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【miRNA-seq】巨噬细胞miR-28-5p-IL-34的反馈环路调节肝细胞癌转移
发布日期:2016-10-17浏览:

晶能生物客户文章-巨噬细胞miR-28-5p-IL-34的反馈环路调节肝细胞癌转移,文章发表于杂志Hepatology

题目:miR-28-5p-IL-34-Macrophage Feedback Loop Modulates Hepatocellular Carcinoma Metastasis

杂志:Hepatology         IF11.055

实验方法:miRNA-sequencing (Illumina HiSeq 2500)

 


摘要:

    作者对miRNA进行测序鉴定出22个参与肝细胞癌(HCC)转移的miRNA。其中 miR-28-5p在肝癌细胞中表达下调,这与肿瘤转移、复发及低的成活率有关。生物功能研究显示在裸鼠体内 miR-28-5p的缺乏促进肿瘤生长和转移,而在体外却不能改变肝癌细胞的生物学特性。通过基因表达谱和生物信息分析,作者鉴定出miR-28-5p的靶基因白介素-34(IL-34),miR-28-5p 缺乏对HCC生长和转移产生的影响依赖于IL-34介导的肿瘤相关巨噬细胞(TAMs)的渗透。另外,作者发现在 HCC细胞中通过转化生长因子-β1(TGF-β1),被miR-28-5p-IL-34介导的TAMs可抑制miR-28-5p的表达,形成一个miR-28-5p-IL-34-macrophage的正反馈循环。在临床HCC样本中,miR-28-5p 的表达水平与 IL-34 的表达和TAMs的数量成负相关。低miR-28-5p 表达量,高IL-34水平及高TAMs数量的病人有更短的存活期(OS)和复发时间(TTR)。

背景:

     大约90%的癌症患者死亡是由癌细胞转移所导致,许多研究显示癌细胞的内部机制和外部微环境因子可导致肿瘤转移,然而由于转移过程复杂,对于这些机制的理解仍是不全面的。miRNA是一种小非编码RNA,研究已经证实其对肿瘤转移的调节起着关键作用,但是目前miRNA对肝细胞癌的影响还没有被完全阐明。

研究方法:

研究结果:

1. miR-28-5p的下调与HCC的转移及预后有关

    本文分别对具有不同转移能力的肝癌细胞系(转移能力:MHCC97L<MHCC97H<HCCLM3)及移植瘤模型组织(HCCLM3-LM)进行miRNA测序,结果在这些样本中共找到22与转移能力相关的显著差异miRNA,其中12个miRNA与样本转移能力呈正相关,10个与样本转移能力呈负相关。有趣的是,miR-28-3p 和 miR-28-5p来自同一个转录本且表达量最丰富,并且与4个样本的转移能力呈负相关,因此作者重点关注这两个miRNA的作用。

    作者评估了miR-28-3p 和miR-28-5p的表达水平,发现只有miR-28-5p在肝癌细胞中的表达量相比于正常细胞明显减少。另外,它在转移性的HCC细胞系中的表达量低于非转移性的HCC细胞系。作者还对miR-28-3p 和miR-28-5p进行了qRT-PCR检测,结果显示相比于正常组织,只有miR-28-5p的表达量在肿瘤中是显著下调的。此外,HCC转移或复发的病人的miR-28-5p表达量低于非转移或复发的病人。因此miR-28-5p表达量较高的病人比表达量低的病人预后效果好。

Figure 1 Downregulation of miR-28-5p correlated withtumor metastasis and prognosis in HCC. (A) A partial list of microRNAs with expression patterns that correlatedwith metastatic potential in MHCC97L, MHCC97H,HCCLM3 and HCCLM3-LM revealed bymiRNA-seq. The metastatic potential of HCC cell lines is as follows: MHCC97L<MHCC97H<HCCLM3.HCCLM3-LM: lung metastatic tissues from a HCCLM3-RFP xenograft model animalexhibited higher metastatic potential than HCCLM3. (B) Expression of miR-28-5pand miR-28-3p in six established HCC cell lines and a non-neoplastic cell linewas determined by qRT-PCR, *P<0.05; **P<0.01 compared with L0-2. (C)qRT-PCR revealed that miR-28-5p but not miR-28-3p expression wassignificantly  decreased, when comparedto corresponding adjacent nontumor tissues (cohort 1, n=228). (D) Expression ofmiR-28-5p and miR-28-3p in HCC tissues with or without pulmonary metastasis andwith or without recurrence after surgical resection was determined by qRT-PCR. (E)Kaplan-Meier analysis showed that HCC patients with overexpression of miR-28-5pbut not with overexpression of miR-28-3p exhibited better overall survival anda lower cumulative recurrence rate compared with those who had low expressionof miR-28-5p. Data depict the mean±SD and are representative of three independent experiments. 

2. 裸鼠体内 miR-28-5p的缺乏促进肿瘤生长和转移,在体外却不能改变HCC细胞的生物学特性

        将带有miR-28-5p 和 anti-miR-28-5p的质粒分别转染到HCC细胞系和裸鼠体内,结果显示miR-28-5p的下调对HCC细胞增殖、转移、凋亡等功能没有产生影响。而在裸鼠体内,当miR-28-5p的表达量降低时,肿瘤体积显著增大,利用活体成像技术证实了这个结果。

Figure 2 An miR-28-5p deficiency promoted tumor growthand metastasis in nude mouse models. (A) Growth curves of tumors in xenograftnude mouse models are shown,*P<0.05; **P<0.01  compared with control. (B) Representativebioluminescence images of mice show subcutaneous tumors at day 35 afterinoculation of HCC cells. The color scale bar depicts the photon flux emittedfrom these mice.

3. IL-34miR-28-5p下游的直接靶基因

        以上的体内外实验显示miR-28-5p 可能通过细胞的非自治机制来行使其肿瘤抑制功能,一种可能的非自治途径是通过调节分泌蛋白(如细胞因子、趋化因子)的产生影响肿瘤微环境,因此作者根据研究miR-28-5p与细胞因子/趋化因子的上下调关系及预测软件寻找靶基因,结果只有 IL-34符合预测条件。miR-28-5p与 IL-34 mRNA的3’UTR具有互补序列,qRT-PCR 和western blot 也证实miR-28-5p 在HCC细胞中过表达可显著减少 IL-34 的表达水平。反过来,miR-28-5p的敲除可明显提高 IL-34 的表达量。另外,miR-28-5p和IL-34 的mRNA、蛋白水平具有显著的负相关性。

 

 

Figure 3 Identification of IL-34 as a direct downstreamtarget of miR-28-5p. (A) Venn diagrams showing the number of genes identified as potential targets of miR-28-5paccording to three groupings: (1) downregulated cytokines in HCCLM3 cellstreated with miR-28-5p; (2) upregulated cytokines in HepG2 cellstreated withanti-miR-28-5p; and (3) genes predicted by TargetScan as potential miR-28-5ptargets. (B) qRT-PCR and Western blot validated that overexpression ofmiR-28-5p significantly reduced IL-34 expression in HCCLM3 cells, whilemiR-28-5p knockdown markedly enhanced the expression of IL-34 in HepG2 cells,**P<0.01 compared with control. (C) Sequences of has-miR-28-5p and itspotential  binding site at the 3’UTR of IL-34 areshown  and the nucleotides mutated inIL-34-3’UTR mutant (upper panel). miR-28-5p significantly  suppressed the luciferase activity of IL-34containing a wild-type 3'-UTR, but showed no effect on the activity of IL-34with a mutant 3'-UTR, while treatment with anti–miR-28-5p increased the luciferase activityof IL-34 (lower  panel), *P<0.05;**P<0.01. (D) Scatter plot depicting the statistically significant inverse correlation between miR-28-5p levelsand IL-34 mRNA or protein levels in HCC samples (cohort 1, n=228). Data depictthe mean±SD and are representative of three independentexperiments. 

4. IL-34激活巨噬细胞中的 FAK  ERK1/2 信号,促进增殖和趋化性转移

       本研究显示 IL-34可以体外诱导巨噬细胞的增殖和趋化性迁移,westernblot分析发现 使用IL-34处理巨噬细胞后 FAK 和 ERK1/2的磷酸化水平提高,而在加入CSF1R 抑制剂,  FAK 抑制剂或 ERK1/2 抑制剂后,巨噬细胞的增殖和迁移能力降低,这说明FAK 和 ERK1/2 信号的活化与 IL-34介导的巨噬细胞的增殖及趋化性有关。

Figure 4 IL-34 activates FAK and ERK1/2 signaling inmacrophages and promotes proliferation and chemotactic migration of macrophages. IL-34 inducedconcentration-dependent (A) proliferation and (B) chemotactic migration ofmacrophages in vitrowithin  the range of0.1-100 nM, *P<0.05; **P<0.01 compared with control. (C) Western blottingshowed that IL-34 treatment  caused asignificant increase in the phosphorylation levels of FAK and ERK1/2 (leftpanel) and following treatment with the CSF1R inhibitor, FAK inhibitor orERK1/2 inhibitor (right panel).

5. IL-34通过诱导TAM渗透促进HCC的生长和迁移

       与 miR-28-5p相似,体外实验显示 IL-34的改变对HCC的生物学活性没有影响。人体内HCC模型分析显示IL-34可促进肿瘤生长和迁移,而IL-34 的下调抑制肿瘤增殖。研究还发现在IL-34含量较高的肝癌细胞中渗透的TAMs 数量也是更高的,而在含有 IL-34 的其他基质细胞中渗透的TAMs 数量并未发生变化。作者在小鼠体内的实验显示TAMs 的消耗几乎完全消除了IL-34对HCC增殖的影响。结果表明在体内 IL-34对HCC增殖的影响是受TAMs调节的。

Figure 5 IL-34 contributes to HCC growth and metastasisby promoting TAM infiltration. (A) The growth curves and (B) bioluminescenceimages oftumors are shown from xenograft nude mouse models, *P<0.05; **P<0.01 compared with control. (C)Representative images of H&E staining of metastatic nodules in lung areshown from different animal groups. (D) Representative images from tumorsampleserial sections stained for IL-34 and F4/80 are shown. (E) The growthcurves and (F) H&E staining of metastatic nodules in lung from differentanimal groups treated with or without clodrolip are shown, *P<0.05;**P<0.01. Data depict the mean±SD (n=6) and are representative of threeindependent experiments. 

6. miR-28-5p的缺乏导致IL-34的上调,从而通过TAM渗透的介导促进肿瘤生长和转移

       体外结果显示miR-28-5p的过表达抑制巨噬细胞的趋化迁移和增殖,与敲除IL-34的作用相同,然而 IL-34的过表达可消除这种影响。这表明miR-28-5p 在HCC中的缺乏可使IL-34上调,从而促进巨噬细胞的趋化迁移和增殖。体内实验结果显示IL-34 过表达或miR-28-5p的敲除会增加肿瘤体积,诱导更明显的F4/80+ TAM渗透,反过来,则会减少TAM的渗透数量。另外还发现TAMs的消耗会消除由于IL-34 的过表达或miR-28-5p的敲除对肿瘤所产生的影响。这表明miR-28-5p缺乏对HCC增殖产生的影响依赖于介导IL-34的TAM渗透作用。

Figure 6 miR-28-5p-IL-34 signaling mediates TAMinfiltration and modulates HCC progression and  metastasis. (A-B) miR-28-5pinhibited macrophage migration and proliferation by targeting  IL-34 in HepG2 cells (A) and HCCLM3 cells(B), *P<0.05; **P<0.01. Data depict the mean±SD  and are representative of three independentexperiments. (C-D) Representative bioluminescence images of animals, H&Estaining of metastatic nodules in lung, and immunostaining of F4/80 in tumorsample serial sections from xenograft nude mouse models derived from HepG2(C)and HCCLM3 (D) cells are shown, **P<0.01. Data depict the mean±SD (n=6) and arerepresentative  of three independentexperiments. 

7. 源自TAMTGF-β1肝癌细胞中抑制miR-28-5p的表达

        先前研究发现TAM参与肿瘤增殖,在HCC细胞中的作用机制也已经被报道过,但作者发现将TAMs和HepG2一起培养后会导致miR-28-5p水平下降, IL-34表达量升高。另外将TAMs 和HCC细胞一起培养使肝癌细胞的趋化迁移和增殖能力提高,这种影响会因miR-28-5p过表达或IL-34的敲除而消失。作者通过ELISA实验在HCC细胞中找到了TAMs分泌的主要细胞因子,而只有TGF-β1通过TAMs显著增加了对miR-28-5p表达的抑制,因此推测在HCC细胞中通过TAMs对miR-28-5p的抑制主要依靠TGF-β1,在HCC细胞中形成一个miR-28-5p-IL-34-macrophage正向反馈循环。

Figure 7 TAM-derived TGF-β1 suppresses miR-28-5p expression in HCCcells. (A) Schematic showing that HCC cells were co-cultured with TAMs intranswell apparatus with 0.4µm pore size for 48h, then these HCC cells werere-plated and cultured, after 12h, CM were obtained and used in posteriorexperiments. (B) qRT-PCR revealed that co-culture with TAMs after 48h inhibitedmiR-28-5p expression in HepG2 and Huh7 cells and increased IL-34 expression,**P<0.01 compared with control. (C) Co-culture with TAMs significantlyenhanced the proliferative effect of HepG2 and Huh7 cells on macrophages,**P<0.01, and miR-28-5p overexpression or IL-34 knockdown in HCC cells abrogatedthis effect, ##P<0.01. (D) Co-culture with TAMs significantly enhanced thechemotactic effect of HepG2 and Huh7 cells on macrophages, and miR-28-5poverexpression or IL-34 knockdown in HCC cells abrogated this effect,**P<0.01. (E) ELISA showed the levels of a panel of cytokines related toTAMs. (F) Anti-TGF-β1treatment significantly alleviated the TAMsuppression of miR-28-5p expression on HCC cells. Data depict the mean±SD and are representative of three independent experiments. 

8. miR-28-5p,IL-34, TAMsHCC病人预后的影响

        作者对 380 HCC病人的预后情况进行调查,发现miR-28-5p,IL-34, TAMs的表达量与存活率、复发时间密切相关,具体如下图所示。

Figure 8 Expression and prognostic value of miR-28-5p,IL-34, and TAMs in HCC samples (cohort 2, n=380). (A) Levels of miR-28-5p,IL-34 and CD68+ TAMs in representative HCC patients  are shown.  Patient  1  had  high-level expression  of  miR-28-5p and  low-level expression of IL-34and CD68, while patient 2 had low-level expression of miR-28-5p and high  expression of  both  IL-34 and  CD68.  (B) Scatterplot  depicts  a significant  inverse correlationbetween miR-28-5p and IL-34 and a significant positive correlation betweenIL-34 and CD68 in cancerous tissues. (C-D) Prognostic values of miR-28-5p,IL-34, and CD68+ TAMs by Kaplan-Meier analysis. I, miR-28-5phigh/IL-34low/CD68low; III, miR-28-5plow/IL-34high/CD68high; II, others. (E)Proposed model illustrates the role of the miR-28-5p-IL-34-macrophage positivefeedback loop in the regulation of HCC metastasis.

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