沉默Ku-70基因对耐药性乳腺癌的治疗价值研究
摘要
乳腺癌是危害妇女健康的主要恶性肿瘤，在全球范围内，每年超过100万妇女被诊断患有乳腺癌，占癌症的十分之一，占女性癌症病例的23％[1]。在美国，该疾病每年死亡人数为40,000人[2]。这已成为目前降低患者生存率的主要因素。解决肿瘤的多药耐药问题对多种肿瘤的治疗具有重要意义，因此，从事肿瘤治疗研究的学者们希望更为透彻的了解恶性肿瘤的多药耐药的原因及机制，为肿瘤的治疗提供新的靶点。
Ku蛋白是一个异二聚体蛋白，由Ku70和Ku80两个亚单位组成。最早在自身免疫性疾病患者血清中被发现、鉴定，后来被证明是一个多功能的调节蛋白，参与DNA修复、端粒的维持、细胞凋亡。因此，Ku蛋白被认为在染色体的完整性及细胞存活中发挥重要的作用。近年来的报道提示Ku蛋白的表达与肿瘤的发展有关[3]，该蛋白的高表达促进肿瘤表型的发生、诱导细胞高度增殖及凋亡抵抗，而该蛋白低表达则导致基因组的不稳定及肿瘤的发生，多种实验的结论指出Ku蛋白同时扮演着肿瘤抑制因子及肿瘤发生因子的双重角色，这使Ku成为抗肿瘤药物研究的一个重要的候选靶点，引起众多肿瘤治疗研究人员的关注。下调Ku70蛋白的表达促进宫颈癌细胞Hela对化疗的敏感性[4]；Pim-1基因敲除通过下调Ku蛋白的表达及核定位，抑制Ku70，Ku80对非同源末端连接DNA修复，增强紫杉醇诱导激素难治性前列腺癌凋亡[5]。双调蛋白通过抑制吉西他滨诱导Ku70蛋白的乙酰化促进非小细胞肺癌对吉西他滨的抵抗[6]。上述材料提示，Ku70在多种肿瘤化疗耐药的治疗中扮演重要的角色。但是Ku70蛋白在乳腺癌中的表达及与乳腺癌耐药的相关性还没有报道。
本实验的目的就是为了揭示Ku70蛋白在乳腺癌中的表达及其在乳腺癌耐药治疗中的作用，具体研究如下：
1实验路线
1.1耐表阿霉素乳腺癌细胞系MCF7-ADR细胞耐药性检测
1.1.1MTS法检测MCF7、MCF7-ADR增殖活性差异。
1.1.2倒置相差显微镜观察MCF7、MCF7-ADR细胞形态差异。
1.1.3不同浓度表阿霉素刺激MCF7、MCF7-ADR细胞，计算药物半数抑制率（IC50）及MCF7-ADR对表阿霉素耐药指数。
1.2RT-PCR检测乳腺癌细胞系MCF7及耐表阿霉素乳腺癌细胞系MCF7-ADR细胞Ku-70mRNA水平。
培养人乳腺癌细胞系MCF7、耐表阿霉素乳腺癌细胞系MCF7-ADR细胞至对数生长期，收细胞，提取总RNA，通过RT-PCR检测两种肿瘤细胞Ku-70mRNA水平。
1.3Western blot检测MCF7、MCF7-ADR细胞Ku-70蛋白表达水平
培养人乳腺癌细胞系MCF7、耐表阿霉素乳腺癌细胞系MCF7-ADR细胞至对数生长期，收细胞，提蛋白，垂直电泳后转膜，抗Ku70单克隆抗体杂交，ECL化学发光、成像系统成像，检测MCF7、MCF7-ADR细胞Ku70蛋白表达水平。
1.4向MCF7-ADR细胞中转染Ku70SiRNA片段，RT-PCR、Western blot检测Ku70基因沉默效果。
1.5不同浓度表阿霉素刺激MCF7、MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞， MTS法检测上述细胞增殖活性差异，计算表阿霉素对不同细胞的半数抑制量，比较Ku70沉默前后MCF7-ADR耐药指数的变化。
1.6普通光学显微镜、Hochest-33258染色观察表阿霉素诱导MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞凋亡时细胞形态、细胞核形态差异。
1.7Annexin V-PI染色、流式细胞术检测表阿霉素诱导MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞凋亡能力差异。
1.8PCR-array检测Ku70基因沉默前后MCF7-ADR细胞凋亡相关基因变化，探讨Ku70基因沉默逆转MCF7-ADR耐药性的可能机制。
2结果
2.1耐表阿霉素乳腺癌细胞系MCF7-ADR细胞耐药性检测
MTS法检测MCF7、MCF7-ADR增殖活性差异，结果显示，两种细胞增殖活性无显著差异，MCF7、MCF7-ADR细胞大小、形态相似，近圆形，细胞核较大，胞浆丰富。采用不同浓度表阿霉素刺激MCF7、MCF7-ADR细胞，表阿霉素对二者的半数抑制量（IC50）分别为4.16+0.72ug/ml和34.38+6.75ug/ml，MCF7-ADR细胞对表阿霉素的耐药指数为8.26。
2.2MCF7、MCF7-ADR细胞Ku-70mRNA表达水平差异
RT-PCR检测乳腺癌细胞系MCF7及耐表阿霉素乳腺癌细胞系MCF7-ADR细胞Ku-70mRNA水平，结果显示，二者Ku-70mRNA表达水平无明显差异。
2.3MCF7、MCF7-ADR细胞Ku-70蛋白表达水平差异
Westernblot检测MCF7、MCF7-ADR细胞Ku-70蛋白表达水平，结果显示，二者Ku-70蛋白表达水平亦无明显差异。
2.4MCF7-ADR细胞转染Ku70SiRNA片段效果检测
RT-PCR、Westernblot检测转染Ku70SiRNA片段24h后MCF7-ADR细胞Ku-70mRNA及蛋白表达水平变化，结果显示，转染Ku70SiRNA片段24h后MCF7-ADR细胞Ku-70mRNA及蛋白水平较非特异SiRNA片段转染组显著降低，说明Ku-70基因沉默效果良好，可以进行后续试验。
2.5Ku70沉默对MCF7-ADR耐药性的影响
MTS法检测不同浓度表阿霉素作用后MCF7、MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞增殖活性，结果显示，表阿霉素对3组细胞的半数抑制量分别为4.16+0.72ug/ml、34.38+6.75ug/ml、13.06+2.62ug/ml。MCF7-ADR细胞对表阿霉素的耐药指数由Ku70沉默前的8.26下降为Ku70沉默后的3.14，提示沉默Ku70能够逆转MCF7-ADR对表阿霉素的耐药性。
2.6形态学比较Ku70沉默前后MCF7-ADR对表阿霉素诱导凋亡敏感性变化
普通光学显微镜观察10ug/ml表阿霉素处理48h后MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞形态差异，结果显示Ku70沉默组细胞数量较空白对照组、阴性对照组显著减少，细胞体积缩小，边缘皱缩，折光度差，部分细胞呈圆球形漂浮于培养液中；Hochest-33258染色后蓝色激发光下观察细胞核，可见无药物处理组MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞核大小无明显差异，均呈长椭圆形，染色质疏松、均匀一致蓝染；MCF7-ADR细胞经表阿霉素作用48h后较无药物处理组细胞核体积缩小，染色质浓集呈亮蓝色，MCF7-ADR/Ku70-SiRNA经表阿霉素作用48h后较无药物处理组细胞核体积明显缩小、染色质固缩呈亮蓝色团块，部分细胞可见明显的凋亡小体。
2.7流式细胞术比较Ku70沉默前后MCF7-ADR对表阿霉素诱导凋亡敏感性变化
Annexin V-PI染色、流式细胞术检测10ug/ml表阿霉素处理24h后MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞凋亡水平差异，结果表明，Ku70沉默后MCF7-ADR对表阿霉素诱导凋亡敏感性较沉默前明显增加，经10ug/ml表阿霉素处理后空白对照组、阴性对照组、Ku70沉默组AnnexinⅤ阳性染色比例依次为4.72+0.56%、4.63+0.78%，16.76+2.93%。
2.8Ku70基因沉默后MCF7-ADR细胞凋亡相关基因APAF1、BBC3、BCL2、BCL2、BIRC6、CASP3、DAPK1、TNFRSF10A表达增加，NAIP、CHUK基因表达减少。
3结论
3.1MCF7-ADR细胞与MCF7细胞相比，其Ku70mRNA及蛋白水平均无明显变化，Ku70不能成为乳腺癌耐药标志物。
3.2Ku70基因沉默能够逆转MCF7-ADR细胞对表阿霉素的耐药性。
3.3Ku70基因沉默能够增强表阿霉素诱导MCF7-ADR细胞凋亡能力，提示Ku70-SiRNA能够作为乳腺癌化疗的增敏剂。
3.4沉默MCF7-ADR细胞Ku70基因后，凋亡相关基因APAF1、BBC3、BCL2、BIRC6、CASP3、DAPK1、TNFRSF10A表达增加，NAIP、CHUK基因表达减少，提示Ku70基因沉默后大部分凋亡信号分子表达增加，说明Ku70基因沉默活化耐表阿霉素乳腺癌细胞中的凋亡信号，从而增加其对表阿霉素的敏感性，逆转其耐药性。
Breast cancer is the major malignant tumour that endangers the health of women,in worldwide, more than100million women are diagnosed with breast cancer,accounting for10%of the patients with cancer, accounting for23%of the femalecancer cases every year[1]. In the United States,40,000people died of breast cancerone year[2]. Breast cancer is one of the most sensitive tumor to chemotherapy in solidtumors, but tumor multidrug resistance (MDR) often leads to the failure ofchemotherapy, which has become a major factor for the lower survival rate ofpatients. Solve the MDR problem of the tumor is important for the treatment of avariety of tumors, therefore, scholars hope to understand more of cancer causes andmechanisms of multidrug resistance in tumor therapy, to provide a new target forcancer treatment.
Ku protein is a heterodimeric protein, composed by Ku70and Ku80two-subunit,and was found and identified earliest in the serum of patients with autoimmunediseases, later proved to be a versatile regulatory protein involved in DNA repair,telomere maintenance, apoptosis. Thus, Ku protein is considered to play an importantrole in the chromosomal integrity and cell survival.
Reports in recent years suggested that the Ku protein expression was associatedwith tumor development[3], the more expression of Ku protein promoted tumorphenotype occurred, induced cell proliferation and apoptosis resistance, on the otherhand, less expression of Ku protein leaded to genomic instability and the occurrenceof tumors, a variety of experiments concluded that Ku protein also plays dual role-atumor suppressor and tumor factors. Ku becomes an important anticancer drugcandidate targets, causing attentions of many cancer treatment researchers. rapysensitivity of cervical carcinoma cell line, Hela[4]; Pim-1gene knockout enhanced
Down-regulating Ku70protein expression could promote chemothepaclitaxelinduced hormone-refractory prostate cancer apoptosis by down-regulating the expression and nuclear localization of Ku protein, inhibiting of Ku70, Ku80pairof non-homologous end-joining DNA repair[5]. Amphiregulin promoted non-smallcell lung cancer resistante to gemcitabine through inhibitiing gemcitabine-inducedacetylation of Ku70protein[6]. The materials above prompts, Ku70play an importantrole in a variety of tumor resistance to chemotherapy treatment. Ku70proteinexpression in breast cancer and correlations with breast cancer resistance has not beenreported.
The purpose of this experiment is to reveal the Ku70protein expression in breastcancer and its role in the treatment of breast cancer resistance, details are as follows:
1The experimental route
1.1Drug resistance test of MCF7-ADR–a breast cancer cell line whichresistant epirubicin.
1.1.1MTS assay detected MCF7, MCF7-ADR proliferative activity.
1.1.2Observed MCF7, MCF7-ADR cell morphological differences via invertedphase contrast microscope.
1.1.3Stimulated MCF7, MCF7-ADR cells with different concentrations ofepirubicin and calculated the drugs half fatality rate (IC50) and drug resistance indexof MCF7-ADR to pharmorubicin.
1.2RT-PCR detected Ku-70mRNA levels in breast cancer cell line MCF7andMCF7-ADR cells
Cultured human breast cancer cell line MCF7and breast cancer cell lineMCF7-ADR which resistance to epirubicin to logarithmic phase, extracted total RNA,then detected Ku-70mRNA of the two tumor cells by RT-PCR.
1.3Western blot detected Ku-70protein levels of MCF7and MCF7-ADR cells.
Cultured human breast cancer cell line MCF7and breast cancer cell lineMCF7-ADR which resistance to epirubicin to logarithmic phase, extracted totalprotein, transferred to a membrane after vertical electrophoresis, hybridized withanti-Ku70antibody, observed Ku70protein expression levels of MCF7ofMCF7-ADR cells via chemiluminescence imaging system after ECL.
1.4Transfected Ku70SiRNA fragment into MCF7-ADR cells, RT-PCR, Westernblot analysis Ku70gene silencing results.
1.5Stimulated MCF7, MCF7-ADR, MCF7-ADR/Ku70-SiRNA with differentconcentration epirubicin, MTS assay tested cell proliferative activity, then calculated the median lethal dose of epirubicin to different cell lines, compared resistant indexchanges of MCF7-ADR after Ku70silenced.
1.6Observed apoptosis morphological differences of MCF7-ADR cells andMCF7-ADR cells knoched out Ku70gene cultured with doxorubicin via opticalmicroscopy and Hochest-33258staining.
1.7Compared the apoptosic cells number of MCF7-ADR cells andMCF7-ADR cells knoched out Ku70gene treated with doxorubicin via flowcytometry after Annexin V-PI staining.
1.8PCR-array detected apoptosis-related gene changes in MCF7-ADR cellsafter knoched out Ku70gene to investigate the possible mechanisms of Ku70genesilence reversed drug resistance of MCF7-ADR to doxorubicin.
2Results
2.1Drug resistance test of MCF7-ADR–a breast cancer cell line whichresistant epirubicin.
MTS assay tested MCF7, MCF7-ADR cells proliferative differences, the resultsshowed no significant proliferative difference between MCF7and MCF7-ADR. Therewere no significant different between MCF7and MCF7-ADR cells as far as cell sizeand shape concernd. The two kinds of cells were nearly round, nuclei were large, andhad abundant cytoplasm. Using different concentrate epirubicin to stimulate MCF7and MCF7-ADR cells, the median inhibitory concentration (IC50) were4.16+0.72ug/ml and34.38+6.75ug/ml individely. The resistance index of MCF7-ADR cellsto epirubicin was8.26.
2.2Ku-70mRNA levels in breast cancer cell line MCF7and MCF7-ADR cells.
RT-PCR detected Ku-70mRNA levels of MCF7and MCF7-ADR cells, theresults showed no significant difference between two cell lines.
2.3Expression difference of Ku-70protein in MCF7and MCF7-ADR cells
Analysis Ku-70protein expression of MCF7and MCF7-ADR cells via Westernblot, the results showed there was no significant difference between two cell lines.
2.4Analysised Ku70gene silencing results after transfected with Ku-70SiRNA
Analysis Ku70gene silencing results via RT-PCR and Western blot assay aftertransfected Ku70SiRNA fragment into MCF7-ADR cells24h, the results showedKu-70expression was significately less than the cells that transfected control SiRNA fragment. The results indicated that Ku-70silencing result was good and followed testcould be carried out.
2.5The effect of Ku70silencing on drug resistance of MCF7-ADR
MTS assay tested cell proliferative activity of MCF7, MCF7-ADR,MCF7-ADR/Ku70-SiRNA stimulated with different concentration epirubicin, theresults showed that MCF7-ADR drug resistance to epirubicin was reversed aftertransfected Ku70-SiRNA, the IC50of the three cell lines was4.16+0.72ug/ml、34.38+6.75ug/ml、13.06+2.62ug/ml individely. Epirubicin resistance index of MCF7-ADRcells decreased from8.26to3.14Ku70after transfected Ku70SiRNA.
2.6Analysis epirubicin-induced apoptosis sensitivity change of MCF7-ADRafter Ku70silence through morphology observe.
Observed morphology changs of MCF7-ADR, MCF7-ADR/Ku70-siRNA treatedwith10ug/ml epirubicin via Hochest-33258staining, the results showed that thenumber of MCF7-ADR transfected Ku70SiRNA decreased than the control group,MCF7-ADR transfected Ku70SiRNA was smaller and cells edge shrinked, chromatincondensated or pyknosis, obvious apoptotic bodies could be found in some cells.
2.7Analysis epirubicin-induced apoptosis sensitivity change of MCF7-ADRafter Ku70silence through flow cytometry. Epirubicin-induced apoptosis sensitivityof MCF7-ADR increaced significately after transfected Ku70SiRNA, AnnexinⅤpositvie stained rate of MCF7-ADR, MCF7-ADR transfected contol SiRNA,MCF7-ADR transfected Ku70SiRNA was4.72+0.56%,4.63+0.78%and16.76+2.93%.
2.8In MCF7-ADR/Ku70SiRNA cells apoptosis-related genes APAF1, BBC3,BCL2, BCL2, BIRC6, CASP3, DAPK1, TNFRSF10A expression increased andapoptosis-related genes NAIP, HUK expression decreased.
3Conclusions
3.1Ku70mRNA and protein levels had no significant diference betweenMCF7-ADR cells and MCF7cells, that indicated Ku70can not be a marker of breastcancer drug resistance.
3.2Ku70gene silencing was able to reverse epirubicin resistance ofMCF7-ADR cells.
3.3Ku70gene silencing enhanced epirubicin induced MCF7-ADR apoptosis, suggesting that Ku70-SiRNA might work as breast cancer chemotherapy sensitizer.
3.4In MCF7-ADR/Ku70SiRNA cells apoptosis-related genes APAF1, BBC3,BCL2, BCL2, BIRC6, CASP3, DAPK1, TNFRSF10A expression increased andapoptosis-related genes NAIP, HUK expression decreased. Ku70gene silencingprompted expression of most apoptosis signaling molecules, indicating that Ku70gene silencing activated apoptotic signaling in breast cancer cells which resistedepirubicin, thereby increased the sensitivity ofMCF7-ADR to epirubicin and reversedthe resistance to epirubicin.
Ku蛋白是一个异二聚体蛋白，由Ku70和Ku80两个亚单位组成。最早在自身免疫性疾病患者血清中被发现、鉴定，后来被证明是一个多功能的调节蛋白，参与DNA修复、端粒的维持、细胞凋亡。因此，Ku蛋白被认为在染色体的完整性及细胞存活中发挥重要的作用。近年来的报道提示Ku蛋白的表达与肿瘤的发展有关[3]，该蛋白的高表达促进肿瘤表型的发生、诱导细胞高度增殖及凋亡抵抗，而该蛋白低表达则导致基因组的不稳定及肿瘤的发生，多种实验的结论指出Ku蛋白同时扮演着肿瘤抑制因子及肿瘤发生因子的双重角色，这使Ku成为抗肿瘤药物研究的一个重要的候选靶点，引起众多肿瘤治疗研究人员的关注。下调Ku70蛋白的表达促进宫颈癌细胞Hela对化疗的敏感性[4]；Pim-1基因敲除通过下调Ku蛋白的表达及核定位，抑制Ku70，Ku80对非同源末端连接DNA修复，增强紫杉醇诱导激素难治性前列腺癌凋亡[5]。双调蛋白通过抑制吉西他滨诱导Ku70蛋白的乙酰化促进非小细胞肺癌对吉西他滨的抵抗[6]。上述材料提示，Ku70在多种肿瘤化疗耐药的治疗中扮演重要的角色。但是Ku70蛋白在乳腺癌中的表达及与乳腺癌耐药的相关性还没有报道。
本实验的目的就是为了揭示Ku70蛋白在乳腺癌中的表达及其在乳腺癌耐药治疗中的作用，具体研究如下：
1实验路线
1.1耐表阿霉素乳腺癌细胞系MCF7-ADR细胞耐药性检测
1.1.1MTS法检测MCF7、MCF7-ADR增殖活性差异。
1.1.2倒置相差显微镜观察MCF7、MCF7-ADR细胞形态差异。
1.1.3不同浓度表阿霉素刺激MCF7、MCF7-ADR细胞，计算药物半数抑制率（IC50）及MCF7-ADR对表阿霉素耐药指数。
1.2RT-PCR检测乳腺癌细胞系MCF7及耐表阿霉素乳腺癌细胞系MCF7-ADR细胞Ku-70mRNA水平。
培养人乳腺癌细胞系MCF7、耐表阿霉素乳腺癌细胞系MCF7-ADR细胞至对数生长期，收细胞，提取总RNA，通过RT-PCR检测两种肿瘤细胞Ku-70mRNA水平。
1.3Western blot检测MCF7、MCF7-ADR细胞Ku-70蛋白表达水平
培养人乳腺癌细胞系MCF7、耐表阿霉素乳腺癌细胞系MCF7-ADR细胞至对数生长期，收细胞，提蛋白，垂直电泳后转膜，抗Ku70单克隆抗体杂交，ECL化学发光、成像系统成像，检测MCF7、MCF7-ADR细胞Ku70蛋白表达水平。
1.4向MCF7-ADR细胞中转染Ku70SiRNA片段，RT-PCR、Western blot检测Ku70基因沉默效果。
1.5不同浓度表阿霉素刺激MCF7、MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞， MTS法检测上述细胞增殖活性差异，计算表阿霉素对不同细胞的半数抑制量，比较Ku70沉默前后MCF7-ADR耐药指数的变化。
1.6普通光学显微镜、Hochest-33258染色观察表阿霉素诱导MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞凋亡时细胞形态、细胞核形态差异。
1.7Annexin V-PI染色、流式细胞术检测表阿霉素诱导MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞凋亡能力差异。
1.8PCR-array检测Ku70基因沉默前后MCF7-ADR细胞凋亡相关基因变化，探讨Ku70基因沉默逆转MCF7-ADR耐药性的可能机制。
2结果
2.1耐表阿霉素乳腺癌细胞系MCF7-ADR细胞耐药性检测
MTS法检测MCF7、MCF7-ADR增殖活性差异，结果显示，两种细胞增殖活性无显著差异，MCF7、MCF7-ADR细胞大小、形态相似，近圆形，细胞核较大，胞浆丰富。采用不同浓度表阿霉素刺激MCF7、MCF7-ADR细胞，表阿霉素对二者的半数抑制量（IC50）分别为4.16+0.72ug/ml和34.38+6.75ug/ml，MCF7-ADR细胞对表阿霉素的耐药指数为8.26。
2.2MCF7、MCF7-ADR细胞Ku-70mRNA表达水平差异
RT-PCR检测乳腺癌细胞系MCF7及耐表阿霉素乳腺癌细胞系MCF7-ADR细胞Ku-70mRNA水平，结果显示，二者Ku-70mRNA表达水平无明显差异。
2.3MCF7、MCF7-ADR细胞Ku-70蛋白表达水平差异
Westernblot检测MCF7、MCF7-ADR细胞Ku-70蛋白表达水平，结果显示，二者Ku-70蛋白表达水平亦无明显差异。
2.4MCF7-ADR细胞转染Ku70SiRNA片段效果检测
RT-PCR、Westernblot检测转染Ku70SiRNA片段24h后MCF7-ADR细胞Ku-70mRNA及蛋白表达水平变化，结果显示，转染Ku70SiRNA片段24h后MCF7-ADR细胞Ku-70mRNA及蛋白水平较非特异SiRNA片段转染组显著降低，说明Ku-70基因沉默效果良好，可以进行后续试验。
2.5Ku70沉默对MCF7-ADR耐药性的影响
MTS法检测不同浓度表阿霉素作用后MCF7、MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞增殖活性，结果显示，表阿霉素对3组细胞的半数抑制量分别为4.16+0.72ug/ml、34.38+6.75ug/ml、13.06+2.62ug/ml。MCF7-ADR细胞对表阿霉素的耐药指数由Ku70沉默前的8.26下降为Ku70沉默后的3.14，提示沉默Ku70能够逆转MCF7-ADR对表阿霉素的耐药性。
2.6形态学比较Ku70沉默前后MCF7-ADR对表阿霉素诱导凋亡敏感性变化
普通光学显微镜观察10ug/ml表阿霉素处理48h后MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞形态差异，结果显示Ku70沉默组细胞数量较空白对照组、阴性对照组显著减少，细胞体积缩小，边缘皱缩，折光度差，部分细胞呈圆球形漂浮于培养液中；Hochest-33258染色后蓝色激发光下观察细胞核，可见无药物处理组MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞核大小无明显差异，均呈长椭圆形，染色质疏松、均匀一致蓝染；MCF7-ADR细胞经表阿霉素作用48h后较无药物处理组细胞核体积缩小，染色质浓集呈亮蓝色，MCF7-ADR/Ku70-SiRNA经表阿霉素作用48h后较无药物处理组细胞核体积明显缩小、染色质固缩呈亮蓝色团块，部分细胞可见明显的凋亡小体。
2.7流式细胞术比较Ku70沉默前后MCF7-ADR对表阿霉素诱导凋亡敏感性变化
Annexin V-PI染色、流式细胞术检测10ug/ml表阿霉素处理24h后MCF7-ADR、MCF7-ADR/Ku70-SiRNA细胞凋亡水平差异，结果表明，Ku70沉默后MCF7-ADR对表阿霉素诱导凋亡敏感性较沉默前明显增加，经10ug/ml表阿霉素处理后空白对照组、阴性对照组、Ku70沉默组AnnexinⅤ阳性染色比例依次为4.72+0.56%、4.63+0.78%，16.76+2.93%。
2.8Ku70基因沉默后MCF7-ADR细胞凋亡相关基因APAF1、BBC3、BCL2、BCL2、BIRC6、CASP3、DAPK1、TNFRSF10A表达增加，NAIP、CHUK基因表达减少。
3结论
3.1MCF7-ADR细胞与MCF7细胞相比，其Ku70mRNA及蛋白水平均无明显变化，Ku70不能成为乳腺癌耐药标志物。
3.2Ku70基因沉默能够逆转MCF7-ADR细胞对表阿霉素的耐药性。
3.3Ku70基因沉默能够增强表阿霉素诱导MCF7-ADR细胞凋亡能力，提示Ku70-SiRNA能够作为乳腺癌化疗的增敏剂。
3.4沉默MCF7-ADR细胞Ku70基因后，凋亡相关基因APAF1、BBC3、BCL2、BIRC6、CASP3、DAPK1、TNFRSF10A表达增加，NAIP、CHUK基因表达减少，提示Ku70基因沉默后大部分凋亡信号分子表达增加，说明Ku70基因沉默活化耐表阿霉素乳腺癌细胞中的凋亡信号，从而增加其对表阿霉素的敏感性，逆转其耐药性。
Breast cancer is the major malignant tumour that endangers the health of women,in worldwide, more than100million women are diagnosed with breast cancer,accounting for10%of the patients with cancer, accounting for23%of the femalecancer cases every year[1]. In the United States,40,000people died of breast cancerone year[2]. Breast cancer is one of the most sensitive tumor to chemotherapy in solidtumors, but tumor multidrug resistance (MDR) often leads to the failure ofchemotherapy, which has become a major factor for the lower survival rate ofpatients. Solve the MDR problem of the tumor is important for the treatment of avariety of tumors, therefore, scholars hope to understand more of cancer causes andmechanisms of multidrug resistance in tumor therapy, to provide a new target forcancer treatment.
Ku protein is a heterodimeric protein, composed by Ku70and Ku80two-subunit,and was found and identified earliest in the serum of patients with autoimmunediseases, later proved to be a versatile regulatory protein involved in DNA repair,telomere maintenance, apoptosis. Thus, Ku protein is considered to play an importantrole in the chromosomal integrity and cell survival.
Reports in recent years suggested that the Ku protein expression was associatedwith tumor development[3], the more expression of Ku protein promoted tumorphenotype occurred, induced cell proliferation and apoptosis resistance, on the otherhand, less expression of Ku protein leaded to genomic instability and the occurrenceof tumors, a variety of experiments concluded that Ku protein also plays dual role-atumor suppressor and tumor factors. Ku becomes an important anticancer drugcandidate targets, causing attentions of many cancer treatment researchers. rapysensitivity of cervical carcinoma cell line, Hela[4]; Pim-1gene knockout enhanced
Down-regulating Ku70protein expression could promote chemothepaclitaxelinduced hormone-refractory prostate cancer apoptosis by down-regulating the expression and nuclear localization of Ku protein, inhibiting of Ku70, Ku80pairof non-homologous end-joining DNA repair[5]. Amphiregulin promoted non-smallcell lung cancer resistante to gemcitabine through inhibitiing gemcitabine-inducedacetylation of Ku70protein[6]. The materials above prompts, Ku70play an importantrole in a variety of tumor resistance to chemotherapy treatment. Ku70proteinexpression in breast cancer and correlations with breast cancer resistance has not beenreported.
The purpose of this experiment is to reveal the Ku70protein expression in breastcancer and its role in the treatment of breast cancer resistance, details are as follows:
1The experimental route
1.1Drug resistance test of MCF7-ADR–a breast cancer cell line whichresistant epirubicin.
1.1.1MTS assay detected MCF7, MCF7-ADR proliferative activity.
1.1.2Observed MCF7, MCF7-ADR cell morphological differences via invertedphase contrast microscope.
1.1.3Stimulated MCF7, MCF7-ADR cells with different concentrations ofepirubicin and calculated the drugs half fatality rate (IC50) and drug resistance indexof MCF7-ADR to pharmorubicin.
1.2RT-PCR detected Ku-70mRNA levels in breast cancer cell line MCF7andMCF7-ADR cells
Cultured human breast cancer cell line MCF7and breast cancer cell lineMCF7-ADR which resistance to epirubicin to logarithmic phase, extracted total RNA,then detected Ku-70mRNA of the two tumor cells by RT-PCR.
1.3Western blot detected Ku-70protein levels of MCF7and MCF7-ADR cells.
Cultured human breast cancer cell line MCF7and breast cancer cell lineMCF7-ADR which resistance to epirubicin to logarithmic phase, extracted totalprotein, transferred to a membrane after vertical electrophoresis, hybridized withanti-Ku70antibody, observed Ku70protein expression levels of MCF7ofMCF7-ADR cells via chemiluminescence imaging system after ECL.
1.4Transfected Ku70SiRNA fragment into MCF7-ADR cells, RT-PCR, Westernblot analysis Ku70gene silencing results.
1.5Stimulated MCF7, MCF7-ADR, MCF7-ADR/Ku70-SiRNA with differentconcentration epirubicin, MTS assay tested cell proliferative activity, then calculated the median lethal dose of epirubicin to different cell lines, compared resistant indexchanges of MCF7-ADR after Ku70silenced.
1.6Observed apoptosis morphological differences of MCF7-ADR cells andMCF7-ADR cells knoched out Ku70gene cultured with doxorubicin via opticalmicroscopy and Hochest-33258staining.
1.7Compared the apoptosic cells number of MCF7-ADR cells andMCF7-ADR cells knoched out Ku70gene treated with doxorubicin via flowcytometry after Annexin V-PI staining.
1.8PCR-array detected apoptosis-related gene changes in MCF7-ADR cellsafter knoched out Ku70gene to investigate the possible mechanisms of Ku70genesilence reversed drug resistance of MCF7-ADR to doxorubicin.
2Results
2.1Drug resistance test of MCF7-ADR–a breast cancer cell line whichresistant epirubicin.
MTS assay tested MCF7, MCF7-ADR cells proliferative differences, the resultsshowed no significant proliferative difference between MCF7and MCF7-ADR. Therewere no significant different between MCF7and MCF7-ADR cells as far as cell sizeand shape concernd. The two kinds of cells were nearly round, nuclei were large, andhad abundant cytoplasm. Using different concentrate epirubicin to stimulate MCF7and MCF7-ADR cells, the median inhibitory concentration (IC50) were4.16+0.72ug/ml and34.38+6.75ug/ml individely. The resistance index of MCF7-ADR cellsto epirubicin was8.26.
2.2Ku-70mRNA levels in breast cancer cell line MCF7and MCF7-ADR cells.
RT-PCR detected Ku-70mRNA levels of MCF7and MCF7-ADR cells, theresults showed no significant difference between two cell lines.
2.3Expression difference of Ku-70protein in MCF7and MCF7-ADR cells
Analysis Ku-70protein expression of MCF7and MCF7-ADR cells via Westernblot, the results showed there was no significant difference between two cell lines.
2.4Analysised Ku70gene silencing results after transfected with Ku-70SiRNA
Analysis Ku70gene silencing results via RT-PCR and Western blot assay aftertransfected Ku70SiRNA fragment into MCF7-ADR cells24h, the results showedKu-70expression was significately less than the cells that transfected control SiRNA fragment. The results indicated that Ku-70silencing result was good and followed testcould be carried out.
2.5The effect of Ku70silencing on drug resistance of MCF7-ADR
MTS assay tested cell proliferative activity of MCF7, MCF7-ADR,MCF7-ADR/Ku70-SiRNA stimulated with different concentration epirubicin, theresults showed that MCF7-ADR drug resistance to epirubicin was reversed aftertransfected Ku70-SiRNA, the IC50of the three cell lines was4.16+0.72ug/ml、34.38+6.75ug/ml、13.06+2.62ug/ml individely. Epirubicin resistance index of MCF7-ADRcells decreased from8.26to3.14Ku70after transfected Ku70SiRNA.
2.6Analysis epirubicin-induced apoptosis sensitivity change of MCF7-ADRafter Ku70silence through morphology observe.
Observed morphology changs of MCF7-ADR, MCF7-ADR/Ku70-siRNA treatedwith10ug/ml epirubicin via Hochest-33258staining, the results showed that thenumber of MCF7-ADR transfected Ku70SiRNA decreased than the control group,MCF7-ADR transfected Ku70SiRNA was smaller and cells edge shrinked, chromatincondensated or pyknosis, obvious apoptotic bodies could be found in some cells.
2.7Analysis epirubicin-induced apoptosis sensitivity change of MCF7-ADRafter Ku70silence through flow cytometry. Epirubicin-induced apoptosis sensitivityof MCF7-ADR increaced significately after transfected Ku70SiRNA, AnnexinⅤpositvie stained rate of MCF7-ADR, MCF7-ADR transfected contol SiRNA,MCF7-ADR transfected Ku70SiRNA was4.72+0.56%,4.63+0.78%and16.76+2.93%.
2.8In MCF7-ADR/Ku70SiRNA cells apoptosis-related genes APAF1, BBC3,BCL2, BCL2, BIRC6, CASP3, DAPK1, TNFRSF10A expression increased andapoptosis-related genes NAIP, HUK expression decreased.
3Conclusions
3.1Ku70mRNA and protein levels had no significant diference betweenMCF7-ADR cells and MCF7cells, that indicated Ku70can not be a marker of breastcancer drug resistance.
3.2Ku70gene silencing was able to reverse epirubicin resistance ofMCF7-ADR cells.
3.3Ku70gene silencing enhanced epirubicin induced MCF7-ADR apoptosis, suggesting that Ku70-SiRNA might work as breast cancer chemotherapy sensitizer.
3.4In MCF7-ADR/Ku70SiRNA cells apoptosis-related genes APAF1, BBC3,BCL2, BCL2, BIRC6, CASP3, DAPK1, TNFRSF10A expression increased andapoptosis-related genes NAIP, HUK expression decreased. Ku70gene silencingprompted expression of most apoptosis signaling molecules, indicating that Ku70gene silencing activated apoptotic signaling in breast cancer cells which resistedepirubicin, thereby increased the sensitivity ofMCF7-ADR to epirubicin and reversedthe resistance to epirubicin.
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