Dear editor,

Ovarian cancer is one of the most prevalent gynecological malignant tumors. Its five‐year overall survival rate is less than 30%, mostly due to advanced metastasis and chemotherapy resistance [1]. However, the mechanisms involved in ovarian cancer metastasis are still poorly understood. Hypoxia is a tumor microenvironment (TME) factor that facilitates tumor progression [2]. Changes in TME and cross‐talk signaling pathway is crucial for ovarian cancer metastasis and indicate that these tumor biological characteristics are ruled by lots of conserved mechanisms [3, 4]. Exosomes are crucial signalosomes between various cell types crosstalk and can carry complex mRNAs and microRNAs (miRNAs) functions between cells [5]. Tumor‐derived exosomes (TDEs) have been shown to not only contribute to TME remodeling but also as a contributor to premetastatic niche formation [6]. There are evidence showing that cancer‐secreted miRNAs may mediate cell‐cell communication in many diseases, including ovarian cancer. For instance, Liu X et al. [7] reported that miR‐199a‐5p, as a tumor suppressor, could inhibit ovarian cancer cell proliferation and invasion by inhibiting the expression of NF‐κB1. Song K et al. [8] reported that emodin had inhibiting effects on ovarian cancer cells colony formation by activating the FOXD3/miR‐199a‐5p axis. These, therefore, suggest that miR‐199a‐5p may be a promising target in ovarian cancer treatment. However, till present, the ovarian cancer cell‐secreted exosomal miR‐199a‐5p interaction with Wnt/β‐catenin in ovarian cancer has not been explored, especially in relation to the TME. In this study, we aimed to explore the role of ovarian cancer cell‐derived exosomal miR‐199a‐5p in ovarian cancer metastasis as a new potential target for ovarian cancer treatment.

We analyzed exosome‐miRNA enriched in ovarian cancer cells under normoxia and hypoxia conditions. The materials and methods for this study are provided in the “supplementary file”. Transmission electron microscopy (TEM) and nano particle tracking analysis (NTA) confirmed the observed round vesicles as exosomes with diameters of 30∼150 nm (Figure 1A, B). Besides, NTA found that A2780 cells cultured under hypoxia condition produced more exosomes than those cultured in normoxia condition (P = 0.03, Figure 1C). The exosome surface‐proteins CD81 and CD63 on the exosomes were positive in Western blotting but negative in cell lysis (Figure 1D). High‐Throughput Sequencing (Illumina PE150) detected a total of 359 differentially expressed miRNAs, of which 153 were up‐regulated (red) and 206 were down‐regulated (green) (Supplementary Fig. S1A). A heatmap was constructed to show the top 50 miRNAs which enriched the up‐regulated top 25 and down‐regulated top 25 miRNA in order of |Fold Change| from large to small via miRNA sequence. (|Fold Change| ≥ 2, corrected P values ≤ 0.050) (Figure 1E). The first 10 significantly downregulated miRNAs were: miR‐10a‐5p, miR‐214‐5p, miR‐199a‐5p, miR‐486‐5p, miR‐200b‐3p, miR‐200a‐3p, miR‐199a‐3p, miR‐199b‐3p, miR‐451a and miR‐203a. Of them, miR‐199a‐5p demonstrated the most significant difference in ovarian cancer cell hypoxia condition compared with normoxia condition (P = 0.003). We further predicted its target gene by using the miRwalk software. The Gene Ontology (GO) analysis mainly enriched the foundation of miR‐199a‐5p in cell‐cell adhesion and transcription factor activity. Subsequently, the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed the enrichment of the TGF‐β signaling pathway, Wnt‐signaling pathway, and more. GO and KEGG were conducted using the DAVID tools (Supplementary Fig. S1 B, C). We detected the level of miR‐199a‐5p in four ovarian cancer cell lines (UWB, HEY, A2780, and Anglne) and the exosomes derived from them using Quantitative‐polymerase chain reaction (Q‐PCR). We found that the expression of miR‐199a‐5p, both in cells and exosomes under hypoxia condition, was dramatically lower than those in normoxia condition (Supplementary Fig. S2A, B). The expression of miR‐199a‐5p expression in ovarian cancer tissue and normal fallopian tube were detected by Q‐PCR and in situ hybridization (ISH), respectively. Q‐PCR results showed that the expression of miR‐199a‐5p in ovarian cancer tissue was significantly lower than in normal fallopian tube (Supplementary Fig. S2C). Consistently, ISH detection of tissue microarrays (TMA) showed the same trend about miR‐199a‐5p expression in ovarian cancer and fallopian tubes (Supplementary Fig. S2D, E). These results demonstrated that the expression of miR‐199a‐5p in ovarian cancer tissue was downregulated. After culturing in hypoxia, the miR‐199a‐5p level in both ovarian cancer cells and exosomes were found to decrease further.

The correlation between miR‐199a‐5p expression and the clinicopathological characteristics of the 57 ovarian cancer on TMA was analyzed by immunohistochemistry. It was identified that the miR‐199a‐5p expression in ovarian cancer was negatively correlated with tumor infiltration (P < 0.001), tumor size (≤ 5 cm vs. > 5 cm, P < 0.001), lymphatic metastasis (absent vs. present, P < 0.001) and TNM stage (T1‐T2 vs. T3‐T4, P < 0.001) (Table 1). However, no statistical significance in miR‐199a‐5p expression was found associated with age (≤ 50 years vs. > 50 years, P = 0.574). These results indicated that the downregulation of miR‐199a‐5p might occur in ovarian cancer progression, and could serve as a promising therapeutic marker for ovarian cancer.

Wound‐healing assays showed that A2780 cells transfected with miR‐199a‐5p plasmid had lower migratory ability than the control cells in hypoxia and normoxia condition (Figure 2A‐C). Meanwhile, the cell migration and Matrigel Transwell assay showed that transfected A2780 cells with miR‐199a‐5p plasmid also had lower invasion (Supplementary Fig. S3A‐D) and migration ability in contrast to the miR‐NC group, while transfection with miR‐199a‐5p inhibitor could promote A2780 migration and infiltration, under normoxia and hypoxia condition. These results demonstrated that the downregulation of miR‐199a‐5p might promote the invasion and migratory ability of ovarian cancer.

The potential target gene of miR‐199a‐5p was predicted using the online software miRwalk which contains twelve online databases. We selected target genes that were associated with hypoxia from the intersection in at least three databases. The potential target gene of miR‐199a‐5p might be the hypoxia‐inducible factor (HIF‐2α) (Figure 2D). We found that the overexpression of miR‐199a‐5p could significantly inhibit the expression of HIF‐2α mRNA, while the miR‐199a‐5p inhibitor had the opposite effects (Figure 2E, F). To further verify this hypothesis, we performed Dual‐luciferase report assay, which showed that has‐miR‐199a‐5p plasmid could significantly suppress the luciferase activity of HIF‐2α wild‐type group compared with HIF‐2α mutant‐type group (P < 0.010) (Figure 2G); hinting that miR‐199a‐5p could directly combine with the 3′UTR of HIF‐2α. Additionally, Western blot and immunohistochemical staining of HIF‐2α levels in ovarian cancer tissues and normal fallopian tissues were tested and the results demonstrated that the relationship between miR‐199a‐5p and HIF‐2α may have a negative correlation (Supplementary Fig. S4A, B, Supplementary Fig. S5A‐D). These results suggested that miR‐199a‐5p could directly regulate HIF‐2α. The Wnt/β‐catenin signaling pathway has been reported to be involved in ovarian cancer progression [9], and in this study, we detected the Wnt3a and β‐catenin expression in A2780 cell and tissues. Western blot analysis showed that Wnt3a and β‐catenin expression were downregulated after miR‐199a‐5p was upregulated in A2780 cells (Figure 2E, F). Meanwhile, the upregulation of Wnt3a and β‐catenin in ovarian cancer tissues and downregulation in normal fallopian tissues indicated that miR‐199a‐5p may be negatively correlated with Wnt3a and β‐catenin expression (Figure 2E, F).

To further study whether ovarian cancer cell‐derived exosomal miR‐199a‐5p could be engulfed by HUVECs, hypoxia ovarian cancer A2780 cell‐secreted exosome was labeled with PKH26 (red fluorescent color) and the miR‐199a‐5p was tagged with fluorescein isothiocyanate (FITC) (green fluorescent color). Then, we co‐cultured endothelial cells with A2780 cell‐derived exosomal miR‐199a‐5p under hypoxic conditions. The internalized exosomal miR‐199a‐5p was shown as yellow fluorescence dots around HUVECs nuclei (Figure 2H). The expression of VE‐cadherin (VE‐Cad) and junctional adhesion molecule‐A (JAM‐A) were detected with Western Blot. The results showed that anti‐NC was cultured with A2780‐derived exosome, resulting in an increased expression of VE‐Cad and JAM‐A in HUVECs. The transfection of anti‐miR‐199a‐5p into HUVECs remarkably reduced the expression of A2780‐secreted exosome to reduce the expression of VE‐cad and JAM‐A in HUVECs (Figure 2I, J). Moreover, ovarian cancer tissues had low VE‐cadherin and JAM‐A staining compared with the normal fallopian tubes according to histologic scoring, meaning that the expression of VE‐cad and JAM‐A in ovarian cancer tissues were downregulated (Supplementary Fig. S5E‐L). These results uncovered the crucial role of ovarian cancer cell‐secreted miR‐199a‐5p on a vascular level and indicated that ovarian cancer cell‐derived exosomal miR‐199a‐5p might enhance the tight junction of HUVECs.

Hypoxia is a hallmark of the TME, and also an underling factor promoting tumor angiogenesis and metastatic progression [2]. Recently, hypoxia has been reported to promote exosomes release from cancer cells [6]. Our findings demonstrated that hypoxia OvCa cell excreted exosomal miR‐199a‐5p played a negative regulatory role in cancer metastasis. In other words, the down‐regulation of exosomal miR‐199a‐5p promoted cancer metastasis. This effect was achieved through HIF‐2α regulating the Wnt/β‐catenin pathway. Exchange of cellular ingredients between cells through paracrine mechanisms is a vital way of intercellular communication and can be mediated by exosomes. Emerging evidence shows that an increment in vascular permeability was linked with an escalation in cancer metastasis [10]. Findings from this study revealed that exosomes mediated the interaction between cancer cells and TME, which increased the endothelial monolayers junction integrity to inhibit cancer metastasis. miR‐199a‐5p secreted by hypoxic ovarian cancer cells acted as a promising tumor suppressor factor. Exosomal miR‐199a‐5p is phagocytized by endothelial cells and increases tight junction in endothelial cells. The crosstalk between ovarian cancer cells and endothelial cells in TME confirms that miR‐199a‐5p may be a potential biomarker, which can be used as a novel therapeutic target for ovarian cancer treatment.

亲爱的编辑，

卵巢癌是最常见的妇科恶性肿瘤之一。其五年总生存率低于30%，主要是由于晚期转移和化疗耐药[ 1 ]。然而，卵巢癌转移所涉及的机制仍知之甚少。缺氧是一种促进肿瘤进展的肿瘤微环境 (TME) 因素 [ 2 ]。TME 和串扰信号通路的变化对卵巢癌转移至关重要，表明这些肿瘤生物学特征受许多保守机制的支配 [ 3, 4 ]。外泌体是各种细胞类型之间重要的信号小体，可以在细胞之间进行复杂的 mRNA 和 microRNA (miRNA) 功能 [ 5]]。肿瘤衍生的外泌体 (TDE) 已被证明不仅有助于 TME 重塑，而且还有助于转移前生态位的形成 [ 6 ]。有证据表明，癌症分泌的 miRNA 可能介导许多疾病中的细胞间通讯，包括卵巢癌。例如，刘 X 等人。[ 7 ] 报道miR-199a-5p作为肿瘤抑制因子，可以通过抑制NF-κB1的表达来抑制卵巢癌细胞的增殖和侵袭。宋克等人。[ 8] 报道大黄素通过激活 FOXD3/miR-199a-5p 轴对卵巢癌细胞集落形成有抑制作用。因此，这些表明 miR-199a-5p 可能是卵巢癌治疗的一个有希望的靶点。然而，到目前为止，卵巢癌细胞分泌的外泌体 miR-199a-5p 与 Wnt/β-catenin 在卵巢癌中的相互作用尚未得到探索，尤其是与 TME 相关的研究。在这项研究中，我们旨在探索卵巢癌细胞衍生的外泌体 miR-199a-5p 在卵巢癌转移中的作用，作为卵巢癌治疗的新潜在靶点。

我们分析了在常氧和缺氧条件下富集在卵巢癌细胞中的外泌体-miRNA。本研究的材料和方法在“补充文件”中提供。透射电子显微镜（TEM）和纳米粒子追踪分析（NTA）证实观察到的圆形囊泡是直径为 30~150 nm 的外泌体（图 1A、B）。此外，NTA 发现在缺氧条件下培养的 A2780 细胞比在常氧条件下培养的细胞产生更多的外泌体（P = 0.03，图 1C）。外泌体上的外泌体表面蛋白 CD81 和 CD63 在蛋白质印迹中呈阳性，但在细胞裂解中呈阴性（图 1D）。高通量测序（Illumina PE150）共检测到 359 个差异表达的 miRNA，其中 153 个上调（红色）和 206 个下调（绿色）（补充图 S1A）。构建热图以显示前 50 个 miRNA，这些 miRNA 按 |Fold Change| 的顺序富集了上调的前 25 个和下调的前 25 个 miRNA。通过miRNA序列从大到小。(|Fold Change| ≥ 2, 修正P值 ≤ 0.050）（图 1E）。前 10 个显着下调的 miRNA 是：miR-10a-5p、miR-214-5p、miR-199a-5p、miR-486-5p、miR-200b-3p、miR-200a-3p、miR-199a-3p、 miR-199b-3p、miR-451a 和 miR-203a。其中，与常氧条件相比，miR-199a-5p在卵巢癌细胞缺氧条件下表现出最显着的差异（P = 0.003）。我们通过使用 miRwalk 软件进一步预测了其目标基因。基因本体论（GO）分析主要丰富了miR-199a-5p在细胞间粘附和转录因子活性方面的基础。随后，Kyoto Encyclopedia of Genes and Genomes (KEGG) 显示了 TGF-β 信号通路、Wnt 信号通路等的富集。GO 和 KEGG 是使用 DAVID 工具进行的（补充图 S1 B、C）。我们使用定量聚合酶链反应 (Q-PCR) 检测了四种卵巢癌细胞系（UWB、HEY、A2780 和 Anglne）及其衍生的外泌体中 miR-199a-5p 的水平。我们发现 miR-199a-5p 在缺氧条件下的细胞和外泌体中的表达显着低于常氧条件下的表达（补充图 S2A，B）。分别通过Q-PCR和原位杂交（ISH）检测miR-199a-5p在卵巢癌组织和正常输卵管中的表达。Q-PCR结果显示，miR-199a-5p在卵巢癌组织中的表达显着低于正常输卵管（补充图S2C）。一致地，组织微阵列（TMA）的 ISH 检测显示出与卵巢癌和输卵管中 miR-199a-5p 表达相同的趋势（补充图 S2D，E）。这些结果表明miR-199a-5p在卵巢癌组织中的表达下调。在缺氧条件下培养后，发现卵巢癌细胞和外泌体中的 miR-199a-5p 水平进一步降低。Q-PCR结果显示，miR-199a-5p在卵巢癌组织中的表达显着低于正常输卵管（补充图S2C）。一致地，组织微阵列（TMA）的 ISH 检测显示出与卵巢癌和输卵管中 miR-199a-5p 表达相同的趋势（补充图 S2D，E）。这些结果表明miR-199a-5p在卵巢癌组织中的表达下调。在缺氧条件下培养后，发现卵巢癌细胞和外泌体中的 miR-199a-5p 水平进一步降低。Q-PCR结果显示，miR-199a-5p在卵巢癌组织中的表达显着低于正常输卵管（补充图S2C）。一致地，组织微阵列（TMA）的 ISH 检测显示出与卵巢癌和输卵管中 miR-199a-5p 表达相同的趋势（补充图 S2D，E）。这些结果表明miR-199a-5p在卵巢癌组织中的表达下调。在缺氧条件下培养后，发现卵巢癌细胞和外泌体中的 miR-199a-5p 水平进一步降低。组织微阵列（TMA）的ISH检测显示出与卵巢癌和输卵管中miR-199a-5p表达相同的趋势（补充图S2D，E）。这些结果表明miR-199a-5p在卵巢癌组织中的表达下调。在缺氧条件下培养后，发现卵巢癌细胞和外泌体中的 miR-199a-5p 水平进一步降低。组织微阵列（TMA）的ISH检测显示出与卵巢癌和输卵管中miR-199a-5p表达相同的趋势（补充图S2D，E）。这些结果表明miR-199a-5p在卵巢癌组织中的表达下调。在缺氧条件下培养后，发现卵巢癌细胞和外泌体中的 miR-199a-5p 水平进一步降低。

通过免疫组织化学分析miR-199a-5p表达与TMA上57例卵巢癌临床病理特征的相关性。结果表明，miR-199a-5p在卵巢癌中的表达与肿瘤浸润（P  < 0.001）、肿瘤大小（≤ 5 cm vs. > 5 cm，P  < 0.001）、淋巴转移（不存在 vs. 存在）呈负相关, P  < 0.001) 和 TNM 分期（T1-T2 与 T3-T4，P  < 0.001）（表 1）。然而，未发现 miR-199a-5p 表达与年龄相关的统计学显着性（≤ 50 岁 vs. > 50 岁，P = 0.574）。这些结果表明 miR-199a-5p 的下调可能发生在卵巢癌的进展过程中，可作为一种有前景的卵巢癌治疗标志物。

伤口愈合试验表明，在缺氧和常氧条件下，转染 miR-199a-5p 质粒的 A2780 细胞的迁移能力低于对照细胞（图 2A-C）。同时，细胞迁移和 Matrigel Transwell 试验表明，转染 miR-199a-5p 质粒的 A2780 细胞与 miR-NC 组相比，也具有较低的侵袭（补充图 S3A-D）和迁移能力，而用 miR-在常氧和缺氧条件下，199a-5p抑制剂可以促进A2780迁移和浸润。这些结果表明miR-199a-5p的下调可能促进卵巢癌的侵袭和迁移能力。

使用包含 12 个在线数据库的在线软件 miRwalk 预测 miR-199a-5p 的潜在靶基因。我们从至少三个数据库中的交叉点中选择了与缺氧相关的目标基因。miR-199a-5p 的潜在靶基因可能是缺氧诱导因子（HIF-2α）（图 2D）。我们发现 miR-199a-5p 的过表达可以显着抑制 HIF-2α mRNA 的表达，而 miR-199a-5p 抑制剂具有相反的作用（图 2E、F）。为了进一步验证这一假设，我们进行了双荧光素酶报告测定，结果表明与 HIF-2α 突变型组相比，has-miR-199a-5p 质粒可以显着抑制 HIF-2α 野生型组的荧光素酶活性（P < 0.010)（图 2G）；暗示 miR-199a-5p 可以直接与 HIF-2α 的 3'UTR 结合。此外，还测试了卵巢癌组织和正常输卵管组织中 HIF-2α 水平的蛋白质印迹和免疫组织化学染色，结果表明 miR-199a-5p 与 HIF-2α 之间的关系可能呈负相关（补充图 S4A， B，补充图 S5A-D)。这些结果表明 miR-199a-5p 可以直接调节 HIF-2α。据报道，Wnt/β-catenin 信号通路与卵巢癌进展有关 [ 9]]，在本研究中，我们检测了 A2780 细胞和组织中 Wnt3a 和 β-catenin 的表达。蛋白质印迹分析显示，在 A2780 细胞中上调 miR-199a-5p 后，Wnt3a 和 β-catenin 表达下调（图 2E，F）。同时，卵巢癌组织中 Wnt3a 和 β-catenin 的上调和正常输卵管组织中的下调表明 miR-199a-5p 可能与 Wnt3a 和 β-catenin 的表达呈负相关（图 2E、F）。

为了进一步研究卵巢癌细胞衍生的外泌体 miR-199a-5p 是否可以被 HUVECs 吞噬，缺氧卵巢癌 A2780 细胞分泌的外泌体用 PKH26（红色荧光色）标记，miR-199a-5p 用异硫氰酸荧光素标记(FITC)（绿色荧光色）。然后，我们在缺氧条件下将内皮细胞与 A2780 细胞衍生的外泌体 miR-199a-5p 共培养。内化的外泌体 miR-199a-5p 显示为 HUVEC 细胞核周围的黄色荧光点（图 2H）。Western Blot检测VE-cadherin（VE-Cad）和连接粘附分子-A（JAM-A）的表达。结果表明，抗 NC 与 A2780 衍生的外泌体一起培养，导致 HUVEC 中 VE-Cad 和 JAM-A 的表达增加。将anti-miR-199a-5p转染到HUVEC中显着降低了A2780分泌的外泌体的表达，从而降低了HUVEC中VE-cad和JAM-A的表达（图2I，J）。此外，根据组织学评分，与正常输卵管相比，卵巢癌组织的 VE-cadherin 和 JAM-A 染色较低，这意味着卵巢癌组织中 VE-cad 和 JAM-A 的表达下调（补充图 S5E- L)。这些结果揭示了卵巢癌细胞分泌的 miR-199a-5p 在血管水平上的关键作用，并表明卵巢癌细胞来源的外泌体 miR-199a-5p 可能增强 HUVEC 的紧密连接。根据组织学评分，与正常输卵管相比，卵巢癌组织的 VE-cadherin 和 JAM-A 染色较低，这意味着卵巢癌组织中 VE-cad 和 JAM-A 的表达下调（补充图 S5E-L） . 这些结果揭示了卵巢癌细胞分泌的 miR-199a-5p 在血管水平上的关键作用，并表明卵巢癌细胞来源的外泌体 miR-199a-5p 可能增强 HUVEC 的紧密连接。根据组织学评分，与正常输卵管相比，卵巢癌组织的 VE-cadherin 和 JAM-A 染色较低，这意味着卵巢癌组织中 VE-cad 和 JAM-A 的表达下调（补充图 S5E-L） . 这些结果揭示了卵巢癌细胞分泌的 miR-199a-5p 在血管水平上的关键作用，并表明卵巢癌细胞来源的外泌体 miR-199a-5p 可能增强 HUVEC 的紧密连接。

缺氧是 TME 的标志，也是促进肿瘤血管生成和转移进展的潜在因素 [ 2 ]。最近，据报道，缺氧会促进外泌体从癌细胞中释放 [ 6 ]。我们的研究结果表明，缺氧 OvCa 细胞分泌的外泌体 miR-199a-5p 在癌症转移中起负调控作用。换言之，外泌体 miR-199a-5p 的下调促进了癌症转移。这种效果是通过 HIF-2α 调节 Wnt/β-catenin 途径实现的。细胞间通过旁分泌机制交换细胞成分是细胞间通讯的重要方式，可以由外泌体介导。新出现的证据表明，血管通透性的增加与癌症转移的增加有关。10 ]。这项研究的结果表明，外泌体介导了癌细胞和 TME 之间的相互作用，这增加了内皮单层连接的完整性以抑制癌症转移。缺氧卵巢癌细胞分泌的 miR-199a-5p 是一种很有前景的肿瘤抑制因子。外泌体 miR-199a-5p 被内皮细胞吞噬并增加内皮细胞的紧密连接。TME 中卵巢癌细胞和内皮细胞之间的串扰证实 miR-199a-5p 可能是一种潜在的生物标志物，可作为卵巢癌治疗的新治疗靶点。