miR-200c靶向UBQLN1调节自噬及乳腺癌的辐射抵抗
摘要
研究背景及目的：
乳腺癌是全球女性中最常见的恶性肿瘤,严重威胁着女性的健康问题。对于大多数接受保乳术的乳腺癌患者来说,放射治疗是主要的治疗手段之一,且具有显著的临床获益,如减少肿瘤局部复发的风险,降低乳腺癌的死亡率。然而,对于一些特定的肿瘤亚型,如基底样癌,肿瘤的局控率仍然很低。对于这一类型的乳腺癌患者来说,治疗失败的主要原因可能是辐射抵抗。因此,阐明乳腺癌辐射抵抗的分子机制可对今后提高乳腺癌的临床治疗效果提供很大帮助。
自噬是细胞内的一种“自食”现象,是一种细胞自身代谢降解的过程,在这一过程中,细胞质成分螯合入一种称为自噬小体(autophagosome)的双膜囊泡中,随后运送至溶酶体进行降解,其中的大分子和细胞器得以进行再循环利用,从而维持细胞的完整性。自噬在正常生理状态下对于维持细胞稳态和细胞器的更新有重要的作用,自噬缺陷与许多人类疾病相关,包括神经退行性疾病和肿瘤。近年来越来越多的研究关注自噬在人类疾病中的作用及其确切的分子机制。最近的研究发现,自噬是肿瘤细胞在如缺氧、营养剥夺、化疗、放疗等各种应激性条件下得以存活的一个重要适应性反应,这也因此引起了肿瘤细胞对治疗的抵抗。然而,在乳腺癌中,自噬介导乳腺癌辐射抵抗的具体分子机制目前仍然不太清楚。
目前的研究已经证实,MicroRNAs (miRNAs)参与调节多种生物学进程,包括细胞增殖、分化及迁移和侵袭。miRNA的表达失调可导致多种人类疾病的发生,其中包括肿瘤。最近的研究表明,miRNAs在自噬过程中可发挥重要的调节作用。有些研究也报道了几种miRNAs可通过调节自噬进程参与一些肿瘤的化疗或辐射抵抗。然而在乳腺癌中,有关自噬在miRNA介导的辐射抵抗中的作用及机制仍不甚明了。此前的研究表明,miR-200家族可参与调节肿瘤干细胞的自我更新、上皮-间质转化(EMT)和化疗抵抗。最近的研究发现,miR-200家族的重要成员miR-200c可通过靶向抑制TBKl和VEGFR2基因的表达来调节肿瘤细胞的辐射敏感性。然而,miR-200c和自噬的调节在乳腺癌辐射抵抗中的关系仍不清楚。
本研究中发现miR-200c可通过抑制辐射诱导的自噬反应来提高乳腺癌细胞对辐射的敏感性。本研究还确定了ubiquilin1蛋白(UBQLN1)是miR-200c的直接靶基因,其表达水平与辐射抵抗及辐射诱导的自噬活性呈正相关。本研究研究为miR-200c在乳腺癌辐射增敏作用中的分子机制提供了新的见解,为miR-200c用于乳腺癌的治疗提供了新的理论依据。
方法：
1.乳腺癌细胞中miR-200c的检测
正常乳腺上皮细胞株MCF-10A,基底型乳腺癌细胞株MDA-MB-231、BT549和管腔型乳腺癌细胞株MCF-7、BT474细胞,依据Trizol Reagent试剂的说明书进行操作,提取细胞总RNA；按照PrimeScript RT reagent kit说明书进行逆转录反应,获得cDNA;以cDNA为模板,采用茎环法依据SYBR Premix Ex Taq Ⅱ试剂盒说明书进行荧光定量PCR反应以检测这5株细胞中miR-200c的表达水平。
2. miR-200c mimic,inhibitor及siRNA和plasmid的转染
采用阳离子脂质体法进行转染,按照lipofectamine2000操作说明进行操作,采用无血清培养基培养细胞,当细胞融合度达到70%左右时,将mimic,inhibitor,siRNA或plasmid稀释于转染专用培养基Opti-MEMI Reduced Serum Medium中,lipofectamine2000同样用Opti-MEMI Reduced Serum Medium稀释后,与mimic, inhibitor, siRNA或plasmid溶液混合,室温下静置20min后,加入培养板中。继续在培养箱中孵育4-6h后,将培养基换成含有血清的完全培养基,转染完成,48h后提取细胞的RNA或蛋白或进行后续的处理。为有效抑制niR-200c的功能簇,miR-200c inhibitor需与miR-429inhibitor同时转染。
3.细胞辐射及克隆形成实验
细胞经消化制成单细胞悬液后,按照预定数量接种于6孔板中,设0,2,4,6,8五个剂量组,每个剂量组三个复孔。照射后将细胞置于37℃、5%C02、饱和湿度培养箱内继续培养12-14天,期间根据培养液pH值变化适时更换新鲜培养液。当培养板孔中出现肉眼可见克隆时,终止培养,弃去培养液,PBS清洗细胞2遍,甲醇固定,1%结晶紫乙醇溶液染色,显微镜下计数含50个细胞以上的克隆数,计算克隆形成率和存活分数,并运用GraphPad Prism5.0软件进行多靶单击模型曲线拟合,并计算辐射增敏因子SER10。
4. Western blot实验
处理后的细胞提取总蛋白,按照BCA法测定蛋白浓度,加入5×SDS-PAGE loading Buffer进行蛋白变性,然后经电泳、转膜、封闭后,孵育一抗和二抗,最后采用ECL法在显影仪上进行显影。
5.构建稳定表达GFP-LC3的MDA-MB-231细胞
将5×105/孔的细胞接种于6孔板中,培养12h待细胞密度达40%左右,加入MAP1LC3B和对照LV5-NC慢病毒悬液100μl/孔(MOI=20),并设2孔加入终浓度为6ug/ml的polybrene以增加感染效率,24h后更换培养基,72h后在荧光显微镜下拍照并计算感染效率。随后采用流式细胞仪分选GFP+细胞,以获得比例较高的GFP+细胞扩大培养,得到稳定表达GFP-LC3的细胞。
6.生物信息学分析预测miR-200c调节自噬的靶基因
利用三大常用miRNA数据库TargetScan,Pictar和miRBase对miR-200c可能的靶基因进行预测分析,将这三个数据库预测到的结果取交集以提高可信度,然后在基因功能分析网站上查询这些基因的功能,并且在Pubmed里进行检索查询这些靶基因的功能是否与自噬相关。
7.检测乳腺癌细胞中miR-200c过表达后UBQLN1的表达
在乳腺癌细胞中通过转染miR-200c mimic或scramble negative control后,通过荧光定量PCR和Western blot检测细胞中miR-200c过表达后UBQLN1的mRNA和蛋白的表达变化。
8.双荧光素酶报告基因检测法验证miR-200c的靶基因
依据阳离子脂质体法将包含miR-200c假定结合位点的UBQLN13'UTR以及突变型UBQLN1的3'UTR的萤光素酶报告质粒和]miR-200c或scramble negative control共同转染乳腺癌细胞。转染完成48h后,依据Promega公司的Dual-Luciferase报告基因检测系统进行样本的萤光素酶活性检测,按照试剂盒说明书进行操作,以萤火虫萤光素酶的活性作为内参,依据海肾萤光／萤火虫萤光的比值(Renilla/Firefly)分析miR-200c抑制UBQLN1的效果。
9.恢复实验证实UBQLN1是miR-200c的功能性靶基因
依据阳离子脂质体法将miR-200c mimic和CMV-UBQLN1plasmid共转染乳腺癌MDA-MB-231细胞。转染完成48h后,Western blot检测转染效率,并进行克隆形成实验检测细胞辐射敏感性的变化,Western blot检测辐射后细胞自噬活性的改变。
10.乳腺癌组织中UBQLN1和LC3的检测
收集乳腺癌组织及瘤周正常组织标本35例,经病理确诊全为浸润性乳腺癌,经4%多聚甲醛固定、包埋、切片后进行免疫组化染色检测组织中UBQLN1和LC3的表达水平。
11.乳腺癌组织中miR-200c的检测
上述乳腺癌组织及瘤周正常组织标本,采用原位杂交的方法检测组织中miR-200c的表达水平。
12.统计分析
采用SPSS13.0软件进行统计分析,两样本均数的比较采用两独立样本的t检验(Independent-Sample T Test)和单样本t检验,两组以上样本均数比较采用完全随机设计资料的方差分析(One-Way ANOVA),方差齐性检验采用Levene's Test。临床标本分析采用Pearson卡方检验和Fisher确切概率法。P值<0.05认为有统计学意义。
结果：
1.乳腺癌细胞株中miR-200c的表达水平
荧光定量PCR结果显示,5株细胞间miR-200c的表达水平有统计学差异(F=130.182, P<0.001), miR-200c在2株管腔型乳腺癌细胞(MCF-7、BT474)中的表达水平高于乳腺正常上皮细胞MCF-10A,但差异无统计学意义(P值分别为0.169和0.696),而在基底型乳腺癌细胞中(MDA-MB-231、BT549), miR-200c的表达水平与MCF-10A相比有统计学差异(P值均为0.026),MDA-MB-231和BT549细胞中miR-200c的表达水平低于MCF-10A。
2. miR-200c增强乳腺癌细胞的辐射敏感性
通过在MDA-MB-231及BT549细胞中过表达miR-200c、在MCF-7细胞中沉默miR-200c的表达,来研究miR-200c对乳腺癌细胞辐射敏感性的影响。结果发现,在MDA-MB-231及BT549细胞中过表达miR-200c后,细胞经辐射后存活分数有所降低,而在MCF-7细胞中敲出miR-200c的表达后,细胞经辐射后的存活分数有所增高,表明miR-200c的过表达可提高乳腺癌的辐射敏感性。
3.辐射可诱导乳腺癌细胞自噬反应
Western blot结果显示,乳腺癌MDA-MB-231和BT549细胞经X线照射后,随着时间的延长,自噬相关蛋白LC3-Ⅱ的表达有所增高,p62蛋白的降解有所下降。这些结果说明辐射诱导了乳腺癌细胞自噬的发生。但在MCF-7细胞中,这种自噬活性的改变没有MDA-MB-231和BT549细胞中明显。
4.自噬在乳腺癌辐射中起保护作用
乳腺癌细胞在辐射前加入自噬抑制剂3-MA或转染自噬相关基因ATG7的siRNA以抑制辐射诱导的自噬反应,来验证自噬对辐射抵抗的影响。Western blot结果显示,3-MA或ATG7siRNA可有效抑制辐射后乳腺癌细胞自噬活性的增加。克隆形成实验结果可见,3-MA或ATG7siRNA处理后,乳腺癌细胞辐射后的细胞存活分数有所下降。这一结果表明,抑制自噬活性可有效增强乳腺癌细胞的辐射敏感性。
5. miR-200c抑制辐射诱导的自噬
通过在稳定表达GFP-LC3的MDA-MB-231细胞中过表达miR-200c后接受X线照射,可见细胞中LC3阳性细胞数要少于对照组(t=13.432,P<0.001),差异具有统计学意义。Western blot的结果可见,空白对照组细胞在接受辐射后24h,p62蛋白较未照射时显著减少,而过表达miR-200c组的细胞中p62蛋白的表达量较未照射时仅有轻度改变,说明miR-200c过表达可使辐射后细胞自噬的活性下降。
6. miR-200c抑制自噬小体的形成
通过加入溶酶体和自噬小体融合的抑制剂Bafilomycin A1后,Western blot结果可以看到MDA-MB-231细胞空转组加了Bafilomycin A1在接受辐射24h后,LC3-Ⅱ蛋白的表达水平比单纯照射组显著增高,而转染了miR-200c mimic组的细胞加入Bafilomycin A1并接受辐射后,其细胞内的LC3-Ⅱ蛋白水平与miR-200c+RT组相比仅有轻度增高表明,miR-200c对自噬的抑制作用是在自噬的起始阶段发挥作用的,也就是抑制了自噬小体的形成,而不是抑制自噬小体的
降解。
7. miR-200c调节辐射诱导的自噬且与乳腺癌辐射抵抗相关
采用自噬抑制剂与辐射联合应用的方式,验证miR-200c辐射增敏的效应与抑制自噬的相关性。克隆形成实验结果可见,在MCF-7细胞中,敲除miR-200c的表达,与空转组比较辐射后细胞存活分数有所增加,而敲除miR-200c联合3-MA后,细胞存活分数较单纯敲除miR-200c组有所降低。MDA-MB-231细胞过表达miR-200c联合3-MA同样可以降低细胞的存活分数,但3-MA的这种增敏效果没有在MCF-7细胞中miR-200c敲除时那样显著。Western blot结果发现,在MDA-MB-231细胞中,过表达miR-200c增强了辐射诱导的凋亡相关蛋白c-PARP和c-caspase3的表达。而在MCF-7细胞中,抑制miR-200c的表达降低了辐射诱导c-PARP的表达。而且,辐射联合自噬抑制剂3-MA后都可增强凋亡相关蛋白的表达。这些结果表明,miR-200c增强乳腺癌细胞的辐射敏感性可能与抑制了辐射诱导的自噬活性有关。
8.生物信息学预测miR-200c调节自噬相关的靶基因
利用三大常用miRNA数据库TargetScan,Pictar和miRBase对miR-200c可能的靶基因进行预测分析,将这三个数据库预测到的结果交集后获得预测评分较高的11个靶基因。然后在Pubmed里进行检索查询这些靶基因的功能是否与自噬相关,筛选出4个基因,分别为RAC1, NPC1, PPP2CA和UBQLN1,依据microRNA.ORG数据库里预测到的miR-200c与这四个靶基因结合的mirSVR score评分和PhastCons score评分(图2-1C),最终选择UBQLN1这一基因作为本研究中miR-200c的靶基因。
9. miR-200c靶向抑制UBQLN1的表达
荧光定量PCR反应和Western blot检测结果可见,在乳腺癌细胞中过表达miR-200c后,UBQLN1基因的mRNA表达水平无明显改变(MDA-MB-231: t=0.641, P=0.587; BT549:t=0.180, P=0.873),而UBQLN1的蛋白表达水平有所下调。双萤光素酶报告基因系统检测结果发现,在乳腺癌细胞中过表达miR-200c后可抑制野生型质粒的萤光素酶活性(MDA-MB-231:t=18.571, P=0.003; BT549:t=24.293, P=0.002),而对突变型质粒的萤光素酶活性无显著影响(MDA-MB-231:t=0.063, P=0.956; BT549:t=1.192, P=0.356)。这些结果提示,miR-200c在转录后水平可直接靶向抑制UBQLN1的表达。
10.干扰UBQLN1的表达增强乳腺癌细胞的辐射敏感性并抑制辐射诱导的自噬活性
克隆形成实验结果发现,沉默UBQLN1的表达后,乳腺癌MDA-MB-231和BT549细胞辐射后的存活分数有所降低,辐射增敏比有所提高。进一步的研究发现,沉默乳腺癌细胞中UBQLN1的表达后,辐射诱导的LC3-Ⅱ蛋白表达减少,p62蛋白降解减少(。这些结果说明,敲除乳腺癌细胞中UBQLN1的表达后,可提高细胞对辐射的敏感性,而且抑制了辐射诱导的细胞自噬活性,与miR-200c在乳腺癌细胞辐射中的作用类似,表示UBQLN1是miR-200c功能性的一个靶基因。
11.UBQLN1是miR-200c的功能性靶基因
通过在乳腺癌MDA-MB-231细胞中共转染miR-200c mimic和UBQLN1的过表达质粒CMV-UBQLN1plasmid后,克隆形成实验发现UBQLN1的过表达可逆转miR-200c对乳腺癌细胞的辐射增敏效果,Western blot实验发现UBQLN1的过表达逆转了miR-200c对辐射诱导自噬的抑制效果。
12.乳腺癌组织中miR-200c的表达与UBQLN1和LC3的表达呈负相关
原位杂交和免疫组化结果发现,在miR-200c低表达的乳腺癌组织中伴有UBQLN1和LC3的高表达,而miR-200c高表达的乳腺癌组织中伴有UBQLN1和LC3的低表达；在瘤周正常组织中,miR-200c呈中度表达,UBQLN1和LC3的表达均为阴性。采用Spearman相关分析结果发现,miR-200c的表达水平与UBQLN1(r=-0.359,P=0.034)和LC3(r=-0.363, P=0.032)均呈负相关。进一步的分析发现,miR-200c和UBQLN1、LC3的表达水平与乳腺癌的临床病例特征无统计学差异(P值均大于0.05),但miR-200c的低表达伴随淋巴结的转移(P=0.020),而UBQLN1和LC3的表达水平与淋巴结转移无统计学差异。这些结果提示,miR-200c的表达水平与UBQLN1的表达呈负相关。
结论：
1. miR-200c在管腔型乳腺癌细胞中的表达水平高于乳腺正常上皮细胞MCF-10A,在基底型乳腺癌细胞中的表达水平低于MCF-10A。
2. miR-200c的过表达可增强乳腺癌细胞的辐射敏感性。
3. miR-200c的表达可抑制辐射诱导的自噬活性且与辐射增敏相关。
4. UBQLN1是miR-200c的一个功能性靶基因。
5. miR-200c在乳腺癌组织中的表达与UBQLN1和LC3的表达呈负相关性。
BACKGROUND AND OBJECTIVE
Breast cancer is the most common malignancy in women in the world and seriously threatens the health of women. Radiotherapy is among the primary strategies for most patients receiving breast-conserving surgery and displays significant clinical benefits, such as decreasing the risk of local recurrence and reducing the risk of mortality due to breast cancer. However, for some subtypes, such as basal-like breast cancer, the local and regional control remains unsatisfactory. A major reason for this failure in treatment may due to its radioresistance. Therefore, understanding the molecular mechanisms involved in the radioresistance of breast tumors may lead to improved clinical outcomes.
Macroautophagy, typically referred to as autophagy, is a homeostatic process involving self-degradation and recycling of macromolecules and cellular organelles and functions as a prosurvival mechanism under stressful conditions by degrading subcellular debris and regenerating metabolic precursors, therefore maintaining cellular integrity. During the last decade, the number of studies on the roles of autophagy in human diseases and its cellular mechanism has become extensive. Recent investigations have demonstrated that autophagy is a critical adaptive response for tumor cells to survive stressful conditions, such as hypoxia, nutrient deprivation, chemotherapy or radiotherapy, thereby providing therapeutic resistance. However, the molecular mechanisms by which autophagy mediates the radioresistance of breast tumors is less clear.
MicroRNAs (miRNAs) have been shown to be key regulators of a variety of biological processes, including cell proliferation, differentiation and invasion. Dysregulation of miRNAs has been reported to contribute to many human diseases, including cancer. Recent studies indicated that miRNAs may function as key regulators of autophagy. Several miRNAs have also been reported to be involved in chemoresistance or radioresistance via modulation of autophagy. However, relatively little is known about the role of autophagy in miRNA-mediated radioresistance in breast cancer. The miR-200family is involved in cancer stem cells self-renewal, the epithelial to mesenchymal transition (EMT) and chemosensitivity. Recent studies indicated that miR-200c, the prevailing member of the miR-200family, can also sensitize cancer cells to radiation by targeting TBK1and VEGFR2. However, the relationship between miR-200c and the modulation of autophagy that induces radioresistance remains unclear.
Here, we show that miR-200c can sensitize breast cancer cells to radiation via a mechanism associated with inhibition of irradiation-induced autophagy. We also identified the protein ubiquilin1(UBQLN1) as a direct target of miR-200c whose expression is positively correlated with radioresistance and irradiation-induced autophagy. Our results provide new insights into the molecular functions of miR-200c in radiosensitivity in breast cancer cells and imply a rationale for enhancing radiosensitivity via miR-200c for the treatment of breast cancer.
METHODS
1. Detection of miR-200c in breast cancer cells
The normal breast epithelial cell line MCF-10A, the luminal phenotype breast cancer cell lines MCF-7and BT474and basal phenotype breast cancer cell lines MDA-MB-231and BT549were used in this study. Total RNA was extracted from the cells using TRIzol reagent according to the manufacturer's protocol. The cDNA library was synthesized using the PrimeScript RT reagent kit following the manufacturer's instruction. For mature miRNA quantification, cDNA was generated using specific stem-loop universal primers. RT-PCR for miR-200c in these five cell lines was performed using SYBR Premix Ex Taq II according to the manufacturer's protocol and was measured using an ABI7500Sequence Detection System. U6was used as the internal control.
2. Oligonucleotide and siRNA transfection
The miRNA mimic, the inhibitors, the control oligonucleotides and UBQLN1siRNA were purchased from RiBoBio. Transfection of oligonucleotides and siRNA was performed using Lipofectamine2000reagent according to the manufacturer's protocol. For efficient inhibition of the miR-200bc/429cluster, equivalent amounts of miR-200c and miR-429inhibitors were combined.
3. Irradiation and clonogenic survival assays
Increasing numbers of cells were seeded on6-well plates (previously transfected with oligonucleotides or siRNA for48hours) in triplicate and exposed to the indicated dose of radiation using6MV X-rays generated from linear accelerators at a dose rate of5Gy/min. After incubation at37℃for10to14days, the cells on the plates were fixed using100%methanol and stained using1%crystal violet. Colonies containing>50cells were counted via microscopic inspection. The surviving fraction was calculated according to the multi-target single-hit model.
4. Western blot analysis
Whole cell protein lysates were extracted using radio-immunoprecipitation assay (RBPA) buffer containing proteinase and phosphatase inhibitor cocktails and quantified using a bicinchoninic acid protein assay kit. Equivalent amounts of total protein were resolved via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then blocked using5%nonfat milk for1hour at room temperature, followed by incubation in the primary antibodies overnight at4℃. After washing three times, the membranes were incubated in HRP-conjugated secondary antibodies for1hour at room temperature. The immunoreactive bands were visualized using an ECL western blot substrate.
5. Preparation of stable GFP-LC3-expressing cells
The recombinant lentivirus containing GFP-LC3was purchased from Genepharma (Shanghai, China). MDA-MB-231cells were infected with lentivirus particles and isolated via fluorescence-activated cell sorting to generate populations stably expressing GFP-LC3.
6. Bioinformatic analysis for predicting miR-200c target genes regulating autophagy
To investigate the molecular mechanism by which miR-200c suppressed irradiation-induced autophagy, we identified potential targets of miR-200c based on three publicly available databases (TargetScan, Pictar and miRBase) and then intersected the results from these databases, followed by searching in the Pubmed to analyze the relationship between these genes and autophagy.
7. Analysis of the expression of UBQLN1mRNA and protein after ectopic expression of miR-200c in breast cancer cells
Quantitative real-time PCR (RT-PCR) and Western blot analysis were used to analyzed the expression of UBQLN1mRNA and protein after transfection with miR-200c mimic.
8. Luciferase reporter assay
The3' untranslated region (UTR) of UBQLN1and mutant3'UTR, which contains the putative miR-200c binding site, were amplified from genomic DNA via PCR and cloned into the Renilla luciferase gene (pLUC-REPORT vector, Promega, Madison, WI, USA). For the luciferase reporter assay, MDA-MB-231cells were co-transfected with a luciferase reporter vector (either pLUC-3'UTR-UBQLN1or pLUC-3'UTR-mut-UBQLN1) and negative control miRNA or the miR-200c mimic. Forty-eight hours after transfection, the cells were assayed for luciferase activity using the Dual-Luciferase Assay Kit (Promega) according to the manufacturer's instructions. For each sample, the relative luciferase activity was normalized to firefly luciferase activity. Three independent experiments were performed in triplicate.
9. Detection of the expression of UBQLN1and LC3in breast cancer tissue.
Immunohistochemical staining (IHC) was performed on3-μm-thick sections excised from paraffin blocks. The sections were fixed using4%formaldehyde overnight and embedded in paraffin, followed by deparaffinization and hydration. Then, the sections were pretreated with sodium citrate buffer in a microwave for antigen retrieval and blocked using goat serum. The sections were stained using rabbit anti-UBQLN1and anti-LC3A/B overnight at4℃. Then, the sections were incubated in biotinylated goat anti-rabbit IgG secondary antibody for1hour, followed by staining with the avidin-biotin peroxidase complex according to the manufacturer's instructions. Scoring of the sections was performed by two independent pathologists.
10. Detection of the miR-200c level in breast cancer tissue
For detection of the expression of miR-200c in breast cancer tissue, in situ hybridization (ISH) was performed.
11. Statistical analysis
In the experimental studies, the data are presented as the means±standard deviation. The samples were analyzed using Student's t test or analysis of variance (ANOVA). The Pearson Chi-square test, Fisher's Exact test or t test was used to analyze the relationship between miR-200c expression and the clinical characteristics. All statistical analysis was performed using SPSS13.0software, and a P value of<0.05was considered to be statistically significant. The qualitative data were representative of more than three independent experiments that were performed in triplicate.
Results:
1. The expression level of miR-200c in breast cancer cell lines
In this study, we first investigated miR-200c expression in four breast cancer cell lines and the normal breast epithelial cell line MCF-10A. The results of quantitative real-time PCR indicated that the expression of miR-200c was higher in luminal phenotype breast cancer cells (MCF-7and BT474) and lower in basal phenotype breast cancer cells (MDA-MB-231and BT549) compared to the normal breast epithelial cell line MCF-10A.
2. MiR-200c sensitizes breast cancer cells to radiation treatment.
Overexpression of miR-200c enhanced the radiosensitivity of the MDA-MB-231and BT549cell lines, while inhibition of the expression of miR-200c led to radioresistance in MCF-7cells. These results suggest that restoring the normal expression level of miR-200c can enhance the radiosensitivity of breast cancer cells.
3. Autophagy is induced by irradiation
Autophagy may contribute to either cytoprotective or cytotoxic effects, depending on the type of cell and stress. Western blot analysis indicated that, after receiving6Gy radiation at various time points, autophagy was activated in three human breast cancer cell lines, as demonstrated by the increased expression of LC3Ⅱ, accompanied by decreased expression of p62. However, the activity of autophagy in MCF-7cells was not significant different compared to the MDA-MB-231and BT549cell lines.
4. Autophagy acts as a protective mechanism in breast cancer cells after irradiation.
Western blot analysis indicated that,3-MA, an autophagy inhibitor that blocks autophagosome formation, can effective inhibited the activity of autophagy induced by irradiation. Clonogenic assays demonstrated that3-MA significantly decreased the survival fraction of three human breast cancer cell lines after irradiation. These results indicate that autophagy is activated due to an adaptive response to promote the survival of breast cancer cells after receiving irradiation.
5. miR-200c inhibited radiation-induced autophagy
To determine whether miR-200c can inhibit irradiation-induced autophagy, we established a MDA-MB-231cell line stably expressing the GFP-LC3fusion protein. After24hours of irradiation, miR-200c overexpression significantly decreased the lipidation of LC3and increased p62protein expression, suggesting a decreased level of autophagy.
6. miR-200c inhibited the formation of autophagosome
To further investigate the specific stage of autophagy that was inhibited by miR-200c, we used bafllomycin Al, a fusion inhibitor of lysosomes and autophagosomes, which can inhibit the degradation of LC3Ⅱ. Twenty-four hours after irradiation, the bafilomycin Al-treated control group displayed markedly increased accumulation of LC3Ⅱ, whereas the miR-200c-treated group displayed only a small alteration. These data indicate that the inhibitory effect of miR-200c on autophagy resulted from the suppression of the early stage of autophagy, the inhibition of autophagosome formation, rather than from the suppression of autophagosome degradation.
7. MiR-200c modulates irradiation-activated autophagy, which is associated with the radioresistance of human breast cancer cells.
Next, we sought to determine whether miR-200c-induced radiosensitization was dependent on suppression of autophagy. Clonogenic survival assay indicated that3-MA significantly increased radiosensitivity, as miR-200c expression was suppressed in MCF-7cells. In MDA-MB-231cells,3-MA also enhanced radiosensitivity when miR-200c was overexpressed, but this effect was not significant when miR-200c expression was suppressed in MCF-7cells. Additionally, we found that miR-200c overexpression increased the irradiation-induced activation of caspase-3and PARP in MDA-MB-231cells. In MCF-7cells, suppression of miR-200c expression decreased the irradiation-induced activation of PARP. Because MCF-7cells are a caspase-3deficient breast tumor cell line, we failed to detect the expression of activation of caspase-3after treatment with irradiation,3-MA or both. Combined treatment of3-MA and radiation significantly increased the activation of apoptosis-related proteins in both groups. These results suggest that miR-200c enhanced the radiosensitivity of breast cancer cells in a manner that may be associated with the suppression of IR-induced autophagy.
8. Identified the autophagy related target genes of miR-200c by bioinformatic analysis
To investigate the molecular mechanism by which miR-200c suppressed irradiation-induced autophagy, we identified potential target of miR-200c based on three publicly available databases (TargetScan, Pictar and miRBase) and then intersected the results from these databases. After this, we predicted11target genes of miR-200c. Then we searched in the Pubmed to analyze the relationship between these genes and autophagy, and screened four genes, RAC1(ras-related C3botulinum toxin substrate1), NPC1(Niemann-Pick disease, type C1), PPP2CA (Serine/threonine-protein phosphatase2A catalytic subunit alpha isoform) and UBQLN1(ubiquilin1). According to the mirSVR score and PhastCons score of prediction from microRNA.ORG database, we choose UBQLN1as the target gene of miR-200c in this study finally.
9. miR-200c inhibit the expression of UBQLNl
Ectopic expression of miR-200c exhibited no effect on the mRNA expression level of UBQLN1, whereas overexpression of miR-200c significantly suppressed the protein expression of UBQLN1. In addition, the luciferase reporter assay revealed that miR-200c significantly suppressed the luciferase activity of the wild-type3'UTR of UBQLN1but not that of the mutant reporter gene, indicating the specificity of miR-200c to target the UBQLNl3'UTR. These results indicated that miR-200c suppressed UBQLN1expression at the post-transcriptional level via translational arrest.
10. Silencing the expression of UBQLNl enhanced the radiosensitivity and suppressed the autophagy activity of breast cancer cells
To confirm that miR-200c enhanced radiosensitivity due to the direct targeting of UBQLN1, we examined the effect of silencing the expression of UBQLN1on radiosensitivity. A clonogenic assay indicated that knockdown of the expression of UBQLN1significantly enhanced the radiosensitivity of MDA-MB-231and BT549cells. Furthermore, silencing of the expression of UBQLN1led to significant inhibition of irradiation-induced autophagy, consistent with the phenotype induced by the overexpression of miR-200c. These results demonstrate that miR-200c suppressed irradiation-induced autophagy by directly targeting UBQLN1.
11. The expression of miR-200c, UBQLNl and LC3are inversely correlated in human breast cancer tissue
To investigate the correlation between the expression of miR-200c, UBQLN1and LC3in human breast cancer tissue, we measured the expression levels of miR-200c, UBQLN1and LC3in human breast cancer specimens via in situ hybridization and immunohistochemistry. We found decreased expression of miR-200c in human breast cancer tissues that exhibited elevated UBQLN1and LC3 expression, and increased miR-200c expression correlated with decreased UBQLN1and LC3expression. In matched adjacent noncancerous breast tissues, we detected moderate expression of miR-200c and did not detect any expression of UBQLN1and LC3. Furthermore, the expression of miR-200c, UBQLN1and LC3displayed little correlation with the clinical and pathological characteristics of breast cancer. However, the miR-200c expression level inversely correlated with lymph node metastasis, but the UBQLN1and LC3expression level displayed little correlation with this characteristic. Whether the expression of UBQLN1influences breast cancer metastasis requires further investigation. These results are consistent with the above finding that decreased expression of miR-200c correlated with increased UBQLN1expression.
Conclusion:
1. The expression of miR-200c was higher in luminal phenotype breast cancer cells and lower in basal phenotype breast cancer cells compared to the normal breast epithelial cell line MCF-10A.
2. Ectopic expression of miR-200c enhanced the radiosensitivity of breast cancer cells.
3. Overexpression of miR-200c suppressed the activity of radiation-induced autophagy, which may associated with radioresistance.
4. UBQLN1is a directly functional target of miR-200c.
5. The expression of miR-200c, UBQLN1and LC3are inversely correlated in human breast cancer tissue.
乳腺癌是全球女性中最常见的恶性肿瘤,严重威胁着女性的健康问题。对于大多数接受保乳术的乳腺癌患者来说,放射治疗是主要的治疗手段之一,且具有显著的临床获益,如减少肿瘤局部复发的风险,降低乳腺癌的死亡率。然而,对于一些特定的肿瘤亚型,如基底样癌,肿瘤的局控率仍然很低。对于这一类型的乳腺癌患者来说,治疗失败的主要原因可能是辐射抵抗。因此,阐明乳腺癌辐射抵抗的分子机制可对今后提高乳腺癌的临床治疗效果提供很大帮助。
自噬是细胞内的一种“自食”现象,是一种细胞自身代谢降解的过程,在这一过程中,细胞质成分螯合入一种称为自噬小体(autophagosome)的双膜囊泡中,随后运送至溶酶体进行降解,其中的大分子和细胞器得以进行再循环利用,从而维持细胞的完整性。自噬在正常生理状态下对于维持细胞稳态和细胞器的更新有重要的作用,自噬缺陷与许多人类疾病相关,包括神经退行性疾病和肿瘤。近年来越来越多的研究关注自噬在人类疾病中的作用及其确切的分子机制。最近的研究发现,自噬是肿瘤细胞在如缺氧、营养剥夺、化疗、放疗等各种应激性条件下得以存活的一个重要适应性反应,这也因此引起了肿瘤细胞对治疗的抵抗。然而,在乳腺癌中,自噬介导乳腺癌辐射抵抗的具体分子机制目前仍然不太清楚。
目前的研究已经证实,MicroRNAs (miRNAs)参与调节多种生物学进程,包括细胞增殖、分化及迁移和侵袭。miRNA的表达失调可导致多种人类疾病的发生,其中包括肿瘤。最近的研究表明,miRNAs在自噬过程中可发挥重要的调节作用。有些研究也报道了几种miRNAs可通过调节自噬进程参与一些肿瘤的化疗或辐射抵抗。然而在乳腺癌中,有关自噬在miRNA介导的辐射抵抗中的作用及机制仍不甚明了。此前的研究表明,miR-200家族可参与调节肿瘤干细胞的自我更新、上皮-间质转化(EMT)和化疗抵抗。最近的研究发现,miR-200家族的重要成员miR-200c可通过靶向抑制TBKl和VEGFR2基因的表达来调节肿瘤细胞的辐射敏感性。然而,miR-200c和自噬的调节在乳腺癌辐射抵抗中的关系仍不清楚。
本研究中发现miR-200c可通过抑制辐射诱导的自噬反应来提高乳腺癌细胞对辐射的敏感性。本研究还确定了ubiquilin1蛋白(UBQLN1)是miR-200c的直接靶基因,其表达水平与辐射抵抗及辐射诱导的自噬活性呈正相关。本研究研究为miR-200c在乳腺癌辐射增敏作用中的分子机制提供了新的见解,为miR-200c用于乳腺癌的治疗提供了新的理论依据。
方法：
1.乳腺癌细胞中miR-200c的检测
正常乳腺上皮细胞株MCF-10A,基底型乳腺癌细胞株MDA-MB-231、BT549和管腔型乳腺癌细胞株MCF-7、BT474细胞,依据Trizol Reagent试剂的说明书进行操作,提取细胞总RNA；按照PrimeScript RT reagent kit说明书进行逆转录反应,获得cDNA;以cDNA为模板,采用茎环法依据SYBR Premix Ex Taq Ⅱ试剂盒说明书进行荧光定量PCR反应以检测这5株细胞中miR-200c的表达水平。
2. miR-200c mimic,inhibitor及siRNA和plasmid的转染
采用阳离子脂质体法进行转染,按照lipofectamine2000操作说明进行操作,采用无血清培养基培养细胞,当细胞融合度达到70%左右时,将mimic,inhibitor,siRNA或plasmid稀释于转染专用培养基Opti-MEMI Reduced Serum Medium中,lipofectamine2000同样用Opti-MEMI Reduced Serum Medium稀释后,与mimic, inhibitor, siRNA或plasmid溶液混合,室温下静置20min后,加入培养板中。继续在培养箱中孵育4-6h后,将培养基换成含有血清的完全培养基,转染完成,48h后提取细胞的RNA或蛋白或进行后续的处理。为有效抑制niR-200c的功能簇,miR-200c inhibitor需与miR-429inhibitor同时转染。
3.细胞辐射及克隆形成实验
细胞经消化制成单细胞悬液后,按照预定数量接种于6孔板中,设0,2,4,6,8五个剂量组,每个剂量组三个复孔。照射后将细胞置于37℃、5%C02、饱和湿度培养箱内继续培养12-14天,期间根据培养液pH值变化适时更换新鲜培养液。当培养板孔中出现肉眼可见克隆时,终止培养,弃去培养液,PBS清洗细胞2遍,甲醇固定,1%结晶紫乙醇溶液染色,显微镜下计数含50个细胞以上的克隆数,计算克隆形成率和存活分数,并运用GraphPad Prism5.0软件进行多靶单击模型曲线拟合,并计算辐射增敏因子SER10。
4. Western blot实验
处理后的细胞提取总蛋白,按照BCA法测定蛋白浓度,加入5×SDS-PAGE loading Buffer进行蛋白变性,然后经电泳、转膜、封闭后,孵育一抗和二抗,最后采用ECL法在显影仪上进行显影。
5.构建稳定表达GFP-LC3的MDA-MB-231细胞
将5×105/孔的细胞接种于6孔板中,培养12h待细胞密度达40%左右,加入MAP1LC3B和对照LV5-NC慢病毒悬液100μl/孔(MOI=20),并设2孔加入终浓度为6ug/ml的polybrene以增加感染效率,24h后更换培养基,72h后在荧光显微镜下拍照并计算感染效率。随后采用流式细胞仪分选GFP+细胞,以获得比例较高的GFP+细胞扩大培养,得到稳定表达GFP-LC3的细胞。
6.生物信息学分析预测miR-200c调节自噬的靶基因
利用三大常用miRNA数据库TargetScan,Pictar和miRBase对miR-200c可能的靶基因进行预测分析,将这三个数据库预测到的结果取交集以提高可信度,然后在基因功能分析网站上查询这些基因的功能,并且在Pubmed里进行检索查询这些靶基因的功能是否与自噬相关。
7.检测乳腺癌细胞中miR-200c过表达后UBQLN1的表达
在乳腺癌细胞中通过转染miR-200c mimic或scramble negative control后,通过荧光定量PCR和Western blot检测细胞中miR-200c过表达后UBQLN1的mRNA和蛋白的表达变化。
8.双荧光素酶报告基因检测法验证miR-200c的靶基因
依据阳离子脂质体法将包含miR-200c假定结合位点的UBQLN13'UTR以及突变型UBQLN1的3'UTR的萤光素酶报告质粒和]miR-200c或scramble negative control共同转染乳腺癌细胞。转染完成48h后,依据Promega公司的Dual-Luciferase报告基因检测系统进行样本的萤光素酶活性检测,按照试剂盒说明书进行操作,以萤火虫萤光素酶的活性作为内参,依据海肾萤光／萤火虫萤光的比值(Renilla/Firefly)分析miR-200c抑制UBQLN1的效果。
9.恢复实验证实UBQLN1是miR-200c的功能性靶基因
依据阳离子脂质体法将miR-200c mimic和CMV-UBQLN1plasmid共转染乳腺癌MDA-MB-231细胞。转染完成48h后,Western blot检测转染效率,并进行克隆形成实验检测细胞辐射敏感性的变化,Western blot检测辐射后细胞自噬活性的改变。
10.乳腺癌组织中UBQLN1和LC3的检测
收集乳腺癌组织及瘤周正常组织标本35例,经病理确诊全为浸润性乳腺癌,经4%多聚甲醛固定、包埋、切片后进行免疫组化染色检测组织中UBQLN1和LC3的表达水平。
11.乳腺癌组织中miR-200c的检测
上述乳腺癌组织及瘤周正常组织标本,采用原位杂交的方法检测组织中miR-200c的表达水平。
12.统计分析
采用SPSS13.0软件进行统计分析,两样本均数的比较采用两独立样本的t检验(Independent-Sample T Test)和单样本t检验,两组以上样本均数比较采用完全随机设计资料的方差分析(One-Way ANOVA),方差齐性检验采用Levene's Test。临床标本分析采用Pearson卡方检验和Fisher确切概率法。P值<0.05认为有统计学意义。
结果：
1.乳腺癌细胞株中miR-200c的表达水平
荧光定量PCR结果显示,5株细胞间miR-200c的表达水平有统计学差异(F=130.182, P<0.001), miR-200c在2株管腔型乳腺癌细胞(MCF-7、BT474)中的表达水平高于乳腺正常上皮细胞MCF-10A,但差异无统计学意义(P值分别为0.169和0.696),而在基底型乳腺癌细胞中(MDA-MB-231、BT549), miR-200c的表达水平与MCF-10A相比有统计学差异(P值均为0.026),MDA-MB-231和BT549细胞中miR-200c的表达水平低于MCF-10A。
2. miR-200c增强乳腺癌细胞的辐射敏感性
通过在MDA-MB-231及BT549细胞中过表达miR-200c、在MCF-7细胞中沉默miR-200c的表达,来研究miR-200c对乳腺癌细胞辐射敏感性的影响。结果发现,在MDA-MB-231及BT549细胞中过表达miR-200c后,细胞经辐射后存活分数有所降低,而在MCF-7细胞中敲出miR-200c的表达后,细胞经辐射后的存活分数有所增高,表明miR-200c的过表达可提高乳腺癌的辐射敏感性。
3.辐射可诱导乳腺癌细胞自噬反应
Western blot结果显示,乳腺癌MDA-MB-231和BT549细胞经X线照射后,随着时间的延长,自噬相关蛋白LC3-Ⅱ的表达有所增高,p62蛋白的降解有所下降。这些结果说明辐射诱导了乳腺癌细胞自噬的发生。但在MCF-7细胞中,这种自噬活性的改变没有MDA-MB-231和BT549细胞中明显。
4.自噬在乳腺癌辐射中起保护作用
乳腺癌细胞在辐射前加入自噬抑制剂3-MA或转染自噬相关基因ATG7的siRNA以抑制辐射诱导的自噬反应,来验证自噬对辐射抵抗的影响。Western blot结果显示,3-MA或ATG7siRNA可有效抑制辐射后乳腺癌细胞自噬活性的增加。克隆形成实验结果可见,3-MA或ATG7siRNA处理后,乳腺癌细胞辐射后的细胞存活分数有所下降。这一结果表明,抑制自噬活性可有效增强乳腺癌细胞的辐射敏感性。
5. miR-200c抑制辐射诱导的自噬
通过在稳定表达GFP-LC3的MDA-MB-231细胞中过表达miR-200c后接受X线照射,可见细胞中LC3阳性细胞数要少于对照组(t=13.432,P<0.001),差异具有统计学意义。Western blot的结果可见,空白对照组细胞在接受辐射后24h,p62蛋白较未照射时显著减少,而过表达miR-200c组的细胞中p62蛋白的表达量较未照射时仅有轻度改变,说明miR-200c过表达可使辐射后细胞自噬的活性下降。
6. miR-200c抑制自噬小体的形成
通过加入溶酶体和自噬小体融合的抑制剂Bafilomycin A1后,Western blot结果可以看到MDA-MB-231细胞空转组加了Bafilomycin A1在接受辐射24h后,LC3-Ⅱ蛋白的表达水平比单纯照射组显著增高,而转染了miR-200c mimic组的细胞加入Bafilomycin A1并接受辐射后,其细胞内的LC3-Ⅱ蛋白水平与miR-200c+RT组相比仅有轻度增高表明,miR-200c对自噬的抑制作用是在自噬的起始阶段发挥作用的,也就是抑制了自噬小体的形成,而不是抑制自噬小体的
降解。
7. miR-200c调节辐射诱导的自噬且与乳腺癌辐射抵抗相关
采用自噬抑制剂与辐射联合应用的方式,验证miR-200c辐射增敏的效应与抑制自噬的相关性。克隆形成实验结果可见,在MCF-7细胞中,敲除miR-200c的表达,与空转组比较辐射后细胞存活分数有所增加,而敲除miR-200c联合3-MA后,细胞存活分数较单纯敲除miR-200c组有所降低。MDA-MB-231细胞过表达miR-200c联合3-MA同样可以降低细胞的存活分数,但3-MA的这种增敏效果没有在MCF-7细胞中miR-200c敲除时那样显著。Western blot结果发现,在MDA-MB-231细胞中,过表达miR-200c增强了辐射诱导的凋亡相关蛋白c-PARP和c-caspase3的表达。而在MCF-7细胞中,抑制miR-200c的表达降低了辐射诱导c-PARP的表达。而且,辐射联合自噬抑制剂3-MA后都可增强凋亡相关蛋白的表达。这些结果表明,miR-200c增强乳腺癌细胞的辐射敏感性可能与抑制了辐射诱导的自噬活性有关。
8.生物信息学预测miR-200c调节自噬相关的靶基因
利用三大常用miRNA数据库TargetScan,Pictar和miRBase对miR-200c可能的靶基因进行预测分析,将这三个数据库预测到的结果交集后获得预测评分较高的11个靶基因。然后在Pubmed里进行检索查询这些靶基因的功能是否与自噬相关,筛选出4个基因,分别为RAC1, NPC1, PPP2CA和UBQLN1,依据microRNA.ORG数据库里预测到的miR-200c与这四个靶基因结合的mirSVR score评分和PhastCons score评分(图2-1C),最终选择UBQLN1这一基因作为本研究中miR-200c的靶基因。
9. miR-200c靶向抑制UBQLN1的表达
荧光定量PCR反应和Western blot检测结果可见,在乳腺癌细胞中过表达miR-200c后,UBQLN1基因的mRNA表达水平无明显改变(MDA-MB-231: t=0.641, P=0.587; BT549:t=0.180, P=0.873),而UBQLN1的蛋白表达水平有所下调。双萤光素酶报告基因系统检测结果发现,在乳腺癌细胞中过表达miR-200c后可抑制野生型质粒的萤光素酶活性(MDA-MB-231:t=18.571, P=0.003; BT549:t=24.293, P=0.002),而对突变型质粒的萤光素酶活性无显著影响(MDA-MB-231:t=0.063, P=0.956; BT549:t=1.192, P=0.356)。这些结果提示,miR-200c在转录后水平可直接靶向抑制UBQLN1的表达。
10.干扰UBQLN1的表达增强乳腺癌细胞的辐射敏感性并抑制辐射诱导的自噬活性
克隆形成实验结果发现,沉默UBQLN1的表达后,乳腺癌MDA-MB-231和BT549细胞辐射后的存活分数有所降低,辐射增敏比有所提高。进一步的研究发现,沉默乳腺癌细胞中UBQLN1的表达后,辐射诱导的LC3-Ⅱ蛋白表达减少,p62蛋白降解减少(。这些结果说明,敲除乳腺癌细胞中UBQLN1的表达后,可提高细胞对辐射的敏感性,而且抑制了辐射诱导的细胞自噬活性,与miR-200c在乳腺癌细胞辐射中的作用类似,表示UBQLN1是miR-200c功能性的一个靶基因。
11.UBQLN1是miR-200c的功能性靶基因
通过在乳腺癌MDA-MB-231细胞中共转染miR-200c mimic和UBQLN1的过表达质粒CMV-UBQLN1plasmid后,克隆形成实验发现UBQLN1的过表达可逆转miR-200c对乳腺癌细胞的辐射增敏效果,Western blot实验发现UBQLN1的过表达逆转了miR-200c对辐射诱导自噬的抑制效果。
12.乳腺癌组织中miR-200c的表达与UBQLN1和LC3的表达呈负相关
原位杂交和免疫组化结果发现,在miR-200c低表达的乳腺癌组织中伴有UBQLN1和LC3的高表达,而miR-200c高表达的乳腺癌组织中伴有UBQLN1和LC3的低表达；在瘤周正常组织中,miR-200c呈中度表达,UBQLN1和LC3的表达均为阴性。采用Spearman相关分析结果发现,miR-200c的表达水平与UBQLN1(r=-0.359,P=0.034)和LC3(r=-0.363, P=0.032)均呈负相关。进一步的分析发现,miR-200c和UBQLN1、LC3的表达水平与乳腺癌的临床病例特征无统计学差异(P值均大于0.05),但miR-200c的低表达伴随淋巴结的转移(P=0.020),而UBQLN1和LC3的表达水平与淋巴结转移无统计学差异。这些结果提示,miR-200c的表达水平与UBQLN1的表达呈负相关。
结论：
1. miR-200c在管腔型乳腺癌细胞中的表达水平高于乳腺正常上皮细胞MCF-10A,在基底型乳腺癌细胞中的表达水平低于MCF-10A。
2. miR-200c的过表达可增强乳腺癌细胞的辐射敏感性。
3. miR-200c的表达可抑制辐射诱导的自噬活性且与辐射增敏相关。
4. UBQLN1是miR-200c的一个功能性靶基因。
5. miR-200c在乳腺癌组织中的表达与UBQLN1和LC3的表达呈负相关性。
BACKGROUND AND OBJECTIVE
Breast cancer is the most common malignancy in women in the world and seriously threatens the health of women. Radiotherapy is among the primary strategies for most patients receiving breast-conserving surgery and displays significant clinical benefits, such as decreasing the risk of local recurrence and reducing the risk of mortality due to breast cancer. However, for some subtypes, such as basal-like breast cancer, the local and regional control remains unsatisfactory. A major reason for this failure in treatment may due to its radioresistance. Therefore, understanding the molecular mechanisms involved in the radioresistance of breast tumors may lead to improved clinical outcomes.
Macroautophagy, typically referred to as autophagy, is a homeostatic process involving self-degradation and recycling of macromolecules and cellular organelles and functions as a prosurvival mechanism under stressful conditions by degrading subcellular debris and regenerating metabolic precursors, therefore maintaining cellular integrity. During the last decade, the number of studies on the roles of autophagy in human diseases and its cellular mechanism has become extensive. Recent investigations have demonstrated that autophagy is a critical adaptive response for tumor cells to survive stressful conditions, such as hypoxia, nutrient deprivation, chemotherapy or radiotherapy, thereby providing therapeutic resistance. However, the molecular mechanisms by which autophagy mediates the radioresistance of breast tumors is less clear.
MicroRNAs (miRNAs) have been shown to be key regulators of a variety of biological processes, including cell proliferation, differentiation and invasion. Dysregulation of miRNAs has been reported to contribute to many human diseases, including cancer. Recent studies indicated that miRNAs may function as key regulators of autophagy. Several miRNAs have also been reported to be involved in chemoresistance or radioresistance via modulation of autophagy. However, relatively little is known about the role of autophagy in miRNA-mediated radioresistance in breast cancer. The miR-200family is involved in cancer stem cells self-renewal, the epithelial to mesenchymal transition (EMT) and chemosensitivity. Recent studies indicated that miR-200c, the prevailing member of the miR-200family, can also sensitize cancer cells to radiation by targeting TBK1and VEGFR2. However, the relationship between miR-200c and the modulation of autophagy that induces radioresistance remains unclear.
Here, we show that miR-200c can sensitize breast cancer cells to radiation via a mechanism associated with inhibition of irradiation-induced autophagy. We also identified the protein ubiquilin1(UBQLN1) as a direct target of miR-200c whose expression is positively correlated with radioresistance and irradiation-induced autophagy. Our results provide new insights into the molecular functions of miR-200c in radiosensitivity in breast cancer cells and imply a rationale for enhancing radiosensitivity via miR-200c for the treatment of breast cancer.
METHODS
1. Detection of miR-200c in breast cancer cells
The normal breast epithelial cell line MCF-10A, the luminal phenotype breast cancer cell lines MCF-7and BT474and basal phenotype breast cancer cell lines MDA-MB-231and BT549were used in this study. Total RNA was extracted from the cells using TRIzol reagent according to the manufacturer's protocol. The cDNA library was synthesized using the PrimeScript RT reagent kit following the manufacturer's instruction. For mature miRNA quantification, cDNA was generated using specific stem-loop universal primers. RT-PCR for miR-200c in these five cell lines was performed using SYBR Premix Ex Taq II according to the manufacturer's protocol and was measured using an ABI7500Sequence Detection System. U6was used as the internal control.
2. Oligonucleotide and siRNA transfection
The miRNA mimic, the inhibitors, the control oligonucleotides and UBQLN1siRNA were purchased from RiBoBio. Transfection of oligonucleotides and siRNA was performed using Lipofectamine2000reagent according to the manufacturer's protocol. For efficient inhibition of the miR-200bc/429cluster, equivalent amounts of miR-200c and miR-429inhibitors were combined.
3. Irradiation and clonogenic survival assays
Increasing numbers of cells were seeded on6-well plates (previously transfected with oligonucleotides or siRNA for48hours) in triplicate and exposed to the indicated dose of radiation using6MV X-rays generated from linear accelerators at a dose rate of5Gy/min. After incubation at37℃for10to14days, the cells on the plates were fixed using100%methanol and stained using1%crystal violet. Colonies containing>50cells were counted via microscopic inspection. The surviving fraction was calculated according to the multi-target single-hit model.
4. Western blot analysis
Whole cell protein lysates were extracted using radio-immunoprecipitation assay (RBPA) buffer containing proteinase and phosphatase inhibitor cocktails and quantified using a bicinchoninic acid protein assay kit. Equivalent amounts of total protein were resolved via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then blocked using5%nonfat milk for1hour at room temperature, followed by incubation in the primary antibodies overnight at4℃. After washing three times, the membranes were incubated in HRP-conjugated secondary antibodies for1hour at room temperature. The immunoreactive bands were visualized using an ECL western blot substrate.
5. Preparation of stable GFP-LC3-expressing cells
The recombinant lentivirus containing GFP-LC3was purchased from Genepharma (Shanghai, China). MDA-MB-231cells were infected with lentivirus particles and isolated via fluorescence-activated cell sorting to generate populations stably expressing GFP-LC3.
6. Bioinformatic analysis for predicting miR-200c target genes regulating autophagy
To investigate the molecular mechanism by which miR-200c suppressed irradiation-induced autophagy, we identified potential targets of miR-200c based on three publicly available databases (TargetScan, Pictar and miRBase) and then intersected the results from these databases, followed by searching in the Pubmed to analyze the relationship between these genes and autophagy.
7. Analysis of the expression of UBQLN1mRNA and protein after ectopic expression of miR-200c in breast cancer cells
Quantitative real-time PCR (RT-PCR) and Western blot analysis were used to analyzed the expression of UBQLN1mRNA and protein after transfection with miR-200c mimic.
8. Luciferase reporter assay
The3' untranslated region (UTR) of UBQLN1and mutant3'UTR, which contains the putative miR-200c binding site, were amplified from genomic DNA via PCR and cloned into the Renilla luciferase gene (pLUC-REPORT vector, Promega, Madison, WI, USA). For the luciferase reporter assay, MDA-MB-231cells were co-transfected with a luciferase reporter vector (either pLUC-3'UTR-UBQLN1or pLUC-3'UTR-mut-UBQLN1) and negative control miRNA or the miR-200c mimic. Forty-eight hours after transfection, the cells were assayed for luciferase activity using the Dual-Luciferase Assay Kit (Promega) according to the manufacturer's instructions. For each sample, the relative luciferase activity was normalized to firefly luciferase activity. Three independent experiments were performed in triplicate.
9. Detection of the expression of UBQLN1and LC3in breast cancer tissue.
Immunohistochemical staining (IHC) was performed on3-μm-thick sections excised from paraffin blocks. The sections were fixed using4%formaldehyde overnight and embedded in paraffin, followed by deparaffinization and hydration. Then, the sections were pretreated with sodium citrate buffer in a microwave for antigen retrieval and blocked using goat serum. The sections were stained using rabbit anti-UBQLN1and anti-LC3A/B overnight at4℃. Then, the sections were incubated in biotinylated goat anti-rabbit IgG secondary antibody for1hour, followed by staining with the avidin-biotin peroxidase complex according to the manufacturer's instructions. Scoring of the sections was performed by two independent pathologists.
10. Detection of the miR-200c level in breast cancer tissue
For detection of the expression of miR-200c in breast cancer tissue, in situ hybridization (ISH) was performed.
11. Statistical analysis
In the experimental studies, the data are presented as the means±standard deviation. The samples were analyzed using Student's t test or analysis of variance (ANOVA). The Pearson Chi-square test, Fisher's Exact test or t test was used to analyze the relationship between miR-200c expression and the clinical characteristics. All statistical analysis was performed using SPSS13.0software, and a P value of<0.05was considered to be statistically significant. The qualitative data were representative of more than three independent experiments that were performed in triplicate.
Results:
1. The expression level of miR-200c in breast cancer cell lines
In this study, we first investigated miR-200c expression in four breast cancer cell lines and the normal breast epithelial cell line MCF-10A. The results of quantitative real-time PCR indicated that the expression of miR-200c was higher in luminal phenotype breast cancer cells (MCF-7and BT474) and lower in basal phenotype breast cancer cells (MDA-MB-231and BT549) compared to the normal breast epithelial cell line MCF-10A.
2. MiR-200c sensitizes breast cancer cells to radiation treatment.
Overexpression of miR-200c enhanced the radiosensitivity of the MDA-MB-231and BT549cell lines, while inhibition of the expression of miR-200c led to radioresistance in MCF-7cells. These results suggest that restoring the normal expression level of miR-200c can enhance the radiosensitivity of breast cancer cells.
3. Autophagy is induced by irradiation
Autophagy may contribute to either cytoprotective or cytotoxic effects, depending on the type of cell and stress. Western blot analysis indicated that, after receiving6Gy radiation at various time points, autophagy was activated in three human breast cancer cell lines, as demonstrated by the increased expression of LC3Ⅱ, accompanied by decreased expression of p62. However, the activity of autophagy in MCF-7cells was not significant different compared to the MDA-MB-231and BT549cell lines.
4. Autophagy acts as a protective mechanism in breast cancer cells after irradiation.
Western blot analysis indicated that,3-MA, an autophagy inhibitor that blocks autophagosome formation, can effective inhibited the activity of autophagy induced by irradiation. Clonogenic assays demonstrated that3-MA significantly decreased the survival fraction of three human breast cancer cell lines after irradiation. These results indicate that autophagy is activated due to an adaptive response to promote the survival of breast cancer cells after receiving irradiation.
5. miR-200c inhibited radiation-induced autophagy
To determine whether miR-200c can inhibit irradiation-induced autophagy, we established a MDA-MB-231cell line stably expressing the GFP-LC3fusion protein. After24hours of irradiation, miR-200c overexpression significantly decreased the lipidation of LC3and increased p62protein expression, suggesting a decreased level of autophagy.
6. miR-200c inhibited the formation of autophagosome
To further investigate the specific stage of autophagy that was inhibited by miR-200c, we used bafllomycin Al, a fusion inhibitor of lysosomes and autophagosomes, which can inhibit the degradation of LC3Ⅱ. Twenty-four hours after irradiation, the bafilomycin Al-treated control group displayed markedly increased accumulation of LC3Ⅱ, whereas the miR-200c-treated group displayed only a small alteration. These data indicate that the inhibitory effect of miR-200c on autophagy resulted from the suppression of the early stage of autophagy, the inhibition of autophagosome formation, rather than from the suppression of autophagosome degradation.
7. MiR-200c modulates irradiation-activated autophagy, which is associated with the radioresistance of human breast cancer cells.
Next, we sought to determine whether miR-200c-induced radiosensitization was dependent on suppression of autophagy. Clonogenic survival assay indicated that3-MA significantly increased radiosensitivity, as miR-200c expression was suppressed in MCF-7cells. In MDA-MB-231cells,3-MA also enhanced radiosensitivity when miR-200c was overexpressed, but this effect was not significant when miR-200c expression was suppressed in MCF-7cells. Additionally, we found that miR-200c overexpression increased the irradiation-induced activation of caspase-3and PARP in MDA-MB-231cells. In MCF-7cells, suppression of miR-200c expression decreased the irradiation-induced activation of PARP. Because MCF-7cells are a caspase-3deficient breast tumor cell line, we failed to detect the expression of activation of caspase-3after treatment with irradiation,3-MA or both. Combined treatment of3-MA and radiation significantly increased the activation of apoptosis-related proteins in both groups. These results suggest that miR-200c enhanced the radiosensitivity of breast cancer cells in a manner that may be associated with the suppression of IR-induced autophagy.
8. Identified the autophagy related target genes of miR-200c by bioinformatic analysis
To investigate the molecular mechanism by which miR-200c suppressed irradiation-induced autophagy, we identified potential target of miR-200c based on three publicly available databases (TargetScan, Pictar and miRBase) and then intersected the results from these databases. After this, we predicted11target genes of miR-200c. Then we searched in the Pubmed to analyze the relationship between these genes and autophagy, and screened four genes, RAC1(ras-related C3botulinum toxin substrate1), NPC1(Niemann-Pick disease, type C1), PPP2CA (Serine/threonine-protein phosphatase2A catalytic subunit alpha isoform) and UBQLN1(ubiquilin1). According to the mirSVR score and PhastCons score of prediction from microRNA.ORG database, we choose UBQLN1as the target gene of miR-200c in this study finally.
9. miR-200c inhibit the expression of UBQLNl
Ectopic expression of miR-200c exhibited no effect on the mRNA expression level of UBQLN1, whereas overexpression of miR-200c significantly suppressed the protein expression of UBQLN1. In addition, the luciferase reporter assay revealed that miR-200c significantly suppressed the luciferase activity of the wild-type3'UTR of UBQLN1but not that of the mutant reporter gene, indicating the specificity of miR-200c to target the UBQLNl3'UTR. These results indicated that miR-200c suppressed UBQLN1expression at the post-transcriptional level via translational arrest.
10. Silencing the expression of UBQLNl enhanced the radiosensitivity and suppressed the autophagy activity of breast cancer cells
To confirm that miR-200c enhanced radiosensitivity due to the direct targeting of UBQLN1, we examined the effect of silencing the expression of UBQLN1on radiosensitivity. A clonogenic assay indicated that knockdown of the expression of UBQLN1significantly enhanced the radiosensitivity of MDA-MB-231and BT549cells. Furthermore, silencing of the expression of UBQLN1led to significant inhibition of irradiation-induced autophagy, consistent with the phenotype induced by the overexpression of miR-200c. These results demonstrate that miR-200c suppressed irradiation-induced autophagy by directly targeting UBQLN1.
11. The expression of miR-200c, UBQLNl and LC3are inversely correlated in human breast cancer tissue
To investigate the correlation between the expression of miR-200c, UBQLN1and LC3in human breast cancer tissue, we measured the expression levels of miR-200c, UBQLN1and LC3in human breast cancer specimens via in situ hybridization and immunohistochemistry. We found decreased expression of miR-200c in human breast cancer tissues that exhibited elevated UBQLN1and LC3 expression, and increased miR-200c expression correlated with decreased UBQLN1and LC3expression. In matched adjacent noncancerous breast tissues, we detected moderate expression of miR-200c and did not detect any expression of UBQLN1and LC3. Furthermore, the expression of miR-200c, UBQLN1and LC3displayed little correlation with the clinical and pathological characteristics of breast cancer. However, the miR-200c expression level inversely correlated with lymph node metastasis, but the UBQLN1and LC3expression level displayed little correlation with this characteristic. Whether the expression of UBQLN1influences breast cancer metastasis requires further investigation. These results are consistent with the above finding that decreased expression of miR-200c correlated with increased UBQLN1expression.
Conclusion:
1. The expression of miR-200c was higher in luminal phenotype breast cancer cells and lower in basal phenotype breast cancer cells compared to the normal breast epithelial cell line MCF-10A.
2. Ectopic expression of miR-200c enhanced the radiosensitivity of breast cancer cells.
3. Overexpression of miR-200c suppressed the activity of radiation-induced autophagy, which may associated with radioresistance.
4. UBQLN1is a directly functional target of miR-200c.
5. The expression of miR-200c, UBQLN1and LC3are inversely correlated in human breast cancer tissue.
引文
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