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

The prognosis for colorectal cancer (CRC) patients with liver metastasis remains poor, and the molecular mechanisms driving CRC liver metastasis are still not fully understood. Hypoxia-induced extracellular vesicles (H-EVs) derived from tumors have emerged as key players in inducing angiogenesis by transferring non-coding RNAs. However, the specific role of CRC-derived hypoxic EVs (H-EVs) in regulating the formation of the pre-metastatic microenvironment (PMN) by inducing angiogenesis remains unclear. Our study demonstrates that H-EVs induce both angiogenesis and liver metastasis. Through microRNA microarray analysis, we identified a reduction in miR-6084 levels within H-EVs. We found that miR-6084 inhibits angiogenesis by being transferred to endothelial cells via EVs. In endothelial cells, miR-6084 directly targets ANGPTL4 mRNA, thereby suppressing angiogenesis through ANGPTL4-mediated JAK2/STAT3 pathway. Furthermore, we uncovered that SP1 acts as a transcription factor regulating miR-6084 transcription, while HIF1A decreases 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 hypoxic EVs promote angiogenesis and liver metastasis through the HIF1A/SP1/miR-6084/ANGPTL4 axis. Additionally, miR-6084 holds promise as a potential diagnostic and prognostic biomarker for CRC liver metastasis. To explore the main microRNAs mediating angiogenesis in CRC-derived hypoxic extracellular vesicles, SW620 cells were cultured under normoxic (20% oxygen concentration) or hypoxic conditions (1% oxygen concentration) in DMEM (Cytiva, USA) plus 10% fetal bovine serum (Excell Bio, HK) and 1% penicillin/ streptomycin (Beyotime, China) for 48 hours. Then, SW620 cell supernatant were collected and EVs were isolated by ultrafiltration plus size exclusion chromatography. Next, total RNA was extracted, and an Affymetrix miRNA 4.0 microarray was used for expression profiling analysis. RNA isolation, adding Poly A tail and biotin labeling, hybridization, Washing, staining, and scanning were performed by Echo biotech (Beijing, China) according to the Affymetrix protocol. Scanned signal was processed following a standard instruction of ThermoScientific Affymetrix pipeline. Raw data (CEL files) were generated using AGCC. Signal summarization and normalization were performed using Affymetrix Expression Console™ software with the RMA+DABG algorithm (Robust Multi-array Average with Detection Above Background).