PeroxiredoxinⅠ对乳腺癌细胞放射敏感性的影响及其作用机制的实验研究
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摘要
乳腺癌是女性最常见的恶性肿瘤,全世界每年约120万妇女发病,50万妇女死于乳腺癌。尽管目前在乳腺癌的综合治疗方面已取得了长足进步,但仍有20%～30%的乳腺癌患者治疗后局部复发和远处转移。因此探索新的治疗手段成为进一步提高乳腺癌治疗效果的突破口。
放射治疗是恶性肿瘤治疗的主要手段之一,而肿瘤细胞的放射敏感性是决定放射治疗疗效的关键因素。近年来,随着对肿瘤放射生物学的深入研究,发现一些基因表达影响着肿瘤细胞的放射敏感性。基因治疗策略可将肿瘤细胞中特异或差异表达的基因敲除,实现特定基因在体内的可控性表达,逆转肿瘤细胞对放化疗的抗拒,从而增加其治疗的敏感性。
Peroxiredoxins(Prxs)是新发现的一类过氧化物酶,在清除体内活性氧中发挥着重要作用,可保护细胞免受过氧化反应造成的损伤。PeroxiredoxinⅠ(PrxⅠ)是Prxs蛋白家族成员之一,在包括乳腺癌在内的多种恶性肿瘤细胞中存在过表达,其表达与肿瘤细胞的增殖、分化、转移、复发及放化疗敏感性密切相关,并可作为诊断肿瘤的分子标志。因此,PrxⅠ可能是肿瘤基因治疗的理想靶点。
DNA双链断裂(Double strand break,DSB)是电离辐射治疗肿瘤的重要机制。DNA损伤后可激活机体内的损伤修复系统,使受损的DNA得以及时修复,以保证机体基因组的稳定。研究发现:DNA损伤感应分子γ-H2AX和DNA损伤修复的关键蛋白Rad51可能在其发挥重要作用。
本研究将基因治疗和放射治疗结合起来,以PrxⅠ为靶点,构建针对PrxⅠ特异性的shRNA真核表达载体,下调乳腺癌MCF-7细胞中PrxⅠ的表达,观察其对放射线的敏感性变化,并探讨相关作用机制,阐明PrxⅠ基因在乳腺癌放射治疗中的作用,为乳腺癌基因治疗提供新的靶位。
方法与结果
第一部分:靶向PeroxiredoxinⅠ基因的shRNA真核表达载体构建及功能鉴定
方法:根据RNA干扰设计原则,设计并合成四条靶向PrxⅠ基因的shRNA干扰序列及阴性对照序列(HK)。先用BbsⅠ及BamHⅠ限制性内切酶双酶切pGPU6/GFP/Neo质粒载体使之线性化,然后通过DNA连接酶将shRNA连接至质粒载体中,经转化、筛选、阳性克隆扩增、抽提重组质粒DNA后,行BamHⅠ、PstⅠ单酶切和DNA测序鉴定;将鉴定正确重组质粒通过脂质体转染入乳腺癌MCF-7细胞。荧光显微镜、流式细胞仪观察并检测转染48h时转染效率;采用RT-PCR及Western blot检测转染前后细胞中的PrxⅠmRNA和蛋白表达变化。
结果:成功构建了四个靶向PrxⅠ基因的shRNA真核表达载体pGPU6-Prx1、pGPU6-Prx2、pGPU6-Prx3、pGPU6-Prx4及阴性表达载体pGPU6-HK,通过脂质体将上述五个重组质粒成功转染入乳腺癌MCF-7细胞中,48h后检测发现各组细胞转染效率均在80%左右。RT-PCR及Western blot检测发现:四个重组质粒均能明显下调乳腺癌细胞MCF-7中的PrxⅠ基因表达,其中pGPU6-Prx3重组质粒的抑制效果最明显,其mRNA及蛋白表达分别抑制了82.6%和80.5%,与对照组和pGPU6-HK组比较差异具有显著性(P<0.05)。成功筛选出抑制效果最佳的一条shRNA,为后续PrxⅠ基因的功能研究奠定了基础。
第二部分:靶向沉默PeroxiredoxinⅠ基因对乳腺癌细胞放射敏感性的影响及其作用机制
方法:将pGPU6-HK、pGPU6-Prx3重组质粒通过脂质体转染入乳腺癌细胞中,通过G418加压筛选建立稳定细胞株,采用RT-PCR、Western blot检测PrxⅠ基因的mRNA和蛋白表达水平。实验分四组:pGPU6-HK组、pGPU6-PrxⅠ组、pGPU6-HK+照射(IR)组、pGPU6-PrxⅠ+IR组。IR组给予单次剂量6Gy的X线照射,在照射48h时通过流式细胞仪检测细胞内活性氧水平、细胞周期、细胞凋亡的变化;MTT法检测细胞增殖能力;Hoechst法观察细胞核形态变化,间接判定细胞凋亡情况;克隆形成实验检测经0、2、4、6、8、10Gy剂量照射后细胞克隆形成率,绘制细胞存活曲线,计算放射生物学参数Do、N、Dq、SF2值,判断细胞的放射敏感性变化;采用Western blot检测DNA双链断裂的标志性蛋白γ-H2AX以及DNA损伤后修复的关键蛋白Rad51表达。
结果:通过G418加压筛选4周后获取了2个稳定细胞株:pGPU6-PrxⅠ和pGPU6-HK。RT-PCR和Western bolt检测发现:与pGPU6-HK组相比,pGPU6-PrxⅠ组中PrxⅠmRNA和蛋白表达量明显减少,抑制率分别为84.8%和83.5%,差异具有显著性(P﹤0.05)。MTT、流式细胞及Hoechst检测结果显示:给予6Gy的X射线照射48h后,pGPU6-PrxⅠ组及联合IR组中细胞内活性氧水平明显升高、G1期增多、S期细胞减少、细胞凋亡显著增加、细胞增殖明显延迟、核浓缩、碎片化等凋亡典型形态改变的细胞增多,与pGPU6-HK比较,差异均具有显著性(P﹤0.05);其中以pGPU6-PrxⅠ+IR组上述变化最明显,与pGPU6-PrxⅠ及pGPU6-HK+IR比较,差异也具有显著性(P﹤0.05)。克隆形成实验结果显示:经不同放射剂量的X线照射后,pGPU6-PrxⅠ组及联合IR组中细胞克隆形成率降低,其中pGPU6-PrxⅠ+IR组细胞的放射生物学参数Do、N、Dq、SF2值分别为1.354、3.106、1.547、0.504,2Gy照射剂量时SER为1.353;Western blot检测结果显示:pGPU6-PrxⅠ组及联合IR组中Rad51蛋白表达明显降低,而γ-H2AX蛋白表达显著升高,与pGPU6-HK组相比,差异具有显著性(P﹤0.01),以pGPU6-PrxⅠ+IR组中更明显,其Rad51蛋白表达下降了79.3%,而γ-H2AX蛋白表达升高了3.7倍,与pGPU6-PrxⅠ组、pGPU6-HK+IR组相比,差异也具有显著性(P﹤0.05)。
第三部分:靶向沉默PeroxiredoxinⅠ基因对乳腺癌裸鼠移植瘤放射敏感性的影响
方法:将稳定筛选的pGPU6-HK、pGPU6-PrxⅠ两株细胞接种于BALB/c裸鼠皮下,以肿瘤长径达5mm作为判断乳腺癌移植瘤模型建立成功标准。实验分四组:pGPU6-HK组、pGPU6-PrxⅠ组、pGPU6-HK+照射(IR)组、pGPU6-PrxⅠ+IR组。IR组给予每次2Gy,隔日一次,总剂量为10Gy,观察移植瘤生长变化,照射第30d处死裸鼠,剥离肿瘤组织,称取瘤重,计算抑瘤率;免疫组化检测肿瘤组织中的PrxⅠ和促凋亡蛋白Caspase3表达;透射电镜观察肿瘤组织细胞的超微结构;Western blot检测肿瘤组织中DNA双链断裂的标志性蛋白γ-H2AX以及DNA损伤后修复的关键蛋白Rad51表达。
结果:成功建立了裸鼠移植瘤模型,pGPU6-HK组及pGPU6-PrxⅠ组细胞成瘤时间分别为10d和14d,pGPU6-PrxⅠ组移植瘤生长相对较慢,肿瘤体积较小,重量较轻,其抑瘤率为37.8%,与pGPU6-HK组相比,差异具有显著性(P﹤0.01)。当给予10Gy的X线剂量照射后,联合IR组裸鼠移植瘤消退速度大于生长速度,肿瘤体积缩小,但在照射结束后第三天肿瘤出现正生长,其生长速度较pGPU6-HK、pGPU6-PrxⅠ组明显缓慢,以pGPU6-PrxⅠ+IR组更为明显,其抑瘤率达到了79.76%,与pGPU6-PrxⅠ(34.92%)、pGPU6-HK+IR(56.94%)比较差异具有显著性(P<0.05)。免疫组化结果及电镜结果显示:pGPU6-PrxⅠ及联合IR组肿瘤细胞中PrxⅠ蛋白表达明显下调,而促凋亡蛋白Caspase3表达显著提高,凋亡、坏死细胞增多。Western bolt结果显示:pGPU6-PrxⅠ及联合IR组肿瘤细胞中的Rad51蛋白表达降低,而γ-H2AX蛋白表达升高,与pGPU6-HK组相比,差异具有显著性(P<0.01)。上述变化pGPU6-PrxⅠ+IR组最为明显,其Rad51蛋白表达抑制了84.8%,γ-H2AX蛋白表达升高5.6倍,与pGPU6-PrxⅠ和pGPU6-HK+IR组相比,差异也具有显著性(P<0.05)。
结论:本研究成功构建了靶向PrxⅠ基因shRNA真核表达载体,从体外、体内证实了靶向沉默PrxⅠ基因后,可有效抑制细胞增殖、调控细胞周期再分布、促进细胞凋亡、降低细胞亚致死损伤修复能力,提高放射治疗增益比,从而增加了乳腺癌MCF-7细胞的放射敏感性,其作用机制可能与细胞内活性氧清除能力减弱、DNA双链断裂增加及DNA损伤后修复能力降低有关。PrxⅠ基因表达与乳腺癌细胞的放射敏感性呈负相关,可作为乳腺癌放射增敏的分子靶点。
Breast cancer is the most common malignancy of women. About 120 million new cases are diagnosed and 50 million women die of breast cancer each year around the world. Despite the comprehensive treatment of breast cancer has made great progress, there are still 20% to 30% of breast cancer patients occur local recurrence and distant metastasis after treatment Therefore, it is the breakthrough to explore new therapeutic tools for further improving the curative effect of breast cancer.
Radiotherapy is one of the major means of treating malignant tumors, however, the radiosensitivity of tumor cells is a key factor to determine radiotherapy effect. In recent years, with thorough study in the radiation biology of the cancer, researchers have found that the expression of some genes at the molecular level affected the radiosensitivity of tumor cells. Gene therapy strategy enable us to knockout these genes that are specific or differential expression between tumor cells and normal cells, and achieve controlled expression of special gene in vivo so as to reverse resistance to the treatment of tumor cell and increase their therapeutic responsiveness.
Peroxiredoxins (Prxs) are a newly discovered peroxidase that play an important role in scavenging reactive oxygen species of the body and protect cells from damaging by overoxidized. PeroxiredoxinⅠ(PrxⅠ),a member of the Prxs family, which is overexpressed in many cancers including breast cancer cells. PrxⅠhave been implicated in regulating many cellular processes such as cell proliferation, differentiation and apoptosis, moreover, the levels of its expression is closely related to the metastasis, recurrence, chemosensitivity and radiosensitivity of tumor cells,and can serve as a molecular marker for tumor diagnosis. Thus, PrxⅠmay be an ideal target for gene therapy.
DNA strand break is an important mechanism which ionizing radiation treat tumor. DNA damage can activate the repair system of body and can be repaired in time to ensure the stability of organism genome. Researches have found thatγ-H2AX and Rad51 proteins may play an important role in DNA damage repair.
We combine gene therapy with radiotherapy in this study: First, we construct specific shRNA eukaryotic expression vector targeting PrxⅠgene in order to down-regulate the expression of PrxⅠ, then we observe the radiosensitivity changes of breast cancer cells MCF-7 cells and investigate its related mechanisms. The purpose of this study is to clarity the role of PrxⅠgene in radiotherapy and provide a new target for gene therapy of breast cancer.
Methods and results:
Part one Construction and identification of shRNA eukaryotic expression vector targeting PrxⅠgene
Methods:According to the principle of RNA interference construction, we designed and synthesized four shRNA sequences targeting PrxⅠand one sequence for negative control. First the shRNA connected to linear pGPU6/GFP/Neo plasmid vector which was digested by BbsⅠand BamHⅠrestriction enzyme. then the recombination plasmids were identified by BamHⅠor PstⅠdigestion and DNA sequencing after transformation, screening,amplifing and extracting,and transfected into breast carcinoma MCF-7 cells by lipofectamine respectively. Transfection efficiency was detected by Fluorescence microscopy and Flow Cytometry, the mRNA and protein expression levels of PrxⅠwere evaluated by RT-PCR and western blot.
Results : we successfully constructed four eukaryotic expression vectors targeting PrxⅠgene:pGPU6-Prx1、pGPU6-Prx2、pGPU6-Prx3、pGPU6-Prx4 and negative expression vectors pGPU6-HK which were successfully transfected into MCF-7 cells by Lipofectamine. Transfection efficiencies were about 80%. The mRNA and protein expressions of PrxⅠin pGPU6-Prx1、pGPU6-Prx2、pGPU6-Prx3 and pGPU6-Prx4 were significantly inhibited compared with control group and pGPU6-HK group(P<0.05). The inhibition rate of pGPU6-Prx3 at mRNA and protein levels were 82.6% and 80.5% respectively. So we chose it for experimental study.
Part two Effects and mechanisms of silencing PrxⅠgene by RNA interference on the radiosensitivity of breast carcinoma cell line MCF-7
Methods:The pGPU6-HK and pGPU6-Prx3 recombinant plasmids were transfected into breast carcinoma cell line MCF-7 by Lipofectamine respectively. Two stable cell lines were establish by G418 screening which were named as pGPU6-HK and pGPU6-PrxⅠrespectively. The mRNA and protein expression of PrxⅠwere detected by RT-PCR and Western blot. Four groups were divided in the experiment: pGPU6-HK group, pGPU6-PrxⅠgroup, pGPU6-HK+IR group and pGPU6-PrxⅠ+IR group. IR groups were irradiated with a single dose 6Gy X-ray. Intracellular reactive oxygen species, cell cycle and cell apoptosis of each group at 48 hour after irradiation were detected by Flow Cytometry. Cell proliferation was disclosed MTT assay. Morphological changes of cell nucleus were observed by hoechst assay to indirectly measure cell apoptosis. Colony efficiency was discovered by colony formation assay after 0、2、4、6、8、10Gy irradiation,cell survival curves were drawn and the values of radiobiological parameters Do, N, Dq and SF2 were calculated to assess cellular radiosensitivity; A landmark proteinγ-H2AX of DNA double-strand breaks and a key protein Rad51 of DNA damage repair were evaluated by Western blot assay.
Results:Two stable cell lines(pGPU6-PrxⅠand pGPU6-HK)were obtained by G418 pressure screening after four weeks. The mRNA and protein expression of pGPU6-PrxⅠwere distinctly decreased compared with pGPU6-HK group(P﹤0.05). The inhibition rates of the mRNA and protein expression were 84.8% and 83.5% respectively. These results of Flow Cytometry, MTT and Hoechst assays demonstrated that ROS levels markedly raised, the G1 phase cell increased,S phase cells decreased,cell apoptosis rate significantly increased,cell proliferation obviously delayed, apoptotic cell which appeared typical apoptotic morphological changes including nuclear condensed and fragmentation also enhanced at 48h after 6Gy irradiation in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK group(P﹤0.05).These changes in pGPU6-PrxⅠ+IR group were more obvious compared with pGPU6-PrxⅠand pGPU6-HK+IR(P﹤0.05).Colony formation assay indicated that colony formation rate reduced after irradiation with different doses and the values of radiobiological parameters Do、N、Dq and SF2 of the pGPU-PrxⅠgroup,were 1.354、3.106、1.547 and 0.504 respectively, lower than the pGPU-HK group. The SER which derived from SF2 was 1.353. The protein expression of Rad51 was distinctly inhibited,while the protein expression ofγ-H2AX was markedly enhanced in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK(P<0.01).These changes of pGPU-PrxⅠ+ IR group were more obvious in which the protein expression of Rad51 decreased 79.3% and the protein expression ofγ-H2AX increased 3.7 times compared with pGPU6-HK. The significant differences were also observed between pGPU-PrxⅠ+IR group and pGPU6-PrxⅠor pGPU6-HK+IR(P﹤0.05).
Part three Effects of silencing PrxⅠgene by RNA interference on the radiosensitivity of nude mice xenografts of breast carcinoma cell line MCF-7
Methods:Two stable cells of pGPU6-HK and pGPU6-PrxⅠwere implanted subcutaneously into BALB/c nude mice to establish xenograft model of breast cancer. It was judged as tumorigenesis when the diameter of tumor reached 5mm. Four groups were divided in the experiment: pGPU6-HK group, pGPU6-PrxⅠgroup, pGPU6-HK+IR group and pGPU6-PrxⅠ+IR group. IR groups were irradiated with a total dose 10Gy. Tumor growth was regularly monitored. All BALB/c nude mice were sacrificed at the thirty day after irradiation and and tumor tissues were stripped, weighted. The inhibitory rate of tumor weight was calculated. The protein expressions of PrxⅠand caspase3 were detected by immunohistochemistry. Ultramicrostructure of tumor cell was observed by electron microscopy. The expressions of a landmark proteinγ-H2AX of DNA double-strand breaks and a key protein Rad51 of DNA damage repair were analyzed by western blot.
Results:Xenograft model in nude mice were successfully established. The time of tumor formation in pGPU6-HK group and pGPU6-PrxⅠgroup was ten days and fourteen days respectively Compared with pGPU6-HK group, tumor volume and weight were smaller and lighter in pGPU6-PrxⅠgroup and inhibition rate of tumor weight was 37.8%(P﹤0.01). Tumor volume of combined with IR emerged negative growth lessened after 10Gy irradiation while showed positive growth at the third day after irradiation end which were significantly slower than pGPU6-HK and pGPU6-PrxⅠ,especially pGPU6-PrxⅠ+IR group. The inhibition rate reached 79.76% that were significant differences compared with pGPU6-PrxⅠ(34.92%) and pGPU6-HK+IR(56.94%)(P<0.05). Immunohistochemistry and electron microscopy illustrated that the expression of PrxⅠprotein significantly down-regulated,whereas the expression of Caspase3 protein markedly up-regulated,cell apoptosis and necrosis increased,the protein expression of Rad51 distinctly decreased, while the protein expression ofγ-H2AX was markedly increased in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK(P<0.01). These changes of pGPU-PrxⅠ+IR group were more obvious compared with pGPU6-PrxⅠgroup and pGPU6-HK+IR group(P < 0.01). The protein expression of Rad51 decreased 84.8% and the protein expression ofγ-H2AX increased 5.6 folds in pGPU-PrxⅠ+ IR group compared with pGPU6-HK.
Conclusion:The shRNA eukaryotic expression vector targeting PrxⅠgene were successfully constructed and xenograft model of breast cancer was successfully established. Silencing PrxⅠgene by RNA interference could significantly enhance the radiosensitivity of breast cancer MCF-7 cells in vitro and vivo through inhibiting cell proliferation,promoting cell apoptosis, regulating cell phase redistribution, improving sensitive enhancement ratio(SER) and its mechanisms might be related to reducing the capacity of scavenging ROS,increasing DNA double-strand breaks and decreasing DNA damage repair capacity. PrxⅠgene showed a negative correlation with the radiosensitivity of breast carcinoma cell line MCF-7 and might act as a molecular target for radionsensitization of breast carcinoma.
放射治疗是恶性肿瘤治疗的主要手段之一,而肿瘤细胞的放射敏感性是决定放射治疗疗效的关键因素。近年来,随着对肿瘤放射生物学的深入研究,发现一些基因表达影响着肿瘤细胞的放射敏感性。基因治疗策略可将肿瘤细胞中特异或差异表达的基因敲除,实现特定基因在体内的可控性表达,逆转肿瘤细胞对放化疗的抗拒,从而增加其治疗的敏感性。
Peroxiredoxins(Prxs)是新发现的一类过氧化物酶,在清除体内活性氧中发挥着重要作用,可保护细胞免受过氧化反应造成的损伤。PeroxiredoxinⅠ(PrxⅠ)是Prxs蛋白家族成员之一,在包括乳腺癌在内的多种恶性肿瘤细胞中存在过表达,其表达与肿瘤细胞的增殖、分化、转移、复发及放化疗敏感性密切相关,并可作为诊断肿瘤的分子标志。因此,PrxⅠ可能是肿瘤基因治疗的理想靶点。
DNA双链断裂(Double strand break,DSB)是电离辐射治疗肿瘤的重要机制。DNA损伤后可激活机体内的损伤修复系统,使受损的DNA得以及时修复,以保证机体基因组的稳定。研究发现:DNA损伤感应分子γ-H2AX和DNA损伤修复的关键蛋白Rad51可能在其发挥重要作用。
本研究将基因治疗和放射治疗结合起来,以PrxⅠ为靶点,构建针对PrxⅠ特异性的shRNA真核表达载体,下调乳腺癌MCF-7细胞中PrxⅠ的表达,观察其对放射线的敏感性变化,并探讨相关作用机制,阐明PrxⅠ基因在乳腺癌放射治疗中的作用,为乳腺癌基因治疗提供新的靶位。
方法与结果
第一部分:靶向PeroxiredoxinⅠ基因的shRNA真核表达载体构建及功能鉴定
方法:根据RNA干扰设计原则,设计并合成四条靶向PrxⅠ基因的shRNA干扰序列及阴性对照序列(HK)。先用BbsⅠ及BamHⅠ限制性内切酶双酶切pGPU6/GFP/Neo质粒载体使之线性化,然后通过DNA连接酶将shRNA连接至质粒载体中,经转化、筛选、阳性克隆扩增、抽提重组质粒DNA后,行BamHⅠ、PstⅠ单酶切和DNA测序鉴定;将鉴定正确重组质粒通过脂质体转染入乳腺癌MCF-7细胞。荧光显微镜、流式细胞仪观察并检测转染48h时转染效率;采用RT-PCR及Western blot检测转染前后细胞中的PrxⅠmRNA和蛋白表达变化。
结果:成功构建了四个靶向PrxⅠ基因的shRNA真核表达载体pGPU6-Prx1、pGPU6-Prx2、pGPU6-Prx3、pGPU6-Prx4及阴性表达载体pGPU6-HK,通过脂质体将上述五个重组质粒成功转染入乳腺癌MCF-7细胞中,48h后检测发现各组细胞转染效率均在80%左右。RT-PCR及Western blot检测发现:四个重组质粒均能明显下调乳腺癌细胞MCF-7中的PrxⅠ基因表达,其中pGPU6-Prx3重组质粒的抑制效果最明显,其mRNA及蛋白表达分别抑制了82.6%和80.5%,与对照组和pGPU6-HK组比较差异具有显著性(P<0.05)。成功筛选出抑制效果最佳的一条shRNA,为后续PrxⅠ基因的功能研究奠定了基础。
第二部分:靶向沉默PeroxiredoxinⅠ基因对乳腺癌细胞放射敏感性的影响及其作用机制
方法:将pGPU6-HK、pGPU6-Prx3重组质粒通过脂质体转染入乳腺癌细胞中,通过G418加压筛选建立稳定细胞株,采用RT-PCR、Western blot检测PrxⅠ基因的mRNA和蛋白表达水平。实验分四组:pGPU6-HK组、pGPU6-PrxⅠ组、pGPU6-HK+照射(IR)组、pGPU6-PrxⅠ+IR组。IR组给予单次剂量6Gy的X线照射,在照射48h时通过流式细胞仪检测细胞内活性氧水平、细胞周期、细胞凋亡的变化;MTT法检测细胞增殖能力;Hoechst法观察细胞核形态变化,间接判定细胞凋亡情况;克隆形成实验检测经0、2、4、6、8、10Gy剂量照射后细胞克隆形成率,绘制细胞存活曲线,计算放射生物学参数Do、N、Dq、SF2值,判断细胞的放射敏感性变化;采用Western blot检测DNA双链断裂的标志性蛋白γ-H2AX以及DNA损伤后修复的关键蛋白Rad51表达。
结果:通过G418加压筛选4周后获取了2个稳定细胞株:pGPU6-PrxⅠ和pGPU6-HK。RT-PCR和Western bolt检测发现:与pGPU6-HK组相比,pGPU6-PrxⅠ组中PrxⅠmRNA和蛋白表达量明显减少,抑制率分别为84.8%和83.5%,差异具有显著性(P﹤0.05)。MTT、流式细胞及Hoechst检测结果显示:给予6Gy的X射线照射48h后,pGPU6-PrxⅠ组及联合IR组中细胞内活性氧水平明显升高、G1期增多、S期细胞减少、细胞凋亡显著增加、细胞增殖明显延迟、核浓缩、碎片化等凋亡典型形态改变的细胞增多,与pGPU6-HK比较,差异均具有显著性(P﹤0.05);其中以pGPU6-PrxⅠ+IR组上述变化最明显,与pGPU6-PrxⅠ及pGPU6-HK+IR比较,差异也具有显著性(P﹤0.05)。克隆形成实验结果显示:经不同放射剂量的X线照射后,pGPU6-PrxⅠ组及联合IR组中细胞克隆形成率降低,其中pGPU6-PrxⅠ+IR组细胞的放射生物学参数Do、N、Dq、SF2值分别为1.354、3.106、1.547、0.504,2Gy照射剂量时SER为1.353;Western blot检测结果显示:pGPU6-PrxⅠ组及联合IR组中Rad51蛋白表达明显降低,而γ-H2AX蛋白表达显著升高,与pGPU6-HK组相比,差异具有显著性(P﹤0.01),以pGPU6-PrxⅠ+IR组中更明显,其Rad51蛋白表达下降了79.3%,而γ-H2AX蛋白表达升高了3.7倍,与pGPU6-PrxⅠ组、pGPU6-HK+IR组相比,差异也具有显著性(P﹤0.05)。
第三部分:靶向沉默PeroxiredoxinⅠ基因对乳腺癌裸鼠移植瘤放射敏感性的影响
方法:将稳定筛选的pGPU6-HK、pGPU6-PrxⅠ两株细胞接种于BALB/c裸鼠皮下,以肿瘤长径达5mm作为判断乳腺癌移植瘤模型建立成功标准。实验分四组:pGPU6-HK组、pGPU6-PrxⅠ组、pGPU6-HK+照射(IR)组、pGPU6-PrxⅠ+IR组。IR组给予每次2Gy,隔日一次,总剂量为10Gy,观察移植瘤生长变化,照射第30d处死裸鼠,剥离肿瘤组织,称取瘤重,计算抑瘤率;免疫组化检测肿瘤组织中的PrxⅠ和促凋亡蛋白Caspase3表达;透射电镜观察肿瘤组织细胞的超微结构;Western blot检测肿瘤组织中DNA双链断裂的标志性蛋白γ-H2AX以及DNA损伤后修复的关键蛋白Rad51表达。
结果:成功建立了裸鼠移植瘤模型,pGPU6-HK组及pGPU6-PrxⅠ组细胞成瘤时间分别为10d和14d,pGPU6-PrxⅠ组移植瘤生长相对较慢,肿瘤体积较小,重量较轻,其抑瘤率为37.8%,与pGPU6-HK组相比,差异具有显著性(P﹤0.01)。当给予10Gy的X线剂量照射后,联合IR组裸鼠移植瘤消退速度大于生长速度,肿瘤体积缩小,但在照射结束后第三天肿瘤出现正生长,其生长速度较pGPU6-HK、pGPU6-PrxⅠ组明显缓慢,以pGPU6-PrxⅠ+IR组更为明显,其抑瘤率达到了79.76%,与pGPU6-PrxⅠ(34.92%)、pGPU6-HK+IR(56.94%)比较差异具有显著性(P<0.05)。免疫组化结果及电镜结果显示:pGPU6-PrxⅠ及联合IR组肿瘤细胞中PrxⅠ蛋白表达明显下调,而促凋亡蛋白Caspase3表达显著提高,凋亡、坏死细胞增多。Western bolt结果显示:pGPU6-PrxⅠ及联合IR组肿瘤细胞中的Rad51蛋白表达降低,而γ-H2AX蛋白表达升高,与pGPU6-HK组相比,差异具有显著性(P<0.01)。上述变化pGPU6-PrxⅠ+IR组最为明显,其Rad51蛋白表达抑制了84.8%,γ-H2AX蛋白表达升高5.6倍,与pGPU6-PrxⅠ和pGPU6-HK+IR组相比,差异也具有显著性(P<0.05)。
结论:本研究成功构建了靶向PrxⅠ基因shRNA真核表达载体,从体外、体内证实了靶向沉默PrxⅠ基因后,可有效抑制细胞增殖、调控细胞周期再分布、促进细胞凋亡、降低细胞亚致死损伤修复能力,提高放射治疗增益比,从而增加了乳腺癌MCF-7细胞的放射敏感性,其作用机制可能与细胞内活性氧清除能力减弱、DNA双链断裂增加及DNA损伤后修复能力降低有关。PrxⅠ基因表达与乳腺癌细胞的放射敏感性呈负相关,可作为乳腺癌放射增敏的分子靶点。
Breast cancer is the most common malignancy of women. About 120 million new cases are diagnosed and 50 million women die of breast cancer each year around the world. Despite the comprehensive treatment of breast cancer has made great progress, there are still 20% to 30% of breast cancer patients occur local recurrence and distant metastasis after treatment Therefore, it is the breakthrough to explore new therapeutic tools for further improving the curative effect of breast cancer.
Radiotherapy is one of the major means of treating malignant tumors, however, the radiosensitivity of tumor cells is a key factor to determine radiotherapy effect. In recent years, with thorough study in the radiation biology of the cancer, researchers have found that the expression of some genes at the molecular level affected the radiosensitivity of tumor cells. Gene therapy strategy enable us to knockout these genes that are specific or differential expression between tumor cells and normal cells, and achieve controlled expression of special gene in vivo so as to reverse resistance to the treatment of tumor cell and increase their therapeutic responsiveness.
Peroxiredoxins (Prxs) are a newly discovered peroxidase that play an important role in scavenging reactive oxygen species of the body and protect cells from damaging by overoxidized. PeroxiredoxinⅠ(PrxⅠ),a member of the Prxs family, which is overexpressed in many cancers including breast cancer cells. PrxⅠhave been implicated in regulating many cellular processes such as cell proliferation, differentiation and apoptosis, moreover, the levels of its expression is closely related to the metastasis, recurrence, chemosensitivity and radiosensitivity of tumor cells,and can serve as a molecular marker for tumor diagnosis. Thus, PrxⅠmay be an ideal target for gene therapy.
DNA strand break is an important mechanism which ionizing radiation treat tumor. DNA damage can activate the repair system of body and can be repaired in time to ensure the stability of organism genome. Researches have found thatγ-H2AX and Rad51 proteins may play an important role in DNA damage repair.
We combine gene therapy with radiotherapy in this study: First, we construct specific shRNA eukaryotic expression vector targeting PrxⅠgene in order to down-regulate the expression of PrxⅠ, then we observe the radiosensitivity changes of breast cancer cells MCF-7 cells and investigate its related mechanisms. The purpose of this study is to clarity the role of PrxⅠgene in radiotherapy and provide a new target for gene therapy of breast cancer.
Methods and results:
Part one Construction and identification of shRNA eukaryotic expression vector targeting PrxⅠgene
Methods:According to the principle of RNA interference construction, we designed and synthesized four shRNA sequences targeting PrxⅠand one sequence for negative control. First the shRNA connected to linear pGPU6/GFP/Neo plasmid vector which was digested by BbsⅠand BamHⅠrestriction enzyme. then the recombination plasmids were identified by BamHⅠor PstⅠdigestion and DNA sequencing after transformation, screening,amplifing and extracting,and transfected into breast carcinoma MCF-7 cells by lipofectamine respectively. Transfection efficiency was detected by Fluorescence microscopy and Flow Cytometry, the mRNA and protein expression levels of PrxⅠwere evaluated by RT-PCR and western blot.
Results : we successfully constructed four eukaryotic expression vectors targeting PrxⅠgene:pGPU6-Prx1、pGPU6-Prx2、pGPU6-Prx3、pGPU6-Prx4 and negative expression vectors pGPU6-HK which were successfully transfected into MCF-7 cells by Lipofectamine. Transfection efficiencies were about 80%. The mRNA and protein expressions of PrxⅠin pGPU6-Prx1、pGPU6-Prx2、pGPU6-Prx3 and pGPU6-Prx4 were significantly inhibited compared with control group and pGPU6-HK group(P<0.05). The inhibition rate of pGPU6-Prx3 at mRNA and protein levels were 82.6% and 80.5% respectively. So we chose it for experimental study.
Part two Effects and mechanisms of silencing PrxⅠgene by RNA interference on the radiosensitivity of breast carcinoma cell line MCF-7
Methods:The pGPU6-HK and pGPU6-Prx3 recombinant plasmids were transfected into breast carcinoma cell line MCF-7 by Lipofectamine respectively. Two stable cell lines were establish by G418 screening which were named as pGPU6-HK and pGPU6-PrxⅠrespectively. The mRNA and protein expression of PrxⅠwere detected by RT-PCR and Western blot. Four groups were divided in the experiment: pGPU6-HK group, pGPU6-PrxⅠgroup, pGPU6-HK+IR group and pGPU6-PrxⅠ+IR group. IR groups were irradiated with a single dose 6Gy X-ray. Intracellular reactive oxygen species, cell cycle and cell apoptosis of each group at 48 hour after irradiation were detected by Flow Cytometry. Cell proliferation was disclosed MTT assay. Morphological changes of cell nucleus were observed by hoechst assay to indirectly measure cell apoptosis. Colony efficiency was discovered by colony formation assay after 0、2、4、6、8、10Gy irradiation,cell survival curves were drawn and the values of radiobiological parameters Do, N, Dq and SF2 were calculated to assess cellular radiosensitivity; A landmark proteinγ-H2AX of DNA double-strand breaks and a key protein Rad51 of DNA damage repair were evaluated by Western blot assay.
Results:Two stable cell lines(pGPU6-PrxⅠand pGPU6-HK)were obtained by G418 pressure screening after four weeks. The mRNA and protein expression of pGPU6-PrxⅠwere distinctly decreased compared with pGPU6-HK group(P﹤0.05). The inhibition rates of the mRNA and protein expression were 84.8% and 83.5% respectively. These results of Flow Cytometry, MTT and Hoechst assays demonstrated that ROS levels markedly raised, the G1 phase cell increased,S phase cells decreased,cell apoptosis rate significantly increased,cell proliferation obviously delayed, apoptotic cell which appeared typical apoptotic morphological changes including nuclear condensed and fragmentation also enhanced at 48h after 6Gy irradiation in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK group(P﹤0.05).These changes in pGPU6-PrxⅠ+IR group were more obvious compared with pGPU6-PrxⅠand pGPU6-HK+IR(P﹤0.05).Colony formation assay indicated that colony formation rate reduced after irradiation with different doses and the values of radiobiological parameters Do、N、Dq and SF2 of the pGPU-PrxⅠgroup,were 1.354、3.106、1.547 and 0.504 respectively, lower than the pGPU-HK group. The SER which derived from SF2 was 1.353. The protein expression of Rad51 was distinctly inhibited,while the protein expression ofγ-H2AX was markedly enhanced in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK(P<0.01).These changes of pGPU-PrxⅠ+ IR group were more obvious in which the protein expression of Rad51 decreased 79.3% and the protein expression ofγ-H2AX increased 3.7 times compared with pGPU6-HK. The significant differences were also observed between pGPU-PrxⅠ+IR group and pGPU6-PrxⅠor pGPU6-HK+IR(P﹤0.05).
Part three Effects of silencing PrxⅠgene by RNA interference on the radiosensitivity of nude mice xenografts of breast carcinoma cell line MCF-7
Methods:Two stable cells of pGPU6-HK and pGPU6-PrxⅠwere implanted subcutaneously into BALB/c nude mice to establish xenograft model of breast cancer. It was judged as tumorigenesis when the diameter of tumor reached 5mm. Four groups were divided in the experiment: pGPU6-HK group, pGPU6-PrxⅠgroup, pGPU6-HK+IR group and pGPU6-PrxⅠ+IR group. IR groups were irradiated with a total dose 10Gy. Tumor growth was regularly monitored. All BALB/c nude mice were sacrificed at the thirty day after irradiation and and tumor tissues were stripped, weighted. The inhibitory rate of tumor weight was calculated. The protein expressions of PrxⅠand caspase3 were detected by immunohistochemistry. Ultramicrostructure of tumor cell was observed by electron microscopy. The expressions of a landmark proteinγ-H2AX of DNA double-strand breaks and a key protein Rad51 of DNA damage repair were analyzed by western blot.
Results:Xenograft model in nude mice were successfully established. The time of tumor formation in pGPU6-HK group and pGPU6-PrxⅠgroup was ten days and fourteen days respectively Compared with pGPU6-HK group, tumor volume and weight were smaller and lighter in pGPU6-PrxⅠgroup and inhibition rate of tumor weight was 37.8%(P﹤0.01). Tumor volume of combined with IR emerged negative growth lessened after 10Gy irradiation while showed positive growth at the third day after irradiation end which were significantly slower than pGPU6-HK and pGPU6-PrxⅠ,especially pGPU6-PrxⅠ+IR group. The inhibition rate reached 79.76% that were significant differences compared with pGPU6-PrxⅠ(34.92%) and pGPU6-HK+IR(56.94%)(P<0.05). Immunohistochemistry and electron microscopy illustrated that the expression of PrxⅠprotein significantly down-regulated,whereas the expression of Caspase3 protein markedly up-regulated,cell apoptosis and necrosis increased,the protein expression of Rad51 distinctly decreased, while the protein expression ofγ-H2AX was markedly increased in pGPU6-PrxⅠand combined with IR groups compared with pGPU6-HK(P<0.01). These changes of pGPU-PrxⅠ+IR group were more obvious compared with pGPU6-PrxⅠgroup and pGPU6-HK+IR group(P < 0.01). The protein expression of Rad51 decreased 84.8% and the protein expression ofγ-H2AX increased 5.6 folds in pGPU-PrxⅠ+ IR group compared with pGPU6-HK.
Conclusion:The shRNA eukaryotic expression vector targeting PrxⅠgene were successfully constructed and xenograft model of breast cancer was successfully established. Silencing PrxⅠgene by RNA interference could significantly enhance the radiosensitivity of breast cancer MCF-7 cells in vitro and vivo through inhibiting cell proliferation,promoting cell apoptosis, regulating cell phase redistribution, improving sensitive enhancement ratio(SER) and its mechanisms might be related to reducing the capacity of scavenging ROS,increasing DNA double-strand breaks and decreasing DNA damage repair capacity. PrxⅠgene showed a negative correlation with the radiosensitivity of breast carcinoma cell line MCF-7 and might act as a molecular target for radionsensitization of breast carcinoma.
引文
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[6] Wu XY, Fu ZX, Wang XH. Peroxiredoxins in colorectal neoplasms[J]. Histol Histopathol. 2010,25(10):1297-1303.
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