MicroRNA-195调控RAF1蛋白的表达增强乳腺癌细胞对阿霉素药物的敏感性的机制研究
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摘要
背景与目的
乳腺癌是妇女中最为常见的恶性肿瘤，并且也是一个重要的全球性的健康问题。据估计全球每年新增近130万的妇女患有乳腺癌，而与乳腺癌相关的死亡病例每年超过45万人。尽管近年来在乳腺癌的预防筛查，早期检测以及治疗等方面已经明显减少了乳腺癌的发病率和死亡率，并有效提高了乳腺癌患者的存活时间。但是，仍有一定数量的人群被诊断为乳腺癌并最终发展为恶性变。当前的治疗策略结合手术切除与辅助治疗，包括细胞毒性抗癌药物，激素治疗，靶向基因的药物治疗等，然而仍有近50%的病人对于初期的化疗无效，对多种化疗药物产生耐药，包括阿霉素耐药（Adriamycin-resistant ADR）。这种现象被称为多重耐药(Mutiple-drug, MDR)，而多重耐药一直是多种恶性肿瘤化疗失败的重要原因。
目前研究发现产生多重耐药的不同机制，如药物活性的失活，细胞内药物泵出胞外，或者是治疗过程中药物靶点的改变等，但是对肿瘤细胞产生耐药的原因以及相关的作用机制研究还很不完善。在乳腺癌细胞对抗癌药物产生多重耐药的机制中发现，肿瘤的发生和发展存在一种信号转导级联反应，作用于下游的表皮因子生长受体，如EGFR或HER2等。在这些受体信号通路中，Ras/Raf/MEK/ERK在肿瘤细胞耐药，以及细胞增值、分化或转移过程中起着重要作用，其中原癌基因所激活的丝/苏氨酸蛋白激酶Raf-1扮演了重要角色。Raf-1能够磷酸化并激活下游的MEK及ERK蛋白激酶，ERK蛋白激酶协同调控多种蛋白的表达，如多重耐药基因MDR所编码的膜P糖蛋白(P-gpglycoprotein)，细胞抗凋亡蛋白BCL-2，从而对细胞周期，细胞迁移﹑凋亡和分化等过程进行调控。最近研究也表明肿瘤耐药性的产生与微小非编码的RNA（MicroRNAs，miRNAs)的表达变化有关。随着miRNA所介导的关于肿瘤耐药性相关研究成果的积累，现在更多的关注于miRNA的靶向调控作为化疗干预的一个新手段。miRNA涉及到许多重要的细胞调控过程，如细胞的增殖﹑凋亡和分化，造血及器官的形成等。同时miRNA也涉及到许多疾病的产生，包括肿瘤的形成。miRNA作为致癌或抑癌基因取决于所作用的靶基因。miR-195在多种实体肿瘤组织中被发现处于低表达状态，如胃癌﹑结肠癌﹑肝癌等。同时，研究也表明miR-195作为一种抑癌基因也参与到肿瘤细胞的增殖或凋亡过程中。然而，对于miR-195所介导的乳腺癌细胞对抗癌药物阿霉素的获得性耐药目前还没有相关研究
因此，本课题的研究目的拟了解乳腺癌耐药组织或细胞中，miR-195的表达与Raf-1的表达变化是否存在关联性；通过构建慢病毒载体介导针对Raf-1的siRNA干扰体系，有效沉默细胞中Raf-1的表达，同时转染miR-195模拟物或抑制物对细胞进行干预后，进行比较观察两种作用方式对细胞增殖或凋亡的影响，明确乳腺癌细胞中miR-195是否直接靶向调控Raf-1的表达；通过调控乳腺癌细胞中miR-195的表达，观察能否有效提高耐药细胞对阿霉素药物的敏感性，以及通过对miR-195的调控后，检测Ras/Raf/MEK/ERK信号通路所介导的下游靶因子相关蛋白BCL-2和P-gp的表达，确定miR-195调控乳腺癌细胞对阿霉素药物敏感性的作用机制。
方法
1﹑通过Western-blot及Q-PCR技术检测乳腺癌组织及乳腺癌耐药组织中Raf-1蛋白及has-miR-195的表达情况，了解二者在乳腺癌组织中的表达关系。
2﹑采用逐步递增阿霉素(Adriamycin, ADR)浓度，间歇作用体外诱导培养人乳腺癌细胞（MCF-7）构建阿霉素耐药细胞株（MCF-7/ADR）模型；采用MTT比色法观察对MCF-7及MCF-7/ADR细胞经不同浓度阿霉素作用不同时间后，两种细胞的增殖情况，以便筛选出最适的干预浓度和作用时间；采用流式细胞术检测两种细胞经特定浓度的阿霉素作用后，对两种细胞凋亡的影响。
3﹑采用Q-PCR技术了解miR-195在人乳腺上皮细胞（HBL-100）﹑乳腺癌细胞（MCF-7）及乳腺癌耐药细胞株（MCF-7/ADR）中的表达情况；采用Western-blot及免疫荧光染色技术了解Raf-1蛋白在三种细胞中的表达及分布情况；初步了解miR-195和Raf-1在乳腺癌细胞中的表达变化；采用Western-blot技术检测两种细胞中耐药相关蛋白P-gp和BCL-2的表达情况。
4﹑设计合成针对Raf-1的siRNA干扰序列，构建带有报告基因GFP的干扰质粒，双酶切及测序鉴定质粒构建成功后，慢病毒载体感染MCF-7及MCF-7/ADR细胞后，通过Q-PCR筛选有效干扰质粒；采用Western-blot技术检测两种细胞中Raf-1蛋白的表达，分析其干扰效果；采用MTT比色法及流式细胞术检测两种细胞在siR-Raf-1干预后，对细胞增殖及凋亡的影响。
5﹑通过生物信息学预测miR-195对Raf-1的靶向作用；转染化学合成的成熟miR-195模拟物（miR-195mimic）及miR-195抑制物（miR-195inhibitor）进入MCF-7及MCF-7/ADR细胞，采用Q-PCR技术检测两种细胞中miR-195的表达变化；采用Western-blot检测转染miR-195合成物后，两种细胞中Raf-1蛋白的表达变化，了解调控miR-195的表达对细胞中Raf-1蛋白的影响；采用MTT比色法及流式细胞术检测两种细胞在转染miR-195合成物后，对细胞的增殖及凋亡的影响。
6﹑分别转染siR-Raf-1干预质粒和miR-195合成物入MCF-7及MCF-7/ADR细胞，再经特定浓度的阿霉素药物作用，通过MTT比色法及流式细胞术检测两种细胞的增殖及凋亡，比较采用两种方式对细胞进行干预后，乳腺癌细胞对阿霉素药物的敏感性变化，以便了解能否通过调控miR-195的表达，有效提高耐药细胞对阿霉素药物的敏感性；采用免疫荧光染色技术观察在转染siR-Raf-1干预质粒及miR-195合成物后，细胞中Raf-1的表达及分布情况；采用Western-blot检测细胞经siR-Raf-1和miR-195合成物干预后，耐药相关蛋白BCL-2和P-gp的表达，以便明确miR-195与Raf-1蛋白激酶在乳腺癌多耐药发生中的作用机制。
结果
1﹑分别在不同组织样本中采用Q-PCR检测miR-195的表达情况，结果发现在乳腺癌组织及乳腺癌耐药组织中miR-195的表达与正常乳腺组织相比较，其miR-195的表达水平明显更低，表达差异有显著性（P<0.01）；同时，miR-195的表达耐药组织比癌组织明显降低，差异有显著性（P<0.05）。而Raf-1蛋白表达（Western-blot）结果显示，乳腺癌组织及乳腺癌耐药组织比正常乳腺组织明显增高的，差异有显著性（P<0.05）；值得注意的是，耐药组织中Raf-1的表达与乳腺癌组织相比较，其表达水平增高近2倍，差异有统计学意义（P<0.05）；在乳腺癌组织中Raf-1的表达与miR-195的表达呈现出明显的差异性，或许这种表达差异性之间存在着某种关联。因此，我们采用Spearman相关性检验分析两者的差异性表达是否存在关联性，结果发现miR-195与Raf-1蛋白在乳腺癌组织中的表达呈现一种显著的负相关关系，其相关性系数达到80%（R=0.8069，P<0.01）。
2﹑成功构建人乳腺癌阿霉素耐药细胞株模型，MCF-7/ADR相对于MCF-7细胞耐药系数RI为10.03；采用MTT比色法分析MCF-7与MCF-7/ADR两种细胞经不同浓度的阿霉素作用不同时间后细胞增殖变化，结果显示增殖比率MCF-7的细胞比MCF-7/ADR降低更为明显，差异有显著性（P<0.01），并筛选出500ng/ml的最适阿霉素作用浓度和48h的作用时间；采用FCM技术分析500ng/ml浓度阿霉素作用前后两种细胞的凋亡率，可见MCF-7经ADR作用后其细胞凋亡比率提高20%，而MCF-7/ADR细胞仅仅提高近10%。
3﹑在MCF-7与MCF-7/ADR细胞中miR-195的表达与HBL-100相比，其miR-195的表达量明显更高，差异有统计学意义（P<0.05），并且MCF-7/ADR与MCF-7相比，miR-195的表达也明显更高，差异有显著性（P<0.01），这与组织中miR-195的表达相符合；MCF-7/ADR与MCF-7两种细胞中Raf-1蛋白的表达与HBL-100相比，其表达均显著上调，差异有统计学意义（P<0.05）；免疫荧光染色也发现Raf-1的荧光表达在MCF-7/ADR与MCF-7中均有较强的荧光表达，而在HBL-100中Raf-1的荧光表达相对较弱；同时在MCF-7/ADR与MCF-7中，发现耐药相关蛋白BCL-2和P-gp的表达与HBL-100相比，表达水平也是显著增加，差异有统计学意义（P<0.01）。
4﹑成功构建针对Raf-1的带有报告基因GFP的两套siRNA干扰质粒pLenR-GPH-Raf-1，经包装293T细胞产毒后能够稳定感染MCF-7和MCF-7/ADR细胞。经Q-PCR及Western-blot检测，结果显示siRNA慢病毒感染组中Raf-1的mRNA和蛋白表达量均显著低于未转染组和对照组（P<0.01），在MCF-7和MCF-7/ADR两种细胞中Raf-1蛋白的抑制比率分别达到73.3%和68.5%；采用MTT比色法分析MCF-7与MCF-7/ADR两种细胞经siRNA慢病毒感染后细胞的增殖变化，结果显示在感染后两种细胞的增殖均显著低于未转染组和对照组（P<0.01），抑制比率分别为23.4%和16.8%；采用FCM技术检测经siRNA慢病毒感染细胞，两种细胞的凋亡比率与对照组相比均明显增高，凋亡率分别提高在MCF-7中为15%和MCF-7/ADR中为12%，差异有显著性（P<0.01）。
5﹑采用不同的生物信息学网址（PITA,TargetScan及miRanda）对miR-195是否靶向作用于Raf-1进行分析，均发现miR-195靶向作用于Raf-1；转染化学合成的成熟miR-195模拟物（miR-195mimic）及miR-195抑制物（miR-195inhibitor）入MCF-7及MCF-7/ADR两种细胞，经Q-PCR检测发现，MCF-7与MCF-7/ADR两种细胞经转染miR-195mimic后，细胞中miR-195的表达水平与未转染组和对照组相比均明显提高，分别升高近5倍和3倍（P<0.01），而转染miR-195抑制物后，两种细胞中的miR-195表达水平与未转染组和对照组相比降低不明显；采用Western-blot检测MCF-7与MCF-7/ADR两种细胞在转染miR-195合成物后Raf-1蛋白的表达水平，结果发现在转染miR-195mimic后，细胞中Raf-1蛋白的表达水平与对照组相比均显著降低（P<0.01），其Raf-1蛋白的抑制比率分别达到68.7%和61.8%，而转染miR-195inhibitor物后，两种细胞中Raf-1的表达没有明显提高；MTT比色法分析MCF-7与MCF-7/ADR两种细胞经转染miR-195合成物后，细胞的增殖变化，结果发现在转染miR-195mimic后，两种细胞的增殖比率与对照组相比均明显降低（P<0.05），抑制比率分别为25.6%和18.2%，而转染miR-195inhibitor后，细胞的增殖并没有明显增加；采用FCM技术观察转染miR-195合成物后细胞的凋亡情况，结果发现MCF-7与MCF-7/ADR两种细胞经miR-195mimic转染后，细胞的凋亡比率显著增加（P<0.05），细胞凋亡比率分别达到25.3%和21.4%，而在转染miR-195inhibitor后细胞凋亡比率与对照组相比，并没有明显变化。
6﹑分别转染siR-Raf-1干扰质粒和miR-195合成物入MCF-7及MCF-7/ADR细胞，再经特定浓度的阿霉素药物作用，MTT比色法分析细胞活力，结果发现在转染miR-195mimic或siR-Raf-1干扰质粒后，细胞再经ADR作用，两种细胞的增殖与对照组相比明显降低（P<0.01），而在转染miR-195inhibitor并经ADR作用后，两种细胞的活力并没有明显变化；采用FCM技术观察MCF-7及MCF-7/ADR细胞转染miR-195mimic或siR-Raf-1干扰质粒后，并经ADR作用，细胞凋亡比率与对照组相比明显增加（P<0.01），其中MCF-7及MCF-7/ADR细胞在转染miR-195mimic后细胞凋亡比率分别达到52.6%和41.2%，与采用ADR药物处理的对照组相比，两种细胞的凋亡比率均提高近20%；同时染siR-Raf-1干扰抑制MCF-7及MCF-7/ADR两种细胞中Raf-1蛋白的表达，也发现与转染miR-195mimic具有相似的效果，两种细胞的凋亡比率明显升高，其凋亡比率分别达到50.3%和40.7%；采用免疫荧光染色观察两种细胞在转染miR-195mimic或siR-Raf-1干扰质粒后，细胞中Raf-1蛋白的荧光表达，结果可见转染后Raf-1蛋白的荧光表达强度与对照组相比明显降低；同时采用Western-blot检测细胞在转染siR-Raf-1干扰质粒和miR-195mimic后，细胞中耐药相关蛋白BCL-2和P-gp的表达与对照组相比，其表达量是明显降低（P<0.01），而两种细胞在转染miR-195inhibitor后，BCL-2和P-gp蛋白的表达与对照组相比没有明显的增加。
结论
1﹑miR-195和Raf-1在乳腺癌组织及乳腺癌耐药组织中分别处于低表达和过表达状态，其表达与正常乳腺组织相比均呈显著差异，且二者的差异性表达呈现显著的负相关性，同时耐药组织与乳腺癌组织相比miR-195的表达明显更低，而Raf-1的表达则明显更高，差异有显著性(P<0.01)。
2﹑建立了人乳腺癌耐阿霉素药物细胞株（MCF-7/ADR），该细胞株具有耐药细胞株的耐药性和生物学特性，是研究乳腺癌细胞对ADR耐药机制较为理想的细胞模型，其MCF-7/ADR细胞与MCF-7细胞的对ADR药物的耐受性比较，MCF-7/ADR细胞对ADR药物作用的耐受性更强。
3﹑MCF-7及MCF-7/ADR细胞与HBL-100细胞相比，miR-195处于明显低表达，而Raf-1的表达则明显上调，这与乳腺癌组织中miR-195和Raf-1的表达相似；同时在两种细胞中耐药相关蛋白BCL-2和P-gp的表达与HBL-100细胞相比明显更高，而在MCF-7/ADR与MCF-7细胞间比较，MCF-7/ADR中这两种蛋白的表达量也明显更高，说明由于MCF-7/ADR细胞中Raf-1蛋白的上调或许使细胞具有更强的耐药性。
4﹑构建针对Raf-1的siRNA干扰质粒可以分别在MCF-7和MCF-7/ADR细胞中特异并有效沉默目的基因Raf-1的mRNA及蛋白表达，并同时能够抑制两种细胞的增殖活力，有效提高两种细胞的凋亡比率。
5﹑通过生物信息学预测发现miR-195对Raf-1具有靶向作用；转染miR-195mimic能够有效上调miR-195在两种细胞中的表达，同时引起Raf-1蛋白在细胞的表达显著下降；并且能够有效抑制细胞的增殖活力，明显提高两种细胞的凋亡比率。说明在乳腺癌细胞中miR-195对Raf-1具有直接的靶向调控作用，通过上调miR-195能够抑制Raf-1在细胞中的表达， miR-195作为抑癌基因，对乳腺癌细胞的促凋亡和抑制增殖的功能部分或至少是通过作用于Raf-1蛋白来实现。
6﹑通过转染miR-195mimic入阿霉素作用的MCF-7及MCF-7/ADR细胞，上调细胞中miR-195的表达，结果显示与单独采用阿霉素处理的对照组细胞相比，细胞的增殖明显减弱，同时两种细胞的凋亡明显增加。另外，我们也采用RNAi干扰抑制MCF-7及MCF-7/ADR两种细胞中Raf-1蛋白的表达，也发现具有相似的效果，两种细胞的增殖活力也呈现明显受抑，而细胞的凋亡比率明显升高。证实上调细胞中miR-195的表达能够有效提高乳腺癌耐药细胞对阿霉素药物的敏感性。并且上调miR-195在细胞中的表达水平，不仅抑制细胞中Raf-1的表达，而且也促使细胞中的耐药相关蛋白P-gp和Bcl-2蛋白的表达降低，说明miR-195对提高乳腺癌耐药细胞药物敏感性的作用机制很可能是通过调控Raf-1蛋白激酶在细胞中的表达，影响下游靶激酶导致转录因子影响下游靶激酶导致转录因子的磷酸化，从而减少这些转录因子对P-gp和Bcl-2基因的启动子序列的刺激作用，降低基因的转录，使乳腺癌细胞中P-gp和Bcl-2蛋白的表达相应下调，从而减少了细胞的抗凋亡活性和对药物的转运能力，明显提高乳腺癌细胞对阿霉素化疗药物的敏感性。
Background and Objectives
Breast cancer is the most common malignancy in women and is asignificant worldwide health problem, accounting for approximately1.3million new cancer cases and more than450,000cancer-related deathsannually in the world. Although recent advancements in prevention,screening, early detection and treatments for breast cancer have thepotential to reduce mortality, still a large number of patients are diagnosedat advanced stages. Current treatment strategies combines surgical removalwith adjuvant therapy including cytotoxic anticancer drugs, hormonaltherapy, targeted drugs. However, nearly50%of such patients frequentlydevelop multidrug resistance ((Mutiple-drug, MDR) to chemotherapeuticagents during the course of treatment.It is estimated that one of two breastcancer patients will fail to respond to initial treatments or will rapidlyacquire resistance to multiple-drugs.
Previous studies have demonstrated different mechanisms responsiblefor drug resistance, such as drug inactivation, extrusion of the drug by enhanced drug efflux pumps, or changes in drug target during the course oftreatment. The precise nature of chemotherapy resistance, and theunderlying mechanism of the acquisition of drug resistance are still poorlyunderstood. One of the various mechanisms have been studied in breastcancer MDR to anticancer agents as well as cancer development is signaltransduction cascades downstream of epidermal growth factor (EGF)receptor (EGFR) isoforms (e.g., EGFR&HER2). Among the signalingpathways downstream of these receptors, the Ras/Raf/MEK/ERK pathwayshave been shown to play a critical role in chemotherapeutic drug resistanceas well as cancer differentiation, cellular transformation and, cellularsenescence and metastasis. In this signal transduction cascade,proto-oncogene serine/threonine-protein kinase (Raf-1) plays a veryimportant role. Once activated, Raf-1will phosphorylate and activateMEK1and MEK2protein kinases and then in turn, phosphorylate andactivate the serine/threonine-specific protein kinases ERK1and ERK2tocontrol expression of various genes (such as BCl-2and P-glycoprotein).
Recent studies have also linked the acquisition of cancer drugresistance to altered expression of microRNAs, which are a novel class ofendogenous non-coding small RNA and function in posttranscriptionalregulation of the target gene expressions by binding to the target mRNA toinhibit its translation and/or to degrade the target mRNA molecules.Evidence of miRNA-mediated drug resistance is accumulating, and much attention is now being focused on targeting miRNAs as a novel strategy fortherapeutic intervention. MicroRNAs (miRNAs) are an abundant class ofshort noncoding RNAs,18–25nt in length, that act as potent regulators ofgene expression. They have been implicated in most major cellularprocesses including proliferation, apoptosis, developmental timing,hematopoiesis and organogenesis. Therefore, it is not surprising thatmiRNAs have also already been linked to a number of diseases includingcancer. microRNAs function as onco-miRs or tumor-suppressor-miRsdepending on their respective target genes. miR-195has been found to bedownregulated in a variety of cancers, including gastric cancer, liver cancer,bladder cancer, and adrenocortical cancer.(31-34).miR-195was also foundsignificantly downregulated in breast cancer cells and in clinical specimensafter surgery(35,36). Li et al also found that overexpression of miR-195was found to be correlated inversely with malignancy of breast cancer,suppressing breast cancer cell proliferation and invasion. Raf-1wereidentified as novel direct targets of miR-195as a major mechanism.
The project is targeted on whether, in drug-resistant tissues or cells, thechanges of miR-195expression are related to the changes of Raf-1expression; it effectively inhibits Raf-1expression in cells throughconstructing an interference system that the medium of slow virus vectorscan influence Raf-1. After the transfection of miR-195mimics or theintervention of inhibitors on cells, the influences that the two methods have on the cell reproduction or apoptosis are compared, which can verifywhether Raf-1is a direct target of miR-195in breast cells. Throughregulating miR-195in breast cancer cells, it is observed that whether it ispossible to enhance the sensibility of drug-resistant cells to Adriamycinmedicines. By regulating miR-195, it is made possible to detect the proteinexpressions of drug-resistant BCL-2and P-gp protein which are regulatedby Ras/Raf/MEK/ERK signal transduction pathway, to verify that Raf-1protein is a target of miR-195regulation, and to study the relevance ofAdriamycin-resistant cells to the verification mentioned above.
Methods
1. Apply Western-blot and Q-PCR to detect Raf-1protein expression andhas-miR-195expression in breast cancer tissue and breast cancerdrug-resistant tissue. Understand the relations between the two kinds ofexpressions in breast cancer tissue.
2. By gradually increasing the density of ADR (Adriamycin), cultivate themodel of human breast cancer cell (MCF-7) and MCF-7/ADR throughintermittent in vitro induction. Apply MTT to observe vitality of thecells when MCF-7and MCF-7/ADR had been acted on for differenttime by different density of Adriamycin, in order to screen out the mostsuitable interference density and functioning time. Apply FlowCytometry to detect the effect on the cell apoptosis after the two kindsof cells had been under the effect of special density of Adriamycin.
3. Apply Q-PCR to know the special expression of miR-195on humanbreast epithelial cell, breast cancer cell and breast cancer drug-resistantcell strain; Apply Western-blot and immunofluorescent dying to knowexpression distribution of Raf-1in three different cells; ApplyWestern-blot to detect P-gp expression and BCL-2expression in twokinds of cells.
4. Design and synthesize the siRNA interference sequence targeting atRaf-1, construct interfering particles with report gene GFP; after thesuccess of double digestion and particle construction and after slowvirus vectors infecting MCF-7and MCF-7/ADR, apply Western-blot todetect Raf-1expression in the two kinds of cells and analyze itsinterference effect; apply MTT and Flow Cytometry to detect the effecton the cell vitality and apoptosis after the two kinds of cells had beeninterfered by siR-Raf-1.
5. Predict that Raf-1is a target of miR-195effect by bioinformatics;transfect miR-195mimic and transfect MCF-7and MCF-7/ADRthrough miR-195inhibitor; apply Q-PCR to detect the expressionchanges of the two kinds of cells in miR-195; apply Western-blot todetect the changes of Raf-1protein expression in the two kinds of cellsand then to know the effect on Raf-1protein through regulatingmiR-195expression; apply MTT and Flow Cytometry to detect thevariation in cell vitality and apoptosis after the two kinds of cells had transfect miR-195synthetics.
6. Respectively transfect siR-Raf-1interfering particle and miR-195synthetics into MCF-7and MCF-7/ADR; after the effect of specialdensity of Adriamycin medicine and by MTT and Flow Cytometry todetect the variation in cell vitality and apoptosis; compare the changesof sensitivity of breast cancer cells to Adriamycin medicine after the twokinds of interference; apply immunofluorescent dying to observe thedistribution and expression of Raf-1in cells after siR-Raf-1interferingparticles and miR-195synthetics had been transfected; applyWestern-blot to detect the expressions of drug-resistance related proteinsBCL-2and P-gp after the cells had been interfered by siR-Raf-1andmiR-195synthetics, in order to clarify the effect mechanism of miR-195and Raf-1protein kinase in frequent cases that breast cancer is drugresistant.
Results
1. After applying Q-PCR to detect miR-195expression in different tissuesamples, compared with that in normal breast cancer cells, miR195expressions in breast cancer tissues&breast cancer drug-resistanttissues are obviously lower and there is a significant difference(P<0.05); compared with that in cancer tissues, miR-195expression indrug-resistant tissues is obviously lower and there is a significantdifference (P<0.05). Similarly, after applying Western-blot to analyze Raf-1’s different expressions in different tissue samples, compared withthat in normal breast tissues, Raf-1expressions in breast cancer tissues&breast cancer drug-resistant tissues are obviously higher and there is asignificant difference (P<0.05). Notably, compared to that in breastcancer cells, the level of Raf-1expression in drug-resistant tissues istwice higher and the difference has statistics significance which isdifferent from the expression level of miR-195in breast cancer cells.When analyzing whether miR-195’s different expressions are related toRaf-1protein expression, we find that miR-195expression has anegative correlation with Raf-1in breast cancer tissues and thecorrelation rate reaches80%(R=0.8069，P<0.01).
2. When human breast cancer Adriamycin drug-resistant cell strain modelwas successfully constructed, it shows that the drug resistance rate RI ofMCF-7/ADR to MCF-7was10.03; after applying MTT to analyzeMCF-7and MCF-7/ADR which were under the effect of differentdensity of Adriamycin, compared with that of MCF-7/ADR, the cellvitality of MCF-7obviously decreased to a lower level and there was asignificant difference (P<0.05); screen out500ng/ml of Adriamycin toact as an effect for48h and the apoptosis rate of MCF-7was found toincrease up by20%and that of MCF-7/ADR was found to increase onlyby10%after the Adriamycin effect.
3. Compared to that in breast cancer epithelium cell strain (HBL-100), the miR-195expression in MCF-7and MCF-7/ADR cells are obviouslyhigher and the difference has statistics significance (P<0.05); andcompared to that in MCF-7, miR-195in MCF-7/ADR is obviouslyhigher and the difference is significant (P<0.01) which corresponds tothe miR-195expression in tissues; compared to that in breast cancerepithelium cell strain (HBL-100), the Raf-1expressions in MCF-7andMCF-7/ADR cells are obviously higher and the difference has statisticssignificance (P<0.05); it is found through immunofluorescent dying thatRaf-1has strong fluorescent expression both in MCF-7andMCF-7/ADR while the expression is relatively weak in HBL-100; in themeantime, compared to HBL-100, the expressions of drug-resistantrelevant proteins (BCL-2and P-gp) increase significantly and thedifference has statistics significance (P<0.05).
4. After successful construction of two sets of siRNA interference particles(pLenOR-GPH-Raf-1) with reporter gene GFP of which Raf-1is atarget,293T virus, when packaged, can inflict stable infection onMCF-7and MCF-7/ADR cells. By Q-PCR and Western-blot detection,it is found that, in siRNA slow viruses infection group, mRNA&protein expressions of Raf-1are significantly lower than the uninfectedgroup and the control group (P<0.01). The inhibition rates of Raf-1protein in MCF-7and MCF-7/ADR cells are73.3%and68.5%respectively; by applying MTT to analyze the variations of vitality of MCF-7and MCF-7/ADR cells after infected by slow viruses, it is foundthat the vitality of the both kinds of cells are significantly lower than theuninfected group and the control group (P<0.05) and the inhibition ratesare23.4%and16.8%respectively; by FCM detection, it is found that,after infected by siRNA slow virus cells, the rates of cell apoptosis ofMCF-7and MCF-7/ADR increased significantly compared to theuninfected group and the control group and the difference is significant(P<0.05).
5. It shows that Raf-1is a target of miR-195effect by the help fromdifferent bioinformatics websites (PITA, TargetScan and miRanda). Weused transfected and chemically synthesized mature miR-195mimic andimplanted miR-195inhibitor into MCF-7and MCF-7/ADR cells; byQ-PCR detection, it shows that miR-195expression levels in MCF-7and MCF-7/ADR cells rose significantly, namely quintupled and tripled(P<0.01), compared to the untransfected group and the control group.After transfected by miR-195inhibitor, miR-195expression levels inMCF-7and MCF-7/ADR cells did not show a significant decreasecompared to the untransfected group and the control group; by applyingWestern-blot to detect the expression level of Raf-1protein whenMCF-7and MCF-7/ADR cells had been transfected by miR-195synthesizers, it is found that, after transfected by miR-195mimic, theexpression level of Raf-1protein decreased significantly (P<0.05) compared to the control group in which the inhibition rate of Raf-1protein reached68.7%and61.8%respectively while after transfected bymiR-195inhibitor the expression level of Raf-1in both the cells did nothave a significant increase; by MTT analysis of the cell vitality ofMCF-7and MCF-7/ADR transfected by miR-195synthesizers, it isfound that, after transfected by miR-195mimic, the cell vitality of theboth cells decreased significantly (P<0.01) compared to the controlgroup, the inhibition rates of which were25.6%and18.2%respectively;while transfected by miR-195inhibitor, the cell vitality of the both cellsdid not have a significant increase; by applying FCM to observe thevariations of cell apoptosis after MCF-7and MCF-7/ADR cells hadbeen transfected by miR-195synthesizers, it is found that, whentransfected by miR-195mimic, the rates of cell apoptosis rosesignificantly (P<0.05) compared to the control group, which were25.3%and21.4%respectively; while transfected b miR-195inhibitor,the rates of cell apoptosis did not have significant decreases.
6. Transfect siR-Raf-1interference particles and miR-195synthesizers andadd them into MCF-7and MCF-7/ADR cells. under the effect of givendensity of Adriamycin, it is found, from the result of MTT analysis, thatthe vitality of the two kinds of cells, transfected by miR-195mimic orsiR-Raf-1interference particles and being under the effect of ADR,decreased significantly (P<0.05) compared to the control group; while transfected by miR-195inhibitor and being under the effect of ADR, thevitality did not show significant changes; by applying FCM to observeMCF-7and MCF-7/ADR cells transfected by miR-195mimic orsiR-Raf-1interference particles and being under the effect of ADR, therate of cell apoptosis increased significantly (P<0.01) compared to thecontrol group. The rates of cell apoptosis of MCF-7were50%and54%respectively, and those of MCF-7/ADR were40%and42%respectively.It is found through immunofluorescent dying that, after the two kinds ofcells were transfected by miR-195mimic or siR-Raf-1interferenceparticles, the intensity of the fluorescent expression of Raf-1proteindecreased significantly compared to the control group; in the meantimeby Western-blot detection, the expressions of drug-resistance relatedproteins BCL-2and P-gp decreased significantly (P<0.01) compared tothe control group; while transfected by miR-195inhibitor, theexpressions of drug-resistance related proteins BCL-2and P-gp did nothave a significant increase compared to the control group.
Conclusions
1. miR-195and Raf-1have low expressions and over expressions in breastcancer tissues and breast cancer drug-resistant tissues and theexpressions are significantly different from those in normal breasttissues. Those two kinds of expressions are in a negative correlation.miR-195obviously has a lower expression level in drug-resistant tissues than in breast cancer tissues while Raf-1obviously has a higherexpression level.
2. The successful building of the cell strain MCF-7/ADR in human breastcancer cells shows that the strain is drug-resistant and has specialbiological quality, which is a quite ideal cell model in the study of themechanism of breast cancer cells’ resistance to ADR. In the comparisonof the cell apoptosis between MCF-7/ADR and MCF-7, MCF-7/ADRhas a stronger resistance of ADR medication.
3. When breast cancer cell MCF-7and drug-resistant MCF-7/ADR arecompared to breast epithelial cell HBL-100, it shows that miR-195expression has a significant decrease while Raf-1expression has asignificant increase, which is similar to their expressions in breastcancer tissues; in the meantime, in the two kinds of cells, thedrug-resistance related protein BCL-2and P-gp expressions increasesignificantly compared to HBL-100cells and their expressions areobviously higher compared to MCF-7/ADR and MCF-7. The fact showsthat the high expression of Raf-1protein in MCF-7/ADR cells makescells have stronger drug-resistance.
4. By making siRNA interference particles targeting at Raf-1, the mRNAand protein expressions of the target gene Raf-1can be specified andeffectively silenced in MCF-7and MCF-7/ADR cells, and reproductionvitality of both MCF-7and MCF-7/ADR cells was inhibited and the rates of their cell apoptosis was effectively raised.
5. Raf-1is a target of miR-195effect by bioinformatics prediction; bytransfecting miR-195mimic, the expression of miR-195in both MCF-7and MCF-7/ADR cells can be effectively raised and there is asignificant decrease of Raf-1protein expression in the cells, which caneffectively inhibit the reproduction vitality of the cells and increase therates of cell apoptosis. The two kinds of cells, however, did not havesignificant changes after miR-195inhibitor was transfected. Thisindicates that Raf-1is a direct target of miR-195effect in breast cancercells. Increasing miR-195can inhibit Raf-1expression in the cells andinfluence cell reproduction and apoptosis. miR-195’s effect to stimulatecell apoptosis and inhibit cell reproduction of breast cancer cells isfulfilled at least partially by its effect on Raf-1protein.
6. If the cells are transfected by miR-195mimic or siR-Raf-1interferenceparticle and then were added with ADR, the vitality of cell reproductioncan be effectively decreased and the rate of cell apoptosis can besignificantly raised. The transfection also inhibits the expression ofRaf-1protein and significantly decrease the expression quantity of thedrug-resistance related protein BCL-1and P-gp in the cells. The factindicates that Raf-1protein kinase in Ras/Raf/MEK/ERK signalpathway influences the expression of both BCL-2and P-gp which arerelated to drug-resistance. Thus by this way breast cancer cells develop a quality of drug-resistance of ADR chemotherapy. By increasingmiR-195’s regulation effect on its target Raf-1and inhibiting Raf-1expression in the cells, drug-resistant cells can develop its sensibility toADR medication.
乳腺癌是妇女中最为常见的恶性肿瘤，并且也是一个重要的全球性的健康问题。据估计全球每年新增近130万的妇女患有乳腺癌，而与乳腺癌相关的死亡病例每年超过45万人。尽管近年来在乳腺癌的预防筛查，早期检测以及治疗等方面已经明显减少了乳腺癌的发病率和死亡率，并有效提高了乳腺癌患者的存活时间。但是，仍有一定数量的人群被诊断为乳腺癌并最终发展为恶性变。当前的治疗策略结合手术切除与辅助治疗，包括细胞毒性抗癌药物，激素治疗，靶向基因的药物治疗等，然而仍有近50%的病人对于初期的化疗无效，对多种化疗药物产生耐药，包括阿霉素耐药（Adriamycin-resistant ADR）。这种现象被称为多重耐药(Mutiple-drug, MDR)，而多重耐药一直是多种恶性肿瘤化疗失败的重要原因。
目前研究发现产生多重耐药的不同机制，如药物活性的失活，细胞内药物泵出胞外，或者是治疗过程中药物靶点的改变等，但是对肿瘤细胞产生耐药的原因以及相关的作用机制研究还很不完善。在乳腺癌细胞对抗癌药物产生多重耐药的机制中发现，肿瘤的发生和发展存在一种信号转导级联反应，作用于下游的表皮因子生长受体，如EGFR或HER2等。在这些受体信号通路中，Ras/Raf/MEK/ERK在肿瘤细胞耐药，以及细胞增值、分化或转移过程中起着重要作用，其中原癌基因所激活的丝/苏氨酸蛋白激酶Raf-1扮演了重要角色。Raf-1能够磷酸化并激活下游的MEK及ERK蛋白激酶，ERK蛋白激酶协同调控多种蛋白的表达，如多重耐药基因MDR所编码的膜P糖蛋白(P-gpglycoprotein)，细胞抗凋亡蛋白BCL-2，从而对细胞周期，细胞迁移﹑凋亡和分化等过程进行调控。最近研究也表明肿瘤耐药性的产生与微小非编码的RNA（MicroRNAs，miRNAs)的表达变化有关。随着miRNA所介导的关于肿瘤耐药性相关研究成果的积累，现在更多的关注于miRNA的靶向调控作为化疗干预的一个新手段。miRNA涉及到许多重要的细胞调控过程，如细胞的增殖﹑凋亡和分化，造血及器官的形成等。同时miRNA也涉及到许多疾病的产生，包括肿瘤的形成。miRNA作为致癌或抑癌基因取决于所作用的靶基因。miR-195在多种实体肿瘤组织中被发现处于低表达状态，如胃癌﹑结肠癌﹑肝癌等。同时，研究也表明miR-195作为一种抑癌基因也参与到肿瘤细胞的增殖或凋亡过程中。然而，对于miR-195所介导的乳腺癌细胞对抗癌药物阿霉素的获得性耐药目前还没有相关研究
因此，本课题的研究目的拟了解乳腺癌耐药组织或细胞中，miR-195的表达与Raf-1的表达变化是否存在关联性；通过构建慢病毒载体介导针对Raf-1的siRNA干扰体系，有效沉默细胞中Raf-1的表达，同时转染miR-195模拟物或抑制物对细胞进行干预后，进行比较观察两种作用方式对细胞增殖或凋亡的影响，明确乳腺癌细胞中miR-195是否直接靶向调控Raf-1的表达；通过调控乳腺癌细胞中miR-195的表达，观察能否有效提高耐药细胞对阿霉素药物的敏感性，以及通过对miR-195的调控后，检测Ras/Raf/MEK/ERK信号通路所介导的下游靶因子相关蛋白BCL-2和P-gp的表达，确定miR-195调控乳腺癌细胞对阿霉素药物敏感性的作用机制。
方法
1﹑通过Western-blot及Q-PCR技术检测乳腺癌组织及乳腺癌耐药组织中Raf-1蛋白及has-miR-195的表达情况，了解二者在乳腺癌组织中的表达关系。
2﹑采用逐步递增阿霉素(Adriamycin, ADR)浓度，间歇作用体外诱导培养人乳腺癌细胞（MCF-7）构建阿霉素耐药细胞株（MCF-7/ADR）模型；采用MTT比色法观察对MCF-7及MCF-7/ADR细胞经不同浓度阿霉素作用不同时间后，两种细胞的增殖情况，以便筛选出最适的干预浓度和作用时间；采用流式细胞术检测两种细胞经特定浓度的阿霉素作用后，对两种细胞凋亡的影响。
3﹑采用Q-PCR技术了解miR-195在人乳腺上皮细胞（HBL-100）﹑乳腺癌细胞（MCF-7）及乳腺癌耐药细胞株（MCF-7/ADR）中的表达情况；采用Western-blot及免疫荧光染色技术了解Raf-1蛋白在三种细胞中的表达及分布情况；初步了解miR-195和Raf-1在乳腺癌细胞中的表达变化；采用Western-blot技术检测两种细胞中耐药相关蛋白P-gp和BCL-2的表达情况。
4﹑设计合成针对Raf-1的siRNA干扰序列，构建带有报告基因GFP的干扰质粒，双酶切及测序鉴定质粒构建成功后，慢病毒载体感染MCF-7及MCF-7/ADR细胞后，通过Q-PCR筛选有效干扰质粒；采用Western-blot技术检测两种细胞中Raf-1蛋白的表达，分析其干扰效果；采用MTT比色法及流式细胞术检测两种细胞在siR-Raf-1干预后，对细胞增殖及凋亡的影响。
5﹑通过生物信息学预测miR-195对Raf-1的靶向作用；转染化学合成的成熟miR-195模拟物（miR-195mimic）及miR-195抑制物（miR-195inhibitor）进入MCF-7及MCF-7/ADR细胞，采用Q-PCR技术检测两种细胞中miR-195的表达变化；采用Western-blot检测转染miR-195合成物后，两种细胞中Raf-1蛋白的表达变化，了解调控miR-195的表达对细胞中Raf-1蛋白的影响；采用MTT比色法及流式细胞术检测两种细胞在转染miR-195合成物后，对细胞的增殖及凋亡的影响。
6﹑分别转染siR-Raf-1干预质粒和miR-195合成物入MCF-7及MCF-7/ADR细胞，再经特定浓度的阿霉素药物作用，通过MTT比色法及流式细胞术检测两种细胞的增殖及凋亡，比较采用两种方式对细胞进行干预后，乳腺癌细胞对阿霉素药物的敏感性变化，以便了解能否通过调控miR-195的表达，有效提高耐药细胞对阿霉素药物的敏感性；采用免疫荧光染色技术观察在转染siR-Raf-1干预质粒及miR-195合成物后，细胞中Raf-1的表达及分布情况；采用Western-blot检测细胞经siR-Raf-1和miR-195合成物干预后，耐药相关蛋白BCL-2和P-gp的表达，以便明确miR-195与Raf-1蛋白激酶在乳腺癌多耐药发生中的作用机制。
结果
1﹑分别在不同组织样本中采用Q-PCR检测miR-195的表达情况，结果发现在乳腺癌组织及乳腺癌耐药组织中miR-195的表达与正常乳腺组织相比较，其miR-195的表达水平明显更低，表达差异有显著性（P<0.01）；同时，miR-195的表达耐药组织比癌组织明显降低，差异有显著性（P<0.05）。而Raf-1蛋白表达（Western-blot）结果显示，乳腺癌组织及乳腺癌耐药组织比正常乳腺组织明显增高的，差异有显著性（P<0.05）；值得注意的是，耐药组织中Raf-1的表达与乳腺癌组织相比较，其表达水平增高近2倍，差异有统计学意义（P<0.05）；在乳腺癌组织中Raf-1的表达与miR-195的表达呈现出明显的差异性，或许这种表达差异性之间存在着某种关联。因此，我们采用Spearman相关性检验分析两者的差异性表达是否存在关联性，结果发现miR-195与Raf-1蛋白在乳腺癌组织中的表达呈现一种显著的负相关关系，其相关性系数达到80%（R=0.8069，P<0.01）。
2﹑成功构建人乳腺癌阿霉素耐药细胞株模型，MCF-7/ADR相对于MCF-7细胞耐药系数RI为10.03；采用MTT比色法分析MCF-7与MCF-7/ADR两种细胞经不同浓度的阿霉素作用不同时间后细胞增殖变化，结果显示增殖比率MCF-7的细胞比MCF-7/ADR降低更为明显，差异有显著性（P<0.01），并筛选出500ng/ml的最适阿霉素作用浓度和48h的作用时间；采用FCM技术分析500ng/ml浓度阿霉素作用前后两种细胞的凋亡率，可见MCF-7经ADR作用后其细胞凋亡比率提高20%，而MCF-7/ADR细胞仅仅提高近10%。
3﹑在MCF-7与MCF-7/ADR细胞中miR-195的表达与HBL-100相比，其miR-195的表达量明显更高，差异有统计学意义（P<0.05），并且MCF-7/ADR与MCF-7相比，miR-195的表达也明显更高，差异有显著性（P<0.01），这与组织中miR-195的表达相符合；MCF-7/ADR与MCF-7两种细胞中Raf-1蛋白的表达与HBL-100相比，其表达均显著上调，差异有统计学意义（P<0.05）；免疫荧光染色也发现Raf-1的荧光表达在MCF-7/ADR与MCF-7中均有较强的荧光表达，而在HBL-100中Raf-1的荧光表达相对较弱；同时在MCF-7/ADR与MCF-7中，发现耐药相关蛋白BCL-2和P-gp的表达与HBL-100相比，表达水平也是显著增加，差异有统计学意义（P<0.01）。
4﹑成功构建针对Raf-1的带有报告基因GFP的两套siRNA干扰质粒pLenR-GPH-Raf-1，经包装293T细胞产毒后能够稳定感染MCF-7和MCF-7/ADR细胞。经Q-PCR及Western-blot检测，结果显示siRNA慢病毒感染组中Raf-1的mRNA和蛋白表达量均显著低于未转染组和对照组（P<0.01），在MCF-7和MCF-7/ADR两种细胞中Raf-1蛋白的抑制比率分别达到73.3%和68.5%；采用MTT比色法分析MCF-7与MCF-7/ADR两种细胞经siRNA慢病毒感染后细胞的增殖变化，结果显示在感染后两种细胞的增殖均显著低于未转染组和对照组（P<0.01），抑制比率分别为23.4%和16.8%；采用FCM技术检测经siRNA慢病毒感染细胞，两种细胞的凋亡比率与对照组相比均明显增高，凋亡率分别提高在MCF-7中为15%和MCF-7/ADR中为12%，差异有显著性（P<0.01）。
5﹑采用不同的生物信息学网址（PITA,TargetScan及miRanda）对miR-195是否靶向作用于Raf-1进行分析，均发现miR-195靶向作用于Raf-1；转染化学合成的成熟miR-195模拟物（miR-195mimic）及miR-195抑制物（miR-195inhibitor）入MCF-7及MCF-7/ADR两种细胞，经Q-PCR检测发现，MCF-7与MCF-7/ADR两种细胞经转染miR-195mimic后，细胞中miR-195的表达水平与未转染组和对照组相比均明显提高，分别升高近5倍和3倍（P<0.01），而转染miR-195抑制物后，两种细胞中的miR-195表达水平与未转染组和对照组相比降低不明显；采用Western-blot检测MCF-7与MCF-7/ADR两种细胞在转染miR-195合成物后Raf-1蛋白的表达水平，结果发现在转染miR-195mimic后，细胞中Raf-1蛋白的表达水平与对照组相比均显著降低（P<0.01），其Raf-1蛋白的抑制比率分别达到68.7%和61.8%，而转染miR-195inhibitor物后，两种细胞中Raf-1的表达没有明显提高；MTT比色法分析MCF-7与MCF-7/ADR两种细胞经转染miR-195合成物后，细胞的增殖变化，结果发现在转染miR-195mimic后，两种细胞的增殖比率与对照组相比均明显降低（P<0.05），抑制比率分别为25.6%和18.2%，而转染miR-195inhibitor后，细胞的增殖并没有明显增加；采用FCM技术观察转染miR-195合成物后细胞的凋亡情况，结果发现MCF-7与MCF-7/ADR两种细胞经miR-195mimic转染后，细胞的凋亡比率显著增加（P<0.05），细胞凋亡比率分别达到25.3%和21.4%，而在转染miR-195inhibitor后细胞凋亡比率与对照组相比，并没有明显变化。
6﹑分别转染siR-Raf-1干扰质粒和miR-195合成物入MCF-7及MCF-7/ADR细胞，再经特定浓度的阿霉素药物作用，MTT比色法分析细胞活力，结果发现在转染miR-195mimic或siR-Raf-1干扰质粒后，细胞再经ADR作用，两种细胞的增殖与对照组相比明显降低（P<0.01），而在转染miR-195inhibitor并经ADR作用后，两种细胞的活力并没有明显变化；采用FCM技术观察MCF-7及MCF-7/ADR细胞转染miR-195mimic或siR-Raf-1干扰质粒后，并经ADR作用，细胞凋亡比率与对照组相比明显增加（P<0.01），其中MCF-7及MCF-7/ADR细胞在转染miR-195mimic后细胞凋亡比率分别达到52.6%和41.2%，与采用ADR药物处理的对照组相比，两种细胞的凋亡比率均提高近20%；同时染siR-Raf-1干扰抑制MCF-7及MCF-7/ADR两种细胞中Raf-1蛋白的表达，也发现与转染miR-195mimic具有相似的效果，两种细胞的凋亡比率明显升高，其凋亡比率分别达到50.3%和40.7%；采用免疫荧光染色观察两种细胞在转染miR-195mimic或siR-Raf-1干扰质粒后，细胞中Raf-1蛋白的荧光表达，结果可见转染后Raf-1蛋白的荧光表达强度与对照组相比明显降低；同时采用Western-blot检测细胞在转染siR-Raf-1干扰质粒和miR-195mimic后，细胞中耐药相关蛋白BCL-2和P-gp的表达与对照组相比，其表达量是明显降低（P<0.01），而两种细胞在转染miR-195inhibitor后，BCL-2和P-gp蛋白的表达与对照组相比没有明显的增加。
结论
1﹑miR-195和Raf-1在乳腺癌组织及乳腺癌耐药组织中分别处于低表达和过表达状态，其表达与正常乳腺组织相比均呈显著差异，且二者的差异性表达呈现显著的负相关性，同时耐药组织与乳腺癌组织相比miR-195的表达明显更低，而Raf-1的表达则明显更高，差异有显著性(P<0.01)。
2﹑建立了人乳腺癌耐阿霉素药物细胞株（MCF-7/ADR），该细胞株具有耐药细胞株的耐药性和生物学特性，是研究乳腺癌细胞对ADR耐药机制较为理想的细胞模型，其MCF-7/ADR细胞与MCF-7细胞的对ADR药物的耐受性比较，MCF-7/ADR细胞对ADR药物作用的耐受性更强。
3﹑MCF-7及MCF-7/ADR细胞与HBL-100细胞相比，miR-195处于明显低表达，而Raf-1的表达则明显上调，这与乳腺癌组织中miR-195和Raf-1的表达相似；同时在两种细胞中耐药相关蛋白BCL-2和P-gp的表达与HBL-100细胞相比明显更高，而在MCF-7/ADR与MCF-7细胞间比较，MCF-7/ADR中这两种蛋白的表达量也明显更高，说明由于MCF-7/ADR细胞中Raf-1蛋白的上调或许使细胞具有更强的耐药性。
4﹑构建针对Raf-1的siRNA干扰质粒可以分别在MCF-7和MCF-7/ADR细胞中特异并有效沉默目的基因Raf-1的mRNA及蛋白表达，并同时能够抑制两种细胞的增殖活力，有效提高两种细胞的凋亡比率。
5﹑通过生物信息学预测发现miR-195对Raf-1具有靶向作用；转染miR-195mimic能够有效上调miR-195在两种细胞中的表达，同时引起Raf-1蛋白在细胞的表达显著下降；并且能够有效抑制细胞的增殖活力，明显提高两种细胞的凋亡比率。说明在乳腺癌细胞中miR-195对Raf-1具有直接的靶向调控作用，通过上调miR-195能够抑制Raf-1在细胞中的表达， miR-195作为抑癌基因，对乳腺癌细胞的促凋亡和抑制增殖的功能部分或至少是通过作用于Raf-1蛋白来实现。
6﹑通过转染miR-195mimic入阿霉素作用的MCF-7及MCF-7/ADR细胞，上调细胞中miR-195的表达，结果显示与单独采用阿霉素处理的对照组细胞相比，细胞的增殖明显减弱，同时两种细胞的凋亡明显增加。另外，我们也采用RNAi干扰抑制MCF-7及MCF-7/ADR两种细胞中Raf-1蛋白的表达，也发现具有相似的效果，两种细胞的增殖活力也呈现明显受抑，而细胞的凋亡比率明显升高。证实上调细胞中miR-195的表达能够有效提高乳腺癌耐药细胞对阿霉素药物的敏感性。并且上调miR-195在细胞中的表达水平，不仅抑制细胞中Raf-1的表达，而且也促使细胞中的耐药相关蛋白P-gp和Bcl-2蛋白的表达降低，说明miR-195对提高乳腺癌耐药细胞药物敏感性的作用机制很可能是通过调控Raf-1蛋白激酶在细胞中的表达，影响下游靶激酶导致转录因子影响下游靶激酶导致转录因子的磷酸化，从而减少这些转录因子对P-gp和Bcl-2基因的启动子序列的刺激作用，降低基因的转录，使乳腺癌细胞中P-gp和Bcl-2蛋白的表达相应下调，从而减少了细胞的抗凋亡活性和对药物的转运能力，明显提高乳腺癌细胞对阿霉素化疗药物的敏感性。
Background and Objectives
Breast cancer is the most common malignancy in women and is asignificant worldwide health problem, accounting for approximately1.3million new cancer cases and more than450,000cancer-related deathsannually in the world. Although recent advancements in prevention,screening, early detection and treatments for breast cancer have thepotential to reduce mortality, still a large number of patients are diagnosedat advanced stages. Current treatment strategies combines surgical removalwith adjuvant therapy including cytotoxic anticancer drugs, hormonaltherapy, targeted drugs. However, nearly50%of such patients frequentlydevelop multidrug resistance ((Mutiple-drug, MDR) to chemotherapeuticagents during the course of treatment.It is estimated that one of two breastcancer patients will fail to respond to initial treatments or will rapidlyacquire resistance to multiple-drugs.
Previous studies have demonstrated different mechanisms responsiblefor drug resistance, such as drug inactivation, extrusion of the drug by enhanced drug efflux pumps, or changes in drug target during the course oftreatment. The precise nature of chemotherapy resistance, and theunderlying mechanism of the acquisition of drug resistance are still poorlyunderstood. One of the various mechanisms have been studied in breastcancer MDR to anticancer agents as well as cancer development is signaltransduction cascades downstream of epidermal growth factor (EGF)receptor (EGFR) isoforms (e.g., EGFR&HER2). Among the signalingpathways downstream of these receptors, the Ras/Raf/MEK/ERK pathwayshave been shown to play a critical role in chemotherapeutic drug resistanceas well as cancer differentiation, cellular transformation and, cellularsenescence and metastasis. In this signal transduction cascade,proto-oncogene serine/threonine-protein kinase (Raf-1) plays a veryimportant role. Once activated, Raf-1will phosphorylate and activateMEK1and MEK2protein kinases and then in turn, phosphorylate andactivate the serine/threonine-specific protein kinases ERK1and ERK2tocontrol expression of various genes (such as BCl-2and P-glycoprotein).
Recent studies have also linked the acquisition of cancer drugresistance to altered expression of microRNAs, which are a novel class ofendogenous non-coding small RNA and function in posttranscriptionalregulation of the target gene expressions by binding to the target mRNA toinhibit its translation and/or to degrade the target mRNA molecules.Evidence of miRNA-mediated drug resistance is accumulating, and much attention is now being focused on targeting miRNAs as a novel strategy fortherapeutic intervention. MicroRNAs (miRNAs) are an abundant class ofshort noncoding RNAs,18–25nt in length, that act as potent regulators ofgene expression. They have been implicated in most major cellularprocesses including proliferation, apoptosis, developmental timing,hematopoiesis and organogenesis. Therefore, it is not surprising thatmiRNAs have also already been linked to a number of diseases includingcancer. microRNAs function as onco-miRs or tumor-suppressor-miRsdepending on their respective target genes. miR-195has been found to bedownregulated in a variety of cancers, including gastric cancer, liver cancer,bladder cancer, and adrenocortical cancer.(31-34).miR-195was also foundsignificantly downregulated in breast cancer cells and in clinical specimensafter surgery(35,36). Li et al also found that overexpression of miR-195was found to be correlated inversely with malignancy of breast cancer,suppressing breast cancer cell proliferation and invasion. Raf-1wereidentified as novel direct targets of miR-195as a major mechanism.
The project is targeted on whether, in drug-resistant tissues or cells, thechanges of miR-195expression are related to the changes of Raf-1expression; it effectively inhibits Raf-1expression in cells throughconstructing an interference system that the medium of slow virus vectorscan influence Raf-1. After the transfection of miR-195mimics or theintervention of inhibitors on cells, the influences that the two methods have on the cell reproduction or apoptosis are compared, which can verifywhether Raf-1is a direct target of miR-195in breast cells. Throughregulating miR-195in breast cancer cells, it is observed that whether it ispossible to enhance the sensibility of drug-resistant cells to Adriamycinmedicines. By regulating miR-195, it is made possible to detect the proteinexpressions of drug-resistant BCL-2and P-gp protein which are regulatedby Ras/Raf/MEK/ERK signal transduction pathway, to verify that Raf-1protein is a target of miR-195regulation, and to study the relevance ofAdriamycin-resistant cells to the verification mentioned above.
Methods
1. Apply Western-blot and Q-PCR to detect Raf-1protein expression andhas-miR-195expression in breast cancer tissue and breast cancerdrug-resistant tissue. Understand the relations between the two kinds ofexpressions in breast cancer tissue.
2. By gradually increasing the density of ADR (Adriamycin), cultivate themodel of human breast cancer cell (MCF-7) and MCF-7/ADR throughintermittent in vitro induction. Apply MTT to observe vitality of thecells when MCF-7and MCF-7/ADR had been acted on for differenttime by different density of Adriamycin, in order to screen out the mostsuitable interference density and functioning time. Apply FlowCytometry to detect the effect on the cell apoptosis after the two kindsof cells had been under the effect of special density of Adriamycin.
3. Apply Q-PCR to know the special expression of miR-195on humanbreast epithelial cell, breast cancer cell and breast cancer drug-resistantcell strain; Apply Western-blot and immunofluorescent dying to knowexpression distribution of Raf-1in three different cells; ApplyWestern-blot to detect P-gp expression and BCL-2expression in twokinds of cells.
4. Design and synthesize the siRNA interference sequence targeting atRaf-1, construct interfering particles with report gene GFP; after thesuccess of double digestion and particle construction and after slowvirus vectors infecting MCF-7and MCF-7/ADR, apply Western-blot todetect Raf-1expression in the two kinds of cells and analyze itsinterference effect; apply MTT and Flow Cytometry to detect the effecton the cell vitality and apoptosis after the two kinds of cells had beeninterfered by siR-Raf-1.
5. Predict that Raf-1is a target of miR-195effect by bioinformatics;transfect miR-195mimic and transfect MCF-7and MCF-7/ADRthrough miR-195inhibitor; apply Q-PCR to detect the expressionchanges of the two kinds of cells in miR-195; apply Western-blot todetect the changes of Raf-1protein expression in the two kinds of cellsand then to know the effect on Raf-1protein through regulatingmiR-195expression; apply MTT and Flow Cytometry to detect thevariation in cell vitality and apoptosis after the two kinds of cells had transfect miR-195synthetics.
6. Respectively transfect siR-Raf-1interfering particle and miR-195synthetics into MCF-7and MCF-7/ADR; after the effect of specialdensity of Adriamycin medicine and by MTT and Flow Cytometry todetect the variation in cell vitality and apoptosis; compare the changesof sensitivity of breast cancer cells to Adriamycin medicine after the twokinds of interference; apply immunofluorescent dying to observe thedistribution and expression of Raf-1in cells after siR-Raf-1interferingparticles and miR-195synthetics had been transfected; applyWestern-blot to detect the expressions of drug-resistance related proteinsBCL-2and P-gp after the cells had been interfered by siR-Raf-1andmiR-195synthetics, in order to clarify the effect mechanism of miR-195and Raf-1protein kinase in frequent cases that breast cancer is drugresistant.
Results
1. After applying Q-PCR to detect miR-195expression in different tissuesamples, compared with that in normal breast cancer cells, miR195expressions in breast cancer tissues&breast cancer drug-resistanttissues are obviously lower and there is a significant difference(P<0.05); compared with that in cancer tissues, miR-195expression indrug-resistant tissues is obviously lower and there is a significantdifference (P<0.05). Similarly, after applying Western-blot to analyze Raf-1’s different expressions in different tissue samples, compared withthat in normal breast tissues, Raf-1expressions in breast cancer tissues&breast cancer drug-resistant tissues are obviously higher and there is asignificant difference (P<0.05). Notably, compared to that in breastcancer cells, the level of Raf-1expression in drug-resistant tissues istwice higher and the difference has statistics significance which isdifferent from the expression level of miR-195in breast cancer cells.When analyzing whether miR-195’s different expressions are related toRaf-1protein expression, we find that miR-195expression has anegative correlation with Raf-1in breast cancer tissues and thecorrelation rate reaches80%(R=0.8069，P<0.01).
2. When human breast cancer Adriamycin drug-resistant cell strain modelwas successfully constructed, it shows that the drug resistance rate RI ofMCF-7/ADR to MCF-7was10.03; after applying MTT to analyzeMCF-7and MCF-7/ADR which were under the effect of differentdensity of Adriamycin, compared with that of MCF-7/ADR, the cellvitality of MCF-7obviously decreased to a lower level and there was asignificant difference (P<0.05); screen out500ng/ml of Adriamycin toact as an effect for48h and the apoptosis rate of MCF-7was found toincrease up by20%and that of MCF-7/ADR was found to increase onlyby10%after the Adriamycin effect.
3. Compared to that in breast cancer epithelium cell strain (HBL-100), the miR-195expression in MCF-7and MCF-7/ADR cells are obviouslyhigher and the difference has statistics significance (P<0.05); andcompared to that in MCF-7, miR-195in MCF-7/ADR is obviouslyhigher and the difference is significant (P<0.01) which corresponds tothe miR-195expression in tissues; compared to that in breast cancerepithelium cell strain (HBL-100), the Raf-1expressions in MCF-7andMCF-7/ADR cells are obviously higher and the difference has statisticssignificance (P<0.05); it is found through immunofluorescent dying thatRaf-1has strong fluorescent expression both in MCF-7andMCF-7/ADR while the expression is relatively weak in HBL-100; in themeantime, compared to HBL-100, the expressions of drug-resistantrelevant proteins (BCL-2and P-gp) increase significantly and thedifference has statistics significance (P<0.05).
4. After successful construction of two sets of siRNA interference particles(pLenOR-GPH-Raf-1) with reporter gene GFP of which Raf-1is atarget,293T virus, when packaged, can inflict stable infection onMCF-7and MCF-7/ADR cells. By Q-PCR and Western-blot detection,it is found that, in siRNA slow viruses infection group, mRNA&protein expressions of Raf-1are significantly lower than the uninfectedgroup and the control group (P<0.01). The inhibition rates of Raf-1protein in MCF-7and MCF-7/ADR cells are73.3%and68.5%respectively; by applying MTT to analyze the variations of vitality of MCF-7and MCF-7/ADR cells after infected by slow viruses, it is foundthat the vitality of the both kinds of cells are significantly lower than theuninfected group and the control group (P<0.05) and the inhibition ratesare23.4%and16.8%respectively; by FCM detection, it is found that,after infected by siRNA slow virus cells, the rates of cell apoptosis ofMCF-7and MCF-7/ADR increased significantly compared to theuninfected group and the control group and the difference is significant(P<0.05).
5. It shows that Raf-1is a target of miR-195effect by the help fromdifferent bioinformatics websites (PITA, TargetScan and miRanda). Weused transfected and chemically synthesized mature miR-195mimic andimplanted miR-195inhibitor into MCF-7and MCF-7/ADR cells; byQ-PCR detection, it shows that miR-195expression levels in MCF-7and MCF-7/ADR cells rose significantly, namely quintupled and tripled(P<0.01), compared to the untransfected group and the control group.After transfected by miR-195inhibitor, miR-195expression levels inMCF-7and MCF-7/ADR cells did not show a significant decreasecompared to the untransfected group and the control group; by applyingWestern-blot to detect the expression level of Raf-1protein whenMCF-7and MCF-7/ADR cells had been transfected by miR-195synthesizers, it is found that, after transfected by miR-195mimic, theexpression level of Raf-1protein decreased significantly (P<0.05) compared to the control group in which the inhibition rate of Raf-1protein reached68.7%and61.8%respectively while after transfected bymiR-195inhibitor the expression level of Raf-1in both the cells did nothave a significant increase; by MTT analysis of the cell vitality ofMCF-7and MCF-7/ADR transfected by miR-195synthesizers, it isfound that, after transfected by miR-195mimic, the cell vitality of theboth cells decreased significantly (P<0.01) compared to the controlgroup, the inhibition rates of which were25.6%and18.2%respectively;while transfected by miR-195inhibitor, the cell vitality of the both cellsdid not have a significant increase; by applying FCM to observe thevariations of cell apoptosis after MCF-7and MCF-7/ADR cells hadbeen transfected by miR-195synthesizers, it is found that, whentransfected by miR-195mimic, the rates of cell apoptosis rosesignificantly (P<0.05) compared to the control group, which were25.3%and21.4%respectively; while transfected b miR-195inhibitor,the rates of cell apoptosis did not have significant decreases.
6. Transfect siR-Raf-1interference particles and miR-195synthesizers andadd them into MCF-7and MCF-7/ADR cells. under the effect of givendensity of Adriamycin, it is found, from the result of MTT analysis, thatthe vitality of the two kinds of cells, transfected by miR-195mimic orsiR-Raf-1interference particles and being under the effect of ADR,decreased significantly (P<0.05) compared to the control group; while transfected by miR-195inhibitor and being under the effect of ADR, thevitality did not show significant changes; by applying FCM to observeMCF-7and MCF-7/ADR cells transfected by miR-195mimic orsiR-Raf-1interference particles and being under the effect of ADR, therate of cell apoptosis increased significantly (P<0.01) compared to thecontrol group. The rates of cell apoptosis of MCF-7were50%and54%respectively, and those of MCF-7/ADR were40%and42%respectively.It is found through immunofluorescent dying that, after the two kinds ofcells were transfected by miR-195mimic or siR-Raf-1interferenceparticles, the intensity of the fluorescent expression of Raf-1proteindecreased significantly compared to the control group; in the meantimeby Western-blot detection, the expressions of drug-resistance relatedproteins BCL-2and P-gp decreased significantly (P<0.01) compared tothe control group; while transfected by miR-195inhibitor, theexpressions of drug-resistance related proteins BCL-2and P-gp did nothave a significant increase compared to the control group.
Conclusions
1. miR-195and Raf-1have low expressions and over expressions in breastcancer tissues and breast cancer drug-resistant tissues and theexpressions are significantly different from those in normal breasttissues. Those two kinds of expressions are in a negative correlation.miR-195obviously has a lower expression level in drug-resistant tissues than in breast cancer tissues while Raf-1obviously has a higherexpression level.
2. The successful building of the cell strain MCF-7/ADR in human breastcancer cells shows that the strain is drug-resistant and has specialbiological quality, which is a quite ideal cell model in the study of themechanism of breast cancer cells’ resistance to ADR. In the comparisonof the cell apoptosis between MCF-7/ADR and MCF-7, MCF-7/ADRhas a stronger resistance of ADR medication.
3. When breast cancer cell MCF-7and drug-resistant MCF-7/ADR arecompared to breast epithelial cell HBL-100, it shows that miR-195expression has a significant decrease while Raf-1expression has asignificant increase, which is similar to their expressions in breastcancer tissues; in the meantime, in the two kinds of cells, thedrug-resistance related protein BCL-2and P-gp expressions increasesignificantly compared to HBL-100cells and their expressions areobviously higher compared to MCF-7/ADR and MCF-7. The fact showsthat the high expression of Raf-1protein in MCF-7/ADR cells makescells have stronger drug-resistance.
4. By making siRNA interference particles targeting at Raf-1, the mRNAand protein expressions of the target gene Raf-1can be specified andeffectively silenced in MCF-7and MCF-7/ADR cells, and reproductionvitality of both MCF-7and MCF-7/ADR cells was inhibited and the rates of their cell apoptosis was effectively raised.
5. Raf-1is a target of miR-195effect by bioinformatics prediction; bytransfecting miR-195mimic, the expression of miR-195in both MCF-7and MCF-7/ADR cells can be effectively raised and there is asignificant decrease of Raf-1protein expression in the cells, which caneffectively inhibit the reproduction vitality of the cells and increase therates of cell apoptosis. The two kinds of cells, however, did not havesignificant changes after miR-195inhibitor was transfected. Thisindicates that Raf-1is a direct target of miR-195effect in breast cancercells. Increasing miR-195can inhibit Raf-1expression in the cells andinfluence cell reproduction and apoptosis. miR-195’s effect to stimulatecell apoptosis and inhibit cell reproduction of breast cancer cells isfulfilled at least partially by its effect on Raf-1protein.
6. If the cells are transfected by miR-195mimic or siR-Raf-1interferenceparticle and then were added with ADR, the vitality of cell reproductioncan be effectively decreased and the rate of cell apoptosis can besignificantly raised. The transfection also inhibits the expression ofRaf-1protein and significantly decrease the expression quantity of thedrug-resistance related protein BCL-1and P-gp in the cells. The factindicates that Raf-1protein kinase in Ras/Raf/MEK/ERK signalpathway influences the expression of both BCL-2and P-gp which arerelated to drug-resistance. Thus by this way breast cancer cells develop a quality of drug-resistance of ADR chemotherapy. By increasingmiR-195’s regulation effect on its target Raf-1and inhibiting Raf-1expression in the cells, drug-resistant cells can develop its sensibility toADR medication.
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