干扰HTERT降低人乳腺癌MCF-7细胞对γ射线引起的DNA损伤反应

 目的 研究HTERT RNA干扰对人乳腺癌MCF-7细胞γ射线照射引起的DNA损伤反应的影响。方法 通过逆转录病毒为载体的HTERT-siRNA,抑制人乳腺癌MCF-7细胞中端粒酶催化亚单位HTERT表达,实时定量RT-PCR及Western blot确认HTERT表达水平、用137Cs放射源以3 Gy剂量γ射线照射细胞,于照射前以及照射后1、2、4、8和12h收集细胞,采用磷酸化53BP1 抗体进行免疫荧光染色,并于照射前、照射后1、4和12h收集细胞,Western blot检测53BP1蛋白磷酸化水平。结果 HTERT-siRNA处理的细胞HTERT表达水平比对照细胞降低3.20 (2-△△Ct)倍, HTERT蛋白水平降低2.56倍。HTERT-siRNA处理的细胞对γ射线引起的DNA损伤反应显著降低。结论 RNA干扰HTERT表达水平,可以降低人乳腺癌MCF-7细胞对γ射线照射引起的DNA损伤反应。     

P53对涎腺腺样囊性癌细胞端粒酶活性的抑制作用

目的为了探讨P53基因对腺样囊性癌细胞端粒酶活性的抑制作用及机制。方法构建携带人野生型P53基因的腺病毒表达载体，以脂质体法瞬时转染腺样囊性癌SACC-83细胞，RT-PCR检测转染细胞P53基因mRNA的表达，TRAP-PCR-ELISA法检测转染细胞端粒酶活性的改变。并将P53基因与含有hTERT启动子核心调控区的荧光素酶报告基因pGL2—630共转染SACC-83细胞，测定转染细胞荧光素酶报告基因活性。结果1．瞬时转染含人野生型P53基因的重组腺病毒表达载体p△E1-P53于腺样囊性癌SACC-83细胞后，其P53基因mRNA的表达明显增强；2．基因转染后，SACC-83细胞内源性端粒酶活性显著降低。3．P53基因与含有hTERT启动子核心调控区的荧光素酶报告基因pGL2-630共转染SACC-83细胞后，其荧光素酶活性显著下降。结论外源性表达P53基因可以降低腺样囊性癌细胞SACC-83细胞端粒酶活性，并可能通过抑制端粒酶hTERT基因启动子的转录活性实现。     

Hsa-miR-133a对人乳腺癌细胞侵袭和迁移的影响

目的 探讨Hsa-miR-133a(miR-133a)对人乳腺癌细胞侵袭、迁移和增殖的影响,并初步分析miR-133a影响乳腺癌细胞侵袭及迁移的可能机制.方法 用脂质体介导的转染方法将miR-133a阻遏物(miR-133a inhibitors)转染人乳腺癌细胞株MCF-7,以inhibitor negative control (inhibitor NC)作为阴性对照.通过MTS试剂盒和Transwell侵袭实验检测细胞增殖能力和侵袭力;采用Transwell迁移实验及划痕试验检测细胞迁移能力;再利用生物信息学方法预测miR-133a的靶基因,并对其靶基因进行基因功能分析.结果 (1)miR-133a inhibitors组与inhibitor NC组间细胞增殖活性差异无显著性(P>0.05);(2)划痕后miR-133a inhibitors组细胞迁移能力比inhibitor NC组明显增强(P<0.05);(3)Transwell侵袭及迁移实验显示转染miR-133a inhibitors后,MCF-7细胞的侵袭及迁移能力均明显增强(P<0.01,P<0.05);④生物信息学方法预测miR-133a的靶基因中,部分发挥了促进细胞侵袭、迁移的生物学功能.结论 (1)MiR-133a对人乳腺癌细胞的侵袭及迁移能力可能存在负性调控作用,可能成为乳腺癌治疗的潜在候选靶点;(2)MiR-133a可能通过多种靶基因发挥其对肿瘤的调控作用.     

miR-204-5p抑制乳腺癌细胞增殖、迁移、侵袭及对上皮-间质转化的影响

目的 探索miRNA-204-5p在乳腺癌组织和细胞中的表达,探讨miR-204-5p对乳腺癌细胞增殖、迁移、侵袭和上皮-间质转化(EMT)的影响.方法 qRT-PCR检测乳腺癌患者癌和癌旁组织,乳腺癌细胞和正常乳腺上皮细胞中miR-204-5p的表达;将miR-204-5p模拟物和阴性对照分别转染MCF-7细胞,并检测转染效率;CCK-8法检测转染对细胞增殖的影响;Transwell迁移实验和侵袭实验分别检测转染miR-204-5p后乳腺癌细胞迁移和侵袭能力的变化;Western blot检测上皮-间质转化(EMT)相关蛋白的表达.结果 miR-204-5p在乳腺癌组织表达水平显著下调,miR-204-5p在多种乳腺癌细胞株如MCF-7细胞表达量下调.miR-204-5p模拟物转染抑制MCF-7细胞增殖、迁移、细胞侵袭,E-cadhesin表达水平升高,Vimentin降低.结论 过表达MiR-204-5p对乳腺癌有潜在的治疗作用.     

miR-129靶向RUNX1对结肠腺癌SW480细胞增殖的影响

目的探讨miR-129对结肠腺癌SW480细胞增殖的影响及可能的机制。方法采用miR-129模拟物转染结肠腺癌SW480细胞,分为空白对照组(未进行转染)、miR-129阴性对照组(转染无关序列)、miR-129模拟物转染组(转染miR-129模拟物),通过实时定量PCR进行验证,Western blot检测miR-129过表达后SW480细胞RUNX1蛋白的表达。采用CCK-8法检测miR-129对SW480细胞增殖的影响,采用生物信息学网站预测RUNX1是否为miR-129潜在的靶基因,应用双荧光素酶报告基因实验进行验证。结果 miR-129过表达,SW480细胞中RUNX1mRNA和蛋白表达下调,抑制SW480细胞体外增殖,生物信息学分析结果显示RUNX1是miR-129的靶基因之一,双荧光素酶实验证实RUNX1为miR-129的下游靶基因。结论 miR-129通过靶向调控RUNX1抑制结肠腺癌SW480细胞增殖。     

miR-7通过调节AEG-1途径对肝癌细胞增殖、侵袭和迁移的影响

目的分析miR-7对肝癌细胞增殖、侵袭、迁移的影响及其作用机制。方法取对数生长期的HepG-2细胞分为对照组、miR-7模拟物组、转染miR阴性组并选择非肿瘤细胞系HL7702作为HL7702组,依照分组转染miR-7及阴性miR;后使用RT-PCR法检测其中miR-7水平,并检测各组细胞增殖活性、细胞侵袭活性、细胞迁移活性及AEG-1蛋白表达情况。结果 miR-7模拟物组miR-7水平明显高于HepG-2对照组及转染miR阴性组(P0.01);24 h或48 h时miR-7模拟物组细胞增殖活力明显高于HepG-2对照组及转染miR阴性组(P0.01);miR-7模拟物组侵袭细胞数、细胞迁移率及AEG-1蛋白明显低于HepG-2对照组及转染miR阴性组(P0.01)。结论在肝癌细胞中miR-7可通过调控AEG-1途径而有效抑制增殖、侵袭和迁移。     

SENP3对大鼠成骨细胞端粒酶活性及端粒长度的影响

目的:研究sentrin特异性蛋白酶3(SENP3)对大鼠成骨细胞端粒酶活性及端粒长度的影响。方法:首先0.2 mmol/L H_2O_2处理体外培养的大鼠成骨细胞后,Western blotting法检测SENP3及特异性蛋白1(Sp1)的表达。pc DNA3.0-SENP3转染成骨细胞,分别于24 h、48 h、72 h后采用四甲基偶氮唑蓝(MTT)法检测细胞活力的变化。转染48 h后,Western blotting法检测Sp1和端粒酶逆转录酶(TERT)的表达,PCR-TRAP法及PCR法检测端粒酶活性及端粒长度;ELISA检测上清中碱性磷酸酶(ALP)和骨桥蛋白(OPN)的含量;放射免疫法(RIA)检测骨钙蛋白(OCN)的含量。最后将pc DNA3.0-SENP3与siRNA-Sp1共转染成骨细胞,并检测以上指标。结果:H_2O_2处理成骨细胞后,SENP3和Sp1的表达显著上升。pc DNA3.0-SENP3转染成骨细胞后,Sp1和TERT的表达显著上升,细胞活力、ALP、OPN及OCN含量也都显著上升;端粒酶活性显著增加及端粒长度缩短显著延缓。而当pc DNA3.0-SENP3与siRNA-Sp1共转染成骨细胞后,细胞活力,ALP、OPN及OCN含量,端粒酶活性及端粒长度均未发生显著变化。结论:SENP1通过上调Sp1的表达促进TERT的表达,增加端粒酶活性上升及延缓端粒长度缩短,从而增强成骨细胞增殖能力。     

miR-128通过RECK靶向作用影响乳腺癌细胞的生物学行为

目的探讨miR-128通过RECK靶向作用影响乳腺癌细胞的生物学行为及其作用机制。方法 Western blot检测miR-128在细胞株中的表达情况及慢病毒转染效率;流式细胞术检测miR-128对乳腺癌细胞凋亡行为的影响;双荧光素酶实验检测miR-128和RECK在乳腺癌细胞中的相互作用;流式细胞术实验检测RECK对miR-128在乳腺癌细胞周期和集落形成过程中的逆转作用。结果 MCF-7细胞中miR-128的表达量是NC组细胞的3. 05倍,慢病毒转染效率明显; miR-128通过诱导细胞凋亡和抑制细胞周期进程来抑制MCF-7细胞的生长; RECK是miR-128的直接靶基因,miR-128可以直接调控RECK的表达活性; RECK异位恢复时,miR-128对乳腺癌细胞的凋亡和细胞周期的抑制也得到了部分恢复。结论 miR-128在乳腺癌的发生发展过程中起重要作用,可以和RECK相互影响调控乳腺癌细胞的生物学行为,提示miR-128和RECK可能成为乳腺癌的潜在治疗靶标。     

胸腹水细胞端粒酶活性定性和定量检测

目的探讨端粒酶活性定量检测在诊断良恶性胸腹水中的应用价值。方法采用TRAP-银染定性方法和rrRAP-PicoGreen定量方法,对102例已确诊患者的胸腹水细胞进行端粒酶活性分析。结果恶性胸腹水细胞端粒酶活性明显高于良性胸腹水细胞,其定性检测诊断率明显高于细胞病理学。乳腺癌患者胸腹水细胞的端粒酶活性明显高于卵巢癌、肝癌患者胸腹水细胞的端粒酶活性;肺癌患者胸腹水细胞端粒酶活性明显高于肝癌。在良性胸腹水中,感染性胸腹水细胞端粒酶活性高于非感染性胸腹水。结论恶性胸腹水细胞端粒酶活性明显升高。端粒酶活性定量检测较定性检测更敏感、简便,对良恶性胸腹水的诊断和鉴别诊断有一定应用价值。     

脂质体介导的cripto反义寡核苷酸抑制结肠癌细胞端粒酶活性

目的：探讨cripto反义寡核苷酸（ASODN）对结肠癌细胞端粒酶活性的影响。 方法: 应用脂质体瞬时转染法介导cripto 反义寡核苷酸，处理人结肠癌细胞系后，分别采用实时定量PCR检测cripto mRNA表达，用TRAP检测端粒酶活性，采用软琼脂集落培养试验检测结肠癌细胞的生长。 结果: Cripto ASODN可有效抑制结肠癌细胞集落生长，且与浓度相关。结肠癌细胞经Cripto ASODN转染后，端粒酶活性明显受到抑制，呈作用浓度和时间依赖性。并与ASODN的浓度和处理时间有关。 结论: cripto基因可能参与对结肠癌细胞端粒酶活性的调控。     

Involvement of miR-30c in resistance to doxorubicin by regulating YWHAZ in breast cancer cells

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression implicated in cancer, which play crucial roles in diverse biological processes, such as development, differentiation, apoptosis, and proliferation. The aim of this study was to investigate whether miR-30c mediated the resistance of breast cancer cells to the chemotherapeutic agent doxorubicin (ADR) by targeting tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ). miR-30c was downregulated in the doxorubicin-resistant human breast cancer cell lines MCF-7/ADR and MDA-MB-231/ADR compared with their parental MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, we observed that transfection of an miR-30c mimic significantly suppressed the ability of MCF-7/ADR to resist doxorubicin. Moreover, the anti-apoptotic gene YWHAZ was confirmed as a target of miR-30c by luciferase reporter assay, and further studies indicated that the mechanism for miR-30c on the sensitivity of breast cancer cells involved YWHAZ and its downstream p38 mitogen-activated protein kinase (p38MAPK) pathway. Together, our findings provided evidence that miR-30c was one of the important miRNAs in doxorubicin resistance by regulating YWHAZ in the breast cancer cell line MCF-7/ADR.     

端粒酶反义腺病毒载体对乳腺癌细胞MCF-7端粒酶的抑制作用

端粒酶ＲＮＡ的反义地人乳腺癌细胞系ＭＣＦ－７细胞端粒酶活性的影响。方法用重组腺病毒转移并表达端粒酶ＲＮＡ的反义ｃＤＮＡ,采用基因重组腺脂质体共转当闰酶反义重组病毒,用Ｓｏｕｔｈｅｒｎ杂交鉴定病毒的整合功能,用ＴＲＡＰ－ 染法检测端粒酶活性。结果ＭＣＦ－７细胞是恶性乳腺癌的典型细胞系。对对照组ＭＣＦ－７、ＭＣＦ－７、ｖＡｄ－ＡＡＶ细胞相比,反义病毒感染后的细胞是恶性乳腺癌的典型细胞系。与对照组ＭＣＦ     

Silencing of TMSG1 enhances metastasis capacity by targeting V-ATPase in breast cancer

TMSG1, as a novel tumor metastasis suppressor gene, has been demonstrated to closely relate to the metastasis and drug-resistant of breast cancer. However, its molecular mechanism is still unclear. In this study, we explored the effect of small interference RNA (siRNA) targeting TMSG1 on the invasion of human breast carcinoma cell line MCF-7 and its molecular mechanisms associated with the extracellular pH. qRT-PCR and Western blot analysis revealed dramatic reduction of the levels of TMSG1 mRNA and protein after transfection of siRNA in MCF-7 cells. Cell migration and invasion were obviously increased by TMSG1 siRNA treatment. The activity of vacuolar ATPase (V-ATPase) and MMP-2 was significantly increased in MCF-7 cells transfected with the TMSG1 siRNA compared with the controls. Furthermore, acidic intracellular environment significantly increased the MMP-2 activity and the capacity of cell migration and invasion. In conclusion, silencing of TMSG1 increased V-ATPase activity, decreased extracellular pH and in turn the activation of secreted MMP-2, which ultimately promoted metastasis capacity of breast cancer cell.     

Inhibitory effect of miR-184 on the potential of proliferation and invasion in human glioma and breast cancer cells in vitro

MiR-184 was an important suppressor to tumor cells proliferation and invasion and some studies show that it was down-regulated in aggressive human tumor cells and a potential tumor therapy target through expression of miR-184 results in reduced tumor cell aggressiveness. In this study, miR-184 showed an inhibitive activity of glioma U87MG cell line and breast cancer MCF-7 cell line in proliferation and invasion by MTS and transwell assay. We found that the miR-184 also could arrest cell cycle and adhesion by up-regulating the expression of p53 and p21 and activity of caspase-3/8, suppressing the expression of SND1, MMP-2/9, CD44 and activity of AKT/NF-κB pathway. The results showed that miR-184 could be a potential target for glioma and breast cancer treatment.     

Bak基因转染对乳腺癌MCF-7细胞增殖及对紫杉醇敏感性的影响

目的 探讨Bak基因转染对乳腺癌MCF-7细胞增殖及对紫杉醇敏感性的影响。 方法 采用Western blotting法和Real-time PCR检测乳腺癌MCF-7细胞转染前后Bak蛋白表达。通过细胞计数试剂盒8（CCK-8）和流式细胞术检测Bak基因转染后,及紫杉醇作用24 h、48 h和72 h后对MCF-7细胞增殖和细胞周期的影响。 结果 Western blotting和 Real-time PCR检测结果显示,转染质粒后,Bak蛋白表达量显著升高,MCF-7 Bak组mRNA表达量为2.15±0.07,明显高于MCF-7 NC组1.03±0.04（t=13.412,P<0.05）。转染48 h、72 h和96 h后,MCF-7 Bak细胞增殖速率为（0.31±0.03）%、（0.37±0.03）%、（0.47±0.04）%,低于MCF-7 NC组的（0.40±0.03）%、0.48±0.04）%、（0.61±0.06）%,差异有统计学意义（t ₄₈=2.145、t ₇₂=3.164、t ₉₆=5.487,P<0.05）。MCF-7 Bak组G2期细胞数是（26.84±2.69）%,显著高于MCF-7 NC组（16.02±1.61）%（t=12.887,P<0.05）。紫杉醇作用24 h、48 h和72 h后,MCF-7 Bak组细胞增殖抑制率为（35.98±4.00）%、（54.66±5.50）%、（80.11±8.00）%,高于MCF-7 NC组的（24.12±2.40）%、（40.12±4.00）%、（61.09±6.09）%,差异有统计学意义（t ₂₄=8.456、t ₄₈=10.547、t ₇₂=13.442,P<0.05）。紫杉醇作用24 h后,MCF-7 Bak组G0/G1期细胞数（73.01±7.02）%高于MCF-7 NC组（63.84±6.68）%（P<0.05）。 结论 上调Bak基因表达可抑制乳腺癌MCF-7细胞增殖,上调G0/G1期比例,增强紫杉醇的敏感性。     

RNA interference-mediated silencing of the p53 tumor-suppressor protein drastically increases apoptosis after inhibition of endogenous fatty acid metabolism in breast cancer cells

Fatty acid synthase (FAS)-dependent endogenous fatty acid synthetic activity is abnormally elevated in a biologically-aggressive subset of breast carcinomas. Remarkably, tumor-associated FAS hyperactivity represents a novel target for anti-metabolic therapy because pharmacological inhibitors of FAS are selectively cytotoxic for tumor cells, triggering their apoptotic cell death. Since the p53 tumor-suppressor protein (TP53) is thought to play a novel role in cellular responses of a variety of non-genotoxic metabolic stresses, we characterized the involvement of TP53 in the response of breast cancer cells to FAS inhibition. MCF-7 breast cancer cells were selected for study because they have an intact TP53 pathway and undergo little apoptosis following FAS blockade. Two chemically distinct inhibitors of FAS (the natural mycotoxin cerulenin and the novel small-molecule inhibitor C75) were studied in parallel to provide a broad picture of consequences suffered by the loss of FAS function on TP53 signaling. Treatment with either cerulenin or C75 induced TP53 protein accumulation at 24 h in MCF-7 cells. To determine whether the up-regulation of TP53 following exposure to cerulenin or C75 was solely due to inhibition of endogenous fatty acid metabolism, we first evaluated the cytotoxic response to chemical FAS blockers on MCF-7 cells in which FAS gene expression was previously silenced by using the highly sequence-specific mechanism of RNA interference. MCF-7 cells became insensitive to C75-induced cytotoxicity when the expression of FAS was specifically suppressed by targeted knock-down with small interfering RNA (siRNA), whereas they partially retained their sensitivity to cerulenin. These results demonstrate that C75-induced cytotoxic damage to breast cancer cells is closely dependent on its ability to inhibit FAS-catalyzed endogenous fatty acid biogenesis, thus ruling out a significant direct effect of C75 on DNA. To determine the functional role of TP53 on breast cancer cell survival after FAS blockade, we evaluated FAS inhibitor-mediated apoptosis in MCF-7 cells transiently transfected with a pool of sequence-specific double-stranded RNA oligonucleotides targeting TP53 gene. In these conditions, TP53 protein levels were unchanged during the period of FAS-inhibitor exposure. Remarkably, siRNA-induced silencing of TP53 gene expression did result in a dramatic increase (approximately 300%) in apoptotic cell death following exposure to C75. Strikingly, there was no apparent relationship between the TP53 mutational status and sensitivity to chemical FAS inhibitor in a panel of human breast cancer cell lines. However, the degree of TP53 mRNA expression was predictive of sensitivity to C75-induced cytotoxicity, with low-TP53 mRNA expressing breast cancer cells showing hypersensitivity to FAS blockade. These findings strongly suggest that: a) TP53 is a novel molecular sensor of energy imbalance after the perturbation of endogenous fatty acid metabolism in breast cancer cells; b) TP53 function closely influences the decision between apoptosis and growth arrest following FAS blockade; and c) pharmacological inhibitors of FAS activity may be clinically useful against breast carcinomas exhibiting mutation or aberrant expression of TP53.     

iASPP对表达野生型p53基因的乳腺癌细胞株MCF-7凋亡的影响

目的： P53凋亡刺激蛋白（ASPP）家族成员能够调节P53诱导的细胞凋亡。其中ASPP家族抑制成员(iASPP)主要通过特异性地和P53蛋白结合抑制肿瘤细胞的凋亡,本实验通过构建iASPP的RNAi质粒,研究在体内外抑制iASPP基因后对表达野生型p53基因的乳腺癌细胞MCF-7凋亡的影响。方法: 采用分子生物学方法构建iASPP的腺病毒RNAi质粒pAd-iASPP-RNAi;体外转染MCF-7细胞,同时建立MCF-7细胞的裸鼠移植瘤模型;分别采用RT-PCR和Western blotting检测转染后瘤细胞iASPP mRNA和蛋白质表达的变化;采用流式细胞术检测转染前后瘤细胞凋亡的变化。结果: 转染iASPP RNAi后,MCF-7细胞的iASPP mRNA及蛋白表达量降低（抑制率分别为95.4%和96.8%,P<0.01）;MCF-7细胞的凋亡百分率、坏死百分率与对照组相比具有显著差异（P<0.01）。裸鼠移植瘤模型经pAd-iASPP-RNAi处理后,iASPP mRNA及蛋白表达量也明显降低（抑制率分别为87.4%和89.2%,P<0.01）;移植瘤细胞的凋亡百分率和坏死百分率显著上升（P<0.01和P<0.05）。结论: 在体内外抑制iASPP基因的表达能够通过p53途径诱导表达野生型p53的乳腺癌细胞凋亡。     

端粒酶反义核酸对乳腺癌细胞生长的抑制作用

为了探讨针对人类端粒酶RNA（hTR）基因的反义寡核苷酸（AS－ODN）对乳腺癌细胞系MCF－7的影响，将AS－OND作用于细胞。进行细胞计数，MTT比色法测细胞生长抑制率，PCR－ELISA法测端粒酶活性，流式细胞仪测细胞周期与凋亡。结果表明该AS－ODN能抑制MCF－7细胞生长，降低端粒酶活性并诱导细胞凋亡。针对hTR的AS－ODN对治疗恶性肿瘤有潜在意义。     

RNA干扰Notch1基因对乳腺癌MCF-7细胞增殖及凋亡的影响

目的探讨RNA干扰技术沉默Notch1基因表达对人乳腺癌细胞增殖和凋亡的影响。方法设计并合成靶向Notch1基因的小分子干扰RNA质粒,在转染试剂Sofast介导下转染人乳腺癌细胞株MCF-7,用RT-PCR和Western blot法检测转染前、后Notch1基因的表达,挑选干扰效率最强的一组表达载体;cck8比色法检测分析各组细胞的存活率;流式细胞术检测细胞凋亡比例;Western blot法检测转染各组MCF-7细胞Notch1、NF-κB及Caspase-3蛋白表达。结果 Notch1-shRNA能有效封闭Notch1基因的表达,Notch1基因和蛋白表达水平明显降低(P<0.05);Notch1-shRNA能明显抑制细胞增殖(P<0.05);转染48h后细胞凋亡比例增加(P<0.01)。NF-κB蛋白水平表达降低,Caspase-3蛋白表达水平增高。结论利用RNA干扰技术沉默Notch1基因的表达可以明显抑制MCF-7细胞的增殖,促进MCF-7细胞凋亡,其机制可能通过NF-κB信号通路调节相关凋亡蛋白的表达,进而影响细胞的凋亡和增殖,靶向Notch1的RNA干扰技术在乳腺癌的基因治疗中具有一定的研究价值。