Cervical squamous cell carcinoma-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis by targeting VASH1

Abstract

Cancer-secreted exosomal miRNAs are emerging mediators of cancer-stromal cross-talk in the tumor environment. Our previous miRNAs array of cervical squamous cell carcinoma (CSCC) clinical specimens identified upregulation of miR-221-3p. Here, we show that miR-221-3p is closely correlated with peritumoral lymphangiogenesis and lymph node (LN) metastasis in CSCC. More importantly, miR-221-3p is characteristically enriched in and transferred by CSCC-secreted exosomes into human lymphatic endothelial cells (HLECs) to promote HLECs migration and tube formation in vitro, and facilitate lymphangiogenesis and LN metastasis in vivo according to both gain-of-function and loss-of-function experiments. Furthermore, we identify vasohibin-1 (VASH1) as a novel direct target of miR-221-3p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of VASH1 could respectively rescue and simulate the effects induced by exosomal miR-221-3p. Importantly, the miR-221-3p-VASH1 axis activates the ERK/AKT pathway in HLECs independent of VEGF-C. Finally, circulating exosomal miR-221-3p levels also have biological function in promoting HLECs sprouting in vitro and are closely associated with tumor miR-221-3p expression, lymphatic VASH1 expression, lymphangiogenesis, and LN metastasis in CSCC patients. In conclusion, CSCC-secreted exosomal miR-221-3p transfers into HLECs to promote lymphangiogenesis and lymphatic metastasis via downregulation of VASH1 and may represent a novel diagnostic biomarker and therapeutic target for metastatic CSCC patients in early stages.

Fig. 1

Upregulation of miR-221-3p in the primary tumor positively correlates with lymph node metastasis of CSCC. a ISH scores (HSCORE) of miR-221-3p were analyzed between the LN-N (LN negative; n = 64) and LN-P (LN positive; n = 43) groups in CSCC specimens. b Staining of miR-221-3p and LYVE1 (lymphatic marker) in serial sections of CSCC specimens. Representative micrographs are shown (left). The tumor cells are indicated by black arrows. The lymphatic vessels are indicated by red arrows. Correlations between miR-221-3p staining and peritumoral lymphatic vessel density (PLVD) were analyzed (right). Scale bar, 50 µm. Error bars represent the mean ± SD of three independent experiments. ***, P < 0.001

Fig. 2

miR-221-3p can be enriched in CSCC-secreted exosomes and transferred to HLECs. a Morphology of Ect1 and Siha-secreted exosomes was confirmed by transmission electron microscopy. Scale bar, 50 nm. b Positive markers (CD63 and CD81) of Ect1 and Siha-secreted exosomes were detected by western blot. c Human lymphatic endothelial cells (HLECs) pre-treated with PKH67-labeled exosomes secreted by Ect1 and Siha for 48 h were stained by phalloidin (red) and DAPI (blue) for confocal microscopy analysis. Scale bar, 20 µm. d Basic miR-221-3p levels in indicated cells and paired exosomes were detected by qRT-PCR. e miR-221-3p levels in HLECs pre-treated with PBS or indicated exosomes for 24 h were detected by qRT-PCR. Exo, exosomes. miR-221, miR-221-3p. Error bars represent the mean ± SD of three independent experiments

Fig. 3

CSCC-secreted exosomal miR-221-3p promotes lymphangiogenesis in vitro. a Cellular and exosomal miR-221-3p levels in Ect1 stably transfected with miR-221-3p overexpression (miR-221) or negative control (miR-NC) lentivectors were detected by qRT-PCR. b Cellular and exosomal miR-221-3p levels in Siha stably transfected with miR-221-3p knockdown (anti-221) or negative control (anti-NC) lentivectors were detected by qRT-PCR. c Transwell migration assay in HLECs pre-treated with indicated exosomes. Average migrated cells per field were calculated. d Tube formation assay in HLECs pre-treated with indicated exosomes. Average length of tubes per field were calculated. e Representative micrographs of migration (upper panel) and tube formation assay (lower panel) in HLECs pre-treated with indicated exosomes are shown. Scale bar, upper panel, 50 µm; lower panel, 100 µm. miR-NC exo, Ect1/miR-NC secreted exosomes. miR-221 exo, Ect1/miR-221-3p secreted exosomes. anti-NC exo, Siha/anti-NC secreted exosomes. anti-221 exo, Siha/anti-221-3p secreted exosomes. Error bars represent the mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001

Fig. 4

CSCC-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis in vivo. a popliteal lymph node metastasis model was established in nude mice by inoculating the footpad with Siha/anti-221-3p (5 × 10⁶) stably expressing mCherry. When footpad tumor size reached 50 mm³, exosomes (10 µg) secreted by Ect1/miR-NC, Ect1/miR-221-3p, Siha/anti-NC, or Siha/anti-221-3p were then injected into the center of the tumors (n = 3/group, repeated twice) twice a week. After five injections, primary tumors reached a comparable size of ~ 150 mm³, and then footpad tumors and popliteal LNs were collected for study. a Staining of miR-221-3p and LYVE1 in serial sections of mice footpad tumors. Representative micrographs of positive staining are shown (left). The tumor cells are indicated by black arrows. The lymphatic vessels are indicated by red arrows. The correlation between miR-221-3p levels and PLVD was statistically analyzed (right). Scale bar, 20 µm. b Staining of mCherry in popliteal LNs from mice treated with the indicated exosomes. Representative micrographs are shown. Metastasis-positive LNs were identified by staining for cancer cell-expressed mCherry. Scale bar, upper panel, 200 µm; lower panel, 20 µm. c The ratio of metastasis-positive to total dissected popliteal LNs from mice treated with the indicated exosomes. ***, P < 0.001

Fig. 5

CSCC-secreted exosomal miR-221-3p targets lymphatic VASH1 to induce lymphangiogenesis in HLECs. a RNA sequence alignment between miR-221-3p and the 3′-UTR of VASH1 (left), and the effect of miR-NC and miR-221-3p on the activity of the luciferase reporter containing either wild type (WT) or mutant type (MT) were tested by dual-luciferase reporter assay (right). b–c RNA and protein levels of VASH1 were, respectively, detected by qRT-PCR and western blot in HLECs transfected with miR-221-3p mimic or negative control (NC) compared with those treated with the indicated exosomes. d–e RNA and protein levels of VASH1 were, respectively, detected by qRT-PCR and western blot in HLECs treated with indicated exosomes in the presence of VASH1 overexpression plasmid (VASH1) or vector control and VASH1 silence fragment (siVASH1) or siRNA control. f–g Overexpression of VASH1 rescued the biologic effects associated with exosomal miR-221-3p, whereas knockdown of VASH1 simulated the biologic effects associated with exosomal miR-221-3p through cell migration and tube formation assays. Error bars represent the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 6

CSCC-secreted exosomal miR-221-3p is associated with VASH1 expression and lymphatic metastasis. a miR-221-3p levels in circulating exosomes of CSCC patients with LN-N (LNs negative; n = 20) and LN-P (LNs positive; n = 20) were detected by qRT-PCR. b Sprout assay in HLECs pre-treated with circulating exosomes from LN-N patients with low miR-221-3p levels or LN-P patients with high miR-221-3p levels in the presence of miR-221-3p inhibitor (anti-221) or negative control (NC). Representative micrographs are shown (left). Average sprouts per spheroid were calculated (right). Scale bar, 50 µm. c Correlation analyses of exosomal miR-221-3p, tumor miR-221-3p, peritumoral lymphatic vessel density and VASH1 levels in the above CSCC patients. CSCC specimens subjected to ISH for tumor miR-221-3p and double-label IF for stromal VASH1 (red) and LVYE1 (green). Representative staining micrographs are shown (upper panel). T, tumor. Scale bar, 20 µm. Paired circulating exosomal miR-221-3p levels in CSCC patients were detected by qRT-PCR. Correlation analyses were calculated between two sets of quantified data as indicated (lower panel). d Illustrative model showing the mechanism whereby CSCC-secreted exosomal miR-221-3p promotes lymphangiogenesis by downregulating lymphatic VASH1 expression that then facilitates lymphatic metastasis. Error bars represent the mean ± SD of three independent experiments. * P < 0.05; ***, P < 0.001