三月week2文献阅读：Cell-Cycle-Targeting MicroRNAs as Therapeutic
Tools against Refractory Cancers
细胞周期靶向MicroRNAs作为治疗难治性癌症的工具
Highlights
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Characterization of human microRNAs which target the cell-cycle machinery
针对细胞周期机制的人类microRNAS的特征
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Profiling cell-cycle-targeting miRNAs against 122 human cancer cell lines from CCLE
分析细胞周期靶向miRNA作用122株CCLE人癌细胞系
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Algorithm to predict the response of tumors to cell-cycle-targeting miRNAs
预测肿瘤对细胞周期靶向miRNAs的反应的算法
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In vivo delivery of cell-cycle-targeting miRNAs inhibits cancer growth
体内传递靶向细胞周期的miRNAs抑制肿瘤生长
In Brief
By performing screens for miRNAs targeting cell-cycle proteins, Hydbring et al. identify a class of miRNAs that target multiple cyclins and CDKs.
Hydbring.等人通过对靶向细胞周期蛋白的miRNAs进行筛选，识别出一类靶向多个周期蛋白和CDKs的miRNAs。
Nanoparticle delivery of these miRNAs inhibits tumor growth in several xenograft models, including treatment-refractory patient-derived xenografts.
这些mirRNAs的纳米颗粒传递在几种异种移植模型中抑制肿瘤生长，包括来源于异种移植的难治疗性病人。
Summary 摘要
Cyclins and cyclin-dependent kinases (CDKs) are hyperactivated in numerous human tumors.
细胞周期蛋白和细胞周期蛋白依赖性激酶(CDKs)在许多人类肿瘤中高度活化。
To identify means of interfering with cyclins/CDKs, we performed nine genome-wide screens for human microRNAs (miRNAs) directly regulating cell-cycle proteins.
为了确定干扰细胞周期蛋白/CDKs的方法，我们对直接调控细胞周期蛋白的人类microRNAs (miRNAs)进行了9个全基因组筛选。
We uncovered a distinct class of miRNAs that target nearly all cyclins/CDKs, which are very effective in inhibiting cancer cell proliferation.
我们发现了一类针几乎靶向所有细胞周期蛋白/CDKs的独特miRNAs，它们在抑制癌细胞增殖方面非常有效
By profiling the response of over 120 human cancer cell lines, we derived an expression-based algorithm that can predict the response of tumors to cell-cycle-targeting miRNAs.
通过分析120多个人类癌细胞株的反应，我们推导出一种基于表达的算法，可以预测肿瘤对细胞周期靶向 miRNAs的反应。
Using systemic administration of nanoparticle-formulated miRNAs, we inhibited tumor progression in seven mouse xenograft models, including three treatment-refractory patient-derived tumors, without affecting normal tissues.
在不影响正常组织的情况下，我们使用纳米颗粒组成的miRNAs系统给药，抑制了7种小鼠异种移植瘤模型的肿瘤进展，其中包括3种难治性患者源性肿瘤。
Our results highlight the utility of using cellcycle-targeting miRNAs for treatment of refractory cancer types
我们的研究结果强调了利用细胞周期靶向miRNAs治疗难治性癌症的有效性
Significance 意义
Targeting cell-cycle machinery represents an attractive anti-cancer therapeutic strategy, and chemical inhibitorsof cyclin-CDK kinases are in clinical trials.
靶向细胞周期机制是一种有吸引力的抗癌治疗策略，cyclinc - cdk激酶的化学抑制剂正在临床试验中。
Here, we identified a class of miRNAs targeting multiple cyclin/CDKs and propose that these miRNAs might be superior to currently available therapeutic compounds.
在此，我们确定了一类靶向多个cyclin/CDKs的miRNAs，并提出这些miRNAs可能优于目前可用的治疗化合物。
We found that cell-cycle-targeting miRNAs are very potent against triple-negative breast cancer, one of the most aggressive breast cancer types.
我们发现细胞周期靶向miRNAs对三阴性乳腺癌非常有效，三阴性乳腺癌是最具侵袭性的乳腺癌类型之一。
We provide an approach to select miRNAs that are particularly efficacious against a given tumor, thereby allowing individualized therapies.
我们提供一种方法来选择对特定肿瘤特别有效的miRNAs，从而允许个体化治疗。
We demonstrate that cell-cycle-targeting miRNAs can be administered to tumor-bearing animals, where they inhibit growth of patientderived tumors that are resistant to available therapies.
我们证明细胞周期靶向miRNAs可以应用于荷瘤动物，在荷瘤动物身上，它们可以抑制对现有疗法产生耐药性的患者源性肿瘤的生长。
Hence, this study suggests a strategy of targeting aggressive human tumors
因此，这项研究提出了一种针对侵袭性人类肿瘤的策略
Introduction 介绍
The proliferation of mammalian cells is driven by cyclins and their catalytic partners, cyclin-dependent kinases (CDKs).
哺乳动物细胞的增殖是由细胞周期蛋白及其催化伙伴细胞周期蛋白依赖性激酶(CDKs)驱动的。
Cyclin CDK complexes phosphorylate cellular proteins, thereby driving cell-cycle progression.
细胞周期蛋白CDK复合物磷酸化细胞蛋白，从而驱动细胞周期的进展。
Stimulation of cells with growth factors induces the expression of D-type cyclins (cyclins D1, D2, and D3), which bind and activate CDK4 or CDK6.
用生长因子刺激细胞可诱导D型细胞周期蛋白(cyclins D1, D2, D3)的表达，这些细胞周期蛋白结合并激活CDK4或CDK6。
Later during the G1 phase, E-type cyclins (cyclins E1 and E2) become upregulated and activate CDK2 (and to a lesser extent CDK1).
在G1期后期，E型周期蛋白(周期蛋白E1和E2)上调并激活CDK2 (CDK1的激活程度较小)。
Cyclin E CDK2 complexes regulate entry of cells into the DNA synthesis (S phase).
细胞周期蛋白E CDK2复合物调控细胞进入DNA合成(S期)。
Further progression of cells through the S phase is driven by cyclin A2, which partners with CDK2.
细胞通过S期的进一步进展是由cyclin A2驱动的，它与CDK2结合。
Later during mitotic prophase, cyclin B translocates to the nucleus and activates CDK1.
在有丝分裂前期后期，细胞周期蛋白B转移到细胞核并激活CDK1。
Cyclin B-CDK1 kinase drives mitotic events such as spindle pole assembly, chromosome condensation, and nuclear envelope breakdown (Malumbres and Barbacid, 2009).
Cyclin B-CDK1激酶驱动有丝分裂事件，如纺锤体极组装、染色体冷凝和核膜破裂(Malumbres和Barbacid, 2009)。（细胞增殖过程的概述）
A comprehensive analysis of human cancers revealed that genes encoding cyclins and CDKs belong to the most frequently amplified loci (Beroukhim et al., 2010).
对人类癌症的全面分析表明，编码细胞周期蛋白和CDKs的基因属于最常见的扩增位点(Beroukhim et al.， 2010)。
The importance of overexpression of D-type cyclins in pathogenesis of human cancer has been particularly well established (Musgrove et al., 2011).
过度表达D型细胞周期蛋白在人类癌症发病机制中的重要性已经得到了特别明确的证实(Musgrove et al.， 2011)。
Analyses of mouse genetic models revealed the requirement for specific cyclins and CDKs in development and maintenance of a wide array of tumor types (Malumbres and Barbacid, 2009)
对小鼠遗传模型的分析揭示了在多种肿瘤类型的发育和维持过程中对特异性细胞周期蛋白和CDKs的需求(Malumbres和Barbacid, 2009)。（细胞周期蛋白和CDKs在细胞增殖中的重要性。）
Collectively, these observations firmly established essential roles for cyclins and CDKs in tumori genesis and led to the development of several CDK inhibitors.
总的来说，这些观察结果坚定地确立了细胞周期蛋白和CDKs在肿瘤发生中的重要作用，并导致了几种CDK抑制剂的开发。
Some of these compounds are currently in clinical trials, and three CDK4/6 inhibitors (palbociclib, abemaciclib, and ribociclib) received a ‘‘Breakthrough Therapy’’ designation from the US Food and Drug Administration and were approved for treatment of estrogen-receptor-positive breast cancers (Asghar et al., 2015;Finn et al., 2015).
其中一些化合物目前正在临床试验中，三种CDK4/6抑制剂(palbociclib、abemaciclib和ribociclib)获得了美国食品和药物管理局(fda)的“突破性治疗”指定，并被批准用于治疗雌激素受体阳性乳腺癌(Asghar et al.， 2015;Finn et al.， 2015)。
However, the clinical success of targeting the cell-cycle machinery has been limited so far.
然而，迄今为止，针对细胞周期机制的临床成功受到了限制。
For instance, palbociclib was shown to significantly prolong progression-free survival of cancer patients, but it had no major impact on overall survival (Finn et al., 2015).
例如，palbociclib被证明可以显著延长癌症患者的无进展生存期，但对总体生存期没有重大影响(Finn et al.， 2015)。
A likely reason for this disappointing outcome is that redundant cyclins and CDKs compensate for the inhibition of CDK4 and CDK6, thereby allowing tumor progression.
造成这一令人失望结果的一个可能原因是，冗余的周期蛋白和CDKs补偿了CDK4和CDK6的抑制，从而允许肿瘤进展。
For this reason, agents that target multiple cyclins and CDKs might offer a therapeutic advantage by preventing compensatory upregulation of cell-cycle kinases
因此，针对多种细胞周期蛋白和CDKs的药物可能通过阻止细胞周期激酶的代偿性上调而提供治疗优势。
（现存药物几种CDK抑制剂的缺陷，靶向作用多种细胞周期蛋白和CDKs的药物的优势）
MicroRNAs (miRNAs) have been recognized for their potential in cancer therapeutics, and multiple miRNAs were suggested to either play tumor-suppressive or tumor-promoting roles (Adams et al.,2014;Hayes et al., 2014).miRNAs bind their target transcripts via the 30 UTRs of mRNAs.
MicroRNAs (miRNAs)已被认为具有潜在的癌症治疗作用，多个miRNAs被认为要么具有肿瘤抑制作用，要么具有肿瘤促进作用(Adams et al.，2014;Hayes et al.，2014)。miRNAs通过30个mRNAs的UTRs与靶转录本结合。
miRNAs do not extinguish expression of their targets but reduce their levels, with individual miRNAs targeting many different transcripts (Bartel, 2009).
miRNAs并不会抑制靶基因的表达，而是会降低靶基因的表达水平，单个miRNAs针对许多不同的转录本(Bartel, 2009)。
We hypothesized that such a broad dampening of expression of several cell-cycle proteins might allow to selectively block proliferation of cancer cells without having major effects on their non-transformed counterparts.
我们假设，如此广泛地抑制几种细胞周期蛋白的表达，可能会选择性地阻止癌细胞的增殖，而不会对未转化的癌细胞产生重大影响。
Moreover, targeting several cell-cycle proteins at once could be beneficial in cancer therapy since it would give less room for cell-cycle compensatory mechanisms that may lead to acquired resistance to CDK inhibition
此外，同时靶向几个细胞周期蛋白可能对癌症治疗有益，因为它能将为可能导致CDK抑制的获得性耐药性的细胞周期补偿机制提供更少的空间。
（文献发现MicroRNAs潜在作用，提出假设靶向抑制几种细胞周期蛋白的表达，可能会选择性地阻止癌细胞的增殖）
Although numerous studies postulated targeting cyclins and CDKs by miRNAs in cancer treatment (Bonci et al., 2008;Johnson et al., 2007;Kota et al., 2009), no attempt was made to systematically delineate miRNAs regulating these proteins.
尽管许多研究假设miRNAs靶向cyclins和CDKs用于癌症治疗(Bonci et al.， 2008;Johnson et al.， 2007;Kota等人，2009)，但是没有尝试系统地描述调控这些蛋白的miRNAs。
In this study, we performed nine genome-wide screens to identify the full range of miRNAs regulating major cell-cycle proteins.
在这项研究中，我们进行了9个全基因组筛选，以确定调控主要细胞周期蛋白的全部miRNAs。
（文献研究对象：9个全基因组筛选，调控主要细胞周期蛋白的全部miRNAs）
Results
Genome-Wide Screens and Their Validation
全基因组筛选及其验证
In order the determine the full repertoire of miRNAs directly regulating cell-cycle cyclins and CDKs, we cloned 3‘UTRs of cyclins D1, D2, D3, E1, E2, as well as CDK1, CDK2, CDK4, and CDK6 downstream of the firefly luciferase gene into a dual luciferase reporter vector.
为了确定直接调控细胞周期周期蛋白和CDKs的miRNAs的全部序列，我们克隆了萤火虫荧光素酶基因下游的周期蛋白D1、D2、D3、E1、E2以及CDK1、CDK2、CDK4和CDK6的3’UTRs，并将其作为双荧光素酶报告载体。
The vector also encoded renilla luciferase driven by the SV40 promoter (Figure 1A).
该载体还编码了由SV40启动子驱动的肾荧光素酶(fig1A)。（fig1A(一)筛选方法。基因描述改编自Ensembl，第87版。基因的黑色部分表示克隆的30UTR区域。）
Vectors containing 3’UTRs of cyclins or CDKs were subsequently stably expressed in U2OS cells, thereby generating nine reporter cell lines.
含有3’utrs的细胞周期蛋白或CDKs载体随后在U2OS细胞中稳定表达，从而产生9个报告细胞系。
Expression of miRNAs targeting the 3‘UTR of a given cyclin or CDK in these cell lines is expected to repress the expression of the firefly luciferase while leaving renilla luciferase levels intact, and hence to decrease the firefly to renilla ratio.
在这些细胞系中，靶向给定cyclin或CDK的3'UTR的miRNAs的表达有望抑制萤火虫荧光素酶的表达，同时保持肾荧光素酶水平不变，从而降低荧光素酶与肾荧光素酶的比率。
（目的：确定直接调控细胞周期周期蛋白和CDKs的miRNAs的全部序列，实验：编码miRNAs的全部序列和肾荧光素酶载体在U20细胞中表达，产生9个报告细胞系）
Reporter cell lines stably expressing each of nine different 3'UTR luciferase constructs were plated in 96-well plates and transfected with human miRNA mimic library containing 885 annotated human miRNAs, one miRNA per well.
稳定表达9种不同3’utr荧光素酶构建物的报告细胞株被镀在96孔板上，并转染含有885个注释人类miRNA的人类miRNA模拟文库，每孔一个miRNA。
The firefly to renilla luciferase ratios were determined 28 hr post transfection (Figure 1A and Table S1).
转染28小时后测定萤火虫荧光素酶和肾荧光素酶的比值(fig1A和表S1)。（实验步骤）
All screens were performed in three technical replicates with all replicates displaying correlations of 0.9 or higher (Figure S1A)
所有屏幕在三个技术复制中执行，所有复制显示0.9或更高的相关性(figS1A)。（实验结果）
Since in our screens all miRNA mimics as well as the reporter system were of the exogenous origin, we considered it unlikely that the choice of the cell line (U2OS cells) would affect the results of the screens.
由于在我们的筛选中所有miRNA模拟物以及报告系统都是外源的，我们认为选择细胞系(U2OS细胞)不太可能影响筛选结果。
Nevertheless, to exclude this possibility,we first determined that there was no correlation between the results of our screens and the levels of endogenous cyclins, CDKs,or miRNAs in U2OS cells (Figures S1B–S1G).
然而，为了排除这种可能性，我们首先确定我们的筛选结果与U2OS细胞内源性cyclins、CDKs或miRNAs水平之间没有相关性(fig S1B-S1G)。
Moreover, we validated our screening results using two additional epithelial cell lines, CAL51 (breast cancer) and A549 (lung adenocarcinoma);please see Figures S2A–S2E for cell-cycle characterization of these cells.
此外，我们使用另外两种上皮细胞系CAL51(乳腺癌)和A549(肺腺癌)验证了我们的筛选结果;这些细胞的细胞周期特征见figS2A-S2E。
As we did for U2OS cells, we engineered CAL51 and A549 cells to stably express the luciferase reporters linked to 3'UTRs of nine cyclins or CDKs.
正如我们对U2OS细胞所做的那样，我们设计了CAL51和A549细胞，以稳定地表达与9种周期蛋白或CDKs的3'UTRs相关的荧光素酶报告基因。
We then compared the response of U2OS-, CAL51-, and A549-based reporter cell lines to 20 selected miRNAs.
然后我们比较了基于U2OS-、CAL51和A549的报告细胞株对20个选定miRNAs的响应。
We found that the results were highly reproducible between the three cell lines.
我们发现这三个细胞系之间的结果具有很高的重复性。
Thus, the overall reproducibility between screening results in U2OS and CAL51 cells was 79.4%, between U2OS and A549 was 78.3%, and between A549 and CAL51 was 75.6% (Figures 1B, 1C, S3A, and S3B).
因此，U2OS和CAL51细胞的筛查结果的总重现性为79.4%，U2OS和A549细胞的总重现性为78.3%，A549和CAL51细胞的总重现性为75.6%(fig1B、1C、S3A和S3B)。
We concluded that the choice of U2OS cells for the screens did not significantly influence the results.
我们的结论是，U2OS细胞的选择对筛查结果没有显著影响。
**（推测：选择细胞系(U2OS细胞)不太可能影响筛选结果 **
**验证：设计了CAL51和A549细胞，U2OS-、CAL51-和a549的报告细胞株 **
结论：U2OS细胞的选择对筛查结果没有显著影响）
We performed unsupervised clustering of the results of all nine screens (Figures 1D and 1E).
我们对所有9个检测的结果进行无监督聚类(fig1D和图1E)。
Strikingly, we found that miRNAs containing a common seed sequence invariably clustered together, i.e., they produced almost identical results across the nine screens, as seen for instance for the miR-34/449 family (Figure 1E, upper panel), and the extended miR-15/16 family (Figure 1E, lower panel)
引人注目的是，我们发现含有共同种子序列的miRNAs总是聚集在一起，即它们在9个屏幕上产生了几乎相同的结果，例如miR-34/449家族(fig1E，上面板)和扩展的miR-15/16家族(fig1E，下面板)。
This observation provided an additional confirmation for the reliability of our screens
这一观察结果进一步证实了我们的检测的可靠性。（实验数据作图验证检测）
We next compared the results of our nine screens with in silico predicted targeting using five softwares: TargetScan, miRanda, miRDB, miRWalk, and TargetSpy.
接下来，我们用TargetScan、miRanda、miRDB、miRWalk和TargetSpy等5个软件，将我们的9个检测的结果与在硅中预测的目标结果进行了比较。
Each of these algorithms predicts targeting of transcripts by miRNAs, based on the 3‘UTR sequences of the genes (Figure S3C).
这些算法都基于基因的3'UTR序列预测miRNAs对转录本的靶向性(figS3C)。
We found TargetScan to be most accurate in its ability to predict targeting, followed by miRanda (Figures S3D–S3F).
我们发现，在预测目标的能力方面，TargetScan是最准确的，其次是miRanda(figS3D-S3F)。
We observed a strong correlation between TargetScan context score (which predicts the likelihood of targeting) and the results of our screens (Figures S3G and S3H).
我们观察到TargetScan上下文评分(预测目标的可能性)和检测结果之间有很强的相关性(figS3G和S3H)。
Despite overall correlation, several miRNAs that were predicted to target a given cyclin or CDK did not score in our screens (Figure 1F), highlighting the limitation of the TargetScan predictive value
尽管存在总体相关性，但在我们的检测中上，预测针对给定cyclin或CDK的几个miRNAs没有得分(fig1F)，这突出了TargetScan预测值的局限性.(用5种软件将检测的结果与在硅中预测的目标结果进行了比较)
A selected number of ‘‘hits’’ from our screens were further validated using mutant miRNAs containing point mutations within their seed sequences (Figure 1G), as well as mutant 3’UTRs with mutations or deletions within miRNA target sequences (Figure 1H).
从我们的筛选中选择的“命中”数量，通过在其种子序列中包含点突变的突变miRNA(图1G)，以及其种子序列中包含点突变的突变miRNA(fig1H)进一步验证。
In all cases examined, these mutations strongly diminished the ability of miRNAs to repress luciferase expression (Figures 1G and 1H).
在所有检测的病例中，这些突变都严重削弱了miRNAs抑制荧光素酶表达的能力(fig1G和1H)。
Lastly, we confirmed the ability of selected miRNAs to reduce the expression of endogenous cyclin and CDK transcripts in 12 cancer cell lines (see below).
最后，我们证实了所选 miRNAs能够降低12个癌细胞系内源性cyclin和CDK转录本的表达(见下文)。
We concluded that miRNAs identified in our screens indeed repress expression of cyclins and CDKs
我们的结论是，在我们的检测中识别的miRNAs确实抑制了细胞周期蛋白和CDKs的表达.
(实验筛选对比其种子序列中包含点突变的突变miRNA，以及其种子序列中包含点突变的突变miRNA
验证：检测中识别的miRNAs确实抑制了细胞周期蛋白和CDKs的表达)
Identification of Cell-Cycle-Targeting miRNAs
细胞周期靶向miRNAs的鉴定
We constructed a heat map of all miRNAs targeting at least one cyclin or CDK.
我们构建了一个针对至少一个cyclin或CDK的所有miRNAs的热图。
These analyses revealed the presence of a group of miRNAs that target all or nearly all cell-cycle proteins analyzed (Figure 2A).
这些分析揭示了一组针对所有或几乎所有的分析的细胞周期蛋白的miRNAs的存在(fig2A)。
Importantly, the number of these cell-cycle-targeting miRNAs was significantly higher than the number predicted from random permutations (Figure 2B).
重要的是，这些细胞周期靶向miRNAs的数量显著高于随机排列预测的数量(fig2B)。
Thus, we found 16 miRNAs targeting 5 cyclins or CDKs, compared with 0.26 predicted from random permutations, 6 miRNAs targeting 6 cyclins/ CDKs (predicted 0.01), 4 miRNAs targeting 7 cyclins/CDKs (predicted 0.0007), 3 miRNAs targeting 8 cyclins/CDKs (predicted 0), and 1 miRNA targeting 9 cyclins/CDKs (predicted 0) (Figure 2B);p values <104 for all cases.
因此，我们发现了16个靶向5个cyclins或CDKs的miRNA，与随机排列个相比(预测的0.26)，6个靶向6个cyclins/CDKs的miRNA(预测0.01)，4个靶向7个cyclins/CDKs的miRNA(预测0.0007)，3个靶向8个cyclins/CDKs的miRNA(预测0)，以及1个靶向9个cyclins/CDKs的miRNA(预测0)(图2B);所有情况的p值均<104。
In total, we enumerated 30 miRNAs targeting at least 5 cyclins/CDKs and 14 miRNAs targeting at least 6 cyclins/CDKs (Figures 2A and 2B;Table S1).
我们总共列举了30个靶向至少5个cyclins/CDKs的mirna，以及14个靶向至少6个cyclins/CDKs的mirna(fig2A和fig2B;tableS1)。
In contrast, the number of miRNAs uniquely targeting individual cyclins or CDKs was not higher than predicted from random permutations (Figure S3I).
相比之下，靶向单个细胞周期蛋白或CDKs的特异性miRNAs的数量并不高于随机排列的预测(figS3I)。
(揭示了一组针对所有或几乎所有分析的细胞周期蛋白的miRNAs的存在,检测和预测间进行了比较。)
Also pairwise comparisons of all screening data revealed that miRNAs targeting one cell-cycle protein were significantly enriched for targeting other cell-cycle proteins (Figure 2C).
对所有筛查数据的两两比较显示，靶向一个细胞周期蛋白的miRNAs显著富集，而靶向其他细胞周期蛋白(fig2C)。
Collectively, these analyses revealed that the mammalian genome contains a previously unanticipated class of miRNAs that target multiple components of the core cell-cycle machinery.
总的来说，这些分析揭示了哺乳动物基因组包含了一种以前没有预料到的miRNAs，它靶向核心细胞周期机制的多个组件。
We called these miRNAs cell-cycle targeting miRNAs.
我们称这些miRNAs为细胞周期靶向miRNAs。
(结论总结：细胞周期靶向miRNAs靶向核心细胞周期机制的多个组件)
In order to provide a quantitative measure of the ability of miRNAs to repress the cell-cycle machinery, for each miRNA we calculated the average repression value across the nine screens.
为了定量测量miRNA抑制细胞周期机制的能力，我们计算了9个检查上每个miRNA的平均抑制值。
Using this criterion, 16 miRNAs repressed all nine 3‘UTRs by an average of 40% or more, while 60 miRNAs showed at least average 30% repression across the nine screens (Table S1).
使用这一标准，16个miRNAs对所有9个3’UTRs平均抑制40%或更多，而60个miRNAs在9个检测上显示了至少30%的平均抑制(表S1)。
（miRNA抑制细胞周期机制的能力，miRNA的平均抑制值）
We next askedwhether cell-cycle-targeting miRNAs might also target the other three major cyclins that were not included into our screens, namely cyclins A2, B1, and B2.
接下来我们问，细胞周期靶向miRNA是否也可以靶向我们检测上没有包括的其他三种主要的周期蛋白，即周期蛋白A2、B1和B2。
To address this point, we generated U2OS reporter cell lines stably expressing luciferase constructs containing 3’UTRs of cyclins A2, B1, or B2, and tested their response to ten randomly selected cell-cycle-targeting miRNAs.
为了解决这个问题，我们生成了稳定表达荧光素酶构建物的U2OS报告细胞系，其中包含3'UTRs的周期蛋白A2、B1或B2，并测试了它们对随机选择的10个细胞周期靶向miRNAs的反应。
We found that several cell-cycle-targeting miRNAs repressed the expression of cyclin A2, B1, and B2 reporter constructs.
我们发现一些细胞周期靶向 miRNAs制了cyclin A2、B1和B2报告基因的表达。
In contrast, ten randomly selected control, non-cell-cycle-targeting miRNAs had essentially no effect (Figures 2D–2F).
相比之下，随机选择10个非细胞周期靶向 miRNAs作为对照，基本上没有效果(fig2D-2F)。
We also determined that cell-cycle-targeting miRNAs were not enriched in targeting other proproliferative pathways, beside cyclins and CDKs (Table S2).
我们还确定，细胞周期靶向miRNAs在靶向细胞周期蛋白和CDKs之外，没有富集在其他促增殖通路(表S2)。
We concluded that the mammalian genome expresses a distinct class of miRNAs that can regulate expression of up to 12 major cyclins and CDKs.
我们的结论是，哺乳动物基因组表达了一类独特的 miRNAs，可以调控多达12种主要的周期蛋白和CDKs的表达。
（猜测：细胞周期靶向mirna是否也可以靶向我们检测上没有包括的其他三种主要的周期蛋白，即周期蛋白A2、B1和B2
实验：
结论：哺乳动物基因组表达了一类独特的 miRNAs，可以调控多达12种主要的周期蛋白和CDKs的表达）
Computational Analyses of Cell-Cycle-Targeting miRNAs
细胞周期靶向 miRNAs 的计算分析
Using data from The Cancer Genome Atlas (TCGA), we analyzed the expression of cell-cycle-targeting miRNAs across 4,807 human tumor samples representing 18 tumor types.
利用癌症基因组图谱(TCGA)的数据，我们分析了代表18种肿瘤类型的4,807个人类肿瘤样本中细胞周期靶向mirna的表达。
We searched for correlation/anti-correlation between the expression of miRNAs and the levels of transcripts encoding cyclins and CDKs.
我们寻找miRNAs表达与编码cyclins和CDKs的转录本水平之间的相关性/反相关性。
We found that miR-195-5p showed the strongest anticorrelation with expression of transcripts encoding cyclins E1, E2, CDK1, CDK2, and CDK4, and also displayed a strong anti correlation with expression of cyclins D1, D2, D3, and CDK6 (Table S3), suggesting that it might play a role in regulating the levels of these proteins in cancer cells.
我们发现mir - 195 - 5 - p与细胞周期蛋白E1,E2, CDK1, CDK2 的转录本编码表达显示最强的反相关性,同时也表现出与细胞周期蛋白D1D2、D3、和CDK6 的表达强烈的反相关性(表S3),这表明它可能发挥作用调节这些蛋白质在肿瘤细胞的水平。
**(计算分析结论：miRNAs表达与编码cyclins和CDKs的转录本关系： **
mir - 195 - 5 - p与细胞周期蛋白E1,E2, CDK1, CDK2 的转录本编码表达显示最强的反相关性,同时也表现出与细胞周期蛋白D1D2、D3、和CDK6 的表达强烈的反相关性(表S3),这表明它可能发挥作用调节这些蛋白质在肿瘤细胞的水平)
We also searched for correlation/anti-correlation between expression of all miRNAs and expression of all protein-coding transcripts in 4,807 human tumor samples.
我们还在4,807个人类肿瘤样本中寻找所有mirna表达与所有蛋白编码转录本表达之间的相关性/反相关性。
The correlating transcripts were then analyzed for enrichment in KEGG pathway categories.
然后分析相关转录产物在KEGG通路类别中的富集情况。
This guilt-by-association analysis revealed that expression of some cell-cycle-targeting miRNAs, such as miR-195-5p and miR-214-5p, strongly anti-correlated with expression of genes belonging to cell-cycle and DNA replication pathways (Figure 3A).
这种关联分析显示，一些细胞周期靶向mirna的表达，如miR-195-5p和miR-214-5p，与细胞周期和DNA复制通路的基因表达具有很强的反相关性(fig3A)。
We hypothesized that cell-cycle-targeting miRNAs, through their virtue of repressing the cell-cycle machinery, might display growth-suppressive properties.
我们假设，细胞周期靶向miRNAs，通过其抑制细胞周期机制的优点，可能显示生长抑制特性。
Consequently, we predicted that expression of some of these miRNAs might be silenced during tumorigenesis.
因此，我们预测其中一些miRNAs的表达可能在肿瘤发生过程中被抑制。
To test this prediction, we used the TCGA database to compare the expression of all cell-cycle-targeting miRNAs in 14 types of human tumors versus adjacent healthy tissue from the same patient.
为了验证这一预测，我们使用TCGA数据库比较了14种人类肿瘤中所有细胞周期靶向miRNAs与同一患者相邻健康组织的表达情况。
We found that of all miRNAs, miR-195-5p was the one most significantly downregulated in 11 of 14 tumor types (Figure 3B； p values from 1.77 3 1012 to 0.011 for significant downregulation, Wilcoxon test)
我们发现所有的miRNAs, mir - 195 - 5 - p是一个最重要的表达，在14肿瘤类型11肿瘤类型下调(fig3 B; p值从1.77 3 1012到0.011,这些Wilcoxon测试差别明显)
(假设：细胞周期靶向miRNAs，通过其抑制细胞周期机制的优点，可能显示生长抑制特性
数据分析验证：使用TCGA数据库比较了14种人类肿瘤中所有细胞周期靶向miRNAs与同一患者相邻健康组织的表达情况
发现：所有的miRNAs, mir - 195 - 5 - p是一个最重要的表达，在14肿瘤类型11肿瘤类型下调)
In addition, we intersected the results of our nine screens with the TCGA registry of deleted regions across many human tumor types and searched for cell-cycle-targeting miRNAs that are commonly deleted in human cancers (Figure 3C).
此外，我们将9个筛选的结果与以CGA注册表中许多人类肿瘤类型中被删除的区域分割，并搜索在人类癌症中通常被删除的细胞周期靶向miRNAs(fig3C)。
These analyses revealed that the miR-193a gene, which encodes one of the most potent cell-cycle-regulating miRNAs, miR-193a-3p, is frequently deleted in several types of human tumors.
这些分析显示，miR-193a基因编码的miR-193a-3p是一种最有效的细胞周期调节miRNAs，在几种人类肿瘤中经常被删除。
We also identified numerous cell lines displaying copy number deletions of miR-193a (Figure S4A).
我们还鉴定了许多显示miR-193a拷贝数缺失的细胞系(图S4A)。
Among them, a lung squamous cell carcinoma cell line, SW900, harbors a very narrow, focal homozygous deletion encompassing the miR-193a gene (Figures S4A and S4B).
其中，肺鳞状细胞癌细胞系SW900包含一个非常狭窄的、局灶性纯合缺失的miR-193a基因(图S4A和S4B)。
（数据分析：将9个筛选的结果与以CGA注册表中许多人类肿瘤类型中被删除的区域分割，并搜索在人类癌症中通常被删除的细胞周期靶向miRNAs
发现：miR-193a基因编码的miR-193a-3p是一种最有效的细胞周期调节miRNAs，在几种人类肿瘤中经常被删除）
To test the response of SW900 cells to re-introduction of the deleted miRNA, we transfected double-stranded mimic of miR- 193a into SW900 cells and analyzed transcript abundance by RNA sequencing.
为了检测SW900细胞对缺失miRNA重新导入的反应，我们将miR- 193a双链模拟物转染到SW900细胞中，通过RNA测序分析转录物丰度。
Consistent with the well-established repressive effect of miRNAs on gene expression, nearly all transcripts that were affected by re-introduction of miR-193a displayed reduced levels (Figure 3D).
与miRNA对基因表达的抑制作用相一致，几乎所有被再次引入miR-193的转录本a都显示出降低的水平(fig3D)
Strikingly, the repressed targets were strongly enriched in the cell-cycle category and included cyclins and CDKs (Figures 3E and S4C).
值得注意的是，被抑制的靶点在细胞周期类别中高度富集，包括周期蛋白和CDKs(fig3E和S4C)。
Consistent with inhibition of the cell-cycle machinery, expression of miR-193a blocked proliferation of SW900 cells (data not shown).
与抑制细胞周期机制一致，miR-193a的表达抑制了SW900细胞的增殖(数据未显示)。
（实验：miR- 193a双链模拟物转染到SW900细胞中，通过RNA测序分析转录物丰度
数据分析：转录本表达水平，抑制表达富集分析
结论：miR-193a的表达抑制了SW900细胞的增殖
）