Fig. 6.

miR‐140‐5p negatively modulates FBN1 expression. Mimic or inhibitor or shRNA was used to interfere with the levels of miR‐140‐5p and PGM5‐AS1 in U2OS cells. (A) Prediction results of the gene targeted by miR‐140‐5p. The ovals represent the predicted results and the upregulated genes in the microarray dataset GSE41445. The overlapping section refers to the intersection of the five ovals. (B) Expression of MMP, FBN1, and KLF9 in U2OS cells with altered PGM5‐AS1 expression detected by RT‐qPCR. (C) FBN1 level in osteosarcoma tissues and adjacent normal bone tissues from 73 osteosarcoma patients tested using RT‐qPCR. (D) The mRNA expression of FBN1 in human normal cell line hFOB 1.19 and five osteosarcoma cell lines U2OS, SaOS‐2, MG63, HOS, and SJSA1. *P < 0.05, vs. human normal cell line hFOB 1.19 cells. (E) Relative expression of FBN1 in U2OS cells with altered miR‐140‐5p expression detected by RT‐qPCR. (F) Protein expression of FBN1 in U2OS cells with altered miR‐140‐5p expression detected by western blot analysis. (G) The binding of miR‐140‐5p and FBN1 confirmed by dual‐luciferase reporter gene assay. (H, I) Luciferase activity of FBN1 measured by dual‐luciferase reporter gene assay. (J) Interaction between miR‐140‐5p and FBN1 in U2OS cells as detected by RIP assay. (K) Interaction between miR‐140‐5p and FBN1 in U2OS cells with altered PGM5‐AS1 expression as measured using RIP assay. *P < 0.05, vs. U2OS cells treated with sh‐NC, adjacent normal bone tissues, IgG treatment, or U2OS cells treated with both sh‐NC and inhibitor NC; ^# P < 0.05, vs. U2OS cells administered with inhibitor NC or AGO2 enrichment in sh‐NC. The measurement data were expressed as mean ± standard deviation. The data in C were analyzed using the paired t‐test. Unpaired t‐test was employed for data analysis between two groups and one‐way ANOVA, as well as Tukey's post hoc test for comparison of data among multiple groups. The experiment was repeated three times.