Cell-Cycle-Targeting miRNAs Inhibit Proliferation of Cancer Cells
细胞周期靶向miRNAs抑制癌细胞的增殖
We asked whether other cell-cycle-targeting miRNAs might also have an anti-proliferative effect on cancer cells.
我们想知道其他细胞周期靶向miRNAs是否也可能对癌细胞具有抗增殖作用。
To test this, we transiently expressed a panel of 11 miRNAs targeting 6 to 9 cyclins/CDKs (Figure 4A) in human osteosarcoma U2OS cells and monitored cell number expansion over 6 days.
为了测试这一点，我们在U2OS人骨肉瘤细胞中瞬时表达了11个miRNAs，目标为6到9个cyclins/CDKs(fig4A)，并在6天内监测细胞数量的扩张。
We found that all 11 cell-cycle-targeting miRNAs strongly inhibited proliferation of U2OS cells (Figures 4B and 4C).
我们发现所有11个细胞周期靶向miRNAs都能强烈抑制U2OS细胞的增殖(fig4B和fig4C)。
Importantly, point mutations within miRNA seed sequences abolished the ability of miRNAs to inhibit cell growth, confirming the specificity of the effect (Figure 4D).
重要的是，miRNA种子序列中的点突变破坏了miRNA抑制细胞生长的能力，证实了这种效应的特异性(fig4D)。
(推测：其他细胞周期靶向miRNAs是否也可能对癌细胞具有抗增殖作用
实验：在U2OS人骨肉瘤细胞中瞬时表达了11个miRNAs，目标为6到9个cyclins/CDKs(fig4A)，并在6天内监测细胞数量的扩张
图分析：表达热图
结论：发现所有11个细胞周期靶向miRNAs都能强烈抑制U2OS细胞的增殖)
Profiling the Response of Human Cancer Cell Lines from CCLE to Cell-Cycle-Targeting miRNAs
分析人类癌症细胞系从CCLE到细胞周期靶向mirna的反应
In order to further investigate the anti-proliferative properties of cell-cycle-targeting miRNAs, we profiled the response of 122 human cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE), representing 12 separate cancer types, to four selected miRNAs.
为了进一步研究细胞周期靶向miRNAs的抗增殖特性，我们分析了来自癌症细胞系百科全书(CCLE)的122个人类癌症细胞系(代表12种不同的癌症类型)对4个选定的miRNAs的反应。
For profiling, we chose miR-195-5p, miR-193a-3p, and miR-214-5p, which were highlighted above, as well as miR-890, the latter being chosen based on its identification as one of the top hits in our screens (Table S1)
为了进行分析，我们选择了miR-195-5p、miR-193a-3p、miR-214-5p(上面突出显示)，以及miR-890，后者是根据其在我们的屏幕中被识别为最热门之一而选择的(表S1)。
（进一步研究实验说明：细胞周期靶向miRNAs的抗增殖特性研究 实验：选择miR-195-5p、miR-193a-3p、miR-214-5p ，miR-890在122个人类癌症细胞系中的反应。）
We transiently expressed each of the four miRNAs in 122 cell lines and monitored for reduction in cell number expansion (Figure 5A), induction of apoptosis (Figure 5B), and induction of cellular senescence (Figures S5A).
我们在122个细胞系中瞬时表达了这四种 miRNAs，并监测它们在细胞数量扩张减少(fig5A)、诱导凋亡(fig5B)和诱导细胞衰老(figS5A)方面的作用。
We found that all four miRNAs had a profound effect on cancer cell proliferation (Figures 5A, 5C, and S5B；Table S4).
我们发现,所有四个miRNAs对癌症细胞增殖产生了深远的影响(fig5、5 c, S5B;表S4)。
In several cell lines expression of cell-cycletargeting miRNAs also triggered cancer cell death (Figure 5B and Table S4).
在一些细胞系中，细胞周期靶向miRNAs的表达也会引发癌细胞死亡(fig5B和表S4)。
None of the cell-cycle-targeting miRNAs were found to induce senescence in a significant fraction of cell lines (Figure S5A and Table S4).
未发现任何细胞周期靶向miRNAs在相当一部分细胞系中诱导衰老(figS5A和表S4)
(数据计算分析：四个miRNAs对癌症细胞增殖产生了深远的影响，在一些细胞系中，细胞周期靶向miRNAs的表达也会引发癌细胞死亡，未发现任何细胞周期靶向miRNAs在相当一部分细胞系中诱导衰老)
Among all cancer types analyzed, cell lines corresponding to gastric and triple-negative breast cancers (TNBCs) displayed the strongest response to expression of cell-cycle-targeting miRNAs, with miR-193a 3p being the most effective in suppressing cell numbers across all tumor types (Figures 5C and S5B).
在分析的所有癌症类型中，胃癌和三阴性乳腺癌(TNBCs)对应的细胞系对细胞周期靶向mirna表达的反应最强，miR-193a 3p是所有肿瘤类型中抑制细胞数量最有效的(fig5C和S5B)。
TNBCs underwent a significant, often over 10-fold reduction in cell number expansion, with several TNBC cell lines also displaying a pronounced apoptotic response (Figures 5A and 5B；Table S4)；see Figure S5C for expression of cell-cycle proteins in TNBC.
TNBCs经历了很大,通常超过10倍减少细胞数量扩张,与几个TNBC细胞系也显示明显的凋亡反应(fig5 a和5 b;表S4);参见figS5C TNBC细胞循环蛋白的表达。
Importantly, we verified that ectopic expression of miR-193a-3p in TNBC led to downregulation of predicted cell-cycle targets (Figures S5D and S5E).
重要的是，我们证实了miR-193a-3p在TNBC中的异位表达导致预测的细胞周期靶点下调(figS5D和S5E)。
(数据分析图解读：所有癌症类型中，胃癌和三阴性乳腺癌(TNBCs)对应的细胞系对细胞周期靶向mirna表达的反应最强，miR-193a 3p是所有肿瘤类型中抑制细胞数量最有效的，证实了miR-193a-3p在TNBC中的异位表达导致预测的细胞周期靶点下调)
To test whether the observed effects on cancer cell expansion and apoptosis were mediated via the ability of cell-cycle-targeting miRNAs to repress the expression of target cyclins/CDKs, we engineered four TNBC cell lines to ectopically express cDNAs encoding cyclins D1, D3, and E2 (Figure S5E), which represent targets of miR-193a-3p (Table S1).
测试是否观察到对癌了细胞扩张和通过细胞周期靶向miRNAs的介导细胞凋亡抑制细胞周期蛋白/ CDKs目标的表达的影响,我们检测四个TNBC细胞系异位表达细胞周期蛋白D1,D3, E2的互补编码(figS5E),它代表mir - 193 a - 3 - p的靶点(表S1)。
Importantly, the three cyclin cDNAs lack 3‘UTRs, and hence they are resistant to targeting by miRNAs.
重要的是，这三个cyclin cDNAs缺乏3’UTRs，因此它们对miRNAs的靶向性有抵抗力。
In contrast to parental cells,where miR-193a-3p blocked cancer cell growth, cells expressing miRNA-resistant cyclins were refractory to the anti-proliferative effect of miR-193a-3p, or, in the case of MDA-MB-468 cells, the anti-proliferative effect was blunted (Figure 5D).
与亲代细胞miR-193a-3p阻断癌细胞生长相比，表达耐mirna的周期蛋白的细胞对miR-193a-3p的抗增殖作用难以耐受，或者在MDA-MB-468细胞中，抗增殖作用减弱(fig5D)。
Moreover, expression of miRNA-resistant cyclins in four breast cancer cell lines largely prevented induction of apoptosis by miR-193a-3p in vitro (Figure S5F), and prevented apoptosis of tumor cells in vivo (see below).
此外，miR-193a-3p在4个乳腺癌细胞系中表达耐mirna的cyclins，在体外对miR-193a-3p诱导凋亡有很大的抑制作用(图S5F)，在体内对肿瘤细胞的凋亡有抑制作用(见下文)。
These observations indicate that targeting of cell-cycle proteins by miR-193a-3p is responsible for inhibition of proliferation and induction of apoptosis in cancer cells.
这些观察结果表明，miR-193a-3p靶向细胞周期蛋白可抑制癌细胞增殖，诱导细胞凋亡。
（提出问题：测试是否观察到对癌了细胞扩张和通过细胞周期靶向miRNAs的介导细胞凋亡抑制细胞周期蛋白/ CDKs目标的表达的影响
实验：检测四个TNBC细胞系异位表达细胞周期蛋白D1,D3, E2的互补编码(figS5E),它代表mir - 193 a - 3 - p的靶点
结果数据分析：miR-193a-3p靶向细胞周期蛋白可抑制癌细胞增殖，诱导细胞凋亡）
We also engineered lung cancer A549 cells and colon cancer LoVo cells to ectopically express cDNAs encoding cyclin D1, CDK2, and CDK6 (Figure S5G), which we found to represent targets of miR-214-5p (Table S1).
我们还检测肺癌A549细胞和结肠癌LoVo细胞体异位外表达编码cyclinD1、CDK2和CDK6的cDNAs(图S5G)，我们发现这些cDNAs代表miR-214-5p的靶点(表S1)。
In contrast to parental cells, where miR-214-5p blocked cell expansion, cancer cells expressing miRNA-resistant cell-cycle targets were refractory to the antiproliferative effect of miR-214-5p (Figure 5E).
与亲代细胞miR-214-5p阻断细胞扩张不同，表达mirna耐药细胞周期靶点的癌细胞对miR-214-5p的抗增殖作用难以抑制(fig5E)。
(更多实验数据检测：还检测肺癌A549细胞和结肠癌LoVo细胞体异位外表达编码cyclinD1、CDK2和CDK6的cDNAs
结论：表达mirna耐药细胞周期靶点的癌细胞对miR-214-5p的抗增殖作用难以抑制)
Given the observed strong effects of cell-cycle-targeting miRNAs on tumor cells, we examined the response of ten human non-transformed cell lines tothe fourmiRNAs.
考虑到细胞周期靶向mirna对肿瘤细胞的强大作用，我们检测了10个人类非转化细胞系对4个mirna的反应。
We foundthatthese miRNAs had a much milder effect on growth of non-transformed cells and that they did not trigger apoptosis (Figures 5F and 5G)
我们发现这些mirna对非转化细胞的生长有更温和的影响，并且它们不会引发细胞凋亡(fig5F和图5G)。
(更多实验情况说明：10个人类非转化细胞系对4个mirna的反应)
Response of Human Breast Cancer Cells to miR-193a-3p Compared with Therapeutic Compounds
与治疗化合物相比，人类乳腺癌细胞对miR-193a-3p的反应
Our CCLE screen demonstrated that TNBC lines are highly sensitive to cell-cycle-targeting miRNAs and that miR-193a-3p had overall the strongest effect across cell lines representing all tumor types.
我们的CCLE筛选显示，TNBC细胞系对细胞周期靶向miRNAs高度敏感，而miR-193a-3p在代表所有肿瘤类型的细胞系中总体效果最强。
In order to further evaluate the therapeutic potential of this miRNA, we compared the response of breast cancer cells to miR-193a-3p expression versus treatment with an inhibitor of CDK4/6 kinase, palbociclib (PD-0332991,Ibrance).
为了进一步评估这一miRNA的治疗潜力，我们比较了乳腺癌细胞对miR-193a-3p表达的响应与CDK4/6激酶抑制剂palbociclib (PD-0332991,Ibrance)的治疗。
Consistent with previous reports (Finn et al., 2009) palbociclib treatment of nine TNBC cell lines had only a very mild effect on cancer cell expansion, and it was totally ineffective in cell lines lacking the functional retinoblastoma protein, RB1 (Figure 6A).
与之前的报道一致(Finn et al.， 2009)， palbociclib对9个TNBC细胞系的治疗对癌细胞的扩增作用非常微弱，在缺乏功能性视网膜母细胞瘤蛋白RB1的细胞系中完全无效(fig6A)。
In contrast, ectopic expression of miR-193a-3p strongly decreased cell numbers in eight out of nine TNBC cell lines.
相比之下，miR-193a-3p的异位表达显著减少了9个TNBC细胞系中的8个细胞数量。
Of particular interest, miR-193a-3p was potent against cell lines harboring deletion of the RB1 gene, pointing to the therapeutic potential of this miRNA against RB1-negative tumors (Figure 6A).
特别值得注意的是，miR-193a-3p对含有RB1基因缺失的细胞系具有很强的抑制作用，这表明该miRNA对RB1阴性肿瘤具有治疗潜力(fig6A)。
(实验目的：进一步评估这一miRNA的治疗潜力
数据分析结果：palbociclib对9个TNBC细胞系的治疗对癌细胞的扩增作用非常微弱，在缺乏功能性视网膜母细胞瘤蛋白RB1的细胞系中完全无效，miR-193a-3p对含有RB1基因缺失的细胞系具有很强的抑制作用)
Chemical inhibition of CDKs was shown to have a profound effect on TNBC, as this tumor type may be particularly dependent on CDK kinases for cancer cell survival (Horiuchi et al., 2012).
化学抑制CDKs被证明对TNBC有深刻的影响，因为这种肿瘤类型可能特别依赖CDK激酶来维持癌细胞存活(Horiuchi et al.， 2012)。
We therefore compared the effects of miR-193a- 3p expression in TNBC cells versus treatment with CDK inhibitors dinaciclib and purvalanol A, which inhibit CDK1, CDK2, and other cell-cycle kinases.
因此，我们比较了miR-193a- 3p在TNBC细胞中的表达与CDK抑制剂dinaciclib和purvalanol A对CDK1、CDK2等细胞周期激酶的抑制作用。
We found that miR-193a-3p was generally equally or more potent than each of the two inhibitors (Figure 6B).
我们发现miR-193a-3p通常比这两种抑制剂的效力相同或更强(fig6B)。
(文献论证：化学抑制CDKs被证明对TNBC有深刻的影响，因为这种肿瘤类型可能特别依赖CDK激酶来维持癌细胞存活(Horiuchi et al.， 2012)
实验：比较了miR-193a- 3p在TNBC细胞中的表达与CDK抑制剂dinaciclib和purvalanol A对CDK1、CDK2等细胞周期激酶的抑制作用
数据分析，图展示结论：miR-193a-3p通常比这两种抑制剂的效力相同或更强)
In order to explore the effect of miR-193a-3p on other types of breast cancers, we compared the response of human estrogen-receptor-positive (ER+) mammary carcinoma cell lines to miR-193a-3p versus palbociclib, which is currently being used in clinical trials for patients bearing ER+ tumors (Asgharet al., 2015；Finn et al.,2015).
为了探索的影响mir - 193 - a - 3 - p在其他类型的乳腺癌,我们比较了反应人类的雌激素受体阳性的(ER +)乳房癌细胞系输入mir - 193 - a - 3 - p反应与接受palbociclib抗体的反应,它目前被用于临床试验轴承ER +肿瘤患者(Asgharet。2015;芬恩et al ., 2015)。
We found that miR-193a-3pwas quite effective against ER+ breast cancer cell lines,although generally to a lesser extent than palbociclib(Figure 6C).
我们发现mir -193a-3p对ER+乳腺癌细胞系非常有效，尽管总体上比palbociclib的作用要小(fig6C)。
(文献举例论证：接受palbociclib抗体的反应,它目前被用于临床试验轴承ER +肿瘤患者(Asgharet。2015;芬恩et al ., 2015)
实验数据作图分析证实结果：mir -193a-3p对ER+乳腺癌细胞系非常有效，尽管总体上比palbociclib的作用要小)
Lastly, we gauged the impact of miR-193a-3p expression on HER2+ breast cancer cell lines and compared it with the effect of lapatinib.
最后，我们测量了miR-193a-3p表达对HER2+乳腺癌细胞系的影响，并将其与拉帕替尼的影响进行了比较。
Lapatinib, a dual HER2/EGFR tyrosine kinase inhibitor, is used for treatment of HER2+ breast cancers.
拉帕替尼是一种双HER2/EGFR酪氨酸激酶抑制剂，用于治疗HER2+乳腺癌。
We found that miR-193a-3p was equivalent to or superior to lapatinib alone in most cases and often had a comparable impact on cell number expansion with the combined effect of lapatinib plus palbociclib (Figure 6D).
我们发现，在大多数情况下，miR-193a-3p与单独的拉帕替尼相当或优于拉帕替尼，并且在拉帕替尼与palbociclib联合作用下，miR-193a-3p对细胞数量扩张的影响往往相当(fig6D)。
Together, these results suggest a therapeutic potential of miR-193a-3p for breast cancers and in particular for TNBCs
总之，这些结果表明miR-193a-3p对乳腺癌，尤其是对TNBCs具有治疗潜力
(实验：测量了miR-193a-3p表达对HER2+乳腺癌细胞系的影响，并将其与拉帕替尼的影响进行了比较
数据分析结论：这些结果表明miR-193a-3p对乳腺癌，尤其是对TNBCs具有治疗潜力)
Nanoparticle-Mediated Delivery of miR-193a-3p to Xenografts of Triple-Negative Breast Cancers
纳米颗粒介导的miR-193a-3p在三阴性乳腺癌异种移植物中的表达
Nanoparticle-Mediated Delivery of miR-214-5p to KRAS-Mutant Cancers
纳米颗粒介导的miR-214-5p在kras突变癌中的表达
To extend these observations to an in vivo setting, we asked whether a systemic administration of miR-193a-3p to mice bearing xenografts of human TNBC would halt tumor progression.
为了将这些观察扩展到体内环境中，我们提出了这样一个问题:将miR-193a-3p系统地应用于携带人类TNBC异种移植瘤的小鼠是否会阻止肿瘤的进展。
To deliver miR-193a-3p to tumor-bearing mice, we utilized nanoparticles based on epoxidederived lipidoid C12-200.
为了将miR-193a-3p传递给荷瘤小鼠，我们使用了基于环氧衍生类脂质C12-200的纳米颗粒。
This type of nanoparticles was shown to avoid immune responses and to improve delivery of large bulky double-stranded nucleic acids to animals in vivo without causing toxicity (Love et al., 2010).
这种类型的纳米颗粒被证明可以避免免疫反应，并在不引起毒性的情况下，改善大体积双链核酸在动物体内的传递(Love et al.， 2010)。
（问题提出：将miR-193a-3p系统地应用于携带人类TNBC异种移植瘤的小鼠是否会阻止肿瘤的进展。
文献支持实验进行：这种类型的纳米颗粒被证明可以避免免疫反应，并在不引起毒性的情况下，改善大体积双链核酸在动物体内的传递(Love et al.， 2010)）
We first subcutaneously injected immunocompromised mice with TNBC CAL51 or HCC1806 cells leading to formation of tumors (see Figure S6A for the endogenous levels of miR-193a-3p in these tumors).
我们首先皮下给免疫缺陷小鼠注射TNBC CAL51或HCC1806细胞导致肿瘤形成的这些肿瘤中内源性miR-193a-3p水平见图S6A)。
Once the animals developed palpable tumors, every 2–3 days we systematically administered nanoparticles containing miR-193a-3p or containing miRNA mimic negative control via tail vein injection(Figure 7A).
一旦动物出现可触及的肿瘤，每隔2-3天，我们通过尾静脉注射系统地给含有miR-193a-3p或含有miRNA药模拟阴性对照的纳米颗粒(fig7A)。
We found that administration of nanoparticles with miR-193a-3p significantly inhibited tumor growth in vivo (Figures 7B and 7C).
我们发现，miR-193a-3p纳米颗粒在体内显著抑制肿瘤生长(fig7B和fig7C)。
Analyses of tumors for 5-bromo-2-deoxyuridine (BrdU) uptake, Ki67 staining, and cleaved caspase-3 revealed significant reduction of proliferation and induction of apoptosis in miR-193a-3p-treated mice (Figures S6B–S6G)
对肿瘤5-溴-2-脱氧尿苷(BrdU)摄取、Ki67染色和裂解的caspase-3的分析显示，mir -193a-3p处理小鼠的增殖和凋亡诱导显著降低(figS6B-S6G)。
(首先：
实验：皮下给免疫缺陷小鼠注射TNBC CAL51或HCC1806细胞导致肿瘤形成的这些肿瘤中内源性miR-193a-3p
实验结果图分析对比说明：mir -193a-3p处理小鼠的增殖和凋亡诱导显著降低）
To ascertain that the observed in vivo effects were mediated through the ability of miR-193a-3p to directly target cyclins/CDKs, we generated xenografts using CAL51 cells engineered to express miRNA-resistant cyclins D1, D3, and E2.
为了确定体内观察到的效应是通过miR-193a-3p直接靶向细胞周期蛋白/CDKs的能力介导的，我们使用工程设计的CAL51细胞生成异种移植，表达耐mirna的细胞周期蛋白D1、D3和E2。
Strikingly, administration of miR-193a-3p to tumor-bearing animals had essentially no inhibitory effect on tumor growth (Figure S6H).
值得注意的是，miR-193a-3p给药给荷瘤动物对肿瘤生长基本没有抑制作用(图S6H)。
In contrast to xenografts derived from parental CAL51 cells, tumor cells expressing miRNA-resistant cyclins did not show reduced BrdU or Ki67 staining and were protected against miR-193a-3p-induced apoptosis (Figures S6I–S6K).
与来自亲代CAL51细胞的异种移植相比，表达耐mirna的cyclins的肿瘤细胞并没有表现出BrdU或Ki67染色的减少，并且对mir -193a-3p诱导的凋亡具有保护作用(图S6I-S6K)。
（进一步实验论证：体内观察到的效应是通过miR-193a-3p直接靶向细胞周期蛋白/CDKs的能力介导的
实验设计：使用工程设计的CAL51细胞生成异种移植，表达耐mirna的细胞周期蛋白D1、D3和E2
实验结果与图分析：miR-193a-3p给药给荷瘤动物对肿瘤生长基本没有抑制作用）
We further confirmed the anti-tumor effect in vivo using mice bearing orthotopic xenografts of a patient-derived TNBC.
我们进一步证实了在体内的抗肿瘤作用，使用小鼠原位异种移植的来源病人的TNBC。
This tumor was refractory to conventional therapy and had progressed after treatment with doxorubicin and paclitaxel.
该肿瘤对常规治疗无效，经阿霉素和紫杉醇治疗后进展。
Administration of miR-193a-3p to mice bearing this aggressive tumor strongly inhibited tumor expansion (Figures 7D and S6L–S6N)
把药miR-193a-3p给具有这种侵袭性肿瘤的小鼠强烈抑制肿瘤的扩张(fig7D和S6L-S6N)
（进一步证实：把药miR-193a-3p给具有这种侵袭性肿瘤的小鼠强烈抑制肿瘤的扩张）
Analyses of nanoparticle-treated mice revealed that both normal organs as well as tumors displayed uptake of the exogenous miR-193a-3p (Figure S6O).
对纳米颗粒处理小鼠的分析显示，正常器官和肿瘤均显示外源性miR-193a-3p的摄取(图S6O)。
Given this broad uptake, we tested the impact of long-term administration of miR-193a-3p on normal tissues.
鉴于这一广泛的摄取，我们测试了长期给药miR-193a-3p对正常组织的影响。
We found that continuous systemic administration of nanoparticles containing miR- 193a-3p to animals had no detectable effect on proliferation rate in the internal organs (Figures S7A and S7B).
我们发现含miR- 193a-3p的纳米颗粒持续全身给药对动物体内器官的增殖率没有明显影响(图S7A和S7B)。
Also, no increased apoptosis was observed in the internal organs of treated mice (Figure S7C).
此外，在处理后的小鼠的内脏器官中没有观察到凋亡的增加(图S7C)。
This is in contrast to tumor cells in nanoparticle-treated mice, where miR-193a-3p reduced proliferation and triggered apoptosis (Figures S6B–S6G and S6L–S6N)
这与纳米颗粒处理小鼠的肿瘤细胞形成对比，在纳米颗粒处理小鼠中，miR-193a-3p减少增殖并触发凋亡(图S6B-S6G和S6L-S6N)
（实验：对比长期给药miR-193a-3p对正常组织的影响
图分析与结论：含miR- 193a-3p的纳米颗粒持续全身给药对动物体内器官的增殖率没有明显影响）
We also analyzed the histopathological appearance of internal organs in mice treated long-term with miR-193a-3p-containing nanoparticles and found no apparent abnormalities (Figure S7D and data not shown).
我们还分析了长期使用含mir -193a-3p纳米颗粒治疗的小鼠内脏组织病理学外观，未发现明显异常(图S7D，未显示数据)。（实验进一步说明：长期使用含mir -193a-3p纳米颗粒治疗的小鼠内脏组织病理学外观，未发现明显异常）
Lastly, we determined that long-term administration of nanoparticles containing miR-193a-3p had no obvious effect on biochemical parameters in the peripheral blood of recipient mice (Figure S7E), no obvious effect on liver function (Figure S7F) and no detectable effect on the function of the hematopoietic system (Figures S7G and S7H).
最后，我们确定，长期给药含miR-193a-3p的纳米颗粒对受体小鼠外周血生化参数无明显影响(图S7E)，对肝功能无明显影响(图S7F)，对造血系统功能无明显影响(图S7G和S7H)。（实验进一步说明：，长期给药含miR-193a-3p的纳米颗粒对受体小鼠外周血生化参数无明显影响(图S7E)，对肝功能无明显影响(图S7F)，对造血系统功能无明显影响）
Collectively, these analyses indicate that nanoparticle-based delivery of miR-193a-3p may represent an efficient strategy against refractory TNBCs in vivo, by selectively targeting cancer cells while sparing normal, non-transformed tissues.
综上所述，这些分析表明，基于纳米颗粒的miR-193a-3p递送可能是一种针对体内难治 TNBCs的有效策略，方法是选择性靶向癌细胞，同时保留正常的非转化组织。（实验结论：基于纳米颗粒的miR-193a-3p递送可能是一种针对体内难治 TNBCs的有效策略，方法是选择性靶向癌细胞，同时保留正常的非转化组织）
Nanoparticle-Mediated Delivery of miR-214-5p toKRAS-Mutant Cancers
纳米颗粒介导的miR-214-5p在kras突变癌中的表达
Analyses of the response of 122 human cancer cell lines to the four cell-cycle-targeting miRNAs allowed us to correlate the observed response with genetic lesions carried by each of these cell lines, which have been annotated in CCLE.
对122个人类癌细胞株对四种细胞周期靶向miRNAs的反应进行分析，使我们能够将观察到的反应与这些细胞株携带的遗传损伤联系起来，这些遗传损伤已在CCLE中进行了注释。
These analyses revealed that miR-214-5p is more potent against cell lines harboring mutations in KRAS than those with wild-type KRAS (p = 0.02 by Wilcoxon test) (Figure 8A).
这些分析表明，miR-214-5p比野生型KRAS更能抵抗KRAS突变株(Wilcoxon test p = 0.02)(fig8A)。
We validated this finding in vivo using nanoparticle-mediated delivery of miR-214-5p to mice bearing xenografts of human KRAS-mutant lung cancer A549 cells or colon cancer LoVo cells (please see Figure S8A for the levels of miR-214-5p in these tumors).
我们在体内通过纳米颗粒介导将miR-214-5p传递给携带kras突变肺癌A549细胞或结肠癌LoVo细胞异种移植瘤的小鼠来验证这一发现(这些肿瘤中miR-214-5p的水平见图S8A)。
We found that a systemic administration of miR-214-5p inhibited growth of KRAS-mutant tumors in vivo (Figures 8B, 8C, and S8B–S8E)
我们发现，在体内系统给予miR-214-5p可以抑制kras突变瘤的生长(图8B、8C和S8B-S8E)。
(进一步实验分析说明：体内系统给予miR-214-5p可以抑制kras突变瘤的生长)
We extended these observations using mice bearing xenografts of patient-derived metastatic KRAS-mutant colon adenocarcinoma.
我们将这些观察结果扩展到携带病人转移性kras突变结肠腺癌异种移植的小鼠。
The primary tumor was refractory to treatments with FOLFOX (folinic acid + 5-fluorouracil + oxaliplatin) and FOLFIRI (folinic acid + 5-fluorouracil + irinotecan) + bevacizumab.
原发性肿瘤对FOLFOX(叶酸+ 5-氟尿嘧啶+奥沙利铂)和FOLFIRI(叶酸+ 5-氟尿嘧啶+伊立替康)+贝伐单抗治疗无效。
Administration of miR-214-5p to mice bearing this aggressive cancer strongly inhibited tumor growth (Figures 8D, S8F, and S8G).
将miR-214-5p给具有这种侵袭性肿瘤的小鼠，可以强烈抑制肿瘤生长(图8D、S8F和S8G)。（实验论证+图结果分析：将miR-214-5p给具有这种侵袭性肿瘤的小鼠，可以强烈抑制肿瘤生长）
As was the case for miR-193a-3p, repeated administration of miR-214-5p to mice had no detectable effect on cell proliferation in the internal organs (Figures S7A and S7B).
与miR-193a-3p的情况一样，反复给药miR-214-5p对小鼠体内器官的细胞增殖没有检测到影响(图S7A和S7B)。
The recipient mice displayed unperturbed histopathological appearance of the internal organs (Figure S7D), and normal blood chemistry, liver function and hematological parameters (Figures S7E–S7H).
受体小鼠内脏组织病理学表现稳定(图S7D)，血液化学、肝功能、血液学参数正常(图S7E-S7H)。（进一步实验结论：反复给药miR-214-5p对小鼠体内器官的细胞增殖没有检测到影响）
Collectively, these observations suggest that nanoparticlebased delivery of miR-214-5p might represent an effective therapeutic strategy against KRAS-mutant tumors, including aggressive, treatment-refractory cases.
综上所述，这些观察结果表明，基于纳米颗粒的miR-214-5p的递送可能代表着对kras突变瘤(包括侵袭性、难治性肿瘤)的有效治疗策略。（实验总结：基于纳米颗粒的miR-214-5p的递送可能代表着对kras突变瘤）
To exclude a possibility that our miRNA anti-tumor effects might have been caused by a non-specific activation of the immune system, we monitored liver expression of a panel of cytokines, 2 days post injection of miR-214-5p.
为了排除我们的miRNA抗肿瘤作用可能是由免疫系统的非特异性激活引起的可能性，我们在注射miR-214-5p 2天后监测了一组细胞因子的肝脏表达。
We detected no major changes in the cytokine levels, essentially ruling out a non-specific immune response as the underlying mechanism (Figure S8H).
我们没有检测到细胞因子水平的重大变化，基本上排除了非特异性免疫反应作为潜在机制(图S8H)。（实验可能其它因素排除：排除了非特异性免疫反应作为潜在机制）
Expression-Based Algorithm to Predict the Response of Tumors to Cell-Cycle-Targeting miRNAs
基于表达的算法预测肿瘤对细胞周期靶向miRNAs的反应
By correlating gene expression profiles of each of the 122 human cancer cell lines available from CCLE with the response to the four cell-cycle-targeting miRNAs, we developed a high-dimensional regression method based on elastic net (Zou and Hastie, 2005), which predicts the response of cancer cells to each of the miRNAs (Table S5).
通过相关基因表达谱的122个人类癌症细胞系可以从CCLE四cell-cycle-targeting microrna的响应,我们开发了一个基于弹性高维回归方法净(邹Hastie, 2005),预测癌细胞的microrna的反应(表S5)。
In order to test whether this algorithm is applicable to patient-derived tumors, we analyzed gene expression of an aggressive dermatofibrosarcoma protuberans that was refractory to conventional therapy and progressed despite treatment with imatinib (Gleevec) and PI3K inhibitor pictilisib (GDC-0941).
为了检验该算法是否适用于患者来源的肿瘤，我们分析了一种侵袭性隆突性皮肤纤维肉瘤的基因表达，这种肉瘤对传统治疗难以耐受，尽管使用了伊马替尼(Gleevec)和PI3K抑制剂pictilisib (GDC-0941)治疗，但仍在进展。
By querying gene expression data with our algorithm, we predicted that this tumor would be responsive to miR-193a-3p (Table S5).
通过我们的算法查询基因表达数据，我们预测该肿瘤将对miR-193a-3p产生应答(表S5)。
To validate this prediction in vivo, we implanted patient-derived tumor cells into immunocompromised mice and systematically delivered nanoparticles containing miR-193a-3p.
为了在体内验证这一预测，我们将患者来源的肿瘤细胞植入免疫缺陷小鼠体内，并系统地递送含有miR-193a-3p的纳米颗粒。
Indeed, administrationofmiR-193a-3psignificantly inhibitedtumorgrowth invivo (Figures 8E, 8F, and S8I–S8K).
事实上，mir -193a-3p在体内显著抑制肿瘤生长(图8E、8F和S8I-S8K)。
These observations suggest that our expression-based algorithm allows one to predict the response of primary tumors to individual miRNAs
这些观察表明，我们基于表达的算法可以预测原发性肿瘤对单个mirna的反应
（开发算法+文献支持：开发了一个基于弹性高维回归方法净(邹Hastie, 2005),预测癌细胞的microrna的反应
算法和实际实现比较分析：mir -193a-3p在体内显著抑制肿瘤生长
结论：基于表达的算法可以预测原发性肿瘤对单个mirna的反应）
Discussion讨论
A role for miRNAs in the pathogenesis of cancer was suggested shortly after their discovery in humans 16 years ago (Lagos-Quintana et al., 2001).
miRNAs在16年前在人类身上发现后不久，就提出了其在癌症发病机制中的作用(Lagos-Quintana et al.， 2001)。
The study of Lu et al. (2005) revealed that miRNAs are generally downregulated in human cancers, suggesting that many miRNAs might play tumor-suppressive roles.
Lu等人(2005)的研究表明，在人类癌症中，mirna普遍下调，提示许多mirna可能发挥抑癌作用。
Indeed, reduced expression of the let-7 family of miRNAs was associated with clinical progression of lung cancers, while the loss of miR-15/16 was linked to progression in chronic lymphocytic leukemia (Calin et al., 2005;Takamizawa et al., 2004).
事实上，mirna -7家族的表达减少与肺癌的临床进展相关，而miR-15/16的缺失与慢性淋巴细胞白血病的进展相关(Calin et al.， 2005;Takamizawa et al.， 2004)。
However, very few individual miRNAs have been validated to play tumor-suppressive roles using genetic systems.
然而，很少有单个的mirna被证实能够利用遗传系统发挥抑制肿瘤的作用。
This migh seem surprising in the light of numerous reports linking miRNAs to cancer but could be explained by the highly redundant function of these small regulatory RNAs in physiological settings (Bartel, 2009).
在众多将mirna与癌症联系起来的报道中，这可能似乎令人惊讶，但这可以用这些小调控rna在生理环境中的高度冗余功能来解释(Bartel, 2009)。
Several groups proposed to use miRNA overexpression systems as an anti-cancer therapeutic strategy, and this approach led to promising outcomes in vivo (Kota et al., 2009;Liu et al.,2011).
几个小组提出使用miRNA过表达系统作为一种抗癌治疗策略，这一方法在体内产生了有希望的结果(Kota et al.， 2009;刘et al ., 2011)。
In particular, a synthetic miR-34a mimic is currently in clinical trials (NCT01829971) for treatment of patients with primary liver cancer or with liver metastases.
特别是，一种合成miR-34a模拟物目前正在临床试验中(NCT01829971)，用于治疗原发性肝癌或肝转移患者。
Interestingly, miRNAs used in these in vivo systems have been reported to regulate, among other targets, also cyclins and/or CDKs, highlighting a potential link between successful miRNA mimic therapy and repression of the cell-cycle machinery.
有趣的是，在这些体内系统中使用的miRNA已被报道调节细胞周期蛋白和/或CDKs等靶点，突出了成功的miRNA模拟治疗和抑制细胞周期机制之间的潜在联系。
To explore this possible therapeutic link, we performed nine genome-wide screens to enumerate miRNAs that directly regulate the cell-cycle machinery.
为了探索这种可能的治疗联系，我们进行了9个全基因组筛选，以列举直接调控细胞周期机制的mirna。
We uncovered a class of miRNAs in the human genome, which target all, or nearly all cyclins and CDKs.
我们在人类基因组中发现了一类miRNAs，它针对所有或几乎所有的周期蛋白和CDKs。
We showed that these cell-cycle-targeting miRNAs effectively inhibit proliferation of a wide array of cancer cell types.
我们证明，这些细胞周期靶向mirna能够有效抑制多种癌症细胞类型的增殖。
We demonstrated that these miRNAs can be systematically administered to animals in the form of nanoparticles where they effectively inhibit proliferation of tumor cells while sparing normal, non-transformed tissues.
我们证明，这些mirna可以以纳米颗粒的形式系统地应用于动物体内，它们可以有效抑制肿瘤细胞的增殖，同时保留正常的、未转化的组织。
We extended our studies to patient-derived xenografts of tumors that were refractory to standard treatments and demonstrated the efficacy of using cell-cycle targeting miRNAs to target these incurable tumors.
我们将研究扩展到对标准治疗难以耐受的肿瘤患者来源的异种移植，并证明了使用细胞周期靶向mirna靶向这些无法治愈的肿瘤的有效性。
Hence,with improved delivery methods to tumor tissues, these miRNAs might offer an effective anti-cancer strategy against deadly, refractory cancer types.
因此，随着对肿瘤组织传递方法的改进，这些mirna可能提供一种针对致命的、难治的癌症类型的有效抗癌策略。
In addition to inducing proliferative arrest, cell-cycle-targeting miRNAs also triggered cell death of several cancer cell lines.
除了诱导增殖抑制外，细胞周期靶向miRNAs还引发了几种癌细胞系的细胞死亡。
This observation is in agreement with published reports that inhibition of cell-cycle kinases or downregulation of cyclins can trigger tumor cell apoptosis, in addition to cell cycle arrest (Choi et al.,2012;Goga et al., 2007;Horiuchi et al. 2012;Molenaar et al,2009Sawai et al., 2012)
这一观察结果与已发表的报道一致，即抑制细胞周期激酶或下调细胞周期蛋白，除了细胞周期阻滞外，还可触发肿瘤细胞凋亡(Choi et al.，2012;Goga等，2007;Horiuchi等，2012;(Molenaar et al.， 2009; sawai et al.， 2012)。
It is not clear why ectopic expression of the four cell-cycle-targeting miRNAs had the strongest effect on TNBCs and gastric cancers.
目前还不清楚为什么这四种细胞周期靶向mirna的异位表达对TNBCs和胃癌的影响最强。
One possible explanation is that TNBCs were shown to be particularly dependent on CDKs for cancer cell survival (Horiuchi et al., 2012).
一种可能的解释是，TNBCs被证明对CDKs对癌细胞存活的依赖性特别强(Horiuchi et al.， 2012)。
Consistent with this hypothesis, expression of cell-cycle-targeting miRNAs triggered apoptosis of TNBC cells in vitro and in vivo, and this response was prevented by ectopic expression of miRNA-resistant cell-cycle targets in cancer cells.
与这一假说一致，细胞周期靶向mirna的表达在体内外均可触发TNBC细胞的凋亡，而肿瘤细胞中mirna耐药细胞周期靶点的异位表达可抑制这种反应。
Our analyses revealed that a higher proportion of KRAS-mutant cancer cell lines was potently inhibited by miR-214-5p, compared with cancer cells expressing wild-type KRAS.
我们的分析显示，与表达野生型KRAS的癌细胞相比，miR-214-5p对KRAS突变株的抑制率更高。
miR-214-5p is not predicted to target KRAS itself, and expression of miRNA-resistant cell-cycle targets (cyclin D1, CDK2, and CDK6) prevented the anti-proliferative effect of this miRNA.
miR-214-5p不被预测靶向KRAS本身，耐miRNA的细胞周期靶点(cyclin D1, CDK2, CDK6)的表达阻止了该miRNA的抗增殖作用。
Of note, highthroughput screens have previously uncovered synthetic lethal interactions between oncogenic KRAS and inhibition of several CDKs (Barbie et al., 2009).
值得注意的是，高通量筛选先前已发现致癌KRAS与抑制几种CDKs之间的合成致命相互作用(Barbie等，2009)。
Moreover, KRAS-mutant tumors were shown to be more sensitive to CDK inhibition than KRAS wild-type tumors (Macias et al., 2007;Puyol et al., 2010), which might explain their increased sensitivity to miR-214-5p.这可能解释了它们对miR-214-5p敏感性的增加。
此外，KRAS突变型肿瘤比KRAS野生型肿瘤对CDK抑制更敏感(Macias等，2007;
Our observation that cell-cycle-targeting miRNAs affected cancer cells while sparing non-transformed tissues was not entirely unexpected.
我们的观察表明，细胞周期靶向mirna影响癌细胞，而保留非转化组织并不完全出乎意料。
Several studies demonstrated that cancer cells are particularly dependent on cyclins and CDKs for their proliferation and survival, and hence inhibition of these proteins is predicted to preferentially affect cancer cells (Campaner et al., 2010;Choi et al., 2012;Puyol et al., 2010).
几项研究表明，癌细胞的增殖和存活特别依赖于细胞周期蛋白和CDKs，因此预测抑制这些蛋白会优先影响癌细胞(Campaner et al.， 2010;Choi等，2012;Puyol等，2010)。
Moreover, expression of miRNAs decreases, but does not fully extinguish, expression of their targets, and such a global dampening of cell-cycle machinery is likely to especially affect transformed cells.
此外，mirna的表达减少，但并没有完全消除其靶细胞的表达，这种细胞周期机制的整体抑制可能尤其会影响转化细胞。
While our studies mostly explored the effects of overexpression of cell-cycle-targeting miRNAs, it might be expected that at least some of them normally play growth-suppressive roles when expressed at physiological levels.
虽然我们的研究主要是探讨细胞周期靶向mirna过表达的影响，但我们可以预期，当这些mirna在生理水平上表达时，至少有一些正常情况下发挥着抑制生长的作用。
Consistent with this notion, our analyses revealed that the gene encoding miR-193a-3p is frequently deleted in human cancers.
与这一观点一致的是，我们的分析显示，编码miR-193a-3p的基因在人类癌症中经常被删除。
miR-193a-3p was previously proposed to play tumor-suppressive functions by repressing EGFR signaling (Uhlmann et al., 2012), by promoting apoptosis via repression of MCL1 (Kwon et al.,2013;Williams et al., 2015), and by suppressing tumor cell migration and invasion through small GTPase Rab27B (Pu et al., 2016) or ERBB4 and S6K2 (Liang et al., 2015;Yu et al., 2015).
miR-193a-3p之前被提出通过抑制EGFR信号通路(Uhlmann et al.， 2012)，通过抑制MCL1促进细胞凋亡(Kwon et al.，2013;以及通过小GTPase Rab27B (Pu et al.， 2016)或ERBB4和S6K2抑制肿瘤细胞迁移和侵袭(Liang et al.， 2015;Yu等，2015)。
The loss of these anti-tumor functions of miR-193a-3p may contribute to the neoplastic transformation in cancers harboring deletion of the miR-193a locus.
miR-193a-3p的这些抗肿瘤功能的缺失，可能是miR-193a位点缺失的癌症发生肿瘤转化的原因之一。
Another cell-cycle-targeting miRNA highlighted by our study is miR-195-5p.
我们的研究强调的另一个细胞周期靶向miRNA是miR-195-5p。
We found that this miRNA might display growthsuppressive properties in vivo, as demonstrated by a significant downregulation of miR-195-5p in 11 types of human tumors (Figure 3B).
我们发现该miRNA可能在体内表现出抑制生长的特性，这可以通过11种人类肿瘤中miR-195-5p的显著下调来证明(fig3B)
Consistent with our findings, downregulation of miR-195-5p was previously reported in human breast and colorectal cancers (Chen et al., 2015;Luo et al., 2014, 2016).
与我们的研究结果一致，miR-195-5p下调在人类乳腺癌和直肠癌中已有报道(Chen et al.， 2015;Luo et al.， 2014, 2016)。
By correlating gene expression profiles of human cancer cell lines versus the responses to individual miRNA, we generated an expression-based algorithm that can predict the response of a particular tumor to a given miRNA.
通过将人类癌细胞的基因表达谱与个体miRNA的反应相关联，我们生成了一种基于表达的算法，可以预测特定肿瘤对特定miRNA的反应。
In the future, this approach can be extended to examine the effects of all miRNAs.
在未来，这种方法可以扩展到检查所有mirna的影响。
By applying similar algorithms to the one described in our study, one will be able to identify a miRNA, or a combination thereof, that would be particularly potent against a given individual human tumor.
通过将类似的算法应用到我们的研究中所描述的，我们将能够识别一种miRNA，或其组合，这将对特定的人类肿瘤特别有效。
This approach might lead to highly individualized miRNA-based anti-cancer therapies.
这种方法可能导致高度个性化的米尔纳为基础的抗癌治疗。
(整个实验流程的回顾，参考文献论证实验结果)
SUPPLEMENTAL INFORMATION 补充材料
Supplemental Information includes eight figures and seven tables and can be found with this article online at http://dx.doi.org/10.1016/j.ccell.2017.03.004.
补充信息包括8个图和7个表，可以通过本文在线http://dx.doi.org/10.1016/j.ccell.2017.03.004找到。
AUTHOR CONTRIBUTIONS 作者贡献
P.H.和P.S.设计了这项研究并撰写了论文。P.H.做了实验。陈怡如,X.L.,C.J.，和Y.L.贡献了由c.l.a.f.指导和监督的计算分析,V.M.,世贸组织,Y.J.C.,J.M.S.动向和D.F.帮助做实验。洛杉矶帮助他们做实验
与屏幕设计。h .阴R.L.B.,K.J.K.,J.Y.， D.G.A.提供了纳米颗粒配方的mirna。信号发生器、H. Yuzugullu和J.J.Z.帮助进行了实验，并为TNBC的异种移植做出了贡献。R.S.提供结肠癌标本，K.N.结肠癌异种移植，E.S.结肠癌异种移植和皮肤纤维肉瘤。kp帮助设计乳腺癌研究。P.S.指导了这项研究。
ACKNOWLEDGMENTS 致谢
这是由NIH资助的5 R01 CA083688, 5 R01 CA132740，和5 P01 CA080111(到P.S.)。P.H.得到了瑞典斯德哥尔摩温纳格伦基金会(Wennergren foundation)、Y.W.和C.L.博士后奖学金的部分资助由北京-清华生命科学中心和国家自然科学基金资助。中国科学基金重点研究资助项目71532001，获MNISW研究基金移动电话sc + j.m.资助。P.S.和K.P.是诺华研究资助的顾问和接收人。我们感谢ICCB-Longwood筛选中心miRNA模拟文库的Caroline Shamu博士，并帮助设计了该筛选器。
收件时间:2016年1月22日
修订日期:2016年12月6日
接受日期:2017年3月9日
出版时间:2017年4月10日
REFERENCES 参考文献 & STAR+METHODS
详细见文献 Cell-Cycle-Targeting MicroRNAs as Therapeutic Tools against Refractory Cancers