Fig 2: miR-548e Inhibits OC Cell Proliferation, Migration, and Invasion by Suppressing CXCR4 Expression(A and B) The proliferation rates of SKOV3 and Caov3 cells with enhanced miR-548e and CXCR4 expression levels. Both the CCK-8 (A) and clone-formation assay (B) were used to detect cell proliferation. (C) The migration capacities of SKOV3 and Caov3 cells transfected with miR-548e mimics and CXCR4-overexpressing plasmids. Cell migration was measured through the wound-healing assay. (D) The invasion capacities of SKOV3 and Caov3 cells with enhanced miR-548e and CXCR4 expression levels. Cell invasion capacity was analyzed with the Transwell system. (E) E-cadherin, N-cadherin, vimentin, MMP-2, and Slug protein levels in SKOV3 and Caov3 cells transfected with miR-548e mimics and CXCR4-overexpressing plasmids. Protein abundance was measured by western blotting. NC, negative control; CXCR4, chemokine receptor 4; MMP-2, matrix metalloproteinases 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig 3: miR‐126‐3p replacement and miR‐221 inhibition reduced lung dissemination by inhibiting CXCR4 levels. (A) Migration index after miR‐126‐3p and miR‐221‐3p modulation of three lung cancer cells (n = 5). (B) Invasive capacity of lung cancer cells transfected with miR‐126/221 (n = 4). (C) Number of transmigrated lung cancer cells through endothelial monolayer after miRNA modulation (n = 3). (D) Luciferase activity for 3′UTR of CXCR4 wild‐type and mutated sequence after miR‐126 and SCR transfection in A549 cells (n = 4). CXCR4 levels after miR‐126‐3p replenishment and miR‐221‐3p inhibition evaluated by qPCR (n = 3) (E) and flow cytometry (n = 3) (F). (G) Representative images and quantification of CXCR4 in all the three cell lines after miRNA modulation (n = 3). (H) Migration, invasion and transendothelial index of lung cancer cells transient transfected with CXCR4 siRNA and controls (n = 3 for each cell line). Statistically significant differences were determined with Student's t‐test when comparing two groups or ANOVA test for multiple comparisons. *P < 0.05 versus controls. Data are expressed as mean ± S.E.M.

Fig 4: Combined replenishment of miR‐126‐3p and inhibition of miR‐221‐3p reduced xenograft growth in vivo. (A) A549 transfected with both miR‐126‐3p mimic (m126) and miR‐221‐3p inhibitor (i221) showed a reduction in tumour growth in immunodeficient mice compared with SCR and CTR controls. Ki‐67, cleaved caspase 3 and phosphor‐AKT quantification and representative images in A549 m126+i221 cells compared with controls. Necrosis was evaluated on haematoxylin & eosin (H&E) staining in subcutaneously implanted xenograft after miRNA transfection (n = 5 for each group) Scale bar: 200 µm (B) miR‐126‐3p replacement and miR‐221‐3p inhibition reduced tumour growth in subcutaneous Calu1 xenografts in immunodeficient mice. Ki‐67, cleaved caspase 3 and phospho‐AKT count and staining revealed a reduction in proliferation rate and increase in apoptosis of these cells. Necrosis was evaluated on H&E staining (n = 5 for each group). Scale bar: 200 µm. (C) Pan‐cytokeratin and CXCR4 staining and quantification in the lungs of mice treated with A549 transfected miR‐126 and/or miR‐221 alone or in combination (n = 4 for each group). Scale bar: 200 µm. (D) Quantification of A549 and A549/CXCR4+ cells in the lungs of mice by flow cytometry (n = 4). (E) Representative images and quantification of pan‐cytokeratin and CXCR4 staining of the lungs from H460 transfected with miR‐126‐3p, miR‐221 inhibitors alone or in combination and control (n = 4 mice for each group). Scale bar: 200 µm. (F) Flow cytometric quantification of H460‐disseminating cells and H460‐CXCR4 positive in the lung of treated mice (n = 4 mice for each group). (G) Tumour growth curves of mice treated with R peptide and CTR (n = 4 for each group). Scale bar: 200 µm. (H) Pan‐cytokeratin staining and flow cytometric quantification of the lungs from H460 treated with R peptide (n = 4 for each group). Data are expressed as mean ± S.E.M. Statistically significant differences were determined with Student's t‐test when comparing two groups or ANOVA test for multiple comparisons *P < 0.05, ***P < 0.0001.

Fig 5: miR-548e Impairs OC Cell Resistance to Cisplatin by Repressing CXCR4 Expression(A) Effects of miR-548e mimics and CXCR4 overexpression on resistance of SKOV3 and Caov3 cells to cisplatin treatments. Viabilities of cells treated with 2, 4, 6, 8, or 10 μM cisplatin were detected using the CCK-8 assay, and IC50 values were calculated to evaluate drug resistance. (B) The cisplatin-induced SKOV3 and Caov3 cell apoptosis after being transfected with miR-548e mimics and CXCR4 overexpressing plasmids. Flow cytometry was used to detect cell apoptosis. NC, negative control; CXCR4, chemokine receptor 4; IC50, half maximal inhibitory concentration; ∗p < 0.05 and ∗∗p < 0.01.