNE1 and CNE2 were 1.398 Gy,0.881 Gy,0.228 Gy-1,0.104 Gy-2,0.403 and 0.926 Gy, 0.266 Gy,0.763 Gy-1,0.066 Gy-2,0.151, respectively.
SOD enzyme activity and SOD2 expression of nasopharyngeal carcinoma cell lines The levels of total SOD activity and SOD2 activity in cell lines CNEl and CNE2 were 234.04 and 146.10 U/108cell,143.20 and 97.82U/108cell, respectively. Compared to CNE2 cells, levels of SOD2 protein expression were increased 1.94-fold in CNE1 cells.
Construction of the plasmid vectors and stable cell lines The plasmid vectors, pcDNATM6.2-GW/EmGFP-imR-SOD2(411,424 and 700), express miRNA for use in RNAi analysis of SOD2 gene in nasopharyngeal carcinoma cells. EmGFP en-codes Green Fluorescent Protein. Following plasmid/LipofectamineTM2000 trans-duction, transduced CNE1, uninduced cell and cell transfected with pcDNATM6.2-GW/EmGFP-imR-neg down-regulated the expression of SOD2 protein to 86.57%, 48.24%,58.64%,100% and 90% respectively; down-regulated the level of SOD2 mRNA to 63%,47.81%,54.88%,100% and 92% respectively. Transfected pcDNATM6.2-GW/Em GFP-imR-SOD2424 inhibited tumor cell proliferation. All transduced plasmids did not influence cell cycles.
Selected for stable cell line transfected with pcDNATM6.2-GW/EmGFP-imR-SOD2700 using Blasticidin. Assay for SOD2 gene knockdown, compare to unin-duced CNE1 cell and cell stably transfected with pcDNATM6.2-GW/EmGFP-imR-neg showed the level of SOD2 mRNA were down-regulated to 27.82%,100% and 98.28%; the protein expression of SOD2 gene were down-regulated to 30.05%, 100% and 94.88%.
The radiobiological parameter of SOD2 gene knockdown NPC cell lines Sur-viving fraction of SOD2 gene knockdown CNE1 was lower than those of uninduced CNE1 at each dose point on radiation cell survival curves. The value of Do, Dq, a, 0, SF2 of both were 1.446 Gy,0.856 Gy,0.256 Gy-1,0.085 Gy-2,0.407 and 0.927 Gy,0.276 Gy,0.790 Gy-1,0.043 Gy-2,0.153, respectively. The radiation cell sur-vival curves of cell stably transfected with pcDNATM6.2-GW/EmGFP-imR-neg was similar to that of uninduced CNE1 cell.
The effect of miRNA interference silencing SOD2 in CNEl cells on efficiency of radiation CNE1 cells transduced with miR-SOD2 or miR-neg control or parental CNE1 cells were irradiated at either 2 or 4 Gy. Cells transduced with miR-SOD2 demonstrated an increased efficiency of treatment of carcinoma cell with ionizing radiation as seen by a significant increase in colony number at both doses
Conclusions CNE1 is more radioresistance than CNE2. The levels of total SOD activity and SOD2 activity in CNEl cell were significantly(1.5-fold) higher than CNE2 cell. There is marked correlation observed between the activity of SOD, SOD2 and radiosensitivity of the nasopharyngeal carcinoma cell lines.
The protein expression of SOD2 in CNE1 cell is significantly (2-fold) higher than that of CNE2 cell. There is marked correlation observed between the quantity of SOD2 protein and radiosensitivity of the nasopharyngeal carcinoma cell lines. x-irradiation can result in increasing in total quantity of SOD2 protein.
We construct successfully the recombination plasmid vectors which target SOD2 gene, the plasmid vectors which express miRNA can down-regulate the expression of SOD2 gene efficiently, the miRNA of SOD2 gene loci from 700 to 721 is effective to down-regulate SOD2 gene.
In vitro, miRNA expression of SOD2 led to a decrease in mRNA and protein of SOD2 in transduced cell in comparison to contols, the Corresponding cell is very lower radioresistance than uninduced CNE1. The results presented suggest that miRNA interference silencing SOD2 gene therapy may be applicable to the radioresistant nasopharyngeal carcinoma, enabling radiosensitivity escalation in cancer radiotherapy.
引文
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