MicroRNA-6084 orchestrates angiogenesis and liver metastasis in colorectal cancer via extracellular vesicles
Yang Zhang, Xuyang Yang, Su Zhang, Qing Huang, Sicheng Liu, Lei Qiu, Mingtian Wei, Xiangbing Deng, Wenjian Meng, Hai-Ning Chen, Yaguang Zhang, Junhong Han, Ziqiang Wang
Yang Zhang, Xuyang Yang, Su Zhang, Qing Huang, Sicheng Liu, Lei Qiu, Mingtian Wei, Xiangbing Deng, Wenjian Meng, Hai-Ning Chen, Yaguang Zhang, Junhong Han, Ziqiang Wang
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Research Article Angiogenesis Gastroenterology Oncology

MicroRNA-6084 orchestrates angiogenesis and liver metastasis in colorectal cancer via extracellular vesicles

Abstract

The prognosis for colorectal cancer (CRC) patients with liver metastasis remains poor, and the molecular mechanisms driving CRC liver metastasis are not fully understood. Tumor-derived hypoxia-induced extracellular vesicles have emerged as key players in inducing angiogenesis by transferring noncoding RNAs. However, the specific role of CRC-derived hypoxic extracellular vesicles (H-EVs) in regulating premetastatic microenvironment (PMN) formation by inducing angiogenesis remains unclear. Our study demonstrates that H-EVs induce angiogenesis and liver metastasis. Through microRNA microarray analysis, we identified a reduction in miR-6084 levels within H-EVs. We found that miR-6084 inhibited angiogenesis by being transferred to endothelial cells via EVs. In endothelial cells, miR-6084 directly targeted angiopoietin like 4 (ANGPTL4) mRNA, thereby suppressing angiogenesis through the ANGPTL4-mediated JAK2/STAT3 pathway. Furthermore, we uncovered that specificity protein 1 (SP1) acted as a transcription factor regulating miR-6084 transcription, while hypoxia-inducible factor 1A (HIF1A) decreased miR-6084 expression by promoting SP1 protein dephosphorylation and facilitating ubiquitin-proteasome degradation in SW620 cells. In clinical samples, we observed low expression of miR-6084 in plasma-derived EVs from CRC patients with liver metastasis. In summary, our findings suggest that CRC-derived H-EVs promote angiogenesis and liver metastasis through the HIF1A/SP1/miR-6084/ANGPTL4 axis. Additionally, miR-6084 holds promise as a diagnostic and prognostic biomarker for CRC liver metastasis.

Authors

Yang Zhang, Xuyang Yang, Su Zhang, Qing Huang, Sicheng Liu, Lei Qiu, Mingtian Wei, Xiangbing Deng, Wenjian Meng, Hai-Ning Chen, Yaguang Zhang, Junhong Han, Ziqiang Wang

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Figure 4

HIF1A decreases miR-6084 expression by regulating SP1 protein ubiquitination and proteasomal degradation.

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HIF1A decreases miR-6084 expression by regulating SP1 protein ubiquitina...
(A) Two databases were applied to predict the potential transcription factors of miR-6084. (B) The DNA target sequence logos of transcription factor SP1 predicted by JASPAR database. (C) A schematic diagram of SP1-binding sites in the promoter of miR-6084. (D) Expression level of SP1 mRNA was detected by RT-qPCR after knockdown of SP1. β-Actin was used as internal control. Expression level of miR-6084 detected by RT-qPCR after knockdown of SP1. U6 was used as internal control. Sh, short hairpin RNA. (E) ChIP assays were performed to verify the binding between SP1, HIF1A, and the miR-6084 promoter in SW620. (F) Expression level of HIF1A mRNA in SW620 under normoxic or hypoxic condition detected by RT-qPCR. Expression level of SP1 mRNA in SW620 under normoxic or hypoxic condition detected by RT-qPCR. β-Actin was used as internal control. (G) Expression level of HIF1A and SP1 in SW620 under normoxic (-N) or hypoxic (-H) condition detected by Western blot. (H) Nucleocytoplasmic separation was performed and expression level of SP1 in SW620 nucleus and cytoplasm under normoxic or hypoxic condition was detected by Western blot. SNP, soluble nuclear protein; H3, histone H3. (I) SW620 cells were treated by CHX (protein synthesis inhibitor) and MG132 (proteasome inhibitor), and SP1 protein stability was measured by Western blot. (J) IP assays were conducted to verify the binding between SP1 and HIF1A, BTRC, or Ub. (K) Expression level of p-SP1 in SW620 under normoxic/hypoxic condition or treated by protein phosphatase 1/2A (PP1/2A) cantharidin detected by Western blot. Each experiment was conducted at least 3 times. Data were presented as mean ± SD. Statistical significance was assessed with 2-tailed unpaired Student’s t test (D–F), * P < 0.05, ** P < 0.01, *** P < 0.001.