Fig 1: miR-138-5p could target SDC3 in OvC cells. (A) TargetScan predicted the two binding sites between miR-138-5p and SDC3. (B) SDC3 was proved to be targeted by miR-138-5p using luciferase assay. WT, wild-type SDC3 3’UTR containing the two binding sites. Mut1, mutant SDC3 mRNA 3’UTR without the binding site 1. Mut2, mutant SDC3 mRNA 3’UTR without the binding site 2. Co-Mut, mutant SDC3 3’UTR without the two binding sites. NC, negative control. Mimic, miR-138-5p mimic. (C) RNA pull-down assay further proved the binding site between SDC3 mRNA 3’UTR and miR-138-5p. Bio-NC, biotin-labeled negative control. Bio-miR-138-5p, biotin-labeled miR-138-5p mimic. (D) RT-qPCR analysis revealed that the SDC3 mRNA expression was upregulated in OvC tissues compared with contralateral normal fallopian tube tissues (cNor FT) and normal fallopian tube tissues from patients with benign gynecological tumor (Nor FT). (E) The negative correlation between SDC3 mRNA expression and miR-138-5p in OvC tissues. (F) IHC assay showed the low-level SDC3 in high-level miR-138-5p tissues. H&E pathological staining was performed to indicate the ovarian tumor type. The tumor type in all images were identified as high-grade serous carcinoma (HGSC), while the tissues in left and right column images was respectively 1c sub-stage and 3c sub-stage. Scale bar, 100 µm. (G) The expression of SDC3 mRNA decreased in CAOV3 and SKOV3 cells with the transfection of miR-138-5p mimic. (H) The expression of SDC3 protein reduced in CAOV3 and SKOV3 cells with the transfection of miR-138-5p mimic. **P<0.001.
Fig 2: Expression of PTN and its receptors. (A) Western blots for the expression level of PTN and its receptors. (B) Determination of the protein level of PTN and its receptors. *P<0.05. The experiments were repeated at least three times independently. PE, preeclampsia; Wt, wild-type; tax, tamoxifen; PTN, pleiotrophin; SDC1,syndecan-1; SDC3,syndecan-3; RPTPß, receptor protein tyrosine phosphatase ß/?; ALK, anaplastic lymphoma kinase; P, PE; N, normal.
Fig 3: The positive effect of miR-138-5p inhibitor on OvC progression was relieved by SDC3 silence. (A) The high transfection efficiency of sh-SDC3 and miR-138-5p inhibitor was detected by qRT-PCR in CAOV3 and SKOV3 cells. (B) The high transfection efficiency of sh-SDC3 and miR-138-5p inhibitor was further measured by western blot analysis in CAOV3 and SKOV3 cells. (C) sh-SDC3 attenuated the promotive effect of miR-138-5p inhibitor on cell viability in CAOV3 and SKOV3 cells. (D) The positive effect of miR-138-5p inhibitor on colony formation was overturned by sh-SDC3. (E) The positive role of miR-138-5p inhibitor in cell migration was relieved by sh-SDC3. (F) The promotion effect of miR-138-5p inhibitor on cell invasion was impaired by sh-SDC3. Blank, blank control. NC, negative control. Sh, sh-SDC3. Inhibitor, miR-138-5p inhibitor. *P<0.05, **P<0.001 compared with blank control. ##P<0.001 compared with co-transfection of sh-SDC3 and miR-138-5p inhibitor.
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