Fig 2: CXCR4 Promotes Cisplatin Resistance of OC Cells through Suppressing the let-7a/BCL-XL/S Axis(A) Relative let-7a expression levels in OC tissues and adjacent non-cancerous ovarian tissues. Expressional changes of let-7a in OC tissues were analyzed by qRT-PCR. (B) Negative correlation of let-7a with CXCR4 expression in OC tissues. (C) Relative let-7a expression in SKOV3, Caov3, OVCAR3, A2780, and CAV644 cells. Its expression in HOSE cells was detected as the control. (D and E) Silencing and elevation of CXCR4 expression in SKOV3 and Caov3 cells with shCXCR4 and pcDNA3.1-CXCR4 plasmids, respectively. Expression levels of CXCR4 in SKOV3 and Caov3 cells were detected by qRT-PCR (D) and western blotting (E). (F) Relative expression of let-7a in SKOV3 and Caov3 cells with silenced or elevated CXCR4 expression by qRT-PCR. (G) Influences of shCXCR4 and let-7a inhibitor on cisplatin resistance of SKOV3 and Caov3 cells. Cell viabilities were detected by the CCK-8 method under treatments with 2, 4, 6, 8, or 10 μM cisplatin, and the resistances were assessed by the IC50 values. (H) Effects of shCXCR4 and let-7a inhibitor on cisplatin-induced SKOV3 and Caov3 cell apoptosis. Flow cytometry was performed to detect cell apoptosis. (I) Abundances of BCL-XL/S proteins in SKOV3 and Caov3 cells transfected with shCXCR4 and let-7a inhibitor. Protein levels were analyzed by western blotting. (J) The binding of let-7a with BCL-XL/S gene sequences predicted by informatics (top) and confirmed by dual-luciferase reporter assay (bottom). NC, negative control; CXCR4, chemokine receptor 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IC50, half maximal inhibitory concentration; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig 3: miR-548e Suppresses CXCR4 Expression in OC via Direct Binding(A) Relative CXCR4 expression in OC tissues and adjacent non-cancerous tissues by qRT-PCR. (B) Relative CXCR4 expression in primary and metastatic ovarian tumor tissues from seven metastatic OC patients by qRT-PCR. (C and D) CXCR4 expressional levels in OC cell lines SKOV3, Caov3, OVCAR3, A2780, COV644, and HOSE cells by qRT-PCR (C) and western blotting (D). HOSE cells were measured as the control. (E) Correlation of CXCR4 expression with OC patient stages based on TCGA data. (F and G) The correlations of CXCR4 expression with ZFAS1 (F) and miR-548e (G) expression in OC tissues. (H) The binding sites of miR-548e with CXCR4 gene 3′ UTR region. (I) The binding validation through dual-luciferase reporter assay in HEK293T cells. (J) Silencing or overexpression of miR-548e expression in both SKOV3 and Caov3 cells with miR-548e inhibitor and mimics, respectively. miR-548e expression was evaluated by qRT-PCR. (K and L) The expression of CXCR4 in SKOV3 and Caov3 cells with silenced or enhanced miR-548e expression. CXCR4 level was detected by qRT-PCR (K) and western blotting (L), respectively. CXCR4, chemokine receptor 4; SD, standard deviation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ZFAS1, zinc finger antisense 1; WT, wild-type; MUT, mutant; UTR, untranslated region; NC, negative control; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig 4: CXCR4 is involved in proliferation, EMT, and stemness characteristics induced by matrix stiffness. mRNA (A) and protein (B) were significantly up-regulated with increasing matrix stiffness. (C) Expression levels of CXCR4 in HCC tissues with normal (groups L, N=25), medium (group M, N=35), and high liver stiffness backgrounds (group H, N=46). (D) Hep3B and Huh7 cells transfected with shRNA were subjected to Western blotting for CXCR4 expression. (E) MTT revealed that stiffness-mediated HCC proliferation was abrogated by CXCR4 gene deletion. HCC cells seeded on low or high stiffness were transduced with NT shRNA or CXCR4 shRNA. Stiffness-mediated HCC (PCNA) and CyclinD1 expression (F), EMT (G) and stemness characteristics (H) were abrogated by CXCR4 knockdown. Bar: 70μM. *P < 0.05, **P < 0.01.

Fig 5: CXCR4 is involved in YAP activation and nuclear localization by matrix stiffness. Subcellular fractionation (A) and IF (B) assay were used to analyze stiffness-induced YAP nuclear accumulation. (C) RT-qPCR and WB (D) analysis of YAP target genes CTGF and ANKRD1 mRNA and protein levels were performed in Hep3B and Huh7 cells on low or high stiffness. Stiffness-mediated YAP nuclear accumulation and target gene expression measured by WB was abrogated by CXCR4 shRNA (E) or inhibitor AMD3100 (100 nM) (F). (G) Stiffness-mediated YAP upregulation and nuclear accumulation were abrogated by CXCR4 shRNA in Hep3B cells. (H, I) Hep3B and Huh7 cells transduced with CXCR4 overexpression vectors were seeded on low or high stiffness for WB and IF. CXCR4 led to YAP upregulation and nuclear accumulation on low stiffness. Bar:20μM.*P < 0.05.